Designing a Polyepitope Prophylactic Vaccine against Human Cytomegalovirus Dasari Vijayendra BSc, MSc School of Biomolecular and Physical Sciences Science, Environment, Engineering and Technology Griffith University Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy March 2012 i ii ABSTRACT Human cytomegalovirus (CMV) is a ubiquitous β human herpes virus that establishes lifelong infection. Primary CMV infection in immunocompetent individuals is generally asymptomatic, but in congenitally infected children and in transplant patients CMV causes significant morbidity and mortality. Based on the life- time cost to the health care system and its impact on human suffering, development of a vaccine to prevent congenital human cytomegalovirus (CMV) infection has been assigned the highest priority by the Institute of Medicine of the National Academy of Sciences (US) and US National Vaccine Program Office. Therefore there is an emerging need for the development of an effective CMV vaccine. The main objective of vaccine development against CMV is to reduce the risk of CMV associated injury to the developing fetus and in immunocompromised individuals such as recipients of solid organ and hematopoietic stem cell transplants. In immunocompetent individuals CMV infection is maintained under strict control by the immune system by a combination of humoral and cellular immune responses. Thus, an effective CMV + + vaccine should be designed to induce virus-specific antibody, and CD4 and CD8 T cell responses. In spite of extensive efforts over the last 30 years, a clinically licensed vaccine formulation with convincing clinical efficacy remains elusive. Natural CMV infection is characterized by a strain-specific neutralising antibody response. This is particularly relevant in clinical settings such as transplantation and pregnancy where re-infection with heterologous strains is occurring, and the immune system does not mount an effective response against the infecting strain due to underlying immunosuppression. There is an emerging argument that a CMV vaccine which induces high titres of cross-neutralizing antibodies will be more effective in protecting individuals from infection with antigenically different iii CMV strains. In addition, induction of cell-mediated immunity offers the additional advantage of targeting virus-infected cells. Initial studies were undertaken to investigate a vaccine formulation to elicit gB-specific cross-neutralisation and cellular immune responses. We found that a novel formulation combining gB with a TLR9 agonist and immune stimulating complexes (AbISCO 100) was able to induce cross- neutralisation and cellular immune responses. Although a gB vaccine can induce antibody and cellular immune responses, due to the complexity of CMV immunity, a single antigen is unlikely to be broadly + effective. Therefore, in subsequent studies we aimed to induce CD8 T cell responses by targeting multiple CMV antigens. We employed a novel platform technology that + targets CMV-specific CD8 T cell responses to multiple epitopes restricted through a range of HLA class I molecules. Using a prokaryotic experssion system, the multiple + minimal HLA class I-restricted CD8 T cell epitopes were expressed as a polyepitope protein. Exogenous loading of human cells with polyepitope protein resulted in the efficent processing and presentation of the epitopes to virus-specific T cells. This polyepitope protein could be reproducibly used to expand CMV-specific T cells from healthy virus carriers. Characterisation of endogenous processing of the polyepitope revealed a novel cross-presentation pathway that is independent of MHC class I peptide transporters, and requires the proteasome and the autophagy dependent pathway. There is now convincing evidence that the successful development of an effective CMV vaccine will require improved formulation and adjuvant selection. To develop a vaccine formulation based on the gB and polyepitope proteins (gB- CMVpoly) we investigated delivery in combination with human compatible TLR agonists. Durable anti-viral neutralising antibody responses, and polyfunctional Th1 iv + + CD4 and CD8 T cell responses could be generated in HLA A2 transgenic mice following formulation with TLR4 and TLR9 agonists. This combination of adjuvants was subseqeuntly shown to induce inflammatory cytokine production by dendritic cell subsets that are critcal for the activation of cellular immunity. We next evaluated the immunogenicity of the gB-CMVpoly vaccine with the ® human compatible adjuvants IC31 , AS01 or AS15, which have been successfully used in various human vaccine formulations with minimal side effects. All three adjuvants consistently induced a strong neutralising gB-specific antibody response, + and long-lived polyfunctional gB-specific CD4 T cells. However, CMV-specific + ® CD8 T cells were only detectable following formulation of gB-CMVpoly with IC31 or AS01. Based on these results, we propose that a CMV vaccine formulation based on ® gB-CMVpoly proteins in combination with IC31 or AS01 should be assessed in human clinical trials. v vi Statement of Originality This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. (Signed)_____________________________ Name of Student Vijayendra Dasari vii viii TABLE OF CONTENTS Chapter 1: Introduction and Review of Literature ............................... 1 1.1 Historical milestones in vaccine development ............................................. 1 1.2 Immunology of Vaccines ............................................................................... 6 1.2.1 Innate immunity .................................................................................................. 7 1.2.1.1. Toll-like receptors .............................................................................................. 8 1.2.1.2 Nod-like receptors ............................................................................................ 10 1.2.1.3 RIG-I-like receptors .......................................................................................... 10 1.2.1.4 C-type lectin receptors ...................................................................................... 11 1.2.2 Adaptive immunity .................................................................................................. 12 1.2.2.1. T lymphocytes ................................................................................................. 12 + 1.2.2.1.1 CD4 T cells............................................................................................... 13 + 1.2.2.1.2 CD8 T cells............................................................................................... 16 1.2.2.1.3 Effector and memory cells differentiation ................................................. 17 1.2.2.1.4 Expansion phase ........................................................................................ 18 1.2.2.1.5 Contraction phase or death phase .............................................................. 19 1.2.2.1.6 Mechanism of action of cytotoxic T lymphocytes (CTLs) ........................ 20 1.2.2.1.7 Memory phase ........................................................................................... 21 ix 1.2.2.2 B lymphocytes .................................................................................................. 24 1.2.2.2.1 B cell activation and antibody response .................................................... 25 1.2.2.3 Antigen recognition and processing via MHC class I pathway ........................ 29 1.2.2.4 Antigen recognition and processing via MHC class II pathway ....................... 31 1.3 Vaccine design .............................................................................................. 33 1.3.1. Subunit vaccines ..................................................................................................... 34 1.3.1.1 Reverse vaccinology ......................................................................................... 35 1.3.1.2 Transcriptomics ................................................................................................ 35 1.3.1.3 Functional genomics ......................................................................................... 36 1.3.1.4 Immunomics ..................................................................................................... 37 1.3.1.5 Structural vaccinology ...................................................................................... 37 1.3.1.6 Vaccinomics ..................................................................................................... 38 1.3.1.7 Proteomics ........................................................................................................ 38 1.3.2 Epitope driven vaccines ........................................................................................... 39 1.3.2.1 Advantages of epitope based vaccines .............................................................. 42 1.4. Adjuvants ......................................................................................................... 43 1.4.1 Adjuvants in clinical practice .................................................................................. 45 1.4.1.1 Aluminium salt adjuvants ................................................................................. 45 1.4.1.2 MF59 ................................................................................................................ 46 x 1.4.1.3 Monophosphoryl Lipid A (MPL) ..................................................................... 47 1.4.1.4 Monophosphoryl lipid (MPL) A combinations ................................................ 48 1.4.1.5 Cholera toxin B subunit (CTB) ......................................................................... 48 1.4.1.6 VLP and IRIV ................................................................................................... 49 1.4.2 Adjuvants in clinical research .................................................................................. 49 1.4.2.1 TLR ligands ...................................................................................................... 49 1.4.2.2 Cell surface TLRs ............................................................................................. 50 1.4.2.3 Intracellular TLRs ............................................................................................. 51 1.4.2.4 IC31 .................................................................................................................. 53 1.4.2.5 Montanides ....................................................................................................... 53 1.4.2.6 Saponins ............................................................................................................ 54 1.4.2.7 Muramyl dipeptide (MDP) ............................................................................... 56 1.5 Adjuvant safety ................................................................................................. 56 1.6 Human cytomegalovirus: Immunobiology and Pathogenesis ...................... 57 1.6.1 Molecular characteristics of human cytomegalovirus ............................................. 57 1.6.2 CMV pathogenesis................................................................................................... 58 1.6.2.1 Congenital infection .......................................................................................... 59 1.6.2.2 CMV pathology in transplant recipients ........................................................... 60 1.6.3 Immunobiology of CMV ......................................................................................... 61 xi 1.6.3.1 Innate immunity against CMV ......................................................................... 62 1.6.3.2 Humoral responses ............................................................................................ 62 1.6.3.3 T cell mediated immune responses ................................................................... 63 1.6.4 Cytomegalovirus vaccine research .......................................................................... 65 1.7 Specific aims of study ....................................................................................... 71 Chapter 2: Materials and Methods ....................................................... 73 2.1 List of Chemicals .............................................................................................. 73 2.2 Equipment ......................................................................................................... 75 2.3 Media and buffers ............................................................................................. 76 2.4 Molecular methods ........................................................................................... 78 2.4.1 Agarose gel electrophoresis ..................................................................................... 78 2.4.2 Transformation of chemically competent E. coli cells ............................................ 78 2.4.3 Plasmid DNA amplification and purification .......................................................... 79 2.4.4 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) ......... 79 2.4.5 Coomassie blue staining .......................................................................................... 80 2.5 Cell culture techniques ..................................................................................... 80 2.5.1 Cell lines and growth medium ................................................................................. 80 2.5.2 Isolation of peripheral blood mononuclear cells by Ficoll gradient ........................ 81 2.5.3 Cryopreservation of cells ......................................................................................... 81 xii 2.5.4 Thawing of cryopreserved cells ............................................................................... 81 2.6 Murine experiments ......................................................................................... 82 2.6.1 Ethics Approval ....................................................................................................... 82 2.6.2 Immunisations.......................................................................................................... 82 2.6.3 Mouse serum and PBMC separation ....................................................................... 82 2.6.4 Splenocytes preparation ........................................................................................... 83 2.6.5 Isolation of mouse dendritic cells ............................................................................ 84 2.6.6 Enzyme-linked immunosorbent (ELISA) assay to determine gB antibody titers of vaccinated mice ................................................................................................................ 85 2.6.7 Micro-neutralisation assay against heterologous strains of CMV ........................... 85 2.7 Flow cytometry ................................................................................................. 87 2.7.1 Reagents ................................................................................................................... 87 2.7.2 Intracellular cytokine staining to assess IFN-γ production by human T cells ......... 88 2.7.3 Multi-parametric intracellular cytokine staining and degranulation assay to assess human T cells responses ................................................................................................... 89 2.7.4 Intracellular cytokine staining to assess IFN-γ response in mouse T cells .............. 89 2.7.5 Multi-parametric flow cytometry to assess the immune responses in vaccinated mice .................................................................................................................................. 90 2.7.6 Intracellular cytokine staining to assess IL-12p70 response in mouse DCs ............ 90 xiii Chapter 3: Recombinant glycoprotein B vaccine formulation with TLR9 agonist and immune stimulating complex induces specific immunity against multiple strains of cytomegalovirus........................ 93 3.1 Abstract ............................................................................................................. 93 3.2 Introduction ...................................................................................................... 95 3.3 Material and Methods ...................................................................................... 97 3.3.1 Generation of CMV gB expression construct and protein purification ................... 97 3.3.2 Generation of recombinant adenovirus encoding CMV gB protein ........................ 97 3.3.3 Animal immunisation .............................................................................................. 98 3.3.4 Assessment of gB-specific antibody avidity ............................................................ 98 3.3.5 T cell responses assessed by intracellular cytokine staining (ICS) .......................... 99 3.3.6 Statistical Analysis................................................................................................... 99 3.4 Results .............................................................................................................. 101 3.4.1 Evaluation of immunogenicity of recombinant gB protein in combination with TLR9 agonist and/or immune stimulating complexes .................................................... 101 3.4.2 Optimisation of CMV gB vaccine dose for vaccination ........................................ 102 3.4.3 Prime-boost immunisation with CMV gB vaccine formulated with AbISCO 100 and CpG ODN1826 dramatically improves immune responses ..................................... 104 3.4.4 Prime-boost immunisation with CMV gB vaccine induces high avidity antibody responses that neutralise multiple strains of CMV ......................................................... 106 xiv 3.4.5 Immunisation with CMV gB in combination with AbISCO 100 and CpG ODN1826 induces polyfunctional CMV-specific T cell response ................................. 107 3.4.6 Immunisation with CMV gB in combination with AbISCO 100 and CpG ODN1826 affords protection against quasi-viral infection ............................................. 109 3.5 Discussion ........................................................................................................ 110 Chapter 4: Expression, Purification and in vitro assessment of the immunogenicity of recombinant .......................................................... 113 CMV polyepitope protein ..................................................................... 113 4.1 Abstract ...................................................................................................... 113 4.2 Introduction ............................................................................................... 115 4.3 Materials and Methods ............................................................................. 118 4.3.1 Construction of CMV polyepitope vectors ............................................................ 118 4.3.2 Protein expression .................................................................................................. 119 4.3.3 CMV polyepitope protein purification .................................................................. 120 4.3.4 In vitro stimulation and expansion of CMV specific T-cells from healthy donors using polyepitope proteins .............................................................................................. 121 + 4.3.5 Analysis of processing and presentation of CD8 T cell epitopes from CMV polyepitope protein by human cells ................................................................................ 122 4.3.6 Enzyme inhibition assays ...................................................................................... 122 4.3.7 Silencing of ATG12 or Sec61 with short hairpin RNA (shRNA) ......................... 123 xv 4.3.8 Western blotting..................................................................................................... 124 4.3.9 Statistical analysis .................................................................................................. 125 4.4 Results .............................................................................................................. 126 4.4.1 Purification and characterisation of CMV polyepitope protein ............................. 126 + 4.4.2 Ex vivo expansion of CMV epitope specific CD8 T cells from PBMC following stimulation with polyepitope protein .............................................................................. 128 + 4.4.3 CD8 T cells expanded following stimulation with polyepitope proteins display polyfunctional profile ..................................................................................................... 130 + 4.4.4 CD8 T cell epitopes from the polyepitope protein are cross-presented through a TAP-independent pathway but involves proteasome and the autophagy dependent pathway ........................................................................................................................... 132 4.5 Discussion ........................................................................................................ 139 Chapter 5: Generation of robust CMV-specific cellular and humoral immunity following immunisation with recombinant viral antigens in combination with TLR4 and TLR9 agonists ...................................... 145 5.1 Abstract ........................................................................................................... 145 5.2 Introduction .................................................................................................... 146 5.3 Materials and methods ................................................................................... 148 5.3.1 Vaccine formulations ............................................................................................. 148 5.3.2 Mice immunisations ............................................................................................... 148 5.3.3 Isolation of murine dendritic cells ......................................................................... 149 xvi + 5.3.4 In vitro stimulation of CD11c DCs with various combinations of adjuvant formulations .................................................................................................................... 150 5.3.5 Statistical analysis .................................................................................................. 150 5.4 Results .............................................................................................................. 151 5.4.1 Evaluation of the immunogenicity of the recombinant gB-CMVpoly proteins in ® combination with CpG ODN1826 and AbISCO 100 .................................................... 151 5.4.2 Assessment of immunogenicity of gB-CMVpoly vaccine with alternative immunoadjuvants ............................................................................................................ 153 5.4.3 Effect of single and combined TLR4 & 9 agonists on the immunogenicity of gB- CMVpoly protein vaccine ............................................................................................... 157 5.4.4 TLR4 and TLR9 agonists synergistically promote the activation of dendritic cells and induce pro-inflammatory cytokine expression ......................................................... 165 5.5 Discussion ................................................................................................................. 167 Chapter 6: Preclinical assessment of the immunogenicity of a glycoprotein B and polyepitope protein based cytomegalovirus vaccine in combination with human compatible adjuvants .............. 171 6.1 Abstract ........................................................................................................... 171 6.2 Introduction .................................................................................................... 173 6.3 Materials and Methods .................................................................................. 175 ® 6.3.1 Preparation of vaccine formulations with IC31 adjuvant .................................... 175 6.3.2 Preparation of vaccine formulations with AS01 and AS15 adjuvants ................... 175 xvii 6.3.3 Mice immunisation ................................................................................................ 175 6.3.4 Statistical analysis .................................................................................................. 176 6.4 Results .............................................................................................................. 177 6.4.1 Immunisation of HHD mice with the gB-CMVpoly proteins in combination with human compatible adjuvants .......................................................................................... 177 6.4.2 Evaluation of the gB-specific antibody response following immunisation with the gB-CMVpoly proteins in combination with human compatible adjuvants .................... 178 6.4.3 Assessment of the gB-specific cellular immune response following immunisation with the CMV vaccine formulations ............................................................................... 180 6.4.4 Evaluation of CMV polyepitope-specific responses following immunisation with the CMV vaccine formulated with human compatible adjuvants ................................... 184 6.5 Discussion ................................................................................................................. 189 Chapter 7: General Discussion and Future Directions ..................... 193 Chapter 8: Bibliography ....................................................................... 203 xviii LIST OF FIGURES Chapter 1 Introduction and Review of Literature Figure 1.1 Differentiation of mouse Th17 cells 15 Figure 1.2 Possible model of memory T cell differentiation 23 Figure 1.3 Extrafollicular and germinal centre response to ptotein antigens 27 Figure 1.4 Antigen presentation on MHC class I & II molecules 30 Figure 1.5 Schematic of how ISCOMATRIX adjuvant may facilitate antigen translocation into the class I MHC pathway 55 Figure 1.6 Schematic representation of human cytomegalovirus showing various components of the virus 57 Chapter 3 Recombinant glycoprotein B vaccine formulation with TLR9 agonist and immune stimulating complex induces specific immunity against multiple strains of cytomegalovirus + Figure 3.1 Assessment of CMV gB-specific antibody and CD4 T cell responses following immunisation with gB protein formulated with various combinations of adjuvant(s) 101 xix + + Figure 3.2 CMV gB-specific antibody, CD4 T cell and CD8 T cell responses following immunisation with various concentrations of gB protein formulated with ® AbISCO -100 and CpG ODN1826 103 Figure 3.3 Short-term and long-term CMV-specific memory humoral and cellular immune responses following immunisation with CMV gB vaccine using a prime boost strategy 105 Figure 3.4 Assessment of antibody avidity and cross neutralising activity of the humoral immune response induced by the CMV gB vaccine. 106 + Figure 3.5 Cytokine expression by CMV-specific CD4 T cells from CMV gB vaccinated mice 108 Figure 3.6 CMV gB vaccine induces protection against challenge with recombinant vaccinia expressing gB 109 Chapter 4 Expression, Purification and in vitro assessment of the immunogenicity of recombinant CMV polyepitope protein Figure 4.1 Illustration of the design of the CMV polyepitope encoding sequence and downstream processing 126 Figure 4.2 Expression and purification of CMV polyepitope proteins 127 Figure 4.3 SDS-PAGE analysis of purified CMV polyepitope proteins 128 Figure 4.4 CMV polyepitope protein solubility test and characterisation129 xx Figure 4.5 Stimulation of CMV-seropositive donor PBMC with the CMV polyepitope protein 13mer 130 Figure 4.6 Stimulation of CMV-seropositive donor PBMC with the CMV polyepitope protein 14 and 15mer 131 Figure 4.7 The magnitude and quality of expanded CMV specific + CD8 T cells following stimulation with CMV polyepitope protein 132 Figure 4.8 Analysis of the processing and presentation of + CD8 T cell epitopes from the CMV polyepitope protein by LCL and HEK 293 cells 133 Figure 4.9 Analysis of the processing and presentation of the + - CMV polyepitope protein by TAP and TAP cells 134 Figure 4.10 The effect of different chemical inhibitors on the processing and presentation of the polyepitope protein 135 Figure 4.11 Effect of Sec61 and autophagy inhibition on the processing and presentation of the polyepitope protein 137 Chapter 5 Generation of robust CMV-specific cellular and humoral immunity following immunisation with recombinant viral antigens in combination with TLR4 and TLR9 agonists Figure 5.1 Schematic representation of the gB-CMVpoly proteins vaccination in combination with or without xxi ® CpG ODN1826 and AbISCO 100 151 Figure 5.2 Assessment of gB-specific and CMV polyepitope-specific immune responses following immunisation with ® AbISCO 100 and CpG ODN 1826 152 Figure 5.3 Illustration of the gB-CMVpoly proteins vaccination study in combination with various combinations of TLR agonists 154 Figure 5.4 Evaluation of gB and CMV polyepitope-specific immune responses following vaccination with various combinations of TLR agonists 155 + Figure 5.5 Assessment of CMV polyepitope-specific CD8 T cell responses following stimulation with NLV and VLE peptides 156 Figure 5.6 Schematic representation of the gB-CMVpoly protein vaccination study with single and double TLR agonists 157 Figure 5.7 gB-specific antibody responses induced following vaccination with gB-CMVpoly proteins in combination with MPL, CpG ODN1826, MPL & CpG ODN1826 or adjuvants alone 158 + Figure 5.8 Evaluation of gB-specific effector and memory CD4 T cell responses following immunisation with the gB-CMVpoly vaccine formulated with MPL, CpG ODN1826, MPL & CpG ODN1826 or adjuvants alone 159 + Figure 5.9 Multiple cytokine expression by gB-specific CD4 T cells from mice immunised with the gB-CMVpoly vaccine formulated with MPL and CpG ODN1826, CpG ODN1826 alone, MPL alone or adjuvants alone 160 xxii + Figure 5.10 Evaluation of gB-specific effector and memory CD8 T cell responses following immunisation with gB-CMVpoly proteins formulated with MPL and CpG ODN1826, CpG ODN1826 alone, MPL alone or adjuvants alone 161 Figure 5.11 Assessment of CMV polyepitope-specific effector and memory + CD8 T cell responses following immunisation with gB- CMVpoly proteins formulated with MPL and CpG ODN1826, CpG ODN1826 alone, MPL alone or adjuvants alone in peripheral blood and spleen 162 + Figure 5.12 Assessment of the CMV polyepitope-specific CD8 T cell responses following in vitro stimulation of splenocytes with NLV and VLE peptides 163 Figure 5.13 Multiple cytokine expression by CMV-polyepitope specific + CD8 T cells 164 Figure 5.14 Assessment of proinflammatory cytokines production following DC stimulation with various adjuvant combinations 166 Chapter 6 Preclinical assessment of the immunogenicity of a glycoprotein B and polyepitope protein based Cytomegalovirus vaccine in combination with human compatible adjuvants xxiii Figure 6.1 Schematic representation of the gB and CMV polyepitope vaccination study in combination with IC31, AS01 or AS15 adjuvants 177 Figure 6.2 Anti gB antibody titers induced following immunisation with gB and CMV polyepitope vaccine in combination with IC31, AS01 or AS15 179 Figure 6.3 Assessment of the virus-neutralising capacity of antibodies induced following immunisation 180 + Figure 6.4 Representative data of gB-specific effector and memory CD4 T cell responses following immunisation with IC31, IC31 & alum or MPL & CpG formulations in peripheral blood and in the spleen 181 + Figure 6.5 Representative data of gB-specific effector and memory CD4 T cell responses following immunisation with AS01 or AS15 formulations in peripheral blood and in the spleen 182 + Figure 6.6 Multiple cytokine expression by gB-specific CD4 T cells from mice vaccinated with gB and CMV polyepitope vaccine formulated with IC31, IC31 and alum, MPL and CpG or without adjuvants 183 + Figure 6.7 Multiple cytokine expression by gB-specific CD4 T cells from mice vaccinated with gB and CMV polyepitope vaccine formulated with AS01, AS15 or without adjuvants 184 + Figure 6.8 Assessment of the CMV polyepitope-specific CD8 T cell responses following immunisation with the gB and CMV polyepitope vaccine in combination with IC31®, IC31® and alum MPL and CpG, without adjuvants, or with xxiv adjuvants alone 185 Figure 6.9 Multiple cytokine expression by CMV polyepitope-specific + CD8 T cells 186 + Figure 6.10 Assessment of CMV polyepitope-specific CD8 T cell responses following immunisation with the gB and CMV polyepitope vaccine in combination with AS01, AS15 or without adjuvants, or with adjuvants alone 187 Figure 6.11 Multiple cytokine expression by CMV polyepitope-specific + CD8 T cells 188 LIST OF TABLES Chapter 1 Introduction and Review of Literature Table 1.1 Outline of the development of live human vaccines and their approximate time of availability 2 Table 1.2 Outline of the development of non living human vaccines and their approximate time of availability 4 Table 1.3 Databases of MHC binding peptides 39 Table 1.4 List of TLR receptors and ligands 51 Table 1.5 Current status of HCMV vaccine development 70 Chapter 2 Materials and Methods Table 2.1 List of antibodies used in the flow cytometry 87 xxv Chapter 4 Expression, Purification and in vitro assessment of the immunogenicity of recombinant CMV polyepitope protein Table 4.1 List of HLA class-1 restricted Epitopes included in the CMVpolyepitope 13, 14 , 15 &20mer 118 Chapter 7 General discussion and future directions Table 7.1 Summary of immune responses generated with gB and gB-CMVpoly vaccines in combination with experimental and human compatible adjuvants 201 xxvi ACKNOWLEDGEMENTS First and foremost, my sincere gratitude goes to my principal supervisor Professor Rajiv Khanna for providing me the opportunity to complete my PhD. It has been an honour to be one of his PhD students. He showed me an incomparable level of guidance and support throughout my PhD. His knowledge of science, patience, encouragement, intellectual input and vision will be affectionately remembered. I always enjoyed his words “please go ahead and do it”. I would also like to thank my associate supervisor Professor Denis Moss. Special thanks must go to my laboratory supervisor Dr Corey Smith for his immense assistance, intellectual input and valuable guidance throughout my PhD and his time and efforts to proof-read my thesis. I would like to thank my external supervisor Dr Jie Zhong for assisting with neutralising antibody assays and virus challenge experiments for chapter 3 and other parts for of my PhD studies. Particular thanks must go to Dr Tania Crough and Dr Emily Edwards for their efforts to proof-read my thesis. I wish to thank Dr Judy Tellam for her technical assistance in molecular biology. I would also like to thank Michelle Martinez for her assistance for conducting the animal experiments throughout my PhD. I would like to thank Sweera Rehan for her efforts to make lentivirus constructs and running western blots for chapter 4. xxvii I am extremely grateful to the members of the Tumour Immunology Laboratory for their good humour and great work environment. I especially like to thank Linda Jones for looking after my needs as PhD student and Siok, Lea and Leone for their assistance. I wish to thank members of the QIMR animal facility, especially David McNeilly, Alyssa Staines and Suzanne Cassidy for their efforts to breed and supply of HHD I mice. I would like to acknowledge members of Intercell for providing their proprietary ® adjuvant IC31 and GlaxoSmithKline (GSK) for providing AS01 and AS15 adjuvants. I wish to thank Lamin Jiang for making pCEP4gB construct and GENEART, Regensburg, Germany for making the gB protein. I would like to thank Gillian Scott and William Rawlinson from Virology Division, Department of Microbiology, SEALS, Prince of Wales Hospital, Randwick, NSW, Australia for providing HCMV 57A strain to conduct neutralising antibody assay in chapter 3. Finally, it cannot be expressed in written words to heartfelt thanks to my family for their continued support. I am extremely thankful to my wife Chaitanya, for understanding the importance of my PhD and helping me throughout my studies. Sincere thanks to my grandparents and sister for their continuous encouragement. xxviii ABBREVIATIONS aa amino acid Ab antibody Ad adenovirus AIDS acquired immune deficiency syndrome APC antigen presenting cell APC (fluorochrome) allophycocyanin BCG Bacillius Calmette-Guerin BMT bone marrow transplantation BSA bovine serum albumin CD cluster differentiated CDC Centers for Disease Control and Prevention cDNA complimentary deoxyribonucleic acid cDCs conventional dendritic cells CLR C-type lectin receptor CMI cell-mediated immunity xxix CMV cytomegalovirus CNS central nervous system CO2 carbon dioxide CTL cytotoxic T lymphocytes DB dense body DCs dendritic cells DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DNase deoxyribonuclease DTT dithiothretiol EBV Epstein-Barr virus ECL enhanced chemiluminesence E. coli Escherichia coli EDTA ethylenediaminetetra-acetic acid ELISA Enzyme-linked immunoSorbent spot ER endoplasmic reticulum ERAP endoplasmic reticulum aminopeptidases xxx et al. et alii, and other people FACS fluorescence-activated cell sorting FCS foetal calf serum FBS foetal bovine serum FDA food and drug administration Fig. figure FITC (fluorochrome) fluorescein-5-isothiocynate g gram gB glycoprotein B GC germinal centre gH glycoprotein H gL glycoprotein L gM glycoprotein M gN glycoprotein N gO glycoprotein O gpCMV guinea pig CMV GVHD graft-versus-host disease HARRT highly active antiretroviral therapy xxxi HBV hepatitis B virus HCMV human B cytomegalovirus HCV hepatitis C virus HIV human immunodeficiency virus HLA human leukocyte antigen hr(s) hours(s) HRP horse radish peroxidase HPV human papilloma virus HSCT Hematopoietic stem cell transplantation ICS intracellular cytokine staining i.d intra dermal IE immediate-early IFN-γ interferon gamma Ig immunoglobulin IL interleukin i.m intramuscular i.p intraperitoneal IRAKs interleukin-1 receptor-associated kinase 1 xxxii ISCOM Immune stimulating complex IU international units i.v intravenous kb kilobases kDa kilodalton L litre LCL Lymphoblastoid cell line LPS lipopolysaccharide M molar mAb monoclonal antibody MCMV murine cytomegalovirus ME mercaptoethanol mg milligram MHC major histocompatibility complex MIE major immediate-early min(s) minutes xxxiii mL millilitre mM millimolar M.O.I. multiplicity of infection mRNA messenger RNA MVA modified vaccinia virus Ankara MW molecular weight MyD88 Myeloid differentiation primary response gene (88) N2 nitrogen NF-kB nuclear factor-kB NIAID National Institute of Allergy and Infectious Diseases NK natural killer NLR NOD-like receptor Nm nanometer NP nuclear protein NS non structural OD optical density xxxiv ONM outer nuclear membrane ORF open reading frame oriP origin of plasmid PAMPs pathogen-associated molecular patterns PBMC peripheral blood mononuclear cells PBS phosphate buffered saline PC5 (fluorochrome) phycoerythrin-cyanin 5 PCR polymerase chain reaction PE (fluorochrome) phycoerythrin pfu plaque forming units pg picograms pH hydrogen ion concentration PI post-immunisation pp phosphoprotein QIMR Queensland Institute of Medical Research R- RPMI-1640 medium xxxv RE restriction endonuclease RLR RIG-I-like receptor RNA ribonucleic acid RNase ribonuclease RPM revolutions per minute RT room temperature s.c. subcutaneous SCT stem cell transplant(ation) SD standard deviation SDS sodium dodecyl sulphate sec seconds SEM standard error of the mean SLE systemic lupus erythematosis SOT solid organ transplant(ation) TAE tris acetate EDTA TAP transporter associated with antigen processing TBE tris borate EDTA xxxvi TCGF T-cell growth factor TCM central memory T cells TCR T-cell receptor TE Tris/EDTA buffer TEM effector memory T cells TEMED N, N, N’, N’,-tetramethylene diamine TH T-helper TIR Toll/interleukin-1 receptor TLR Toll-like receptor TNF tumour necrosis factor TR terminal repeats U units US unique short domain USFDA United States Food and Drug Administration UL unique long domain UV ultraviolet V volts xxxvii VA viral antigen VLP virus like particle v/v volume per volume VV vaccinia virus W weeks WHO World Health Organisation w/v weight per volume - negative + positive % percentage α alpha β beta δ delta ε epsilon γ gamma β2m β-2 microglobulin o C degree Celcius xxxviii µg microgram xg relative centrifugal force µL microlitre µm micrometer µM micromo xxxix xl Chapter 1: Introduction and Review of Literature 1.1 Historical milestones in vaccine development th The sciences of immunology and vaccinology date back to the 7 century, where Indian Buddhists consumed snake venom to induce toxoid-like immunity, the th chinese attempted four forms of inoculation and variolation in the 10 century (deBary, 1972). The field of vaccinology was ultimately born with the invention of the smallpox vaccination by Edward Jenner. He demonstrated that inoculation of human skin with cowpox virus was protective against smallpox, a procedure he termed “vaccine” (Donald A. Henderson, 2008). The principles learned from Jenner’s work were fundamental to the sciences of both vaccinology and immunology and have subsequently led to the control and eradication of 12 important diseases including smallpox, diphtheria, tetanus, yellow fever, pertussis, Haemophilus influenza type b disease, poliomyelitis, measles, mumps, rubella, typhoid and rabies in the various parts of the world (Table 1.1) (Plotkin, 2005b). Since the time of Edward Jenner and Louis Pasteur until recently, attenuation or the inactivation technique was used for vaccine development. Initially, attenuation of infectious pathogen was conducted by heat, chemical agents and oxygenation (Susan L. Plotkin, 2008). Later, several passages of pathogens in an animal host or embryonated hen’s egg or tissue culture was followed by attenuation. Vaccines against rabies, anthrax and yellow fever were developed by this method (Plotkin, 2005b). In the second strategy (Table 1.2), inactivation of an organism was carried out by the killing of the pathogen without losing its immunogenicity. This strategy was implemented for the development of typhoid, cholera, pertussis, influenza and th hepatitis vaccines. In the first half of the 20 century, chemical inactivation of 1 diphtheria and other bacterial toxins led to the development of the first diphtheria and tetanus vaccines (Enders et al., 1949). In 1923, Alexander Glenny and Barbara Hopkins demonstrated that formalin could convert diphtheria toxin to a less toxic toxoid. Later in 1926, tetanus toxoid was developed in the same manner by Ramon and Christian Zoeller (Susan L. Plotkin, 2008). The Bacille Calmette Guerin (BCG) vaccine developed by Albert Calmette and Camille Guerin was the first live bacterial vaccine. They attenuated Mycobacterium bovis through 230 passages in 13 years and in 1927, this vaccine became available for human use (Susan L. Plotkin, 2008). Table 1.1: Outline of the development of live human vaccines and th The second half of the 20 century was considered the golden age of vaccine development. In 1928, virus propagation in stationary cell culture using tissue culture flasks was introduced by Hugh and Mary from Manchester University. They grew vaccinia virus in sterile cultures of minced chicken kidney in media supplemented with chicken serum and mineral salts (Parish, 1965). Subsequently, George Gey 2 improved this technique by continually rolling the tubes and thus increasing the oxygenation of the stationary cells (Chase, 1982). In 1940, at the Boston Children’s Hospital John Enders, Thomas Weller and Fred Robbins grew Lansing type II poliovirus in human fibroblast cells from the skin and muscle tissue of infants (Enders et al., 1949). However, safety issues associated with the whole-cell, inactivated and attenuated vaccines remained major concerns. By 1977, due to adverse reactions associated with whole-cell pertussis vaccination, the rates of vaccination decreased to 33% in the UK and Japan (Baker, 2003). Such draw backs in vaccine design and safety diverted focus from the classical methods to genetic engineering methods plus recombinant technology to produce subunit vaccines. A major breakthrough in vaccine discovery occurred in 1981 in the US when Hilleman and colleagues discovered and purified non-infectious particles of hepatitis B surface antigen (HBsAg) from infected individuals and licensed this vaccine (Krugman, 1982; Krugman et al., 1967, 1971; Prince, 1968). However, in the same year the human immunodeficiency virus (HIV) epidemic arrived and there were concerns that products derived from human blood may be infected with HIV. Another drawback of this technique was that although hepatitis could be treated using purified non-infectious particles from infected individuals, this technique was unable to meet the need of the worlds population and was not effective. This led to an interest in applying genetic engineering to develop a HBsAg vaccine. Genetic engineering to produce recombinant antigens requires knowledge of the genomic sequence, either deoxyribonucleic acid (DNA), compelementary DNA (cDNA) or even ribonucleic acid (RNA) to amplify the desired gene. The HBsAg gene was cloned and expressed in yeast and mammalian cells, and then delivered with an 3 Table 1.2: Outline of the development of non living human vaccines and their approximate time of availability[adapted from:(Plotkin, 2005b)] alum adjuvant as these particles were similar to the plasma-derived vaccine, and offered great protection against hepatitis B virus (McAleer et al., 1984a; McAleer et al., 1984b; Michel et al., 1984; Scolnick et al., 1984; Valenzuela et al., 1982). Since then the majority of recent developments in vaccine design have used recombinant DNA technology in order to overcome safety, ethical and regulatory concerns. Recombinant DNA technology has led to the development of several subunit vaccines including HIV vaccine, Lyme disease and human papilloma virus (HPV). These were shown to be capable of eliciting humoral and adaptive immune responses and also 4 provided additional tools for producing bacterial proteins, polysaccharides and protein-conjugated polysaccharides for vaccine development. The Lyme disease vaccine was developed by expressing its outer surface protein in Escherichia coli. This vaccine was developed by GlaxoSmithKline and approved by the US Food and Drug Administration (FDA) in 1999 (Smith and Takkinen, 2006). Another landmark event in the development of subunit vaccines was achieved in 2006 by Merck, which developed and marketed a recombinant quadrivalent HPV vaccine by expressing virus like particles in yeast (Kirnbauer et al., 1992; Zhou et al., 1991). Genetic engineering further led to the development of recombinant viruses and bacteria by making live vaccines non-pathogenic, or attenuating the non-pathogenic organisms to use as live vaccines, vectored vaccines and DNA vaccines (McDonnell and Askari, 1996). Vector based vaccines are designed by combining the physiology of one microorganism and the DNA of another. Through this method immunity can be induced against diseases that have complex infection processes (Bartlett et al., 2009; Plotkin, 2005b). Many viral and bacterial vectors have been proposed and tested, including poxviruses, adenoviruses, vaccinia virus vectors and BCG (Mackett et al., 1992; Plotkin, 2005a). The development of DNA vaccines evolved following the expression of specific viral genes under eukaryotic enhancer promoters and polyadenylation signals. When DNA vaccines are injected into muscle, DNA is taken up by surrounding cells and transported to the nucleolus. Eukaryotic promoters allow appropriate transcription, followed by translation of specific genes, which can be processed and presented as foreign antigens by antigen presenting cells (APCs) (Nabel, 2008; Pachuk et al., 2000). In addition, the applications of DNA based vaccines have been tested in a variety of animal models and shown to be effective in inducing protective immunity 5 against influenza virus, malaria, tuberculosis and rabies (Lewis and Babiuk, 1999; Lodmell et al., 1998; Montgomery et al., 1993; Sedegah et al., 1994; Tang et al., 1992; Tascon et al., 1996; Ulmer et al., 1993). These viral and DNA vector vaccines were often employed in homologous and heterologous prime boost strategies. Homologous prime boost is where the immune system is primed with proteins expressed by injected DNA plasmids or vectors, and then boosted with the same strategy, whereas in a heterologous prime boost concept the immune system is boosted with same proteins but expressed in another vector (Amara et al., 2002; Excler and Plotkin, 1997; Moore and Hill, 2004; Robinson, 2003). In the 1990’s the use of reassortants in cell culture became an important technology for designing viral vaccines with segmented genomes. The development of influenza and rotavirus vaccines were achieved by the ability to mix RNA segments from attenuated strains, with RNA encoding protective antigens from circulating wild strains. For example, both live and killed influenza vaccines and rotavirus vaccines are dependent on reassortments. Live influenza vaccine contains replicating reassortants, whereas inactivated vaccines are produced from live reassortant seeds (Webster et al., 1986). The development of two of the three rotavirus vaccines have depended on reassortants containing the vp7 genes of human strains with genes from animal rotaviruses which are non-pathogenic for humans (Kapikian et al., 1996). 1.2 Immunology of Vaccines Vaccination is the most appropriate way to control the transmission and prevention of infectious diseases; however, to generate vaccine-mediated protective efficacy against pathogens is a complex mechanism. Historically vaccine-induced protective efficacy was shown to be conferred by the induction of high avidity antigen 6 specific antibodies produced by B cells (Siegrist, 2008). B cells produce antibodies that limit disease by neutralising a toxin or preventing the spread of the pathogen (Robinson and Amara, 2005). However, it has been since determined that T cells are both integral to the recognition and elimination of infected cells, as well as the activation of B cells (Robinson and Amara, 2005). During vaccination, a cascade of immune responses are induced by interaction of professional antigen presenting cell (APC), T cells and B cells (Delves and Roitt, 2000). In addition, T cells play an important role in generation of immune memory cells capable of rapid and effective reactivation upon subsequent exposure to a specific antigen (Siegrist, 2008). An increased understanding of the underlying molecular and cellular mechanisms of infectious diseases, immunology and vaccinology is very important in our efforts to develop successful vaccine strategies. 1.2.1 Innate immunity The first line of defence to any pathogen is innate immunity, which takes place within minutes of infection, followed by the activation of the adaptive immune response, which may take several days to weeks (Medzhitov and Janeway, 1997). The innate immune system involves several mechanisms to eliminate pathogenic microorganisms from the infected host, and the activation of the adaptive immune response. Some of these mechanisms are: lectin dependent complement activation (Fujita et al., 2004), intracellular pathogen killing by oxygen burst and removal of cell debris (Lu et al., 2002) cells of innate immunity produce cytokines, chemokines, adhesion molecules, alpha defensins and enhances major histocompatibility complex (MHC) 7 independent elimination of pathogens (Blach-Olszewska, 2005; Chang et al., 2005) killing of infected cells by natural killer cells (NK) (Carayannopoulos and Yokoyama, 2004). Cells of the innate immune system express various families of non-phagocytic receptors on their surface (Palsson-McDermott and O'Neill, 2007) such as toll like receptors (TLRs), nucleotide binding and oligomerisation domain (NOD) like receptors (NLRs), retinoic acid inducible gene 1 like receptors (RLRs) and C-type lectin receptors (CLRs) (Palsson-McDermott and O'Neill, 2007), and phagocytic receptors such as scavenger receptors, mannose receptors and β-glucagon receptors that induce phagocytosis by antigen presenting cells mainly macrophages (Gordon, 2002). In the process of innate immunity activation, TLRs and CLRs recognise exogenous antigens where as NLRs and RLRs recognise endogenous antigens (Fritz et al., 2006; Palsson-McDermott and O'Neill, 2007). These receptors are directed against evolutionarily conserved pathogen associated molecular patterns (PAMPs). PAMPs are absent in self-antigens and thus readily identified as ‘danger’ by cells of the innate immune system, which in turn produce proinflammatory cytokines and chemokines (Barton and Medzhitov, 2002; Iwasaki and Medzhitov, 2004). 1.2.1.1. Toll-like receptors TLRs play a crucial role in the sensing of highly diverse PAMPs associated with a broad range of pathogens. To date, 10 families of TLRs in humans and 13 murine TLRs have been identified and their ligands range from lipids to lipopeptides, proteins and nucleic acids (Kawai and Akira, 2006). Based on the cellular localisation, TLRs have been divided into two groups. The first group, consisting of TLR1, 2, 4, 5 and 6 8 are present on the cell surface and are believed to recognise pathogen surface markers. The second group, which consists of TLR3, 7, 8 and 9 are present intracellularly on endosomes and recognise nucleic acids derived from the virus and bacteria (McGettrick and O'Neill). TLRs are type 1 integral membrane glycoprotein with extracellular and cytoplasmic domains. The extracellular domain consists of leucine rich receptors (LLRs) which enhance pathogen binding, and a conserved 200 amino acid long cytoplasmic domain identical to interleukin-1 (IL-1) known as Toll-IL-1 receptor domain (TIR), which triggers the downstream signalling cascade. TIR domains associate either directly with the adaptor protein known as myeloid differentiation factor 88 (MyD88) or through an intermediate adaptor molecule know as the TIR domain containing adaptor protein (TIRAP). Downstream events enhances the binding of the amino terminal death domain of MyD88 to the death domain of another family of signal transducers called IL-1 receptor associated kinases (IRAKs), which in turn binds tumour necrosis factor receptor associated factor 6 (TRAF6). Activation and phosphorylation of IRAKs triggers the activation of TRAF6 and other adaptor proteins know as TAK1 (transforming growth factor β-activated kinase) and TAB2 (TAK1/MAP3K7 binding protein 2). This allows the IKK (inhibitor of nuclear factor kappa β kinase) complex to become active, which leads to the activation of transcription factor NF-kB (nuclear factor kappa β) and interferon regulator factor 3 (IRF3) and consequently results in the expression of inflammatory cytokines and other cellular activation events (Akira and Takeda, 2004; Akira et al., 2003; O'Neill et al., 2003). 9 1.2.1.2 Nod-like receptors Nod-like receptors (NLRs) are exclusively found intracellularly and are involved in the recognition of pathogens and their products. One example is Muramyl dipeptide peptidoglycans from bacteria (Pashine et al., 2005). NLRs are part of a family made up of at least 23 members whose defining motives include a ligand binding C-terminal leucine rich, pattern-recognition domain similar to TLRs; a central nucleotide binding domain that is essential for oligomerisation and subsequent signalling, and an N-terminal effector domain composed of a caspase- recruitment domain (CARD) or a pyrin domain (PYD) (Fritz et al., 2006). The NLR family members Nod1 (CARD4) and Nod2 (CARD15) detect distinct substrates from bacterial peptidoglycans, and activate NF-kB through the interaction of receptor- interacting serine/threonine kinase (RICK) with CARD. Subsequent activation of NF- kB leads to the expression of various inflammatory cytokines and chemokines such as TNF-∞ (tumour necrosis factor-∞), IL-6, IL-8 and membrane cofactor protein 1, which enhance stimulation and recruitment of additional effector cells during host defence (Chamaillard et al., 2003; Chen et al., 2009; Girardin et al., 2003). 1.2.1.3 RIG-I-like receptors RLRs are cytosolic and are exclusively viral sensors. The best characterised RLRs are RIG-I (retinoic acid inducible gene-1), which detects uncapped 5’- triphosphate dsRNA (double stranded RNA) or ssRNA (single stranded RNA) and melanoma differentiation-associated gene 5 (MDA5), which detects poly (I: C) and dsRNA. Similar to NLRs, RIG-1 and MDA5 contains a CARD and a helicase domain and induce the production of type I interferon (Palsson-McDermott and O'Neill, 2007). 10 1.2.1.4 C-type lectin receptors CLRs mediate endocytosis and/or phagocytosis, and are thought to be involved in the immune response to fungal pathogens (Brown, 2006). Dectin-1 is a member of the CLRs subgroup, which recognises the fungal component zymosan, through a single extracellular C-type lectin like domain (CTLD). Following ligand recognition, the ITAM (immunoreceptor tyrosine-based activation motif) of dectin-1 recruits the tyrosine kinase Syc, which leads to the activation of NF-kB [through the adaptor CARD9, MALT1 (mucosa-associated lymphoid-tissue lymphoma-translocation gene- 1) and Bcl-10 (B-cell lymphoma/leukaemia 10)], another transcription factor NFAT (nuclear factor of activated T-cells) and p42/p44 MAPK (mitogen activated protein kinase). Activation of these pathways leads to an increased production of pro- inflammatory cytokines (Brown, 2006). The activation of transcription factors NF-kB and IRF3 initiates the host immune defence. The NF-kB pathway controls the expression of proinflammatory cytokines such as IL-1β and TNF. The IRF pathway induces the production of antiviral type 1 interferons (IFN-α and IFN-β) (Akira and Takeda, 2004) and other cytokines including IL-8, monocytes chemoattractant proteins and macrophage inflammatory proteins. As a result of this, vascular endothelial cells may alter their surface expression of selectins and intracellular cell adhesion molecules, which leads to the extravasation and selective retention of some leukocytes at the inflamed site (Pashine et al., 2005). The inflammatory microenvironment consists of activated monocytes, granulocytes and NK cells. Consequently, monocytes differentiate into macrophages and immature DCs (dendritic cells) are activated (Pashine et al., 2005). 11 1.2.2 Adaptive immunity The adaptive immune response is a critical component of the immune system and plays a major role in eliminating pathogens in the late phase of infection by discriminating between self and non-self antigens, and generating pathogen-specific immunological memory. This is in contrast to the innate immunity response, which is comparatively non-specific and does not induce immune memory. The lymphocytes involved in the adaptive immune response have undergone clonal selection from a large repertoire of lymphocytes bearing antigen-specific receptors. During the adaptive immune response lymphocytes, T and B cells play a vital role in mounting a specific immune response followed by the development of immunological memory against intracellular and extracellular pathogens. T and B lymphocytes are derived from the hematopoietic stem cell in the bone marrow; progenitor T lymphocytes migrate from the bone marrow to the thymus to proliferate and differentiate into mature T cells, whereas progenitor B lymphocytes remain in the bone marrow to differentiate into mature B cells (reviewed in (Ikuta et al., 1992). The quality and quantity of the cells of the adaptive immune system (T and B lymphocytes) depends on the dynamics of the activation of the innate immune response, induction of co-stimulatory molecules and secretion of cytokines and chemokines (Busch et al., 1998). 1.2.2.1. T lymphocytes T cells are a major component of the adaptive immune system. Based on the expression of the coreceptors cluster of differentiation 4 (CD4) and CD8, T lymphocytes are divided into two subpopulations. They originate from progenitor cells in the bone marrow a process called haematopoiesis, then migrate to the thymus where 12 they undergo differentiation and maturation. In the thymus T cell differentiation is thought to occur through T cell receptor (TCR) signalling in thymocyte progenitor cells. According to the kinetic signalling model, persistent signalling through the TCR in the thymus drives thymocytes to express the transcription factor ThPOK, which + subsequently drives the differentiation of CD4 helper cells. On the contrary, disruption of TCR signalling and induction of the transcription factor allows cytokine + signals to drive differentiation of thymocytes into CD8 cytotoxic T cells (Park et al.)2010). Once T cells mature in the thymus, they enter into the blood circulation to elicit their essential immune functions. Additionally, their significant roles in the adaptive immune responses have led to an understanding of the complex mechanisms through which they function. Based on the nature of TCR, T lymphocytes can be divided into two populations αβ TCRs whose TCR is a heterodimer of α and β polypeptides, and γδ TCRs which consist of a γ and δ polypeptide heterodimer. γδ + TCRs make up only 0.5% to 15% of T cells in peripheral blood. Conversely, αβ CD4 + T cells and CD8 T cells comprise the majority of T cells in the periphery and lymphoid organs, and they have the ability to recognise either endogenously or 15 exogenously-processed antigens. TCRs can theoretically identify about 10 distinct epitopes (Sim et al., 1995). Similar to immunoglobulin, TCRs are formed via rearrangements of variable, diverse and joining (V, D and J) genes. They specifically recognise peptide epitopes through TCR interaction with peptide MHCon APCs (Pennycook et al., 1993) . + 1.2.2.1.1 CD4 T cells + CD4 helper T cells (Th), regulate both humoral and cell mediated immune responses. The CD4 molecule is a 55 kDa glycoprotein, which contains four 13 extracellular immunoglobulin-like domains (Littman, 1987). The first two domains are called D1 and D2, and these are joined by a flexible hinge to the third (D3) and fourth domains (D4). The amino terminal domain of D1 is similar to an immunoglobulin Vdomain, and CD4 interacts with MHC class II molecule through the D1. The cytoplasmic domain of the CD4 molecule interacts with the tyrosine kinase called Lck. When the TCR binds its peptide:MHC class II ligand, gene expression that is critical to the activation of T cells is triggered (Weiss and Littman, 1994). Based on the + cytokine secretion profile, CD4 T cells have been divided into two subsets T helper 1 (Th1) and T helper 2 (Th2) [reviewed in (Mosmann and Coffman, 1989)]. Recently two additional lineages of CD4 T cells have been described, namely Th17 and antigen- induced Treg (iTreg) cells (Figure 1.1) [reviewed in (Miossec et al., 2009; Sallusto and Lanzavecchia, 2009)]. Th1 cells develop preferentially during infections with intracellular pathogens, and in response produce IFN-γ, IL-2 and TNF-α to mediate the induction of cell mediated immunity (Mosmann and Coffman, 1989). On the other hand, Th2 differentiation is mediated by IL-4 and the transcription factors GATA-3 and c-Maf (Zheng and Flavell, 1997). Th2 cells predominate during extracellular pathogen infections (Ivanov et al., 2006), and they produce many cytokines including IL-4, IL- 5, IL-10 and IL-13, and regulate the induction of antibody responses (Mosmann et al., 1986; Romagnani, 1994). 14 Figure1.1: Differentiation of mouse Th17 cells. Naïve mouse T cells can differentiate in to one of three effector helper T-cell (Th) subgroups. Each pathway is under control of a different cytokines . The Th17 pathway is under the control of a transforming growth factor β (TGF- β) plus IL-6 and IL-1 or TGF-βplus IL-21 followed by IL-23. This pathway is inhibited by IFN-γ and IL-4. The transcription factor (t-bet, ROR-γt, or GATA3) characteristic of each pathway is shown. R denotes receptor. [adapted from (Miossec, Korn et al. 2009). Th-17 cells are considered a third subset of T helper cells. The cytokines required for Th-17 differentiation are not clearly defined, but the transcription factor ROR-γt (retinoid-related orphan receptor γt) is thought to play a crucial role in mouse and human Th-17 cells differentiation (Chen et al., 2003; Ivanov et al., 2006). Initially, IL-6 and TGF-β were known to promote Th-17 cells differentiation in mice, but subsequent studies have reported that IL-23 can expand Th-17 cells in vitro (Harrington et al., 2005; Park et al., 2005) . Immune responses triggered by Th-17 cells are considered extremely important at mucosal and epithelial surfaces, where they contain infection with pathogenic bacteria and fungi (Aujla et al., 2008). Furthermore, Th-17 cells can rapidly initiate neutrophil mediated inflammatory responses by producing cytokines such as IL-17A, IL-17F, IL-21, and IL-22 against a number of pathogens, which include gram-positive bacteria (Propionibacterium 15 acens), gram-negative bacteria (Citrobacter rodentium, Klebsiella pneumonia), borrelia species, (Mycobacterium tuberculosis) and fungi [reviewed in (Miossec et al., 2009)]. The regulatory T (Treg) cell subset, also known as suppressor T cells were first described by Gershon and Kondo in 1971. Fundamentally, there are two types of Treg cells; adaptive regulatory T cells (iTreg) and natural Treg (nTreg). iTreg cells develop outside the thymus during subimmunogenic antigen presentation and chronic inflammation, whereas nTreg cells develop within the thymus (Curotto de Lafaille and Lafaille, 2009). The role of cytokines during nTreg cells differentiation is less clear + however, differentiation of naïve CD4 T cells into iTreg cells in both mice and humans requires TCR stimulation, the cytokines TGF-β and IL-2, and forkhead box P3 + (Foxp3) transcription factor expression (Chen et al., 2003). Naive CD4 T cell stimulation with cooperative action between TGF-β and NFAT induces Foxp3 expression, which subsequently leads to the acquisition of anergic and suppressive activity in vitro, and in an experimental asthma model they showed inflammation suppression (Fantini et al., 2004). These cells play a crucial role in mucosal immune tolerance, control of severe chronic allergic inflammation, eradication of tumours and autoimmunity inhibition. + 1.2.2.1.2 CD8 T cells + CD8 T cells comprise the second subset of αβ T cells. They are generally characterised as cytotoxic T lymphocytes (CTL). The CD8 molecule is a heterodimer of α and β chains, covalently linked by a disulfide bond. They have a very similar structure to immunoglobulin, each having a ‘V’ domain with a polypeptide chain + anchoring to the cell membrane. They differ primarily from CD4 T cells by their 16 recognition of antigen presented via the MHC class I pathway. The CD8 molecule interacts with the MHC class I molecule through the α and β chains. Similar to CD4 T cells, the cytoplasmic tail of the CD8 α chain binds Lck and brings it into close proximity with the TCR. The main function of CTL is to recognise and eliminate + pathogen infected cells. Therefore, CD8 T cells play an important role in the + clearance of intracellular pathogens. Once CD8 T cells are activated, their precursor frequency can increase from 0.001% in the naïve repertoire to 3% to 50% in the effector/memory repertoire (Butz and Bevan, 1998). 1.2.2.1.3 Effector and memory cells differentiation One of the major criteria for vaccine development is to establish antigen- specific long lived immunological memory, which ideally persists for the life of the + + host. The memory T cell pool consists of both CD4 T and CD8 T cells which are differentiated into effector memory T cells (TEM) and central memory T cells (TCM). The memory T cells can rapidly attain effector functions to kill infected cells, and also secrete inflammatory cytokines to limit the replication of intracellular pathogens. TEM mediate protective memory, they migrate to inflamed peripheral tissue and display + immediate effector functions, and effector CD4 T cells also help B cell responses and + CD8 T cells differentiation, through the activation of APCs or secretion of cytokines such as IL-2, IL-4 and IL-5 [ reviewed in (Kaech et al., 2002)]. In contrast, TCM mediate reactive memory, they reside in secondary lymphoid organs, have little or no effector function, but upon antigen restimulation readily proliferate and differentiate into effector cells [reviewed in (Sallusto et al., 2004)]. In the differentiation process, T cells pass through three different stages, the expansion phase, contraction or death + phase and memory phase. In addition, CD8 T cells modulate effector functions 17 mainly through the perforin-granzyme pathway, Fas-dependent pathway and production of IFN-γ and TNF. 1.2.2.1.4 Expansion phase The expansion phase is initiated in the lymphoid tissue, where naïve T cells interact with antigen, clonally expand and differentiate into effector T cells. Through synergistic mechanisms, effector T cells secret inflammatory cytokines and eliminate infected cells, and typically viral infections can be controlled within a few days. + + During the differentiation into effector cells, CD4 and CD8 T cells undergo reprogrammed gene expression by changes in chromatin structure and active transcription factors. These changes lead to higher levels of mRNA (messenger RNA) for effector molecules such as IFN-γ, IL-4, TNF or perforin (Agarwal and Rao, 1998). The naïve Th differentiation into functional Th1 is driven by IL-12 produced by APCs, which acts in conjunction with signal transducer and activator of transcription 4 (STAT4) and T bet (a Th1-specific T box transcription factor), and is characterised by production of IFN-γ. Conversely, Th2 differentiation is controlled by IL-4, STAT6 and the transcription factor GATA-3 (Lee et al., 2001; Mullen et al., 2001; Szabo et + al., 2000). CD8 T cells develop more rapidly into effector cells; they can undergo at least 7-10 cell divisions within 2-24 hours of primary stimulation by professional + APCs. In contrast, CD4 T cells require a minimum of six hours of antigenic stimulation or 24 hours if co-stimulation is lacking to induce effector differentiation [reviewed in (Kaech et al., 2002)]. 18 1.2.2.1.5 Contraction phase or death phase During the contraction phase, when infection is cleared, more than 90% of effector cells are eliminated, these cells are programmed to undergo apoptosis by a default pathway (Sprent and Tough, 2001). The exact molecular mechanism involved in apoptosis is still unclear however, it was thought that cell death is triggered through multiple ways including deletions or mutations of Fas and Fas ligand, PD-1 (programmed death-1), CTLA4 (Cytotoxic T-Lymphocyte Antigen 4), TNF, TNF receptors I and II, and costimulatory molecules such as CD40 and CD40 ligand (Seder and Ahmed, 2003; Sprent and Surh, 2001). Sprent and colleagues reported that negative signalling by CTLA4 and PD-1 receptors for costimulatory signals leads to the activation of the Fas death pathway by dissociation of cFLIP from Fas, and ligation of Fas with Fas ligand triggers the activation of cytolytic caspases (Irmler et al., 1997). In addition, effector molecules such as perforin and IFN-γ are also known to be involved in the regulation of effector T cell response. Mice deficient in perforin + or IFN-γ exhibit increased numbers of CD8 T cells in the expansion and contraction phase (Badovinac et al., 2000). Furthermore, IFN-γ deficient mice have been shown to + contain 30-40% increase in number of CD4 T cell numbers following mycobacterium infection (Dalton et al., 2000; Refaeli et al., 2002). All these observations clearly indicate that IFN-γ is a potential regulator of cell death for both + + CD4 and CD8 effector T cells. Once activated cells enter into effector phase. They become cytolytic by acquiring antiviral effector functions, including the ability to rapidly produce cytokines such as IFN-γ and TNF-α and up-regulates the expression of granzyme and perforin [reviewed in (Wherry and Ahmed, 2004)]. 19 1.2.2.1.6 Mechanism of action of cytotoxic T lymphocytes (CTLs) + CD8 T cell responses are integral to the control of tumour protection, graft rejection and intracellular pathogens by their cytotoxic activity. Inactivated viral or + bacterial vaccines induce limited CD8 T cell responses, but these limitations can be overcome by delivering antigens with adjuvants, live-vectored or DNA vaccines. + CD8 T cells induce cytotoxic activity via two independent cytolytic pathways; namely perforin-granzyme pathway and Fas-dependent pathway. In the perforin-granzyme pathway, elimination of infected cells requires direct + contact between the CD8 T cell and target cells; which is an affinity dependent + process (Levy et al., 1996; Rubbert et al., 1997). Activation of the CD8 T cells following specific recognition of a target cells leads to an accumulation of granules containing perforin and granzyme at the interface between the effector T cell and 2+ target cell (Kagi et al., 1996). The elevated free Ca in the extracellular space induces conformational changes of the perforin molecules. Multiple perforin molecules come together to form polyperforin pores, which insert into the target cell membrane and make the cell membrane permeable to water and small ions (Kagi et al., 1996). This permeabilisation leads to the entry of granzyme A or B in to the cytoplasm of target 2+ cells. Granzyme A and B are two serine proteases, which are Ca dependent and capable of inducing cell lysis, oligonucleosomal DNA fragmentation and apoptosis (Heusel et al., 1994; Shi et al., 1992). The Fas-dependent pathway is the other major pathway which functions in the 2+ absence of extracellular Ca (Rouvier et al., 1993). In the Fas pathway cytotoxicity depends on the interaction of TCR-MHC I-peptide, which upregulates Fas ligand (FasL) expression on the T cell. FasL belongs to the tumour necrosis factor family and is a 40 kDa class II transmembrane protein (Suda et al., 1993). Interaction and cross- 20 linking of the Fas ligand molecules with Fas molecules on the target cells induces an apoptotic pathway that leads to target cell lysis probably involving interleukin-1β converting enzyme (Kagi et al., 1996). 1.2.2.1.7 Memory phase All surviving effector T cells enter into memory phase and these cells can persist more than 15 years (Ahmed and Gray, 1996; Amara et al., 2004; Badovinac et al., 2003; Banchereau and Steinman, 1998; Hammarlund et al., 2003). Sprent and colleagues proposed that memory T cell generation is a default pathway, whereby the majority of the T cells generated during the primary response are not programmed to die but to survive. However, the lineage of memory T cell development is not fully - understood (Sprent and Surh, 2001). TEM cells are CCR7 (chemokines receptor 7) and are present in the blood, spleen, and non lymphoid tissues. They undergo rapid activation upon re-encounter with specific antigen by producing effector molecules. In + contrast, TCM cells are CCR7 and are found in lymph nodes, spleen, and blood. After restimulation with specific antigen, they decrease CCR7 expression and migrate to the peripheral organs where they differentiate into TEM cells (Seder and Ahmed, 2003). The mechanism of effector cells survival to become memory T cells is not yet clearly + understood, however CD8 T cells survival is possibly related to cytokine control, primarily IL-7 and IL-15. Schluns and colleagues reported that IL-7 is required for + survival and generation of memory CD8 T cells following infection with vesicular stomatitis virus (Schluns et al., 2000). Another study indicated that the effector cytokine IL-15 secreted by APCs seems to play an important role in the survival of effector cells (Sprent et al., 2000). Experiments carried out in vitro and in vivo + indicated that IL-15 can induce strong proliferation of CD8 memory T cells but poor 21 + stimulation of CD4 memory T cells (Sprent and Surh, 2001; Zhang et al., 1998). + Additionally, selective action of IL-15 on CD8 memory T cells increases the + expression of the IL-15 receptor IL-2Rβ (CD122) but remains much lower on CD4 memory T cells (Ku et al., 2000; Zhang et al., 1998). This data suggest that IL-15 is required for the induction of intermittent proliferation and maintenance of viability of + CD8 memory T cells. Much less is known about the role of cytokines in the controlling of CD4+ memory T cells survival. Memory T cell generation is a progressive process that is triggered long after the clearance of infection. These changes correspond to both phenotypic and functional changes. During this process, precursors of memory T cells gradually lo lo lo hi hi hi convert from a CD62L , CCR7 , and CD27 to CD62 , CCR7 , and CD27 and also acquires the ability to rapidly proliferate in response to antigen and produce IL-2 (Wherry et al., 2003). Furthermore, activated and resting memory T cells can be distinguished in terms of cytokine profile. Activated memory T cells display high levels of granzyme B and low levels of Bcl2 (B-cell lymphoma protein 2), whereas resting T cells are characterised by low granzyme B and high levels of Bcl-2 (Kaech et al., 2003). In the memory phase memory T cells undergo slow but steady cell divisions with the help of the cytokines IL-7 and IL-15. This process is called homeostatic + turnover, and results in the formation of long-lived antigen independent memory CD8 T cells. 22 Figure 1.2: Possible model of memory T cell differentiation. [Adapted from (Ahmed, Bevan et al. 2009)] The lineage relationship between naïve T cells, effector and memory T cells is considered a crucial factor for vaccine design to enhance memory T cell responses during chronic infections and in cancer. There are several key issues in the determining the ontogeny of memory T cells. The most fundamental one is the genetic programme of memory T cells and their gene expressions during the acute phase of the + + T cell response. Another key issue includes the ontogeny of CD4 T cells and CD8 T cell memory T cells. Ahmed et al. stated that the detailed understanding in the process + of memory T cells differentiation by the heterogeneity of CD4 T such as Th1, Th2, Th17 and iTreg is the important factor (Ahmed et al., 2009). 23 Steven L. Reiner has proposed three differentiation models, namely linear differentiation, bifurcative differentiation and self-renewing effector model (Figure 1.2). In the linear differentiation model, naïve T cells differentiate into effector T cells upon co-stimulatory signals and inflammatory cytokines, and later become either senescent terminally differentiated T cells, which die by apoptosis after the pathogen is cleared, or they differentiate into TEM cells that express low levels of CD62L and do not express CCR7. These cells undergo homeostatic proliferation in lymphoid tissues to give rise to long lived CD62L and CCR7 expressing TCM. In the bifurcative differentiation model, antigen stimulation of naïve T cells can give rise to two daughter cells namely distal and proximal cells. These cells have different differentiation fates: distal daughter cells can give rise to TCM cells, and the proximal daughter cells can give rise to TEM. In the third proposed model, naïve T cells can develop into a TCM cell or TEM that can self-renew upon antigen stimulation. These cells can home and undergo homeostatic proliferation in lymphoid tissues. Additionally, these cells have the ability to give rise to TEM cells that can migrate to sites of infection but which do not self-renew (Ahmed et al., 2009) . 1.2.2.2 B lymphocytes B lymphocytes play an important role in antibody secretion and induction of humoral immune responses. The immature B cells are produced in the bone marrow and express a membrane bound antigen specific antibody molecule called a B cell receptor. In general, the majority of the B cells express and present antigens through + MHC class II molecules to CD4 T cells [reviewed in (Pierce et al., 1988)]. In addition, recent studies suggest that B cells also express TLRs involved in the host innate immune system (Gururajan et al., 2007). Upon receptor-mediated interaction 24 and exposure to antigen with T cells; B cells divide and mature into plasma cells that produce low-affinity germ line immunoglobulin (Ig) in the extrafollicular reaction [reviewed in (MacLennan et al., 2003)]. The majority of plasma cells are short lived and die within approximately two to three days. However, 10% of plasma cells survive to become long-lived antigen-specific memory B cells. Initially, activated B cell secretes IgM. However, during the differentiation process, Ig class switching takes place from IgM (immunoglobulin M) towards IgG, IgA or IgE [reviewed in (Siegrist, 2008)]. 1.2.2.2.1 B cell activation and antibody response B cell activation can occur in a T cell-dependent (Td) or T cell-independent (Ti) manner (Coffman et al., 1993). Through the T cell-dependent B cell activation pathway, mature naïve B cells develop in the bone marrow and circulate until they encounter a protein antigen to which their specific surface IgM receptor binds. Exposure to antigen and subsequent B cell activation leads to the up-regulation of CCR7 chemokine receptor on their surface for T zone chemokines CCL19 (chemokine ligand 19) and CCL21, and this drives antigen-specific B cells towards the outer T cell zone of secondary lymphoid tissues (Reif et al., 2002). In the lymph node, antigen specific B cells interact with recently activated DCs and T cells that have up-regulated specific surface molecules and thus provide B cell activating signals. Activated T cells rapidly drive the differentiation of the B cells into plasma cells which initially produce low affinity germline antibodies, in a process termed extrafollicular reaction (MacLennan et al., 2003). Immunoglobulin class switching from IgM to IgG, IgA or IgE takes place during the differentiation of B cells through the up-regulation of the 25 activation-induced cytidine deaminase (AID) enzyme and production of a group of cytokines including IL-4, IFN-γ and TGF-β (Deenick et al., 2005; Muramatsu et al., 2000). AID induces class switching from IgM to IgA, IL-4 induces switching to IgG1 and IgE, IFN-γ induces IgG2a, and TGF-β switches IgG2b and IgA (Deenick et al., + 2005). This class switching is triggered by the helper function of CD4 Th1 and Th2 cells following interaction of their CD40L molecules with CD40 on B cells (Deenick et al., 2005). B cells with moderate stimulation or interaction with Th cells restricts the extrafollicular response and promotes germinal center (GC) formation. The activated antigen-specific B cells up-regulate their expression of CXCR5 (C-X-C chemokine receptor 5) and migrate towards B cell follicles to attract the CXCL13 (C-X-C ligand 13) expressing follicular dendritic cells (FDCs). FDCs play an important role in B cell activation by attracting antigen-specific B cells and T cells and also retaining antigen for extended periods. Antigen-bearing FDCs and attracted B cells become the founder of germinal centres (GCs); these B cells undergo massive clonal proliferation by receiving additional activation and survival signals from FDCs and T cells. Each GC is constituted by the progeny of a single antigen-specific B cell. This intense proliferation is responsible for two major events: Ig class-switch from IgM to IgG, IgA or IgE, and maturation of B cells to produce antibodies which have a higher antigen- binding capacity. The maturation of antigen-specific B cells results from somatic hypermutations within the variable-region segments of immunoglobulin genes. Introduction of mutations in the B cell Ig genes increases the affinity of their surface to antigens; this process takes place only in a small portion of B cells, as the majority inadvertently produce antibodies with declining affinity. This property enhances B cells to efficiently compete for binding to lower concentrations of antigens that are associated to the surface of FDCs. These antigens are processed into small peptides by 26 Figure 1.3: Extrafollicular and germinal center response to protein antigens. In response to a protein antigen reaching lymph nodes or spleen, B cells capable of binding to this antigen with their surface immunoglobulins (1) undergo a brisk activation. In an extrafollicular reaction (2), B cells rapidly differentiate in plasma cells (3) that produce low- affinity antibodies (of the IgM +/- IgG/IgA isotypes) that appear at low levels in the serum within a few days after immunisation (4). Antigen-specific helper T cells (5) that have been activated by antigen-bearing dendritic cells trigger some antigen-specific B cells to migrate towards follicular dendritic cells (FDCs) (6), initiating the germinal center (GC) reaction. In GCs, B cells receive additional signals from follicular T cells (Tfh) and undergo massive clonal proliferation, switch from IgM towards IgG, IgA, or IgE, undergo affinity maturation (7) and differentiation into plasma cells secreting large amounts of antigen-specific antibodies (8). At the end of the GC reaction, a few plasma cells exit nodes/spleen and migrate to survival niches mostly located in the bone marrow, where they survive through signal provided by supportng stromal cells. [Adapted from (Siegrist 2008). B cells and are presented on their surface by MHC II molecules. A specific subset of + CD4 T cells, follicular helper T cells (Tfh) recognise the MHC peptide complex on the B cell surface (Fig. 1.3), and upon recognition they express CXCR5 and migrate towards CXCL-13 expressing FDCs (Vinuesa et al., 2005). The cellular interaction between the antigen-specific GC B cells, antigen-bearing FDCs and antigen-specific Tfh cells enhances B cell proliferation and survival, selection of high affinity B cells and also provides the signals required for the subsequent differentiation of GC B cells into antibody-secreting plasma cells or memory B cells. In addition, the development of the GC reaction requires a couple of weeks to produce hypermutated IgG antibodies against protein antigens (Flehmig et al., 1997). Furthermore, the GC reactions are terminated by a feedback mechanism within 3-6 weeks, by which period 27 a large number of antigen-specific plasma cells will have been generated. In general, protein antigens can activate both B and T cells, and trigger highly efficient B cell differentiation through GCs. In GCs, antigen-specific B cells differentiate into antibody-secreting plasma cells or memory B cells. In contrast, polysaccharide antigens that fail to activate T cells do not enhance GCs; therefore they elicit a weaker antibody and no memory response. The intensity of B cell differentiation into plasma cells is influenced by the quality of DC, B cell, and Tfh cell interaction with B cells. The peak of the IgG antibody response against a vaccine antigens occurs 4-6 weeks after primary immunisation [reviewed in (Siegrist, 2008)]. In the T cell-independent B cell activation pathway, thymus independent (TI) antigens stimulate naïve B cells into antibody producing cells in the absence of peptide-specific T cell help. The special properties of TI antigens deliver the prolonged and persistent signals to the naive B cells. In addition, NKcells and/or NK cell derived lymphokines, granulocyte macrophage colony-stimulating factor (GM- CSF) and IFN-γ are believed to provide necessary help to naive B cells in their differentiation and antibody production [ reviewed in (Mond et al., 1995)]. TI antigens can be divided into two classes: TI-1 antigens, which possess an intrinsic activity that can directly activate both immature and mature B cells, and TI-2 antigens, which contain no intrinsic B cell stimulating activity, and activate only mature B cells. During the week following immunisation, TI-2 antigens such as bacterial (S. pneumoniae, N. meningitides, H. influenza, S. typhi) polysaccharides (PS) gradually reach the marginal zone of the spleen/node through blood, then they bind to marginal zone B cells, cross-link the Ig receptors on the B cell surface, and activate B cells in the extrafollicular foci [reviewed in (MacLennan et al., 2003)]. Activated B cells differentiate into plasma cells, secret low-affinity antibodies and undergo isotype switching from IgM to IgG and IgA. As PS antigens do not trigger the formation of 28 GCs, PS immunisation induces a low level of somatic hypermutations in the variable regions of Ig, and only intermediate–affinity IgG antibodies are secreted and a memory B cell response is not elicited (Baker et al., 1971; Lucas and Reason, 1999). The PS-specific cells B cells after differentiation in extracellular pathway migrate towards the red pulp of the spleen, where they persist for a short time prior to apoptosis. 1.2.2.3 Antigen recognition and processing via MHC class I pathway The MHC class I pathway is active in almost all nucleated cell types, and is the pathway by which endogenous proteins are processed into peptide antigens, which are + presented on the surface to CD8 T cells. In the classical pathway, endogenous antigen found within the cytoplasm, is catabolically cleaved by the proteasome and other enzymes into shorter peptides 2-25 amino acids in length (Fig. 1.4) (Kloetzel, 2004; Rock et al., 2004; Shastri et al., 2002). The processed peptides are actively transported into the lumen of the endoplasmic reticulum (ER) with the help of heterodimeric transporter associated with antigen processing (TAP) (Harding, 1995; Harding et al., 1995a; Harding et al., 1995b). In the ER, the ubiquitously expressed endoplasmic reticulum amino peptidase ERAP (or ERAP1) enhances amino-terminal trimming of longer peptides delivered by TAP (Saric et al., 2002; Serwold et al., 2002; York et al., 2002). The MHC class I heavy chain joins first with β2 microglobulin and then with the peptide through a series of cascade events triggered by the components of the MHC class I peptide-loading complex. The transmembrane protein tapasin is one of the components of the MHC class I peptide-loading complex, which physically bridges the dimer of heavy chain and β2 microglobulin to TAP, and enables transport to the endoplasmic reticulum. It is also known to have a key role in peptide editing 29 (Elliott and Williams, 2005; Lewis and Elliott, 1998; Sijts and Pamer, 1997). Other proteins in the peptide loading complex include soluble chaperone, calreticulin and ERp57 which help in quality control for exported peptide-MHC complexes (Elliott and Williams, 2005). ERp57 is a thiol oxidoreductase that forms a disulfide bond with tapasin and maintains proper disulphide structure during the peptide loading (Dick et al., 2002). The MHC class I-peptide complex, which comprises a transmembrane heavy chain, β2 microglobulin, and the antigenic peptides, is transported through Golgi cysternae to the cell surface through a constitutive secretory pathway for + presentation to CD8 T cells (Harding et al., 1995a). Figure 1.4: Antigen presentation on MHC class I and II molecules. Adapted from http://www.lesc.ic.ac.uk/projects/appp_MHC.png 30 1.2.2.4 Antigen recognition and processing via MHC class II pathway The MHC class II pathway is continuously active in professional APCs such as B cells, macrophages, DCs and endothelial cells. DCs are considered the most professional APC for inducing naïve CD4 helper T cells and CD8 T cell differentiation into cytotoxic T cells (Banchereau et al., 2000). Immature DCs freshly derived from bone marrow are avidly endocytic, express low levels of surface MHC class II molecules, and have high levels of MHC class II accumulated in the lysosomal compartment. Upon stimulation with TLR ligands, inflammatory cytokines, T cell ligands (CD40) or NK cells, immature DCs undergo a maturation process (Jensen, 2007; Trombetta and Mellman, 2005) and up regulate MHC class II on the surface. DCs represent a heterogeneous population of cells. There are at least three different subsets of DCs based on their origin and phenotypic characteristics, namely those derived from myeloid, plasmacytoid and langerhans cells (Shortman and Liu, 2002). The myeloid DCs express CD11c on their surface and produce IL-12 whilst plasmacytoid DCs (pDC) produce IFN-α (Liu, 2001). DCs generally enhance endocytosis based on the size of cargo or mechanism of internalisation: phagocytosis, macropinocytosis, clathrin-dependent receptor mediated endocytosis and caveolae mediated endocytosis (Conner and Schmid, 2003; Mellman, 1996). DCs typically present exogenous antigens in complex with MHC class II molecules (Nuchtern et al., 1990). These antigens are rapidly cleaved in lysosomal compartments into smaller peptides (Kornfeld and Mellman, 1989). Immature DCs constantly accumulate MHC class II molecules in lysosomes compartments with multivascular and multilamelar structure (Kleijmeer et al., 1995; Nijman et al., 1995). MHC class II molecules are hetrodimers and consist of two homologous α and β chains. The α and β chains are produced and complexed with a polypeptide known 31 as the invariant chain (Ii) during the synthesis of the MHC class II molecule in the ER (Cresswell, 1996; Jensen et al., 1999; Watts, 2004). The Ii fits into the MHC II peptide groove and acts as a surrogate peptide to stabilise the protein, prevent binding from endogenous peptides, and to facilitate the export of MHC class II from ER to the golgi (Jensen, 2007). In immature DCs antigens and macromolecules gain access to slightly acidic prelysosomal MHC class II rich compartments, where MHC class II Ii complexes accumulate. The captured antigen is directed towards the MHC class II rich compartments containing the catalyst-chaperone protein HLA-DM (Human Leukocyte Antigen-DM) which enhances the peptide binding to MHC class II molecules by promoting a series of catalytic events for removal of the class II associated Ii chain peptide. The catalytic degradation of the Ii chain is regulated by the ratio between cathepsin S and its endogenous inhibitor cystatin C. The cathepsin S promotes cleavage and separation of Ii cytoplasmic tail endosomal retention signal from the MHC class II αβ heterodimer. This process leaves only a short peptide from Ii called CLIP (MHC class II- associated invariant-chain peptide) bound to the peptide binding groove which is protective of the action of proteases. In MHC II compartments at acidic pH, HLA-DM accelerates the peptide exchange and CLIP is replaced by antigenic peptides which range from 12-24 amino acids in length. These peptides are + then transported to the surface of the cell for presentation to the CD4 T cells (Cresswell et al., 1990; Jensen et al., 1999). 32 1.3 Vaccine design Over the last two centuries the field of vaccinology experienced the introduction of numerous new technologies including variolation, attenuation and genetic engineering. Although conventional techniques have been successful against diseases like polio and small pox, their utility has been limited in the vaccine development against pathogens like mycobacterium and HIV due to the low level of immunogenicity or failure to induce humoral and cell mediated immune responses. Another major drawback of the live-attenuated technique is the risk of reversion into their original pathogenic form. The application of modern molecular biology techniques over the last two decades has shifted the focus from the classical whole-cell vaccines comprising of live-attenuated or killed pathogens, chemically detoxified toxins, and polysaccharide conjugated proteins to subunit vaccines. This subsequently led to the development of two efficacious recombinant vaccines namely the hepatitis B vaccine based on a highly purified surface antigen, and Bordetella pertussi, which is based on three highly purified proteins (Andre, 1990; Pizza et al., 1989). In recent years, a new paradigm of vaccine design has emerged through the development of bioinformatics and immune-informatics tools (De Groot et al., 2002). These tools simply use the pathogen’s genomic sequence to predict either full length potent antigens or B cell and T cell epitopes that may enable the development of a subunit vaccine or epitope based vaccine. Since this project is intended to develop a recombinant based vaccine with the combination of full length antigen and T cell epitopes, a significant importance has been given to subunit and T cell epitope based vaccine technology. 33 1.3.1. Subunit vaccines Subunit vaccines are second generation vaccines, which differ considerably from whole cells or whole virus vaccines. In a subunit vaccine, only the elements of the infectious organism required to elicit a protective immune response are incorporated in the vaccine. Subunit vaccines offer several advantages over the traditional vaccines including precise immune targets, greater easy of manufacturing and enhanced safety. In addition, as they are not infectious, they can be given safely to immunocompromised individuals. However, there are some potential limitations of subunit vaccines. Since recombinant antigens lack the intrinsic adjuvants to enhance immunogenicity, they generally need to be administered in combination with an adjuvant or adjuvants. In addition, specific antigens may have shorter in vivo half-life. The majority of subunit vaccines are based on the epitopes, proteins, glycoproteins, glycolipids and polysaccharides that have been shown to contain protective epitopes [reviewed in (Hansson et al., 2000)], which have been identified by genetic, biochemical and immunological analyses. Following identification of potential antigens classical subunit vaccine development applies recombinant DNA technology, and in vitro cultivation of pathogens to dissect the antigens followed by large scale purification of the desired antigens. Furthermore there are issues of time constraint and the fact that most of the pathogens cannot be grown in vitro and many pathogens fail to express potential antigens. However, the more recent introduction of genomic technology has rapidly changed the identification of essential vaccine candidates. As a result, identification of novel vaccine antigens become easier through high-throughput genomic, transcriptomics, functional genomics, immunomics, structural vaccinology, vaccinomics and proteomic analysis. Advanced molecular biology techniques and bioinformatics have documented at least one genome sequence for each human 34 pathogen, more than 880 bacterial genome sequences had been completed by August 2009 and more than 2700 are in progress (Rinaudo et al., 2009). These tools aid and enhance the process of antigen selection, and as such the paradigm of vaccine development shifted from conventional culture based methods to high throughput genome based approaches. In the following sections various high throughput screening methods have been outlined. 1.3.1.1 Reverse vaccinology In this approach, the genome sequence of the microorganism provides the fundamental knowledge of its antigens, from which potential vaccine candidates are selected through rapid screening processes such as genome sequencing, in silico analysis, proteomics (two-dimensional (2D) gel electrophoresis and mass spectrometry), DNA microarray, in vivo expression technology (IVET) and signature tagged mutagenesis (STM) (Adu-Bobie et al., 2003). The Neisseria meningitides serogroup B is the first vaccine being developed by using this technique, and entered into clinical trials by Novartis vaccines. They identified all essential vaccine candidates in 18 months, whereas 40 years of applying conventional methods failed to produce a comprehensive vaccine (Pizza et al., 2000). 1.3.1.2 Transcriptomics Transcriptomics refers to the collection of all mRNA molecules produced by the genome at any one time. The basic principle of this technique relies on microarrays, which are a new and powerful tool that allows the simultaneous analysis of nucleic acid hybridization in a rapid and efficient fashion. In this technique vaccine 35 candidates which are important for pathogenesis and/or survival of the host can be identified, and analysed by expressing the complete set of RNA transcripts under specified conditions (Dhiman et al., 2001). As of now, nearly 39 microarrays for different pathogens are available free of cost from the J. Craig Venter Institute: Pathogen Functional Genomics Resource Center; http//pfgrc.jcvi.org/index.php/microarray/available_microarrays.html These resources have enabled further advances in the rapid sequencing of cDNA, and quantification of sequence reads in vaccine development (Morozova and Marra, 2008). The transcriptomics approach has been employed recently in the vaccine development against Neisseria meningitides serogroup B (Grifantini et al., 2002), vibrio cholera (Merrell et al., 2002) and Staphylococcus aureus (Cassat et al., 2006). 1.3.1.3 Functional genomics Functional genomics makes use of genomic sequences and focuses on gene transcription, translation and protein-protein interactions. High throughput functional screens can be performed on a genomic scale by combining whole-genome microarrays and comprehensive ordered libraries of mutants to identify specific vaccine candidates based on genes essential for microorganism survival and/or pathogenesis. Furthermore, these screens help in assigning functions to several uncharacterised ORFs (open reading frames) identified in genome sequences and in identifying mutants which could serve as live vaccines or delivery system for heterologous antigens (Mazurkiewicz et al., 2006). Functional genomics approaches are mainly based on the inhibition of genes using transposon mutagenesis (which utilises the transposon to insert into a gene and inactivate it) such as STM, genome analysis and mapping by in vitro transposition and transposon site hybridisation 36 (GAMBIT), followed by screening the mutants in animal models or cell culture to identify attenuated clones [ reviewed in (Saenz and Dehio, 2005; Scarselli et al., 2005)]. To date, STM has been applied to more than 30 pathogens which including Neisseria meningitides serogroup B, where 65 genes potentially causing septicaemia in infant rats were identified (Sun et al., 2000). In the case of Helicobacter pylori, 47 genes were identified which may cause colonisation of the gerbil stomach (Kavermann et al., 2003). 1.3.1.4 Immunomics Immunomics is the study of immunomes, which are a detailed map of immune reactions of a given host interacting with a foreign antigen (Sette et al., 2005). In general, the host immune system elicits a humoral immune response to secret antibodies during the course of infection against a set of pathogen specific proteins. Immunomics combines genomic/proteomic-based approaches and serological analysis to identify and validate potential vaccine candidates (Sinha et al., 2005). This approach led to the selection of immunoreactive antigens from pathogens including Klebsiella pneumonia, Streptococcus pyogenes, S. pneumonia, S. suis and Clostridium difficile [reviewed in (Rinaudo et al., 2009)]. 1.3.1.5 Structural vaccinology The continuous progress in the field of genome and proteome data, improved protein expression, purification and structural determination technologies which enabled rapid development of the field of structural vaccinology (Lundstrom, 2007). Structural vaccinology enables the determination and understanding of the structural 37 basis of immunodominant and immunosilent pathogenic antigens (Serruto and Rappuoli, 2006). 1.3.1.6 Vaccinomics In addition to pathogen genomics, human genomics also play an important role in vaccine design. At the individual or population level, variations in humoral, cell- mediated and/or innate immune responses may be induced by heterogeneity in host genetic markers (Poland et al., 2008; Poland et al., 2007). The availability of the human genomic sequence and comparison of genetic similarities and differences between humans has enabled the development of human leukocyte antigen (HLA)- restricted epitope vaccines (Bazhan et al., 2010; Zhong and Khanna, 2009). 1.3.1.7 Proteomics Proteomics are considered a complement to genomics. Proteome approaches are rapidly developing and are considered to be more effective technologies compared to classical genomic based approaches for discovering surface-associated immunogenic proteins as potential vaccine candidates. The complete protein profile of the microorganism, including protein localisation, protein-protein interactions, posttranslational modifications and differential expression in specified conditions can be analysed by implementing techniques such as 2D-PAGE coupled to mass spectrometry, chromatographic techniques and protein arrays [reviewed in (Rinaudo et al., 2009)]. 38 1.3.2 Epitope driven vaccines Significant progress in the fields of molecular biology, immunology and computational biology has enabled the mapping of HLA restricted T cell epitopes for the induction of epitope-specific protective T cell responses [reviewed in (Ellis, 1999)]. These T cell epitopes can be restricted to alleles of the human MHC class I molecules, HLA-A, HLA-B and HLA-C or MHC class II molecule include HLA-DR, HLA-DP or HLA-DQ alleles. The first step in developing an epitope-driven vaccine is to identify allele- specific or promiscuous peptides. In general, there are two different approaches for identifying T-cell epitopes. The first one is a standard overlapping peptide approach whereby epitopes are identified by constructing partially overlapping peptides (10 to 20 amino acids long) to cover the entire amino acid sequence (Maecker et al., 2001), followed by individual peptide screening to determine their ability to bind HLA molecules and induce T cell responses. Although this method is less complicated than the alternative bioinformatics approach, it possesses several disadvantages such as the 39 fact that T cell epitopes that fall in between the overlapping can be overlooked, it is labour intensive and is prohibitively expensive (Sbai, 2004). Further research by DeLisi and Berzofsky, and Rothbard and Taylor led to epitope predictive models by identification of patterns of amino acids residues within the epitopes. These predicative models clearly provided several advantages than classical methods but they failed to provide binding patterns in an allele specific manner (DeLisi and Berzofsky, 1985; Rothbard and Taylor, 1988). Later, technological improvement in the crystallographical studies of MHC molecules in complex with peptides, sequencing of naturally occurring MHC-peptide ligands by Edman degradation and the use of tandem mass spectrometry helped to develop computer-driven algorithms (Sbai, 2004). In 1991, Falk, et al. proposed the use of allele-specific “anchor-based” MHC binding motifs to identify T cell epitopes (Falk et al., 1991). Additionally, a number of different scientists proposed matrix- based approaches for T cell epitope mapping by assigning either a positive or negative coefficient to each possible amino acid that could occupy each position in a peptide or through computational estimates of the free energy of binding (De Groot et al., 1997; Hammer et al., 1994; Parker et al., 1995; Sette et al., 1989). Currently, computational biological methods are the preferred method and hundreds of different studies have identified the epitopes from a number of viral, bacterial, parasitic and cancer targets. Several databases of MHC binding peptides now exist on the web, some of which are tabulated in the above table (Table 1.3). The above information is adapted from the website (http://www.hiv.lanl.gov/content/immunology/pdf/2002/1/Lund2002.pdf). Various theories have been proposed, each of which with the aim of maximising the immunogenicity of epitope vaccines. One option is to present multiple epitopes as a string of beads with or without any spacer sequences to separate individual epitopes. In this regard, some studies have reported that flanking sequences 40 do not influence the presentation of epitopes (An and Whitton, 1997). Additionally, in a recent study involving replication-deficient adenovirus, human cytomegalovirus + + (CMV) polyepitope vaccine elicited strong CMV-specific CD8 T cell and CD4 T cell responses without any spacer sequences (Zhong et al., 2008). However, other studies have indicated that in a “string of beads” construct without any flanking sequences, individual epitopes are usually very closely apposed, which in turn may compromise appropriate proteolytic processing (Moudgil et al., 1998). For example, a study by Velders and his colleagues revealed that the addition of flanking sequences between the epitopes was crucial for epitope-induced tumour specific protection (Velders et al., 2001). Furthermore, a study conducted by Bazhan et al. revealed in an epitope based vaccine study, that the introduction of proteasomal liberation amino acids and TAP recognition motifs between the individual epitopes induced a higher immune response than epitopes without flanking residues (Bazhan et al.)2010). A major consideration during development of an epitope-based vaccine is the adequate population coverage in relation to MHC polymorphism. Failure to consider this may result in an ethnically-biased population coverage (Gulukota and DeLisi, 1996). HLA restriction relies on the selection of epitopes that can be grouped by HLA super-types. One hundred percent of the population can theoretically be covered by targeting the five super-types (Sette and Sidney, 1998, 1999). Additionally, the HLA super-types are not restricted to human class I molecules as studies have shown that nonhuman primates, such as chimpanzee (Bertoni et al., 1998), gorilla (Urvater et al., 2001), and macaques (Dzuris et al., 2000) also express MHC class I molecules that are functionally comparable to human class I super-types. Therefore, nonhuman primate animal models provide an opportunity to test epitope-based vaccines destined for human use. Furthermore, a number of transgenic murine models have been developed to evaluate HLA-restricted epitope based vaccines. Transgenic murine models such as 41 b the A2K model containing the human HLA A*201 with murine α3 chain (Kb) and the HHD-II model containing the human HLA A*201 with disrupted murine MHC molecule are well known models for the investigation of peptide recognition of epitope-based vaccine formulation (Firat et al., 1999; Gallez-Hawkins et al., 2003). Another crucial factor that needs to be considered in epitope-based vaccine development is pathogen heterogeneity. As a high degree of heterogeneity has been documented in pathogens such as HIV, HCV, Plasmodium falciparum and pnemococcus, such high levels of variability pose tough challenges in vaccine development. To overcome such problems, “customised” vaccines directed against one specific pathogen or cancer or combining antigens from all of the important pathogenic strains (Sbai, 2004). 1.3.2.1 Advantages of epitope based vaccines Immunisation with whole organisms or proteins does not always induce appropriate immunity as they predominantly trigger immune responses against the immunodominant epitopes (Udhayakumar et al., 1994). In fact, immune responses against subdominant epitopes which are highly conserved and crucial for pathogen survival may be useful for the vaccine design, and a number of recent viral vaccine studies indicated that CTL response to subdominant epitopes can provide efficient protection (Gegin and Lehmann-Grube, 1992). Additionally, vaccination with whole proteins or microorganisms with their pathogenic functional antigens may lead to toxicity and potential complications in the host (Levy, 1993) . Furthermore, in a study associated with setting of cancer therapeutics revealed that protein based vaccine induce immunity against an oncogenic self-protein (Disis et al., 1996). Since whole microorganism and protein based vaccines cause several adverse reactions, T cell 42 epitope based vaccines may be an ideal choice for the induction of protective immunity and that excludes epitopes that can trigger inappropriate immune response. 1.4. Adjuvants Vaccines are considered to be one of the most effective ways of preventing many infectious diseases. However, many significant obstacles remain unresolved: including antigen selection and presentation of antigens to stimulate appropriate immunity. Live or killed vaccines contain endogenous molecules capable of activating the innate immune system, but safety issues are major concerns of these vaccines. To some extent problems associated with classical vaccines can be overcome by subunit vaccines, but due to poor immunogenicity, the use of subunit vaccines has been limited. Subunit vaccines generally lack the essential capability to induce innate immune responses. Inclusion of immune potentiators (also called adjuvants) along with subunit antigens in a vaccine formulation is necessary to enhance the immunogenicity: many of these adjuvants play a crucial role in triggering early innate immune responses, which in turn aid in the generation of robust adaptive immune responses. Adjuvants are molecules, compounds or macromolecules which boost the quality, quantity or duration of immune responses (Wack and Rappuoli, 2005). The addition of adjuvants to vaccines provides several advantages such as sustained antigen delivery, reduction in antigen concentration and number of doses, and enhancement of potency in newborns and immune-compromised individuals (Kenney and Edelman, 2003). All these factors directly reflect on cost reduction of the vaccine. Adjuvants are classified into immunostimulants and vehicles. Immunostimulants such as TLR ligands, cytokines, Saponins, and bacterial exotoxins directly act on the 43 immune system to increase the innate response. Vehicles provide an organised way of antigen presentation to immune cells by antigen depot and delivery systems (Reed et al., 2009). While the recombinant technology has facilitated the development of vaccines against complex pathogens such as HIV, malaria and tuberculosis, this has + + also increased the need for adjuvants to elicit antibody, and CD4 and CD8 T cell responses. The immune system recognises PAMPs through pathogen recognition receptors (PRR) like TLRs, CLRs, NLRs, and RLRs. These receptors have affinity towards microbial ligands such as cell wall components, lipoproteins, proteins, lipopolysaccharides, DNA, and RNA of infectious organisms (Palsson-McDermott and O'Neill, 2007; Pashine et al., 2005). PAMP-PRR interaction on the surface of immune cells initiates a cascade of events to activate the immune system. Various types of adjuvants have been approved for use in humans, including aluminium hydroxide, MF59, monophosphoryl lipid A, virus like particles (VLPs), immunopotentiating reconstituted influenza virosome (IRIV) and cholera toxin; and others are in development, viz. montanides, saponins, MPL, muramyl dipeptide, immunostimulatory oligonucleotides, TLR ligands and E.coli heat-liabile exotoxin. An understanding of the adjuvant mechanism of action is one of the major criteria in vaccine development. The choice of adjuvant depends on the nature of antigen and its functional properties, and whether the antigen is used to enhance innate immunity, adaptive immunity or both. Some of the characteristics of adjuvants and their mechanism of action have been summarised below. 44 1.4.1 Adjuvants in clinical practice 1.4.1.1 Aluminium salt adjuvants Aluminium hydroxide (Alum), in use since 1926, is the only adjuvant that is widely used in humans. Alum is a stoichiometric compound composed of Al-OH and Al-OH-Al surface groups, which can either accept a proton resulting in a positive surface charge or donate a proton resulting in a negative surface charge. At pH 7.4, Alum exhibits a positive surface charge (Hem, 2008). In general, antigens can be absorbed by electrostatic attraction, hydrophobic forces or ligand exchange. In the case of Alum, electrostatic attraction is the best adsorption mechanism. Optimisation of electrostatic attraction can be done by determining the isoelectric point of the antigen and then selecting an adjuvant with opposite charge (Seeber et al., 1991). Based on the isoelectric point (iep) of antigens, either aluminium hydroxide (iep =11.4) or aluminium phosphate (iep=4.0) should be considered as an adjuvant (Seeber et al., 1991). Initially, Alum was thought to activate innate immunity by precipitating soluble antigen and sustained release of precipitated antigen to activate the APCs (Marrack et al., 2009). However, this historical hypothesis was ruled out by recent studies, which revealed that cell damage or apoptosis at the injection site causes release of saturated concentrations of uric acid into the extracellular space. This is followed by formation of monosodium urate (MSU) crystals which are phagocytosed by resident cells. In these cells, MSU and aluminium salts disrupt the lysosomes which leads to the release of cathepsin B. Cathepsin B induces potassium efflux to activate the NLRP3 inflammasome (NOD like receptor NLR family), activating the production of pro-IL-1β, pro-IL-18 and pro-IL-33. Later, NLRP3 inflammasome activates caspase 1 which cleaves all pro molecules into active molecules (Agostini et 45 al., 2004; Martinon and Tschopp, 2007; Ting et al., 2008). Accumulation of uric acid + + and MSU promotes CD4 T cells, CD8 T cells (Shi et al., 2000), and antibody responses (Behrens et al., 2008). Additionally, two different studies revealed that vaccinated mice with MyD88 and TRIF deficiencies (TIR-domain containing adaptor protein inducing IFNβ) generate antigen specific response of all isotypes, indicating that Alum does not act through TLR, and that activation is a receptor-independent mechanism (Gavin et al., 2006; Schnare et al., 2001). 1.4.1.2 MF59 MF59 was the second adjuvant to be licensed for use in humans. In Europe, MF59 is currently used in the influenza vaccine. MF59 consists of an oil in water microfluidized emulsion composed of droplets with a size <250nm (Wadman, 2005) and two surfactants, polyoxyethylene sorbitan mono-oleate (Tween 80) and sorbitan trioleate [reviewed in (Hem, 2008)]. MF59 was initially developed as a vehicle for Muramyl peptide adjuvant, but was itself later found to have marked adjuvant properties. Several vaccine formulations have been developed with MF59; these include HSV, hepatitis B virus, and HIV. MF59 has acceptable safety, and probably acts by depot effect and by directly stimulating secretion of cytokines and chemokines by monocytes, macrophages and granulocytes. Importantly, Novartis influenza vaccine with MF59 (Fluad) showed higher potency than Alum (Heineman et al., 1999; Hem, 2008; McFarland et al., 2001; Plotkin, 2008a; Straus et al., 1997). It triggers higher antibody titers with a more balanced IgG1:IgG2a response than Alum, and is also a potent stimulator of cellular and humoral responses to subunit vaccines in both animal and clinical studies (Ott et al., 1995). Furthermore, MF59 shows increased 46 immunogenicity of co-administered antigens in different age groups (Podda and Del Giudice, 2003). The safety and adjuvanticity of MF59 has been tested in toddlers and infants with the recombinant HIV (gp120 vaccine) and HCMV glycoprotein B. In another study, infants born to HIV infected mothers vaccinated with a HIV vaccine elicited higher cellular mediated immune responses than a vaccine formulation prepared with Alum (Borkowsky et al., 2000). These observations reveal that MF59 is safe, compatible and efficacious with various antigens. 1.4.1.3 Monophosphoryl Lipid A (MPL) MPL is derived from the lipopolysaccharide (LPS) of Salmonella minnesota. MPL formulation can either be carried out in an aqueous vehicle or oil-in water emulsion (Baldridge and Crane, 1999). The LPS consists of two basic structures: a hydrophilic polysaccharide portion to enhance solubility and a hydrophobic lipid moiety (lipid A) responsible for endotoxic activity (Johnson, 2008). The mechanism of MPL action is TLR4-mediated activation of APCs, inducing the secretion of immunomodulatory cytokines including IL-12, IL-1, TNF-α and GM-CSF, and subsequent secretion of IL-2 and IFN-γ, which is characteristic of a Th1 helper cell- mediated immune response (Evans et al., 2003; Salkowski et al., 1997). At present, MPL has been used in several vaccine formulations such as hepatitis B virus (HBV) and HPV, and has been shown to activate T cell responses (Baldridge and Crane, 1999). AS04 is an aqueous formulation of MPL and Alum which generates higher titers of specific antibody and efficacy with fewer injections. Other studies have included AS01B (liposomes plus 3-O-desacyl-4'-monophosphoryl lipid A plus QS21) and AS02A (oil-in-water emulsion, 3-O-desacyl-4'-monophosphoryl lipid A, and a saponin derivative, QS21) in malaria, tuberculosis, leishmania, HIV, vesicular 47 stomatitis and cancer vaccines [reviewed in(Reed et al., 2009)]. MPL has been approved for use in Europe because it can downregulate the Th2 responses to allergens, and was observed to be safe, well tolerated and potent (Alderson et al., 2006; Wheeler and Woroniecki, 2004). 1.4.1.4 Monophosphoryl lipid (MPL) A combinations MPL combinations include MPL-SE, AS01, AS02, and AS04. MPL-SE is an excellent inducer of Th1 responses; it contains MPL and squalene oil, excipients and water to produce a stable oil-in-water emulsion. MPL-SE has been evaluated in several clinical trials (Goto et al., 2009; Trigo et al.). AS02 promotes humoral and Th1 responses; it is an oil-in water emulsion with a composition of MPL and QS21. AS02 has been evaluated for malaria, HPV, HBV, tuberculosis and HIV vaccine formulations (Reed et al., 2009). AS01, is a liposomal formulation and it enhances both humoral and CTL responses. AS04 contains a combination of MPL and Alum. A formulation combination of HBsAg with AS04 showed higher antibody titers as well as stronger cellular immunity (Boland et al., 2004). The AS04 adjuvant is used in Cervarix (GSK Bio) and the L1 VLP vaccine used to prevent HPV infections (Harper et al., 2006). 1.4.1.5 Cholera toxin B subunit (CTB) CTB is used in oral vaccines to enhance mucosal responses; it can deliver antigens to M cells of the Peyer’s patch. CTB belongs to the AB class of bacterial toxins; it contains a pentameric B oligomer and enzymatically active A subunit. The B oligomer binds to GM-1 receptor on the surface of intestinal epithelial cells, the A 48 subunit is responsible for toxicity. Recombinant CTB (rCTB) consists of the non-toxic B component of the cholera toxin with similar adjuvant properties to natural CTB. In one study, CTB or rCTB co-administered with antigen intranasally enhanced IgA and systemic IgG secretion (Wu and Russell, 1998). rCTB has been used in licensed whole cell oral cholera vaccine, but this vaccine only induces short lived protection (Hill et al., 2006). 1.4.1.6 VLP and IRIV VLP contains one or more self-assembling viral proteins of 20-100 nm. There are some commercially available VLP based vaccines such as the HBV vaccine which is based on expression of surface antigen and the HPV vaccine which comprises the major capsid protein L1. IRIVs are proteoliposomes composed of phospholipids, influenza hemagglutinin and a selected target antigen. Liposomes are an extensively studied vaccine delivery system. They are known to be safe and versatile, and induce humoral and cell mediated immunity to antigens (Gluck et al., 2005). In a clinical study comparing IRIV with Alum, IRIV generated faster immune response and less injection site adverse reactions (Holzer et al., 1996). 1.4.2 Adjuvants in clinical research 1.4.2.1 TLR ligands TLRs play an important role in both innate and adaptive immunity. TLRs can recognize pathogens through PAMPs or microbe associated molecular patterns (MAMPs) (Akira, 2006; Koropatnick et al., 2004). TLR ligands include synthetic compounds mimicking PAMPs, which are capable of activating professional APCs, 49 and result in secretion of inflammatory cytokines and chemokines. Each TLR recognises various microbial components and activates different pathways through various adaptor molecules and transcription factors such as NF-kB, activating protein- 1 (AP-1) and interferon regulatory factors to drive specific immune responses (Akira, 2006; Kawai and Akira, 2006). Based on their cellular localisation TLRs are divided into two groups, surface TLRs and intracellular TLRs. 1.4.2.2 Cell surface TLRs TLR1, 2, 4, 5, and 6 are expressed on the plasma membrane and with the exception of TLR5 recognise lipid structures, TLR5 recognises bacterial flagellin (Medzhitov and Janeway, 2000b). TLR2 is expressed on the plasma membrane and early endosomes of resting monocytes. TLR2 can form dimers with either TLR1 or TLR6, which are structurally similar and arise from a gene duplication event (Hughes and Piontkivska, 2008). TLR4 is the best characterised cell surface TLR, it recognises LPS with the help of other surface proteins, including LPS binding protein (LBP), CD14 and MD2 (Park et al., 2009). During LPS induced activation, MD2 a small secreted glycoprotein helps in the translocation of TLR4 from the golgi to the plasma membrane (Nagai et al., 2002). Subsequent binding of LPS to TLR4 induces translocation from the plasma membrane to the endosome (Tanimura et al., 2008). TLR expression, expressed cell type and ligand specificity have been summarised in the following table (Table 1.4). 50 Table 1.4: List of TLR receptors and ligands Extracellular TLRs Receptor ligand Expressed cell type Reference Recognize triacylated (Hughes and lipopeptides, such as TLR1 Piontkivska, the synthetic ligand 2008) Pam3CSK4, Monocytes, mature Gram-positive bacteria, macrophages, dendritic TLR2 including lipoteichoic cells and acid (LTA) mast cells Monocytes, mature Lipopolysaccharide macrophages and dendritic (Hennessy et al.; TLR4 (LPS) from Gram- cells, mast cells and Latz et al., 2002) negative bacteria intestinal epithelium Intestinal epithelium monocytes, dendritic (Medzhitov and TLR5 Binds flagellin cells and macrophages Janeway, 2000b) Diacylated lipopeptides TLR6 like MALP-2 TLR10 Un known Macrophages/monocytes Intracellular TLRs (Muzio et al., 2000; Zarember TLR3 Double stranded RNA Dendritic cells and Godowski, 2002) Monocytes, macrophages TLR7 Single stranded RNA (Caron et al., dendritic cells and TLR8 from viruses 2005) mast cells Unmethylated CpG Monocytes, macrophages TLR9 found in bacterial and and plasmacytoid (Yi et al., 1998) viral DNA dendritic cells 1.4.2.3 Intracellular TLRs TLR3, 7, 8, and 9 comprise the family of intracellular TLRs and recognise nucleic acids derived from viruses and bacteria (McGettrick and O'Neill). TLR3 is primarily expressed on the endosome of DCs and it recognises dsRNA produced by replicating viruses. The synthetic molecule poly I:C is also a ligand for TLR3. (Muzio et al., 2000; Zarember and Godowski, 2002). The small molecule nucleoside analogues such as imiquimod and resiquimod are ligands for TLR-7 and TLR-7/8 (Gorden et al., 2005). Keith et al. revealed that TLR7 agonists can directly activate 51 purified pDCs and to lesser extent monocytes, whereas TLR8 agonists directly activate purified myeloid DCs, monocytes and monocytes-derived DCs (Gorden et al., 2005). TLR9 is present on the endosomes of macrophages, monocytes and pDCs and acts as a receptor for unmethylated DNA (CpG). CpG contains unmethylated CpG dinucleotides, which are present at a 20% higher frequency in bacterial DNA than mammalian DNA (Krieg et al., 1995). Unmethylated CpG is obtained from bacterial synthetic DNA and contain phosphorothioate linkages. CpG induces innate immune responses by enhancing B cell proliferation and Ig secretion, cytokine secretion by monocytes, and activates NK cell lytic activity and IFN- secretion in vivo and in vitro. In humans, CpG dinucleotides bind TLR9, which is expressed intracellularly within the ER, endosomes, multivesicular bodies and lysosomes of NK, B, and pDC (Klinman, 2003). Upon activation of TLR9, APCs are induced to produce Th1 and proinflammatory cytokines including IL-1, IL-6, IL-8, TNF and IFN-γ. CpG adjuvanticity is compatible with parenteral or mucosal routes of administration (McCluskie et al., 2000a; McCluskie et al., 2000b; Verthelyi et al., 2002). In addition, CpG is known to induce a Th1 bias and potentially redirect immune responses that have a natural Th2 bias (Weeratna et al., 2000). In a comparison between CpG and Alum with HBsAg, CpG induced a Th1-biased response while Alum induced a Th2- biased response in both young and adult animals (Brazolot Millan et al., 1998; Davis et al., 1998). Additionally, CpG has been found to be safe and effective when formulated with vaccines against cancer (Wang et al., 2008), rabies (Wang et al., 2008), and hepatitis B (Weeratna et al., 2000). 52 1.4.2.4 IC31 IC31 is a two component synthetic adjuvant; it contains deoxy-inosine/deoxy- cytosine (ODN1a) and the antimicrobial peptide KLKL5KLK and signals through the MyD88 dependent pathway. CpG motifs or dsRNA such as poly (I:C) are negatively charged immunostimulatory nucleic acids (Alexopoulou et al., 2001; Bauer et al., 2001), and are ligands of TLR9 and TLR3 (Eriksson and Holmgren, 2002). Cationic antimicrobial peptides, such as KLKL5KLK are positively charged molecules that are able to induce adaptive immune responses (Oppenheim et al., 2003). They have the ability to induce protein and peptide specific type I cellular and humoral immune response (Schellack et al., 2006). In a recent study, influenza vaccine formulated with IC31 showed strong influenza vaccine specific humoral and cellular immune responses (Riedl et al., 2008). 1.4.2.5 Montanides Montanides (ISA51 and ISA720) contain mannide-mono-oleate as an emulsifier, produce a water-in-oil emulsion, and are promising adjuvants for vaccine delivery. Montanides are biodegradable and were developed to overcome concerns associated with incomplete Freund’s adjuvant (IFA) (Aucouturier et al., 2006). Previous studies have shown that co-administration of CTL epitopes with Montanides ISA720 can offer protection against murine CMV (Scalzo et al., 1995). Moreover, Montanides have been used in experimental malaria, HIV and cancer formulations (Kenney and Edelman, 2003). They induce potent immune responses but require complicated emulsification procedures [reviewed in (Reed et al., 2009)]. 53 1.4.2.6 Saponins Saponins are triterpene glycosides isolated from plants. Among the Saponins, Quil-A and its derivatives have been used extensively in adjuvant research. Quil-A is extracted from the bark of Quillaja saponaria (Kensil et al., 1995), and it contains a heterogeneous mixture of triterpene glycosides with variation in adjuvanticity and toxicity. Partially purified fractions of Quil-A have been used in immunostimulating complexes (ISCOM) composed of antigen, phospholipids, cholesterol and Quil-A. ISCOMATRIX is similar in composition to ISCOM but without the incorporated antigen (Pearse and Drane, 2005). These are 40nm cage like structures (Skene and Sutton, 2006) that bind protein antigen through hydrophobic interactions. ISCOMATRIX accommodates non-hydrophobic antigens, and is thus not limited to hydrophobic membranes (Pearse and Drane, 2005). ISCOMATRIX has been shown to induce broader immune responses, including a range of subclasses of antibodies as + + well as CD4 and CD8 T cell responses (Drane et al., 2007; Maraskovsky et al., 2009). ISCOMs are directly targeted to APCs via endocytosis (Fig. 1.5), and saponins may initiate higher uptake and more efficient antigen presentation by targeting of DEC-205 on the surface of DCs (Jiang et al., 1995; Mahnke et al., 2000). Once inside cells, antigens can be processed through classical or cross-presentation pathways (Ackerman et al., 2003; Guermonprez et al., 2003; Harding et al., 1991; Houde et al., 2003; van Binnendijk et al., 1992). The translocation of antigen depends on acidification of the endosome and IL-4-driven differentiation of monocytes into DCs. These ISCOMATRIX-stimulated DCs induce cross-presentation of CTL epitopes restricted by diverse HLA alleles (Schnurr et al., 2009). A number of studies have shown that ISCOMATRIX adjuvant is safe, well tolerated and increases vaccine potency (Drane et al., 2007). 54 Another natural Saponin-based adjuvant is QS-21 derived from the tree Quillaja saponaria Molina, which has low toxicity and significantly improved adjuvanticity. In various vaccine formulations such as protein, glycoprotein, and polysaccharides with QS-21, humoral and cellular immune responses were induced (Evans et al., 2001; White et al., 1991). Several clinical trials are in progress with QS- 21, alone or in conjunction with carriers and other immunostimulants for vaccines against HSV, HIV, HBV, malaria and cancers (Reed et al., 2009). 55 1.4.2.7 Muramyl dipeptide (MDP) MDP is the mycobacterium cell wall complex with adjuvant activity (Reed et al., 2009). MDP is the ligand for NOD2 receptor, and induces low levels of TNFα, and high levels of IL-8 (van Heel et al., 2005). Several synthetic analogs of MDP have been generated, e.g., muramyl tripeptide phosphatidylethanolamine (MTP-PtdEtn) and they exhibit a wide range of adjuvant potency and side effects(Reed et al., 2009). 1.5 Adjuvant safety The selection of an adjuvant for a vaccine formulation to enhance immunogenicity must be balanced against the potential risk of inducing adverse reactions. In general, there will be some local reactions with adjuvanted vaccine: including inflammation at the injection site, formation of granulomas and the formation of sterile abscesses (Bernier et al., 1981). Adjuvants can cause systemic reactions which include fever, adjuvant arthritis and anterior chamber uveitis (Allison and Byars, 1991). Although, these reactions are not life threatening and resolve over time, they remain as barriers to community acceptance of routine prophylactic vaccinations. Adjuvant safety concerns are more applicable to paediatric vaccines, and any reactogenicity of the adjuvant may cause physical resistance to vaccination (Principi and Esposito, 2004). Recently, detailed guidelines on adjuvants in vaccines for human use have been defined by the USFDA, National Institute of Allergy and Infectious Diseases (NIAID), European Medicines Evaluation Agency (EMEA), Committee for Medicinal Products for Human Use (CHMP) (Hem, 2008). 56 1.6 Human cytomegalovirus: Immunobiology and Pathogenesis 1.6.1 Molecular characteristics of human cytomegalovirus Human cytomegalovirus (CMV) is a ubiquitous human pathogen of the β herpesviridae group (Weller, 1971). CMV is a linear double stranded DNA (dSDNA) virus and the largest among the human herpesviruses with a genome of 250kb encoding approximately 165 genes (Davison et al., 2003). The genome of CMV comprises covalently linked long (L) and short (S) segments, each consisting of a unique region (UL and US), flanked by terminal and internal inverted repeats (TRL and IRL, TRS and IRS) that yields the overall genome orientation TRL-UL-IRL-IRS-US-TRS [reviewed in (Crough and Khanna, 2009)]. Productive infection triggers the coordinated synthesis of proteins in three time based overlapping phases, namely, immediate early (IE) (0 to 2 h), delayed- early (<24 h) and late (>24 h) (Stinski, 1978). The CMV virion (Fig. 1.6) comprises three major layers, the icosahedral nucleocapsid containing the linear dsDNA, a proteinaceous matrix called the tegument that envelopes the icosahedral nucleocapsid and a lipid bilayer containing a number of glycoproteins which envelopes the tegument (Chen et al., 1999). 57 The nucleocapsid is made up of five viral proteins that are encoded by the UL86, UL85, UL80, UL48.5 and UL46 genes, and is assembled in the nucleolus (Daniel N. Streblow, 2006). The tegument contains the majority of the virion proteins. There are about 20-25 virion associated tegument proteins, including phosphoprotein (pp) 65, virion transactivator pp71, core virion maturation protein pp150, the largest tegument protein (UL 48 gene product) and the UL99 encoded pp28. The majority of these proteins are phosphorylated and their roles are not yet clearly understood (Mocarski, 2007). The envelop contains an incompletely defined number of viral derived glycoproteins. The glycoprotein B (gB) complex, gM/gN complex, and gH/gL/gO complex are abundant, and are known to be involved in cell attachment and viral infectivity (Vanarsdall et al., 2008; Varnum et al., 2004). 1.6.2 CMV pathogenesis CMV infection is ubiquitous in nature, it is estimated that seroprevalence generally ranges between 30 to 70% in developed countries, and up to 90% in developing countries. Transmission of CMV occurs via saliva, sexual contact, placental transfer, blood transfusion, breast feeding, solid organ transplantation (SOT) or hematopoietic stem cell transplantation (SCT) (Sia and Patel, 2000). Similar to other herpesviruses, after primary infection CMV establishes a latent infection with intermediate reactivation in the normal host (Sinclair and Sissons, 2006). In general, CMV primary infection and reactivation from latency in an immunocompetent host is usually asymptomatic. However, reactivation in an immunocompromised host, particularly transplant patients and individuals with HIV often causes serious disease (Drew, 1988). In addition, CMV infection in utero or in newborns with an immature immune system can cause congenital infection. 58 1.6.2.1 Congenital infection Congenital infection in newborns leads to significant neurologic disorders such as hearing loss, mental retardation, cerebral palsy, seizure disorders and developmental delay. CMV has been considered the leading infectious cause of damage to the developing fetus in utero. In the United States, Europe and other developing countries development of a vaccine is a high priority because of the cost and burden of congenital CMV infection. In the United States, congenital CMV infection occurs in approximately 1 in 50 live births and causes permanent disabilities like hearing and vision loss, and cognitive impairment. Every year, 40,000 children are born with congenital CMV infection, causing 400 deaths and leaving approximately 8000 children with permanent disabilities in the United States alone. Approximately 10% of infants are infected by the age of 6 months following transmission from their mothers via the placenta or by breastfeeding (Landolfo et al., 2003). The cost of healthcare in the US due to CMV infection in 1990 alone was estimated to be $1.9billion, with a cost per affected child of over $300,000. In developed countries, CMV infection is the most common congenital infection with 0.5% and 2% of newborns infected in utero. The concern is particularly acute for CMV-seronegative women of child bearing age, with 27,000 estimated new infections in the United States each year. A tenth of the children born to them suffer from visceral organomegaly, microcephaly with intracranial calcifications, chorioretinitis and skin lesions including petechiae and purpera (Shenk and Stinski, 2008). Furthermore, in Europe CMV infection affects 1-3% of new born babies, causing sensorineural hearing loss and costing 260,000euros per infected child (Caroppo et al., 2005). Anti-CMV drugs cannot be administered to gravid women with congenital infection because of potential toxicity to the fetus, and there is a clear need for effective strategies that minimise 59 infection in the mother, transplacental transmission of the virus, and/or fetal disease (Barry et al., 2006). 1.6.2.2 CMV pathology in transplant recipients Over the last two decades significant progress has been made in treatment for a number of terminal illnesses through bone marrow (BMT), hematopoietic stem cell transplantation (HSCT) and SOT. However, in these transplantation settings CMV is a major cause of graft versus host disease as well as viral disease. Virus infection or reactivation occurs in 44-85% of transplant recipients, and symptomatic disease occurs in 8, 29, 25, 50, 22 and 39% of kidney, liver, heart, pancreas/kidney, pancreas, human small bowel and heart-lung transplantation recipients, respectively (Ho, 1994; Reyes et al., 1992). CMV infection is associated with significant morbidity, mortality and graft loss (Baldanti et al., 2008). Well documented evidence reveals that three kinds of CMV transmission take place in SOT recipients. Primary infection progresses when a CMV-sero-negative individual receives an organ from a sero-positive donor followed by reactivation of latent virus. Secondary infection develops when endogenous latent virus is reactivated after post-transplantation in CMV seropositive individuals. Super infection occurs when a sero-positive recipient receives latently infected cells from a seropositive donor due to the reactivation of virus from donor origin (Chou, 1987). Approximately 20-60% of all transplant recipients develop symptomatic CMV infection. The Donor (D) +/ Recepient (R)- group is at of severe infection during the first 3 months following transplantation compared to the D+/R+ group. However, the D+/R+ group has worse graft and patient survival compared to the D+/R- group (Brennan, 2001). In the bone marrow/stem cell transplant recipients, reactivation of CMV occurs in the majority of patients who are seropositive before BMT. D+/R- 60 patients develop infection about 30% of the time. CMV can cause multiorgan disease such as pneumonia and gastrointestinal manifestations after BMT (Boeckh and Boivin, 1998). In HSCT patients, pneumonia is the most common manifestation. CMV infection has also been associated with atherosclerosis and chronic rejection, and two consequences of this are late graft loss (chronic rejection) and cardiovascular dysfunction. Latent CMV infection is also associated with an increased rate of restenosis after coronary angioplasty in non-transplant seropositive individuals. CMV causes serious opportunistic infection in individuals with a compromised immune system due to HIV infection. During the highly active antiretroviral therapy + -3 (HAART), these individuals will have CD4 T cell count below 100 mm which causes a high risk of CMV disease (Steininger et al., 2006). On reactivation in HIV patients, CMV causes CD3 receptor-mediated T cell hypo-responsiveness which cannot be reversed by IL-2. Common manifestation in HIV patients with CMV infection are retinitis, oesophagitis, colitis, gastritis, hepatitis and encephalitis (Gallant et al., 1992). CMV also causes faster progression to AIDS and death (Sabin et al., 2000). Yust et al. observed that the median survival of patients with CMV retinitis and extraocular disease was 7 to 11 months, (Yust et al., 2004). 1.6.3 Immunobiology of CMV CMV triggers strong antiviral responses upon infection. Controlling the viral infection in vivo requires coordination between the innate and adaptive arms of the host immune system. 61 1.6.3.1 Innate immunity against CMV During viral infection the innate immune response is stimulated by TLR signalling. Pathogens like CMV activate transduction pathways to induce the secretion of inflammatory cytokines that recruit cells of the innate immune system and up regulate UL48 gene product costimulatory molecules such as CD80 and CD86 (Boehme and Compton, 2004; Compton et al., 2003). Murine CMV (MCMV) has been shown to activate the innate immune system through TLR9 and TLR3 signalling (Tabeta et al., 2004). CMV has also been shown to activate and signal through the interaction of gB/gH and TLR2, which leads to the production of inflammatory cytokines (IC) and type I IFNs (IFN-α/β) by DCs and macrophages and also activates NK cells (Boehme et al., 2006; Compton et al., 2003). CMV recognition by TLR2 also leads to the activation of the type I IFN pathway and IC secretion by fibroblasts (Juckem et al., 2008). Both classes of molecules are important in the innate immune response and contribute significantly to controlling viral infections (Sen, 2001; Stark et al., 1998). In addition, NK cells are shown to be involved in the clearance of experimental MCMV infection and the adoptive transfer of NK cells can provide protection against MCMV (Bukowski et al., 1985). Furthermore, in renal transplant patients NK cell activity increases during primary and recurrent CMV infection, indicating that NK cells are involved in recovery from CMV infection (Venema et al., 1994). 1.6.3.2 Humoral responses CMV is a potent antigen that can trigger strong humoral and T cell mediated immune responses. The humoral response to CMV is dominated by responses to outer 62 envelope glycoproteins. Among these glycoproteins, gB which is involved in cell attachment and penetration, is the major target of neutralising antibodies. In CMV seropositive individuals approximately 40 to 70% of the total serum neutralising antibodies are specific for gB (Britt et al., 1990). Additionally, gH is another target of the humoral immune response, although less than 10% of normal CMV positive individuals have been shown to develop gH-specific antibodies (Rasmussen et al., 1991). During CMV infection neutralising antibodies control infection by preventing the interaction of viral glycoproteins with surface receptors. A study carried out by Nigro and his colleagues revealed that intravenous administration of CMV hyper immunoglobulin (HIG) in two different dose regimens, a “therapy” regimen or a “prevention” regimen significantly lowered the risk of congenital CMV infection (p=0.04) (Nigro et al., 2005). A recent study using a recombinant CMV gB vaccine formulated with MF59 adjuvant showed 50% efficacy and potentially reduced the burden of congenital CMV infection (Pass et al., 2009). These studies strongly indicate that gB is a potent target of neutralising antibodies and key epitopes are contained within the conserved regions of gB. 1.6.3.3 T cell mediated immune responses Although primary infection can induce potent humoral immune responses, latent virus is not eliminated from the infected host. In persistently infected + + individuals, CMV specific CD8 T cells, CD4 T cells and γδ T cells are crucial for controlling and restricting viral replication [reviewed in (Crough and Khanna, 2009)]. + In healthy virus carriers 10-40% of CD8 T cells in the peripheral blood can be specific for CMV antigens (Crough et al., 2005; Elkington et al., 2003; Khan et al., 63 + 2002; Manley et al., 2004b). The CMV specific CD8 T cell response is considerably diverse; responses are generated against a variety of structural, early, and late antigens in addition to CMV-encoded immunomodulators. An ex vivo T cell assay using + + overlapping 15-mer peptides from all 213 ORFs showed CD8 and/or CD4 T cells responses are directed towards more than 70% of ORFs (Elkington et al., 2003; + Sylwester et al., 2005). However, most immunodominant CMV specific CD8 T cells responses are directed towards UL123 (IE1), UL122 (IE-2) and UL83 (pp65) [reviewed in (Crough and Khanna, 2009)]. + There is clear evidence that CD4 T cells also play a crucial role in controlling CMV infection. During primary infection in asymptomatic and symptomatic + individuals CD4 T cell responses have been shown to influence the outcome of disease (Komanduri et al., 1998). In healthy seropositive individuals, high frequencies + of CD4 T cells are committed to CMV immunity. A median of 9.1% of circulating + CD4 memory T cells were shown to be specific for CMV (Sylwester et al., 2005). However, this proportion may increase up to 40% in some donors (Sester et al., 2002). + Antigen specificity analysis of the CD4 T cells has showed broad antigen recognition. + In some healthy individuals >30% of CD4 T cells are gB specific, and occasionally, in <5% of individuals higher frequencies of precursors for TRL14 and UL16 can be detected (Sylwester et al., 2005). + + Apart from CD4 and CD8 T cells, γδ T cells also play a significant role in controlling CMV infection. Whilst γδ T cells comprise <6% of the lymphocyte population in the blood of healthy humans, a substantial increase in the frequency of γδ T cells is evident in areas of the body exposed to the external milieu, such as the intestinal mucosa (Dechanet et al., 1999b). In MCMV infected mice, accumulation of γδ T cells has been shown to occur in the salivary glands (Cavanaugh et al., 2003; Ninomiya et al., 2000), and the depletion of these cells led to significantly increased 64 MCMV titers (Ninomiya, Takimoto et al. 2000). A study of renal transplant patients revealed that a marked increase in the number of circulating γδ T cells from <5% up to 40% of total T cells was coincident with active CMV infection (Dechanet et al., 1999a). A sustained expansion of γδ T cells is associated with prolonged and elevated antigenemias and increased severity of CMV infection (Lafarge et al., 2001). All of the observations from these studies clearly demonstrate the involvement of γδ T cells in the anti-CMV immune response. 1.6.4 Cytomegalovirus vaccine research A number of vaccines have been explored over the last four decades that mainly target three groups of individuals: neonates, transplant and immunosuppressed individuals, as well as subjects for universal immunisation (Table 1.5) (Griffiths et al., 2011; Khanna and Diamond, 2006; Kharfan-Dabaja et al., 2012; Pass, 2009; Schleiss, 2005; Whitley, 2004). As a first attempt, a live attenuated vaccine was developed from the AD169 strain of CMV by extensively passaging the isolate in human fibroblasts. This vaccine was found to be safe in seronegative adults. A few individuals developed minor adverse reactions such as fever, headache, fatigue and myalgia. Regarding immunogenicity, it showed a good antibody response in CMV seronegative individuals, but no significant change was observed in seropositive individuals (Elek and Stern, 1974; Neff et al., 1979; Stern, 1984). Later in the 1970s, a potential live attenuated vaccine candidate was developed from the Towne strain originally isolated from a congenitally infected two-month-old infant. This strain was extensively passaged in WI-38 human diploid fibroblasts (Elek and Stern, 1974; Neff et al., 1979). More than 800 individuals were tested with the Towne vaccine, the formulation was + + found to be safe except with minor side-effects, and induced CD4 and CD8 T cell 65 and antibody responses. However, the responses declined over the course of 12 months (Jacobson et al., 2006), and antibody responses were variable between studies. One subsequent study reported a stronger neutralising antibody response with the Towne strain compared to natural infection (Adler et al., 1998). Other studies using the Towne vaccine in seronegative renal transplant recipients demonstrated an 85% reduction in CMV disease (Balfour, 1991; Plotkin et al., 1984). Other studies have demonstrated that immunisation of seronegative women with this vaccine does not protect from CMV infection. However, CMV seropositive women were protected from re-infection (Adler, 1995). An efficacy study of the Towne vaccine in seronegative adult men later challenged with low passage wild type (Toledo strain) CMV(Plotkin et al., 1989) showed no protection from challenge. All these observations suggest that the Towne vaccine is safe and well tolerated, and able to induce both humoral and adaptive immune responses but is less potent than natural infection. This could be due to over-attenuation, presumably as a result of mutations rendering it of suboptimal efficacy. A large deletion was observed in the attenuated viral genome at the right end known as the ULb region (Huang et al., 1980; Mocarski et al., 1987; Prichard et al., 2001). This region spans a number of viral genes including those involved in immune modulation, hypothetically affecting immunogenicity (Cha et al., 1996; Hahn et al., 2004; Murphy et al., 2003; Wang and Shenk, 2005). The deleted region contains at least 19 ORFs (UL 133,151), including UL144, 146 and 147, which encode proteins homologous to CXC (∞) chemokines, IL-8, and the TNF receptor. There is evidence suggesting that these gene products may be essential for viral infection in vivo (He et al., 2006; Ma et al., 2006; Prichard et al., 2001). In another approach to improve the immunogenicity of the Towne vaccine, a series of recombinant vaccines were generated by replacing the deleted regions of the Towne strain with the Toledo strain of CMV. The Towne/Toledo chimeric construct was 66 found to be more immunogenic than the Towne vaccine (Heineman et al., 2006). However, safety concerns such as establishing a latent infection and vaccine safety in pregnant women remains a major concern for regulatory authorities (Gandhi and Khanna, 2004) . In subsequent studies, researchers concentrated on subunit vaccines to reduce the safety concerns associated with live and attenuated CMV vaccines. There have been numerous attempts at the generation of a potent glycoprotein vaccine. Of the several viral glycoproteins, gB is responsible for about half of neutralising activity in seropositive humans. However, additional immunisation with gH and gN were shown to strengthen the immunogenicity of a subunit gB vaccine (Britt et al., 1990; Gonczol and Plotkin, 2001). Several strategies have been followed for the development of a gB subunit vaccine; including full length and truncated forms of gB inserted into various expression vectors for protein expression or for use as a live vector. The protein has been expressed in whole or truncated forms in Chinese hamster ovary (CHO) cells (Simmons et al., 1998), or via adenovirus 5, vaccinia, alphavirus replicon system and canarypox (ALVAC) (Gonczol et al., 1995; Gonczol et al., 1991; Marshall et al., 1990). Delivery of gB through live viral vectors showed convincing results in murine models but human studies were disappointing (Adler et al., 1999). The CHO-derived gB, combined with MF59, elicited neutralising antibodies (titre of 1:60) in seronegative individuals after three doses (Gonczol and Plotkin, 2001; Pass et al., 1999) and elicited high titres (1:638) in toddlers. In many of the gB formulations both mucosal IgG and secretary IgA antibodies were elicited. However, whilst overall titres in adults were similar to natural infection, titres declined over a 12 month period from st th 1:115 in the 1 month to 1:67 in the 12 month. Additionally, according to recently published results by Pass and colleagues, CMV gB/MF59 vaccine shown 50% efficacy for prevention of CMV infection in young mothers on the basis of infection rates per 67 100-person-years (Pass et al., 2009) and in seronegative transplant patients with seropositive donors, where duration of viraemia was significantly reduced (Griffiths et al., 2011). Nevertheless, one major hurdle in the development of a CMV gB vaccine is the persistence of antigenic variability in the gB coding region and the presence of four distinct genotypes of gB (Chou and Dennison, 1991; Wang et al., 1996). Increasing evidence suggests that for the generation of potent immunity against CMV, a vaccine should elicit both humoral and cellular immune responses. Indeed, the adoptive transfer of CMV specific CTL from bone marrow donors to high risk transplant patients tremendously increases anti-CMV cytotoxic activity in recipients (Walter et al., 1995). Recent developments have focussed on pp65 and IE1, both of which induce strong CTL responses (McLaughlin-Taylor et al., 1994; Wills et al., 1996). Seronegative individuals administered an ALVAC vaccine expressing pp65 + developed CD8 CTL response similar to naturally infected seropositive individuals (Berencsi et al., 2001). In another approach, Reap et al. used an attenuated strain of an alphavirus, venuzuelan equine encephalitis, to produce virus like replicon particles expressing various combinations of CMV pp65, IE1 or gB. They were able to induce CMV specific neutralising antibodies and a CTL response (Reap et al., 2007a). In recent years another method of CMV vaccine development has employed DNA-based vaccination. At present, a trivalent DNA vaccine and a trivalent VRP vaccine consisting of gB, pp65 and IE1 are in a phase I study (Reap et al., 2007b; Vilalta et al., 2005). DNA vaccination generated antibody responses to gB, and CTL responses to pp65 in mice (Endresz et al., 1999; Gonzalez Armas et al., 1996; Hwang et al., 1999; Pande et al., 1995; Selinsky et al., 2005) but these vaccines have just entered into phase I trials, and efficacy data are not yet available [reviewed in (Gandhi and Khanna, 2004)]. In addition, a DNA vaccine formulation consisting of two plasmids encoding CMV gB and pp65, delivered through poloxamer (non-infectious 68 nanoparticles) was evaluated in HSCT CMV positive recipients. Results showed improved CTL and antibody responses that were sustained for one year (Richard T. Kenney, 2010 ). Furthermore, in a recent phase 2 trial this vaccine significantly reduced the occurrence and recurrence of cytomegalovirus viraemia, and improved the time-to- event for viraemia episodes compared with placebo in HSCT patients (Kharfan-Dabaja et al., 2012). Another provocative approach for the development of a CMV vaccine is the use of dense bodies (DBs). DBs are formed during viral replication as replication defective enveloped particles; they contain dominant immune antigens for both humoral and adaptive immune responses. In preclinical studies, DBs were shown to elicit antibody and CTL responses (Pepperl-Klindworth et al., 2003; Pepperl et al., 2000). In animal studies the DB vaccine was safe, but efficacy data are not available. Another approach uses a replication deficient adenoviral vector encoding 46 CMV T cell epitopes covalently linked to glycoprotein. Preclinical assessment in HLA-A2 mice and humans showed that this formulation is capable of inducing CTL and + + humoral immunity and also recalls CD8 and CD4 T cell memory responses (Zhong et al., 2008). However, concerns like pre-existing immunity to the adenoviral vector may compromise the use of this vaccine formulation (Barouch et al., 2004; Ophorst et al., 2004; Sumida et al., 2005). 69 Table 1.5: Current status of CMV vaccine development Vaccine Type Target population Efficacy Reference of study First isolation of CMV (Craig et al., 1957) from human tissue Women Prevent disease not infection Towne live attenuated Clinical Children Prevent disease (Plotkin et al., 1991) Seronegative adults Prevent disease To be Toledo recombinant Clinical Seropositive adults (Heineman et al., 2006) determined Canarypox-gB Clinical Seronegative adults Low efficacy (Adler et al., 1999) To be Canarypox-pp65 Clinical Seronegative adults (Berencsi et al., 2001) determined Modified vaccinia virus Data not Preclinical (Wang et al., 2007) Ankara-pp65/IE-1 available To be Adults (Pass et al., 1999) determined Data Not Children Clinical available gB/ MF59 subunit 50% efficacy Young women (Pass et al., 2009) reduced Transplant recipients (Griffiths et al., 2011) viraemia Data not (Selinsky et al., 2005) available Bivalent DNA plasmid Clinical Seronegative adults (gB+pp65) reduced (Kharfan-Dabaja et al., viraemia 2012) Trivalent DNA plasmid Data not Clinical Seronegative adults (Morello et al., 2005) (gB+pp65+IE-1) available Peptide vaccine (CTL- To be Clinical Transplant recipients (La Rosa et al., 2002) T-helper epitope) determined Data not (Pepperl-Klindworth et Dense-body vaccine Preclinical available al., 2002) Showed (Zhong and Khanna, Polyepitope vaccine Preclinical protection 2009) 70 1.7 Specific aims of study Research Plan The hallmark of many highly effective viral vector-based vaccines (e.g. smallpox and yellow fever) is the induction of robust cellular and humoral immune responses. However, achieving such a response with synthetic/recombinant protein- based vaccines has been a challenge. The limitation for many recombinant protein- based vaccines is that currently available adjuvants do not stimulate a strong cellular immune response. My PhD project is aiming to formulate a new vaccine against CMV which will be effective against virus-associated diseases in newborn babies and transplant patients. In this project, I am proposing a novel strategy based on the activation of TLRs, which have been shown to play a crucial role in shaping the immune response to various pathogens. Hypothesis A prophylactic vaccine based on recombinant glycoprotein B and multiple T cell epitopes as a polyepitope protein induces both humoral and cellular CMV immunity. The humoral immunity induced by this vaccine is capable of neutralising CMV and the cell mediated immune response is capable of eliminating CMV-infected cells. 71 Relevant Aims 1. Developing a new formulation strategy for CMV encoded gB vaccine which induces both humoral and cellular immunity. 2. To express multiple T cell epitopes from CMV as a polyepitope protein using a prokaryotic expression system and assess its immunogenicity in vitro. 3. To develop and test a prophylactic vaccine formulation based on a combination of CMV-encoded gB and polyepitope proteins. 4. Preclinical assessment of the immunogenicity of a glycoprotein B and polyepitope protein based cytomegalovirus vaccine in combination with human compatible adjuvants 72 Chapter 2: Materials and Methods 2.1 List of Chemicals Acetic Acid glacial BDH chemicals, Poole, UK Acrylamide and Bis-acrylamide Bio-Rad Laboratories, CA, USA Agarose, DNA grade Lonza, Cologne, Germany Ammonium persulphate Bio-Rad Laboratories, CA, USA Ampicillin Sigma-Aldrich, St.Louis, MO, USA Bromophenol blue Sigma-Aldrich, St.Louis, MO, USA Chloroform Sigma-Aldrich, St.Louis, MO, USA Coomassie brilliant blue Bio-Rad Laboratories, CA, USA Dimethyl sulphoxide (DMSO) Sigma-Aldrich, St.Louis, MO, USA DNA molecular weight marker New England Biolabs, MA, USA Dulbecco’s Modified Eagle Medium (DMEM) Invitrogen, NY, USA Ethanol Sigma-Aldrich, St.Louis, MO, USA Ethidium bromide Sigma-Aldrich, St.Louis, MO, USA Ethylene diamine tetra acetic acid (EDTA) APS Chemicals LTD, NSW, Australia 73 Ficoll-Hypaque Sigma-Aldrich, St.Louis, MO, USA Glycerol APS Chemicals LTD, NSW, Australia Glycine APS Chemicals LTD, NSW, Australia Hydrochloric acid Ajax Chemicals, NSW, Australia Isoamyl alcohol Sigma-Aldrich, St.Louis, MO, USA 2-β-mecaptoethanol Sigma-Aldrich, St.Louis, MO, USA Methanol Fronine Pty LTD, NSW, Australia Molecular weight marker kit (10-250 kDa) Bio-Rad Laboratories, CA, USA Penicillin G Invitrogen, NY, USA Phenol Sigma-Aldrich, St.Louis, MO, USA Potassium Chloride Ajax Chemicals, NSW, Australia Protease inhibitors cocktail tablets Roche Diagnostics, Mannheim, Germany Propan-2-ol (iso-propyl alcohol) BDH Chemicals, Poole, UK Skim milk powder Bio-Rad Laboratories, CA, USA Sodium acetate BDH Chemicals, Poole, UK Sodium carbonate BDH Chemicals, Poole, UK Sodium chloride Ajax Chemicals, NSW, Australia Sodium dodecyl sulphate Bio-Rad Laboratories, CA, USA Sodium hydrogen carbonate BDH Chemicals, Poole, UK 74 di-sodium hydrogen orthophosphate BDH Chemicals, Poole, UK Sodium hydroxide BDH Chemicals, Poole, UK Streptomycin sulphate Sigma-Aldrich, St.Louis, MO, USA N,N,N′,N′-Tetramethylethylenediamine (TEMED) Sigma-Aldrich, St.Louis, MO, USA Tris Invitrogen, NY, USA Triton X-100 Sigma-Aldrich, St.Louis, MO, USA Tryphan blue ICN Biochemicals, NSW, Australia Trypsin CSL LTD, VIC, Australia Tween 20 Sigma-Aldrich, St.Louis, MO, USA 2.2 Equipment Analytical balance, Sartorius Sartorius AG, Gottingen, Germany Autoclave, Tomy ES-315 Tomy, Tokyo, Japan Bench top centrifuge, Sigma D-37520 Sigma, Osterode am Harz, Germany CO2 incubator-Steri-Cult 200 Forma Scientific, Marietta, USA Cell counting microscope, Olympus, CX41 Olympus, Tokyo, Japan o Cryo 1 C freezing container Nalgene, NalgeNunc, Rochester, USA 75 Flow Cytometer, FACS cantoII Becton Dickinson, San Diego, USA o Freezer -70 C Thermo Forma, Merietta, USA Haemocytometer Sigma-Aldrich, Stenheim, Germany Haemocytometer coverslips Menzel-Glaser, Menzel, Germany Dry Block heater, Ratek DBH10 Ratek Instruments, Melbourne, Australia Inverted microscope, Olympus CK40 Olympus, Tokyo, Japan Laminar air flow, Clede Apac BH200 Clyde-Apac, Adelaide, Australia Microcentrifuge, Eppendorf 5415D Eppendorf, Hamburg, Germany Milli-Q water supply Millipore corporation, Billerica, USA Hot air oven Sanyo Corporation, Tokyo, Japan pH meter, Ionode Denver Instruments, Denver, USA Spectrophotometer, BioMate3 Thermo Spectronic, New York, USA Water bath Grant Instruments Inc., Shepreth, UK. 2.3 Media and buffers Media was prepared by the Queensland Institute of Medical Research (QIMR) media department. After preparation media was sterilised by filtration through a 0.22 µm membrane filter or if desired, buffers and solutions were sterilised by autoclaving o 2 o at 121 C (15 lb/inch ) for 20 min and stored at 4 C. A class II biological safety cabinet was used for the preparation of media and cell culture buffers. 76 Phosphate buffer saline (PBS) was prepared by dissolving 2.85 g of Na2HPO4.2H2O, 0.625 g of NaH2PO4.2H2O and 8.7 g of NaCl in 1L of Milli Q water and the pH was adjusted to 7.4. Foetal Bovine Serum (FBS) was supplied by JRH Biosciences, Lenexa, USA. Prior o o to use the FBS was heat inactivated at 56 C for 60 min, aliquoted and stored at -20 C. RPMI-1640 (R-) Medium contained L-glutamine and 100 U/mL of penicillin and streptomycin (Gibco, Grand Island, USA). R-10 Medium consisted of R-medium with 10% FBS. T-cell medium consisted of R-medium supplemented with 10% FBS, 30% T-cell growth factor (TCGF), 30 to 100 IU of rIL-2, 100 IU/mL penicillin and 200 µg/mL streptomycin sulphate. Dulbecco’s Phosphate buffer saline contained 0.13 M NaCl, 2.7 mM KCl, 8.7 mM Na2HPO4 and 1.5 mM KH2PO4. Dulbecco’s Modified Eagle Medium (DMEM) consisted of liquid DMEM (Invitrgen/Gibco BRL) supplemented with 10% FBS, 100 IU/mL penicillin and 200 µg/mL streptomycin sulphate. DMEM with high glucose for mouse T cell culture (mouse T cell culture medium) consisted of liquid DMEM supplemented with 10% FBS, 100 IU/mL penicillin, 200 µg/mL streptomycin sulphate, β-mercaptoethanol, non-essential amino acids and sodium pyruvate. Cell freezing medium consisted of RPMI 1640 containing 10% FBS and 10% Dimethyl sulfoxide (DMSO). Trypan blue was prepared as a 0.4% (W/V) solution. 77 Trypsin/versene consisted of 0.5 mM Versene (EDTA) and 0.1% Trypsin. 50X TAE buffer was prepared by dissolving 242g of Tris in distilled water, adding 57.1 mL of glacial acetic acid and 100 mL of 0.5 M EDTA pH 8.0 and the final volume was made up to 1000 mL. 2.4 Molecular methods 2.4.1 Agarose gel electrophoresis DNA samples were analysed using agarose gel electrophoresis. The desired percentage (W/V) of agarose gel was prepared in 1X TAE buffer. To visualise the DNA samples under ultraviolet (UV) light 1 µL of ethidium bromide (10 mg/mL) stock solution was added to the molten agarose just before the solidification. The DNA samples were mixed with 6X loading buffer (New England BioLabs, MA, USA) and electroporated at 80-100 V for 45-60 min on a horizontal agarose slab. To quantify the DNA size, DNA molecular weight markers were run in parallel with the DNA samples. DNA was visualised on a UV light transilluminator (Gene Genus Bio- Imaging System, Medos Co, Australia). 2.4.2 Transformation of chemically competent E. coli cells Unless otherwise stated, purified DNA plasmids were transformed into chemically competent E. coli DH5α or BL21 (DE3) E. coli (Invitrogen, NY, USA) cells using the heat shock method. In the heat shock method, 100 ng of Plasmid DNA (in a volume not exceeding 10% of the volume of the competent cells) was added to 100 µL of the competent cells, incubated on ice for 30 min followed by heat shock at o 42 C for 45 seconds. The cells were immediately cooled on ice for 2 min, 900 µL of 78 o Luria broth medium (Invitrogen, NY, USA) was added and the cells incubated at 37 C for 1 hour on a shaker at 200 rpm to allow expression of the antibiotic resistance genes encoded by the plasmid. Following incubation, approximately 100-200 µL of transformed E. coli cells were spread on a Luria-Bertani agar (LB agar) plate o containing an appropriate antibiotic and incubated at 37 C overnight in an inverted position. 2.4.3 Plasmid DNA amplification and purification A single colony from a transformed plate was inoculated into 5 mL of LB o broth containing an appropriate antibiotic and grown at 37 C overnight on a shaker at 200 rpm. Based on the quantity of the plasmid requirement the overnight culture was scaled up to 5 mL for a plasmid DNA miniprep, 50-100 mL for a plasmid DNA midiprep or 500-1000 mL for a plasmid DNA maxiprep. Plasmid DNA was purified using a QIAGEN (Hilden, Germany) a miniprep, midiprep or maxiprep kit by following the manufacturer’s protocol. 2.4.4 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Based on the molecular weight of the protein being assessed, 8-12% resolving gels and 4% stacking SDS-PAGE gels were prepared from a 30% (W/V) acrylamide:bisacrylamide stock solution. In addition to acrylamide:bisacrylamide stock solution, 0.4 M Tris-HCl, pH 8.8, 0.1% SDS, 0.025% of (V/V) TEMED and 0.7% ammonium sulphate (APS) was added to polymerise the gels. Samples were prepared in SDS-PAGE loading buffer (50 mM Tris-HCl, pH 6.8, 2% V/V SDS, 10% V/V glycerol, 0.1% W/V Bromophenol blue and 4.5% DL-Dithiothreitol or β 79 o mercaptoethanol (as a reducing agent), heated at 95 C for 10 min, cooled to room temperature (RT) and loaded on the gels. To estimate the molecular weight of the proteins a prestained molecular weight marker (Bio-Rad, CA, USA) was included in each gel. Electrophoresis was performed in a Mini-Protein II Cell gel apparatus (Bio- Rad, CA, USA) at RT in Tris-glycine buffer (25 mM Tris, 0.25 M glycine and 0.1% SDS) at 50 volts through the stacking gel and 100 volts through the resolving gel. 2.4.5 Coomassie blue staining Following electrophoresis, the SDS-PAGE gels were stained with 0.25% Coomassie blue (dissolved in 50% (V/V) methanol and 10% (V/V) acetic acid) for 1 hour at RT. The protein gels were destained using a destaining solution 40% (V/V) methanol and 10% (V/V) acetic acid in water. 2.5 Cell culture techniques 2.5.1 Cell lines and growth medium o MRC5 cells were grown in R-10 medium at 37 C and 6.5% CO2. EBV transformed lymphoblastoid cells lines (LCLs) were grown in R-10 medium o at 37 C and 6.5% CO2. o CEM.T1 and CEM.T2 were grown in R-10 medium at 37 C and 6.5% CO2. Human embryonic kidney 293 cells (HEK 293) were grown in R-10 medium at o 37 C and 6.5% CO2. o HEK 293 T cells were grown in R-10 medium at 37 C and 6.5% CO2. 80 2.5.2 Isolation of peripheral blood mononuclear cells by Ficoll gradient Peripheral blood samples were drawn into heparinised tubes, diluted with an TM equal volume of R- medium and layered over Ficoll-Paque (GE Healthcare Biosciences, Uppsala, Sweden) at the ratio of 20mL:10mL. The Ficoll gradient was centrifuged at 450 x g without acceleration and break. The leukocyte layer between R- media and Ficoll-Paque was collected, diluted with R- medium and centrifuged at 200 x g for 10 minutes. The cells were washed with R-medium (200 x g for 10 minutes), resuspended in R-10 medium, counted using the Trypan blue exclusion method and directly used for T cell expansion or cryopreserved in liquid nitrogen. 2.5.3 Cryopreservation of cells Prior to cryopreservation cells viability was assessed using the Trypan Blue exclusion technique before being pelleted by centrifugation at 200 x g for 5 minutes. Freezing medium was prepared prior to use and chilled on ice. Following centrifugation cells were resuspended in an appropriate volume of freezing medium and aliquoted into 1 mL cryovials. The cryovials were transferred immediately into a o prechilled cryo-freezing container and stored at -70 C for 24 hours before being transferred to liquid nitrogen. 2.5.4 Thawing of cryopreserved cells o Cells were removed from liquid nitrogen storage, thawed directly in a 37 C water bath, transferred into a 10 mL tube containing R-10 medium and centrifuged at 81 200 x g for 5 minutes. The cells were washed again with R-10 medium by centrifuging at 200 x g for 5 minutes and resuspended in an appropriate volume for R-10 medium. Cell viability was determined using the Trypan Blue exclusion technique. 2.6 Murine experiments 2.6.1 Ethics Approval All murine immunisation studies were performed in compliance with the QIMR animal ethics committee under project number P158 and protocol number A03601M. 2.6.2 Immunisations To carryout intramuscular immunisation mice were anesthetised with Isoflurane (Pharmachem, Eagle Farm, Australia), and 50 µL of vaccine formulation was administered into each thigh muscle using a 1 mL insulin syringe with a 27 gauge needle. Subcutaneous immunisations were carried out at the base of the tail in a 100 µL volume using a 1 mL insulin syringe with a 27 gauge needle. 2.6.3 Mouse serum and PBMC separation Mice were bled regularly after primary and secondary immunisation to assess antibody and cell mediated immune responses. Blood was collected from the tail vein. 82 For serum collection, blood was left at room temperature for 3-4 hours to allow retraction of clots and then centrifuged at 5000 rpm for 10 minutes. Serum was o o separated, heat inactivated at 56 C for 30 minutes and stored at -70 C. For PBMC separation, mouse blood was collected in DMEM supplemented with the anticoagulant heparin and layered over Ficoll-Paque. PBMC were separated o by centrifugation at 450 x g for 15 minutes at 20 C. The leukocyte layer between the sample and the Ficoll-Paque was harvested and cells were washed twice with 10 mL of DMEM and resuspended in the desired volume of mouse T cell culture medium (DMEM supplemented with 10% FBS, penicillin, streptomycin, non-essential amino acids, sodium pyruvate and β-mercaptoethanol). 2.6.4 Splenocytes preparation Mice were sacrificed by CO2 asphyxiation and spleens were collected in 3 mL of mouse T cell culture medium. Single cell suspensions were prepared by gently mashing the spleen with a plunger of a syringe. Cells were centrifuged at 1200 rpm for 5 minutes, resuspended in 3 mL of ammonium chloride and Tris buffer (0.017M Tris base in 0.89% ammonium chloride, pH7.4) then incubated for five minutes at room temperature to deplete red blood cells. Cells were centrifuged, washed twice with PBS containing 2% FBS and resuspended in 5 mL of mouse T cell culture medium. To remove excess tissue and cellular debris, the final cell suspension was filtered through a 70 µm cell strainer (Becton Dickinson, San Diego, USA). Cell viability was then determined using the Trypan Blue exclusion method. 83 2.6.5 Isolation of mouse dendritic cells Dendritic cells (DCs) were purified from the spleens of HD mice. Spleens were placed in 7 mL of enzymatic digestion mix (R-medium with 2% FBS, 1 mg/mL collagenase and 20 µg/mL DNase I) in a Petri dish, tissue was then cut into small fragments and transferred into a 10 mL polypropylene tube. Tissue was digested by o frequently mixing with a wide bore pasture pipette for 25 minutes at 25 C and then 600 µl of 0.1 M EDTA was added to disrupt any DC-T cell complexes and mixing was continued for another 5 minutes. Undigested tissue fragments were removed by filtration through a 70 µM cell strainer. Cells were washed two times with R-2% FBS to recover the cells from the digest, counted and resuspended in 400 µL of buffer 8 (PBS, 0.5% FBS and 2 mM EDTA) per 10 total cells. To isolate CD11c positive DCs, 100 µL of CD11c MicroBeads (Miltenyi Biotech GmbH, Bergisch Gladbach, 8 o Germany) per 10 total cells were added and incubated at 4 C for 15 minutes. Cells 7 were washed by adding 1-2 mL of buffer per 10 cells, centrifuged at 200 x g for 10 8 minutes and resuspended up to 10 cells in 500 µL of buffer. CD11c DCs were separated by positive selection using autoMACS, cells were washed and resuspended in DC growth medium (R-5% FBS, penicillin, streptomycin, non-essential aminoacids, sodium pyruvate and β-mercaptoethanol). The purity of enriched CD11c DCs was determined by surface staining with AF700 conjugated anti-CD11c antibody. Surface stained cells were acquired on a BD FACSCanto II and analysed using FlowJo software. 84 2.6.6 Enzyme-linked immunosorbent (ELISA) assay to determine gB antibody titers of vaccinated mice Anti gB antibody titres were determined using ELISA as previously described (Zhong et al., 2004). Briefly, polystyrene 96-well half area plates (Costar, Corning, o NY) were coated with 1µg/mL of recombinant CMV gB protein and incubated at 4 C overnight. Serially diluted sera from vaccinated mice, control (placebo) mice or pooled serum from healthy seropositive individuals were added to the wells and incubated for 2 hours at RT. 25μL of HRP-conjugated goat anti-mouse Ig (H+L) antibody (Southern Biotech, Birmingham, Alabama 35260 USA) was added to each well followed by incubation for 1 hour at RT followed by addition of 25μL/well of substrate 3.3’, 5.5’- tetramethylbenzidine substrate reagent (eBioscience, San Diego, CA). The reaction was stopped by adding 25μL of 1N HCL and the optical density (OD) at 450 nm was determined. 2.6.7 Micro-neutralisation assay against heterologous strains of CMV Neutralising activity was determined against four different strains of CMV which include Towne (gB1 type), AD169 (gB2 type), Toledo (gB3 type) and 57A (gB4 type). Assay procedure was followed as described previously (Wang et al., 2004). In brief, human fibroblast MRC-5 cells were plated in 96 well flat bottom plates. The next day complement inactivated serum samples from gB vaccinated mice, control mice (placebo) or pooled serum from healthy seropositive individuals were serially diluted and added to a standard amount of virus particles (1000pfu/well) diluted in 30μL of R0 (RPMI with no serum) in 96 well ‘U’ bottom plates and o incubated for 2 hours at 37 C, 5% CO2. As a positive control, virus without serum and 85 a negative control serum without virus were also included in the test. The serum/CMV o mixture was then added to the MRC5 cells and incubated at 37 C, 5% CO2 for 2 hours. After incubation contents from the wells were discarded and washed gently five times with RPMI medium containing 10% FBS (R10) and a final volume of 200μL of R10 o added to each well followed by incubation for 16-18 hours at 37 C, 5% CO2. After incubation cells were fixed with 100μL of chilled methanol, incubated with peroxidase block (Dako, Denmark), followed by mouse anti-CMV IE-1/IE2 monoclonal antibody (Chemicon, CA, USA) for 3 hours at RT. Cells were then incubated with 50μL/well HRP-conjugated goat anti-mouse Ig (1:200 diluted in PBS) for 3 hours at RT. In the final step cells were stained with 20μL/well DAB plus substrate (Dako, CA, USA) for 10 mins at RT and positive nuclei with dark brown colour were counted. The percentage of neutralisation of viral infectivity was calculated using the following formula: [(number of IE1+ nuclei of CMV infected cells – number of IE1+ nuclei of serum treated CMV infected cells/ number of IE1+ nuclei of CMV infected cells) ×100]. 86 2.7 Flow cytometry 2.7.1 Reagents Flow cytometry antibodies list used in this study Table 2.1 List of antibodies used in the flow cytometry Specificity Species Conjugate Supplier Human IFN-γ mouse PE Becton Dickinson, San Diego, USA Human CD3 mouse APC Becton Dickinson, San Diego, USA Human CD4 mouse FITC Becton Dickinson, San Diego, USA Human CD4 mouse AF700 Becton Dickinson, San Diego, USA Human CD8 mouse PerCP-Cy5.5 eBioscience, San Diego, USA Human TNF mouse PE-Cy7 Becton Dickinson, San Diego, USA Human IL-2 mouse APC Becton Dickinson, San Diego, USA Human CD107 mouse FITC Becton Dickinson, San Diego, USA Human MIP1β mouse PE Becton Dickinson, San Diego, USA Mouse IFN- γ Rat PE Becton Dickinson, San Diego, USA Mouse CD3 hamster APC Becton Dickinson, San Diego, USA Mouse CD4 Rat FITC Becton Dickinson, San Diego, USA Mouse CD8 Rat PerCP-Cy5.5 Becton Dickinson, San Diego, USA Mouse IL-2 Rat APC Becton Dickinson, San Diego, USA Mouse TNF Rat PECy-7 Becton Dickinson, San Diego, USA Mouse CD11c hamster AF700 eBioscience, San Diego, USA Mouse PDCA-1 Rat FITC eBioscience, San Diego, USA Mouse IL-12 (p40/70) Rat PE Becton Dickinson, San Diego, US 87 FACS wash buffer for staining and washing the cells contained PBS with 2% FBS FACS buffer for fixing the cells was prepared by dissolving 1% paraformaldehyde o powder (Sigma-Aldrich, St Louis, USA) in PBS at 37 C in a tube covered with aluminium foil to protect paraformaldehyde from light. Following preparation, the o solution was stored in the dark at 4 C. 2.7.2 Intracellular cytokine staining to assess IFN-γ production by human T cells 5 Approximately 2x10 cells in 50 μL of R-10 medium were added to the required wells of a 96 well V-bottom plate, followed by the addition of 150 μL/well of R10 containing 0.3μL of Brefeldin A (BD Pharmingen, San Diego, CA) and 0.2 μg of o the desired CMV peptide. Cells were incubated at 37 C, 6.5% CO2 for four hours, then washed twice with PBS containing 2% FBS (wash buffer), resuspended in 50µL of wash buffer containing APC-conjugated anti-CD3, FITC-conjugated anti-CD4 and o PerCP-Cy5.5 conjugated anti-CD8 monoclonal antibodies and incubated at 4 C for 30 minutes. Cells were washed twice with wash buffer, fixed with 100 μL/well of Cytofix/Cytoperm solution (BD Pharmingen) and permeabilised by washing twice with 1X Perm/Wash buffer (BD Pharmingen). Cells were then incubated with PE o conjugated anti-IFN-γ monoclonal antibody diluted in 1X Perm/Wash buffer at 4 C for 30 minutes. Cells were washed again twice with Perm/wash buffer, resuspended in PBS containing 1% paraformaldehyde and samples acquired on a FACSCanto II. Data was then analysed using FlowJo Software (TreeStar) 88 2.7.3 Multi-parametric intracellular cytokine staining and degranulation assay to assess human T cells responses 5 Approximately 2x10 cells in 50 μL of R-10 medium were added to the required wells followed by addition of 150 μL/well of R10 containing 0.3 μL of Brefeldin A and 0.15 μL of monensin (BD Pharmingen, San Diego, CA), 10μL of FITC conjugated anti-CD107a antibody and 0.2 μg of the desired CMV peptide. Cells o were incubated at 37 C, 6.5% CO2 for four hours. After stimulation cells were washed and surface stained with PerCP-Cy5.5 conjugated anti-CD8 and AF700 conjugated o anti-CD4 antibodies for 30 minute at 4 C. After washing, fixing and permeabilising, cells were stained intracellularly with PE-conjugated anti-MIP1β, PE-Cy7 conjugated anti-IFN-γ and APC conjugated anti-TNF. Cells were acquired on a BD FACSCantoII and FACS data was analysed using FlowJo software. 2.7.4 Intracellular cytokine staining to assess IFN-γ response in mouse T cells 5 Following vaccination approximately 2x10 mouse PBMC or splenocytes in 50 μL of mouse T cell culture medium were added to the required wells. To stimulate these cells, 10 μg of gB protein or 0.2 μg of CMV peptides were added and incubated o for two hours at 37 C, 10% CO2. Following incubation, 150 μL of DMEM containing 0.3 μL of Brefeldin A (BD Pharmingen, San Diego, CA) was added to each well and o incubated overnight at 37 C, 10% CO2. Cells were washed twice with wash buffer, surface stained with APC-conjugated anti-CD3, FITC-conjugated anti-CD4 and PerCP-Cy5.5 conjugated anti-CD8 monoclonal antibodies resuspended in wash buffer o and incubated at 4 C for 30 minutes. Cells were washed twice with wash buffer, fixed 89 with 100 μL/ well of Cytofix/Cytoperm and washed twice with Perm/Wash buffer. Cells were then intracellularly stained with PE conjugated anti-IFN-γ monoclonal o antibody at 4 C for 30 minutes, cells were washed twice Perm/Wash buffer and acquired on a BD FACSCanto II. 2.7.5 Multi-parametric flow cytometry to assess the immune responses in vaccinated mice Following vaccination PBMC or splenocytes were stimulated ex vivo as mentioned above. Cells were surface stained with FITC conjugated anti-CD4 and o PerCP-Cy5.5 conjugated anti-CD8 for 30mins at 4 C. After washing, fixing and permeabilising, cells were stained intracellularly with PE-conjugated anti-IFN-γ, PE- Cy7 conjugated anti-TNF and APC conjugated anti-IL2 antibodies. Cells were acquired on a BD FACSCanto II and data was analysed using FlowJo software and Boolean gate analysis. 2.7.6 Intracellular cytokine staining to assess IL-12p70 response in mouse DCs Production of IL-12p70 and TNF cytokines was analysed by intracellular staining following stimulation with the various adjuvant combinations. After harvesting supernatants cells were resuspended in the remaining 100 µL of medium, transferred to 96 well ‘V’ bottom plates and another 100 µL of medium containing the protein transport inhibitor, Brefeldin A was added. The cells were incubated for 6 hours, washed twcie, then incubated with AF700 conjugated anti-CD11c and FITC conjugated anti-PDCA-1 monoclonal antibodies. After washing, fixing and permeabilising, cells were stained intracellularly with PE-conjugated anti-IL-12p70 90 antibody. Cells were acquired on a FACSCanto II and analysed using FlowJo software. 91 92 Chapter 3: Recombinant glycoprotein B vaccine formulation with TLR9 agonist and immune stimulating complex induces specific immunity against multiple strains of cytomegalovirus 3.1 Abstract Natural human cytomegalovirus (CMV) infection is characterised by a strain- specific neutralising antibody response. This is particularly relevant in clinical settings such as transplantation and pregnancy where re-infection with heterologous strains is occurring, and the immune system does not mount an effective response against the infecting strain due to underlying immunosuppression. There is an emerging argument that a CMV vaccine which induces high titres of cross-neutralising antibodies will be more effective in protecting individuals from infection with antigenically different CMV strains. In addition, induction of cell-mediated immunity offers the additional advantage of targeting virus-infected cells. Here, we present a novel formulation of the CMV vaccine that, by combining recombinant soluble gB protein with a TLR9 agonist (CpG ODN 1826) and immune stimulating complexes (AbISCO 100), was able to elicit strong polyfunctional CMV-specific cellular and cross-neutralising humoral immune responses. Our data demonstrate that prime-boost immunisation of HLA A2 mice with gB protein in combination with CpG ODN1826 and AbISCO 100 induced + + durable CMV-specific CD4 and CD8 T cell and humoral responses. Further, these responses neutralise infection with multiple strains of CMV expressing different gB genotypes, and afforded protection against challenge with recombinant vaccinia virus encoding gB protein. These observations argue that this novel vaccine strategy, if 93 applied to humans, should facilitate the generation of a robust, pluripotent immune response which may be more effective in preventing infection with multiple strains of CMV. 94 3.2 Introduction Human cytomegalovirus (CMV) is a β-herpes virus that can cause primary infection through transmission via multiple routes such as saliva, sexual contact, breastfeeding, placental transfer, blood transfusion, solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT), then establishes latency with periodic reactivation in the host (Crough and Khanna, 2009; Gandhi and Khanna, 2004). In immunocompetent individuals, primary infection is mostly asymptomatic; however, it can lead to symptomatic illness such as infectious mononucleosis and splenomegaly in some individuals. Due to the disease burden of primary CMV and reinfection during pregnancy, development of a vaccine to prevent congenital CMV was given highest priority by the US Institute of Medicine (Arvin et al., 2004). The potential target population for vaccination could be women of child bearing age and transplant recipients prior to immunosuppressive treatment. Recently, a subunit vaccine based on CMV glycoprotein B (gB) formulated with MF59 adjuvant successfully concluded phase II clinical trials (Pass et al., 2009). The gB/MF59 vaccine showed 50% efficacy in young mothers and congenital CMV infection occurred in 1/81 (1%) in the vaccinated group and 3/97 (3%) in the placebo group (Pass et al., 2009). The primary objective of this vaccine was to elicit neutralising antibodies in vaccinated individuals similar to natural CMV infection (Pass, 2009) and results from this study clearly indicated that this vaccine provides some protection similar to the natural CMV infection. There is an emerging argument that to improve the efficacy of the CMV gB vaccine, it will be essential to induce a humoral and cellular immune response. Indeed previous studies by Klein and colleagues have shown that human sera from naturally infected individuals often fail to neutralise heterologous CMV isolates (Klein et al., 1999). Furthermore, previous studies carried 95 out in pregnant women and transplant recipients have also suggested the importance of virus neutralising antibodies in limiting virus dissemination and reinfection with heterologous strains (Boppana et al., 2001; Ishibashi et al., 2007; Ross et al., 2010). In addition, the loss of T cell immunity in advanced HIV-AIDS and transplant recipients significantly compromises the ability of the host to restrict viral infection and replication (Barry et al., 2007; Harari et al., 2004; Reusser et al., 1991). Based on these observations, we explored the capacity of different formulations of the CMV gB vaccine to induce humoral and cellular immune responses and control multiple strains of CMV expressing different gB genotypes. Here we show that a formulation of CMV gB vaccine including a TLR9 agonist and immune stimulating complexes is highly effective in inducing strong antiviral humoral and cellular immune responses. These responses are capable of neutralising multiple strains of CMV with differing gB genotypes, and providing protection against challenge with recombinant vaccinia virus expressing gB. 96 3.3 Material and Methods 3.3.1 Generation of CMV gB expression construct and protein purification An expression vector encoding the soluble form of gB protein from Ad169 strain (with the transmembrane region of the sequence deleted and furin cleavage site mutated from Arg433 Gln433, Arg435 Thr435 and Arg436 Gln436) with a tissue plasminogen signal sequence was cloned into the pCEP4 vector (Invitrogen, Carlsbad, CA, USA). The resulting plasmid, pCEP4gB, was purified using Qiagen maxiprep kit (Qiagen, Valencia, CA, USA) and transiently transfected into freestyle HEK293 embryonal human kidney cells using Invitrogen’s 293Fectin reagent. Protein TM o expression was carried out in Freestyle expression medium (Invitrogen) at 37 C, 5% CO2 in Erlenmeyer flasks for 48 to 72 h and protein was purified by affinity chromatography using a gB-specific monoclonal antibody (Singh and Compton, 2000). 3.3.2 Generation of recombinant adenovirus encoding CMV gB protein DNA sequence encoding gB was amplified from the AD169 virus stock by PCR using gene specific primers. This PCR product was designed to encode gB sequence from the alanine residue at position 31 to valine at position 700 with the deletion of the + signal sequence. Following amplification the DNA was cloned into pBluescript II KS phagemid and confirmed by DNA sequence analysis. The assembly and production of the recombinant adenovirus encoding gB was carried out as described previously (Zhong et al., 2008). 97 3.3.3 Animal immunisation HLA A2 transgenic mice (Animal Resource Center, Canning Vale, WA, Australia) containing the human HLA A*0201 with murine α3 chain (referred to as b HLA A2/K ) (Engelhard et al., 1991; Newberg et al., 1992) were maintained in a pathogen–free animal facility at the Queensland Institute of Medical research (QIMR). All protocols were followed in compliance with the QIMR animal ethics committee. Six to eight weeks old female mice were immunised intramuscularly with varied ® concentrations of gB protein and AbISCO 100 (ISCONOVA, Uppsala, Sweden) and/or CpG ODN 1826 (Invivogen, San Diego, California, USA) followed by determination of CMV specific humoral and cellular immune response at various time points. In addition, mice immunised intramuscularly with an adenovirus vector 8 encoding CMV gB (referred to as Ad-gB 7.5x10 pfu/mice) were used as positive control. 3.3.4 Assessment of gB-specific antibody avidity CMV gB-specific avidity was evaluated as previously described (Marshall and Adler, 2003). In brief, serial dilutions of sera from immunised animals were carried out in duplicate and to induce dissociation 5M urea in PBST (PBS containing 0.05% Tween20), or PBST alone as a control, were added to the appropriate wells and then incubated with HRP-conjugated goat anti-mouse Ig for one hour. The plates were then developed as outlined for the ELISA in chapter2. 98 3.3.5 T cell responses assessed by intracellular cytokine staining (ICS) + 6 To determine the gB CD4 T cell response, approximately 1x10 splenocytes (50μL/well) in 96-well ‘V’ bottom plates were stimulated with 2.5μg of gB or DMEM + (as a negative control). The CD8 T cell responses were assessed by stimulating 50μL of splenocytes with 2μg/mL of gB peptides WQGIKQKSLVELERLANRSS, SMESVHNLVYAQLQFTYDTL and GRCYSRPVVIFNFANSSYVQ. These peptides were identified from previous experiments in which we stimulated gB vaccinated mice splenocytes with a gB matrix (polls of 20mer peptides overlapping by 10 amino acids) that, together, cover the full length of gB protein and should contain all possible T cell o epitopes. After incubation with these peptides for 2 hours at 37 C, 10% CO2, 150μL/well of DMEM containing 0.3μL of Brefeldin A (BD Pharmingen, San Diego, CA) was added and plates incubated overnight. Cells were then stained with APC- conjugated anti-CD3 (BD Pharmingen), FITC-conjugated anti-CD4 (BD Pharmingen) and PerCP-conjugated anti-CD8 (BD Pharmingen) monoclonal antibodies o resuspended in PBS containing 2% FBS, and incubated at 4 C for 30 minutes. Cells were fixed with cytofix (BD Pharmingen) and permiabilised by washing twice with 1X cytoprem (BD Pharmingen). Cells were then incubated with PE conjugated anti- IFN-γ monoclonal antibody (BD Pharmingen) for interferon-γ staining or PECy7 conjugated anti-TNF-α (BD Pharmingen) and APC conjugated anti-IL-2 (BD Phramingen) for multiple cytokine analysis, and analysed by FACS Canto. 3.3.6 Statistical Analysis Statistical analyses were done using Microsoft Office Excel 2007 and GraphPad Prism 4 software. For antibody titers, neutralising antibody titers and cellular immune 99 responses, the means and standard deviation were calculated, and p values were determined by student T test. Error bars represent standard error. 100 3.4 Results 3.4.1 Evaluation of immunogenicity of recombinant gB protein in combination with TLR9 agonist and/or immune stimulating complexes To evaluate the best compatible adjuvant combination for the CMV gB b vaccine, groups of HLA A2/K mice (5 mice in each group) were immunised with 50µg of recombinant gB protein in combination with 50µg/dose CpG ODN1826 (TLR9 agonist) and/or 13µg/dose AbISCO 100 intramuscularly. B4 3.5 Abisco 100+CpG ODN1826 gB + AbISCO 100 3 gB+Abisco 100+CpG ODN1826 A gB +/- adjuvants 2.5 gB alone CpG ODN1826 gB + CpG ODN18262 + AbISCO 100 Ad-gB 1.5 gB + 1 AbISCO 100 0.5 gB + CpG ODN1826 0 + AbISCO 100 HLA A2/Kb gB alone Serum dilutionsC CpG ODN1826 + AbISCO 100 gB + CpG ODN1826 gB + AbISCO 100 Ad-gB gB + CpG ODN1826 + AbISCO 100 gB alone gB + CpG ODN1826 Ad-gB 0 0.05 0.1 0.15 0.2 % of IFN-γ expressing CD4+ T cells Fig. 3.1 : Assessment of CMV gB-specific antibody and CD4+ T cell responses following immunization with gB protein formulated with various combinations of adjuvant(s). Panel A: Seven groups of HLA A2/Kb mice immunised intramuscularly with gB protein alone or formulated with AbISCO®-100 and/or CpG ODN1826, adjuvants alone or adenovirus vector encoding CMV gB protein. These mice were sacrificed 10 days post immunisation. Panel B: CMV gB specific antibody titers were evaluated by ELISA. The data represents the mean ± SEM from five mice in each group. Panel C: Assessment of CMV gB-specific CD4+ T cell responses following immunization. Splenocytes from these mice were stimulated with gB protein and then assessed for IFN-γ production using intracellular cytokine assays. Data presented in the figure represents the mean ± SEM from five mice in each group. In addition, animals immunised with adjuvants or gB alone were used as negative control, while mice immunised intramuscularly with an adenovirus vector encoding 101 OD at 450nm p=0.01 p=0.04 p=0.002 p=0.04 8 CMV gB (referred to as Ad-gB 7.5x10 pfu/mice) were used as positive control (Fig. 3.1A). These animals were sacrificed 10 days post immunisation and assessed for CMV gB-specific T cell immunity using intracellular cytokine analysis, and humoral responses were assessed using gB ELISA. All mice vaccinated with CMV gB protein formulated with AbISCO 100, with or without CpG ODN1826, showed high levels of antibody titres comparable with those generated with the Ad-gB vaccine (Fig. 3.1B). Low or undetectable gB-specific antibody responses were observed in mice immunised with gB alone or adjuvants (AbISCO 100 and CpG ODN1826) alone. In contrast, the assessment of gB-specific T cell responses using an IFN-γ ICS assay, revealed that gB vaccine formulated with both AbISCO 100 and CpG ODN1826 were the only combinations to induce significant cellular immunity. All other combinations induced low or undetectable T cell responses (Fig. 3.1C). The T cell responses induced by the gB vaccine formulated with both AbISCO 100 and CpG ODN1826 were significantly higher when compared to the Ad-gB vaccine. Taken together, these analyses suggested that a gB vaccine formulation with AbISCO 100 and a TLR9 agonist can elicit strong humoral and cellular responses. 3.4.2 Optimisation of CMV gB vaccine dose for vaccination Having established that a CMV gB vaccine can be potentially formulated to induce both humoral and cellular immune responses, we next conducted a series of experiments to identify the optimal dose of gB protein for this vaccine formulation. b HLA A2/K transgenic mice were immunised with varying doses of gB protein (5 g, 10 g, 50 g and 100 g) in combination with AbISCO 100 and CpG ODN1826. Ten days after vaccination, these animals were assessed for CMV-specific antibody and T 102 cell responses (Fig. 3.2A). Data on antibody responses and T cell responses are presented in Figures 3.2B & C respectively. These analyses showed that 5 g of gB protein in combination with AbISCO 100 and CpG ODN1826 was sufficient to + induce optimal antibody (Fig. 3.2B) and CD4 T cell response (Fig 3.2C). To explore + the generation of CD8 cytotoxic T lymphocyte responses, mice were assessed for reactivity to previously mapped T cell epitopes from the gB protein (WQGIKQKSLVELERLANRSS, SMESVHNLVYAQLQFTYDTL and + GRCYSRPVVIFNFANSSYVQ). Optimal CD8 T cell responses were also generated with 5 g of gB (Fig 3.2C). Assess gB-specific A antibody, gB-specific gB + CpG ODN1826 HLA A2/Kb CD4+ and CD8+ T cell + AbISCO 100 responses 10 days after vaccination B C 2.5 Adjuvant alone CD4+ T cells Adjuvant gB (5 g) CD8+ T cells alone 2 gB (10 g) gB (5 g) 1.5 gB (50 g) gB (100 g) gB (10 g) 1 gB (50 g) 0.5 gB (100 g) 0 0 0.1 0.2 0.3 0.4 0.5 % of IFN-γ expressing T cells Serum dilutions Fig. 3.2: CMV gB-specific antibody, CD4+ T cell and CD8+ T cell responses following immunisation with various concentrations of gB protein formulated with AbISCO®-100 and CpG ODN1826. Panel A: Five groups of mice were immunised intramuscularly with 5µg, 10µg, 50 µg or 100µg of CMV gB protein formulated with AbISCO®-100 and CpG ODN1826. Ten days after vaccination, these animals were sacrificed and assessed for humoral and cellular immune responses. Panel B: CMV gB-specific antibody titers were evaluated by ELISA. Panel C: Assessment of CMV gB-specific CD4+ T cell and CD8+ T cell responses following immunization. Splenocytes from these mice were stimulated with gB protein or peptide epitopes and then assessed for IFN-γ production using intracellular cytokine assays. Data presented in the figure represents the mean SEM from five mice in each group. 103 OD at 450nm p<0.001 The level of antibody and T cell responses were comparable to those generated in mice immunised with 50 or 100 g of gB. No detectable gB-specific immune response was observed in control mice immunized with AbISCO 100 and CpG ODN1826 alone. 3.4.3 Prime-boost immunisation with CMV gB vaccine formulated with AbISCO 100 and CpG ODN1826 dramatically improves immune responses To further evaluate this novel CMV gB vaccine formulation, we tested the b immunogenicity of this vaccine using a prime-boost strategy. HLA-A2/K mice were immunised with gB formulated with AbISCO 100 and CpG ODN1826 and then boosted with the same formulation on day 21 (Fig. 3.3A). Animals immunised with either a prime or boost dose alone, or with a prime-boost of adjuvant alone were included as controls for this analysis. CMV gB-specific humoral and cellular immune responses were initially assessed 10 days after the boost injection. Data presented in Figures 3.3B-D show prime-boost vaccination increased both gB-specific antibody + and CD4 T cell responses, although no significant differences were evident in the + CD8 T cell response. Mice were sacrificed 54 days after the booster immunisation (i.e. 75 days after initial priming) to assess the impact of prime-boost immunisation on long-term humoral and cellular memory responses. Although the overall antibody and T cell responses were lower on day 75 compared to day 30, these responses were significantly higher than those observed in mice given prime immunisation alone (Figures 3.3E-G). To further confirm the T cell responses detected following prime- boost immunisation, splenocytes from these mice were stimulated in vitro with previously mapped T cell epitopes from the gB protein (WQGIKQKSLVELERLANRSS, SMESVHNLVYAQLQFTYDTL and 104 GRCYSRPVVIFNFANSSYVQ) and cultured for 10 days in growth medium supplemented with IL-2. On day 10, these expanded T cells were assessed for gB- + specific CD8 T cell responses using intracellular cytokine assays. Data A Day 0 Day 21 Day 30 and Day 75 prime with boost with assess gB-specific HLA A2/KbgB + CpG ODN1826 gB + CpG ODN1826 antibody, CD4+ and + AbISCO 100 + AbISCO 100 CD8+ T cell responses Day 30 4.5 B C D 4.0 Prime-boost Prime-boost 3.5 p<0.0001 Placebo 3.0 Prime alone Boost alone Boost alone 2.5 Boost alone 2.0 Prime-Boost prime alone prime alone 1.5 1.0 Placebo Placebo 0.5 0 0 0.5 1 1.5 2 0 0.1 0.2 0.3 % of CD4+ T cells producing IFNγ % of CD8+ T cells producing IFNγ Antibody dilutions Day 75 4 E F G Placebo 3.5 Placebo Placebo prime alone 3 2.5 Prime-boost Prime-boost Prime-boost 2 p<0.0001 1.5 1 prime alone prime alone 0.5 0 0 0.5 1 0 0.1 0.2 0.3 % of CD4+ T cells producing IFNγ % of CD8+ T cells producing IFNγ Antibody dilutions m1 m2 m3 m4 m5 H 55 5 5 510 0.66 10 0.64 10 101.13 103.17 2.53 4 4 4 4 4 10 10 1010 10 3 3 3 3 3 no pep 10 10 10 10 10 2 2 2 2 10 210 10 10 10 0 0 00 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 2 3 4 5 0 10 10 10 100 10 10 10 10 0 10 10 10 10 5 10 5 5 5 5 1.39 10 10 10 101.27 1.44 5.93 5.37 4 10 4 4 4 410 10 10 10 + pep 3 10 3 3 3 310 10 10 10 2 10 2 2 2 210 10 10 10 0 0 0 0 0 2 3 4 5 0 10 10 10 10 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 50 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 CD8+ T cells Fig. 3.3 Short-term and long-term CMV-specific memory humoral and cellular immune responses following immunisation with CMV gB vaccine using a prime boost strategy. Panel A: Multiple groups of HLA A2/Kb mice were immunised intramuscularly with 5µg CMV gB protein formulated with AbISCO®-100 and CpG ODN1826 or adjuvants alone. A booster dose was given on day 21 to mice designated for prime-boost strategy and then sacrificed 10 days (day 30) or 54 days (i.e. day 75) after the final immunization. Panel B & E: Evaluation of CMV gB specific antibody titers using ELISA. Panel C, D, F & G: Assessment of CMV gB-specific CD4+ T cell and CD8+ T cell responses following immunization. Splenocytes from these mice were stimulated with gB protein or peptide epitopes and then assessed for IFN-γ production using intracellular cytokine assays. Data presented in the figure represents the mean ± SEM from five mice in each group. Panel H: Splenocytes from the prime-boost group (75 days post immunization) were stimulated in vitro with gB peptide epitopes as outlined in the Material and Methods section and cultured for 10 days in the presence of rIL-2. These cells were assessed for gB-specific T cell reactivity using intracellular cytokine assays. Data in the dot plots represents the percentage of IFN-γ producing CD8+ T cells from individual mice (referred to as m1-m5). 105 OD at 450nm OD at 450nm IFNγ p=0.0009 p=0.001 p=0.001 p=0.001 p=0.01 + presented in Figure 3.3H, shows strong expansion of gB-specific CD8 T cells (range 0.31% - 2.84%) from animals immunised with the CMV gB vaccine using a prime- boost strategy. Taken together, these observations clearly demonstrate that prime- boost immunisation with recombination CMV gB protein in combination with AbISCO 100 and CpG ODN1826 generates both humoral and cellular responses against gB which can be sustained long-term. 3.4.4 Prime-boost immunisation with CMV gB vaccine induces high avidity antibody responses that neutralise multiple strains of CMV Generation of a high avidity antibody response against the gB protein is crucial for the successful control of CMV infection in vivo. In the next set of experiments, we assessed the avidity of the gB-specific antibody response induced in mice immunised A B Prime alone Placebo Towne (gB1) Prime alone Prime-boost Boost alone Human serum AD169 (gB2) Prime-boost Toledo (gB3) Prime alone 57A (gB4) Prime-boost 0 10 20 30 40 50 60 70 80 90 100 1 4 16 64 512 1024 % of gB specific antibodies 50% neutralizing titers Fig. 3.4: Assessment of antibody avidity and cross neutralising activity of the humoral immune response induced by the CMV gB vaccine. Panel A: Avidity maturation of CMV gB-specific antibody response in mice immunised with the gB vaccine. Data represents the percentage of antibodies with gB specific avidity. Panel B: Virus neutralizing capacity of antibody responses induced following immunisation with CMV gB vaccine. Serum samples from these mice were pre-incubated with four different strains of CMV, Towne (gB1), AD169 (gB2), Toledo (gB3) and 57A (gB4). Serum treated virus preparations were then used to infect MRC-5 cells. Following overnight incubation virus infectivity was assessed using IE-1/IE-2 expression as outlined in the “Material and Methods” section. with CMV gB vaccine. Data presented in Figure 3.4A shows that although prime- boost immunisation increases the antibody titre compared to prime alone, it did not alter gB-specific antibody avidity. Next we assessed whether this high avidity antibody 106 30 days 75 days Post-immunization Post-immunization response was capable of neutralising CMV strains expressing different genotypes of gB protein (i.e. gB1, gB2, gB3 and gB4), because previous observations have suggested that neutralising antibodies are not necessarily cross-reactive to multiple CMV strains following natural infection (Klein et al., 1999). Antiviral neutralising activity in serum samples from animals receiving prime alone or prime-boost immunisations were assessed using micro-neutralising assays against the four different strains of CMV. Data presented in Figure 3.4B shows that serum samples from prime- boost animals displayed higher anti-CMV neutralising titres when compared to prime alone or placebo groups. Furthermore, the neutralising titres in prime-boost animals were comparable to those seen in serum samples from healthy virus carriers (Fig. 3.4B). More importantly, the antibody response generated in prime-boost showed strong neutralising capacity against multiple strains of CMV expressing different genotypes of gB. Data from these experiments clearly shows that antibodies from mice immunised with the prime-boost strategy can induce a high avidity antibody response which is capable of neutralising multiple genotypes of CMV. 3.4.5 Immunisation with CMV gB in combination with AbISCO 100 and CpG ODN1826 induces polyfunctional CMV-specific T cell response A number of recent studies have implicated a role for T cell polyfunctionality, including the capacity to generate multiple cytokines (IL-2, IFN- and TNF), in protection against CMV infection (Almeida et al., 2007; Harari et al., 2007; Precopio et al., 2007). To determine whether the CMV gB vaccine formulation induced a + polyfunctional T cell response, CMV-specific CD4 T cells from vaccinated mice were analysed for their ability to produce IFN-γ, TNF and IL-2 following ex vivo stimulation with gB protein. Splenocytes from these mice were isolated on days 30 107 (effector phase) and 75 (memory phase), stimulated with gB protein and the frequency + of CD4 T cells producing multiple cytokines was analysed by FACS and calculated by ‘Boolean gating’ analysis. Analysis of three cytokines allowed identification of + + seven distinct populations of gB specific CD4 T cells and the proportion of CD4 T cells expressing various combinations of cytokines. Data from this analysis is presented in Figure 3.5. This analysis revealed that the majority of the gB-specific + + + + effector and memory CD4 T cells were triple (IFN-γ , TNF and IL-2 ) and double + + - (IFN-γ , TNF and IL-2 ) cytokine producers. The proportion of triple cytokine + producers increased as the CD4 T cells moved from the effector phase to the memory phase of the immune response, indicating that the CMV gB vaccine formulation is highly effective in inducing and maintaining a polyfunctional virus-specific T cell response. Another interesting feature of these virus-specific T cells was that a large proportion of these cells produced IL-2 which suggests that these cells may support + the expansion of CD8 T cells following secondary antigen encounter. 4% 6% 8% 0% IFN+ TNF+ IL2+ IFN+TNF+IL2- 14% 25%43% IFN+TNF-IL2+ IFN+TNF-IL2- 5% 52% 2% IFN-TNF+IL2+ 1% 2% IFN-TNF+IL2- 13% IFN-TNF-IL2+ 25% Fig. 3.5: Cytokine expression by CMV-specific CD4+ T cells from CMV gB vaccinated mice. Ex vivo expression of IFN-γ, TNF-α and IL-2 by antigen-specific CD4+ T-cells from mice immunized with CMV gB vaccine formulation. Splenocytes were prepared from HLA A2/Kb mice 30 days and 75 days post-vaccination and cultured with gB protein. Brefeldin A was added during the last 6 h of incubation, followed by T cell surface marker and intracellular cytokine staining. The proportion of CD4+ T cells producing multiple cytokines on days 30 and 75 were split into seven different populations based on Boolean gating analysis of IFN-γ, TNF and IL-2. Each of the different populations expressing distinct sets of cytokines is indicated with a different colour. 108 3.4.6 Immunisation with CMV gB in combination with AbISCO 100 and CpG ODN1826 affords protection against quasi-viral infection Having established the immunogenicity of the CMV gB vaccine formulation, the next experiment was designed to determine protective efficacy of this vaccine. Due b to the species restriction of CMV, we challenge immunised HLA A2/K mice with recombinant vaccinia encoding gB (Vacc.gB) protein (Ad169 CMV strain) to evaluate the protective efficiency of the CMV gB vaccine (Fig. 3.6A). Data presented in Figure b 3.6B shows that HLA A2/k mice immunised with CMV gB vaccine showed significant reduction in the virus load following challenge with Vacc.gB when compared to the control mice which were mock immunised with adjuvants alone. A 2- 4 fold reduction in viral load was observed in animals immunised with the CMV gB vaccine. A Boost with gB gB + CpG ODN1826 + CpG ODN1826 Challenge with recombinant vaccinia + AbISCO 100 HLA A2/K b + AbISCO 100 encoding gB protein and then (day 21) assessed viral load after 3 days B 6 p=0.0003 5 4 3 2 Immunized Placebo Fig. 3.6: CMV gB vaccine induces protection against challenge with recombinant vaccinia expressing gB. HLA A2 transgenic mice were immunised with the CMV gB vaccine formulation or mock immunized with adjuvant alone and 21 days after booster immunization these mice were challenged intraperitoneally with recombinant vaccinia encoding gB (Vacc.gB) at 107pfu/mouse. Ovaries were collected four days later and used to assess viral load. Virus titres in the ovaries of mice immunized with the CMV gB vaccine or adjuvant alone are presented in the figure. All statistical analyses were conducted using GraphPad Prism 4 software. 109 Log (Vaccinia-gB load in ovaries/mouse) 3.5 Discussion Data presented in this study clearly shows that a CMV gB vaccine formulation which combines a TLR9 agonist and immune stimulating complexes can be successfully used to generate a strong cross-neutralising humoral and cellular immune responses. We also showed that a prime-boost vaccination strategy can dramatically enhance the effector and long-term memory T cell response. It is important to stress that these immune responses were induced over a wide range of gB concentrations (5- 100 g) and are well within the range of concentration used for human trials (Pass et al., 2009). One of the interesting features of the cellular immune response induced by this novel CMV gB vaccine was the polyfunctionality of antigen-specific T cells. A + + large proportion of T cells generated following vaccination were triple (IFN-γ , TNF + + + - and IL-2 ) and double (IFN-γ , TNF and IL-2 ) cytokine producers. More importantly, this polyfunctional profile was sustained long-term after vaccination. These cytokines play a vital role in protection and in the development of memory immune response. TNF plays a crucial role in establishment and maintenance of microarchitecture in secondary lymphoid organs (Kuprash et al., 1999) and protection against infectious pathogens (Flynn et al., 1995; Suresh et al., 2004). Of particular interest was the expression of IL-2 by the majority of these effector cells which indicates that these cells have high proliferative capacity and also provide help for the + generation and maintenance of anti-viral CD8 T cell responses (Cousens et al., 1995; Smith, 1988; Willerford et al., 1995). CMV displays a broad host cell range and is able to infect various cell types such as endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, leukocytes and dendritic cells (Hahn et al., 2004; Percivalle et al., 1993; Riegler et al., 2000; Sinzger et al., 1995). Infection of endothelial cells by CMV has been regarded as a 110 possible virulence factor that might influence the clinical course of infection (Gerna et al., 2002; Sinzger et al., 1999). Recent studies have shown that epithelial entry- specific neutralising antibodies titres from the gB/MF59 vaccinated individuals were several folds lower when compared to CMV natural infection (Cui et al., 2008). Another major challenge in designing an effective CMV vaccine is that the immune response induced following vaccination should be able to prevent reinfection with heterologous strains of CMV. Indeed, recent studies have shown that maternal reinfection by new strains of cytomegalovirus is a major source of congenital infection in a highly CMV-immune population (Boppana et al., 2001; Mussi-Pinhata et al., 2009; Ross et al., 2010). On the basis of sequence variation in the UL55 gene encoding gB protein, CMV can be classified into four major gB genotypes (gB1, gB2, gB3 and gB4). A number of studies have suggested that genetic variation of CMV strains may correlate with their pathogenicity in CMV-associated diseases (Nogueira et al., 2009; Roubalova et al., 2010). Although there is no direct evidence, it has been proposed that antibody responses directed towards one particular genotype may not be effective against other genotypes and thus will be ineffective in preventing reinfection with heterologous strains of CMV (Boppana et al., 1999; Boppana et al., 2001; Urban et al., 1992). It is important to stress here that our gB vaccine formulation does not target other potential neutralising components of CMV (e.g. UL128-131) which are crucial for the entry of virus in endothelial cells, epithelial cells and dendritic cells (Hahn et al., 2004; Percivalle et al., 1993; Riegler et al., 2000; Sinzger et al., 1995), + + we were successful in inducing a robust cellular immunity (CD8 and CD4 T cells) which may overcome this potential limitation and thus improve the protective efficacy of the CMV gB vaccine. Indeed, recent studies by Hansen and colleagues have shown + that anti-viral CD8 T cell responses play crucial role in preventing superinfection of rhesus CMV-infected rhesus macaques (Hansen et al., 2010). 111 Taken together, data presented in this study shows that a CMV gB vaccine formulated with AbISCO 100 and CpG ODN1826 induces a humoral immune response with cross-neutralising activity against heterologous strains of CMV expressing different genotypes of gB. Furthermore, the immune response induced by this vaccine was also effective in providing strong resistance against viral infection as demonstrated by the challenge with recombinant vaccinia virus expressing gB protein. These observations are also supported by earlier studies by Wang and colleagues who also showed that prior immunisation with modified vaccinia Ankara encoding gB protein can induce neutralising antibodies against CMV strains of different genotypes (Wang et al., 2004). Future studies should be designed to compare various gB vaccine formulations (including recombinant gB protein with MF59 and alum) to determine the most efficient formulation for the prevention of CMV infection. Most importantly, the data presented in this study provide an insight for the design of protein-based vaccines against viral infections aiming to induce both humoral and cellular immune responses and also overcome the potential limitations on the use of viral vector based vaccines with respect to their safety concerns in healthy human population. 112 Chapter 4: Expression, Purification and in vitro assessment of the immunogenicity of recombinant CMV polyepitope protein 4.1 Abstract Based on the life-time cost to the health care system and its impact on human suffering, development of a vaccine to prevent congenital human cytomegalovirus (CMV) infection has been assigned the highest priority by the Institute of Medicine of the National Academy of Sciences (US) and US National Vaccine Program Office. In spite of numerous attempts over the last three decades, successful licensure of a CMV vaccine formulation remains elusive. One of the major roadblocks in CMV vaccine design has been the inability of any vaccine formulations to induce sustainable virus- + specific CD8 T cell responses which play a crucial role in controlling both primary and persistent viral infection. This chapter describes a novel platform technology + which allows the activation of CMV-specific CD8 T cell responses directed towards multiple epitopes restricted through a range of HLA class I molecules. Using a prokaryotic experssion system, we have sucecssfully expressed multiple minimal HLA + class I-restricted CD8 T cell epitopes as a polyepitope protein. Exogenous loading of human cells with polyepitope protein results in endogenous processing of each of the epitopes and these cells are efficently recognized by virus-specific T cells. More importantly, this polyepitope protein can be reproducibly used to expand CMV- specific T cells from healthy virus carriers. Finally, in depth characterization of endogenous processing revealed a novel cross-presentation pathway for polyepitope 113 protein which is independent of MHC class I peptide transporters but involves both the proteasome and the autophagy dependent pathway. 114 4.2 Introduction Primary CMV in healthy individuals is generally asymptomatic, establishing a latent state with occasional reactivation and shedding from mucosal surfaces. In some cases primary CMV infection is accompanied by clinical symptoms of a mononucleosis-like illness, similar to that caused by Epstein-Barr virus. There are two important clinical settings where CMV causes significant morbidity and mortality. These include congenital infection, and primary infection or reactivation of virus in immunosuppressed adults. In the congenital setting, CMV is the leading cause of mental retardation and other abnormalities such as deafness in children, and this impact has been emphasised by its categorisation by the Institute of Medicine as a Level I vaccine candidate [i.e. most favourable impact–saves both money and quality- adjusted life years (QALYs) (Arvin et al., 2004). CMV-associated complications in immunocompromised individuals such as HIV-infected individuals is often seen in + patients with CD4 T cell counts below 50/ l (Palella et al., 1998; Salmon-Ceron et al., 2000). In addition, the impact of CMV in transplant patients, including both solid organ transplant and allogeneic hematopoietic stem cell transplant recipients, is well recognised. Primary exposure to CMV results in the induction of a strong primary immune response, which is maintained as a long-term memory response, and serves to restrict viral replication following reactivation. There is now firm evidence that both humoral and cellular immune responses play a crucial role in controlling CMV infection. Studies carried out in murine CMV models provided the initial evidence on the importance of T cell immunity, where a loss of T cell function was co-incident with increased reactivation and dissemination of viral infection (Mutter et al., 1988; Reddehase et al., 1985). Furthermore, the reconstitution of virus-specific T cell 115 immunity was coincident with recovery from acute viral infection. Subsequent studies in humans under different clinical settings have further emphasised the role of virus- specific T cells. These studies showed that allogeneic stem cell transplant patients, who had insufficient anti-viral T cell immunity, demonstrated an increased risk of developing CMV-associated complications. Convincing evidence for the role of cellular immunity in the control of CMV-disease came from studies carried out by Stan Riddell and colleagues, who showed that adoptive transfer of donor derived + CMV-specific CD8 T cells not only restored antigen-specific cellular immunity, but also prevented CMV-associated clinical complications in allogeneic stem cell transplant patients (Riddell et al., 1992; Walter et al., 1995). Taking these studies into consideration, a variety of CMV vaccines have been evaluated in preclinical and clinical trials. These CMV vaccine strategies have assessed glycoprotein B (gB), pp65 and IE-1 as potential targets, and they have been delivered by numerous delivery platforms, including the attenuated CMV Towne strain (Jacobson et al., 2006), recombinant viral vectors encoding full length antigens and epitopes (Bernstein et al., 2009; Zhong and Khanna, 2009), DNA (Wloch et al., 2008), dense body (Frankenberg et al., 2002), and subunit (Drulak et al., 2000) vaccines. However none of these approaches have shown convincing clinical efficacy and have not entered into clinical practice. The majority of these vaccine delivery platforms, in particular, live-attenuated vaccines and viral vector based vaccines have raised several regulatory concerns such as perceived long-term theoretical health risks (Anderson and Schneider, 2007; Liu; Soderberg-Naucler, 2006). Thus, a need remains for the development of a CMV vaccine using safe vaccine delivery platform technology to reduce the risk of CMV associated injury to the developing fetus, and immunologically compromised individuals such as recipients of solid organ and hematopoietic stem cell transplants, and patients with advanced HIV disease. 116 Having successfully developed a vaccine formulation based on gB protein in ® combination with CpG ODN1826 and AbISCO 100 (Dasari et al., 2011), in this + chapter we have explored the possibility of improving this vaccine by including CD8 T cell epitopes from CMV antigens. We have previously shown that polyepitope + technology is one of the most efficient strategies for the delivery of multiple CD8 T cell epitopes (Zhong et al., 2008). The polyepitope technology is designed to express + minimal HLA class I restricted CMV CD8 T cell epitopes as a “string of beads” and thus, avoids any potential deleterious effects associated with the delivery of full-length antigens. Although polyepitope sequences have been successfully expressed using viral or DNA vectors, here we have developed a novel platform technology to express multiple CMV epitopes as polyepitope proteins using a prokaryotic expression system. The protein-based expression system overrides any safety concerns of viral or DNA- based delivery systems. Extensive in vitro characterisation of the polyepitope protein showed that the majority of epitopes included in the polyepitope protein is efficiently processed by human cells, and this protein can also be used for rapid expansion of CMV-specific T cells. 117 4.3 Materials and Methods 4.3.1 Construction of CMV polyepitope vectors Table 4.1: List of HLA class-1 restricted Epitopes included in the CMVpolyepitope 13, 14 , 15 &20mer Epitopes included in HLA HCMV Epitope Amino acid Abbreviated Restriction antigens sequence location code 13mer 14mer 15mer 20mer + + HLA A1 pp50 VTEHDTLLY 245-253 VTE + + + + + + pp65 NLVPMVATV 495-503 NLV HLA A2 + + + + IE-1 VLEETSVML 316-324 VLE + + IE-1 KLGGALQAK 184-192 KLG HLA A3 + + pp150 TTVYPPSSTAK 945-955 TTV + + + + HLA 11 pp65 GPISHGHVLK 16-24 GPI + + + HLA A23/24 pp65 AYAQKIFKIL 248-257 AYA + + + + HLA A24 pp65 QYDPVAALF 341-349 QYD + + + + HLA B7 pp65 TPRVTGGGAM 417-426 TPR + + + + pp65 RPHERNGFTVL 265-275 RPH + + + + HLA B8 IE-1 QIKVRVDMV 88-96 QIK + + + + IE-1 ELRRKMMYM 199-207 ELR + HLA B27 DNAse ARVYEIKCR 274-282 ARV + + + + pp65 FPTKDVAL 188-195 FPT + + + + HLA B35 pp65 IPSINVHHY 123-131 IPS + pp65 CPSQEPMSIYVY 103-114 CPS + HLA B40/60 pp65 CEDVPSGKL 232-240 CED + HLA B41 gB YAYIYTTYL 153-161 YAY + + + + HLA B44 pp65 QEFFWDANDIY 511-521 QEFF + HLA B57 pp65 QAIRETVEL 331-339 QAI + + HLA cw6 pp65 TRATKMQVI 211-219 TRA A series of CMV polyepitope inserts were designed to encode multiple HLA class I restricted T-cell epitopes from five different antigens (pp65, IE-1, pp50, pp150 and gB).These polyepitope sequences encoded 13, 14, 15 or 20mer CD8+ T cell epitopes for several HLA class I-restricted alleles (see Table 4.1). 118 The polyepitope sequences were designed in such a way that each epitope sequence was preceded by a proteasome liberation amino acid sequence (AD or K or R) and a TAP recognition motif (RIW, RQW, NIW or NQY). In addition, a hexa- histidine tag was inserted at the c-terminus of each polyepitope protein to allow purification using a nickel-nitrilotriacetic acid (Ni-NTA) column. The amino acid sequence of each construct was translated into DNA sequence based on E. coli codon utilisation and inserts were synthetically constructed (DNA2.0, California, USA) and cloned into an expression plasmid (pJexpress 404) under an isopropyl–β-D- thiogalactopyraniside (IPTG) inducible promoter. These synthetically designed polyepitope constructs were transformed into chemically competent E. coli DH5α (Invitrogen, Carlsbad, CA, USA) and plasmids were purified using a QIAGEN maxi prep kit (QIAGEN, Hilden, Germany). 4.3.2 Protein expression Chemically competent E. coli BL21 (DE3) pLysS (Invitrogen, California, USA) was transformed with the CMV polyepitope expression vector. Transformed cells were plated on Luria Bertani (LB) agar supplemented with 100 μg/mL of o ampicillin (LB-Amp) and plates were incubated overnight at 37 C. An isolated colony o was picked and inoculated into 10 ml of LB-Amp broth and grown in a shaker at 37 C and 200 rpm overnight. A small amount of overnight culture was inoculated into 50 mL of LB-Amp broth and grown for 12 hours, then 1% of culture was transferred into 2 L of LB-Amp broth that was then grown until the O.D. reached 0.6 at 600nm. CMV polyepitope protein induction was carried out by adding 1 mM/mL of IPTG. These cells were allowed to grow for an additional 4 hours and protein expression levels 119 were determined by analysing un-induced and induced samples on 12-15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). 4.3.3 CMV polyepitope protein purification At the end of the induction phase, E. coli cultures were harvested by centrifugation at 10,000 rpm for 15 minutes, the cell pellet was resuspended in 80 mL of lysis buffer (25 mM Tris pH 7.4, 0.5% TritonX100, 150 mM NaCl, 0.5 mg/mL lysozyme) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany) and incubated on the ice for 30 minutes. Cell lysis was carried out by sonication on ice for 4 x 5 minutes cycles with a 10 minute break between each cycle. The lysate was centrifuged at 13,000 rpm for 30 minutes and supernatant and pellet fractions were analysed using SDS-PAGE. Since the majority of the protein was found in the pellet fractions in the form of inclusion bodies (IBs), IBs were washed once with lysis buffer (without lysozyme) under stirring for two hours at RT and solubilised in 150 mL of solubilisation buffer (100 mM NaH2PO4, 10 mM Tris, 8 M urea pH 8.0) o overnight at 4 C. The soluble protein was clarified by centrifugation at 13,000 rpm for 30 minutes and supernatant was used for purification of polyepitope proteins. To purify the CMV polyepitope proteins we used 5 mL of Ni-NTA (QIAGEN, Hilden, Germany) metal-affinity chromatography matrix. The matrix was washed with 5 column volumes of distilled water followed by equilibration with 3 column volumes of solubilisation buffer. The soluble protein was loaded on the column and the flow rate was adjusted to 1 mL/minute. The unbound protein and impurities were washed- out with 10 column volumes of wash buffer 1 (100 mM NaH2PO4, 10 mM Tris, 8 M urea pH 6.3) and 20 column volumes of wash buffer 2 (100 mM NaH2PO4, 10 mM Tris, 8 M urea pH 5.9). The bound protein was eluted with elution buffer (100 mM 120 NaH2PO4, 10 mM Tris, 8 M urea pH 4.3) and the eluted fractions were analysed using SDS-PAGE. The positive fractions were pooled together and CMV polyepitope protein estimation was carried out using a Bradford assay kit (Bio-Rad, Hercules, California, USA) following the manufacturer’s instructions. Purified protein was subjected to a solubility test (to identify the right buffer composition for storing the protein in the soluble form) in which 80 μL of purified protein was diluted into 800 μL of various compositions of buffers with different pH ranges. These include (a) 25 mM MES (2-(N-morpholino) ethanesulfonic acid) buffer pH 5.6; (b) 25 mM MES buffer pH 3.2; (c) 25 mM MES pH 4.5; (d) 25 mM MES pH 4.5 and 400 mM L-arginine; (e) 10 mM Tris and 100 mM NaH2Po4 pH 4.3; (6) 10 mM Tris, 100 mM NaH2Po4 and 400 mM L- arginine pH 4.3; (f) PBS, 50 mM L-arginine and 50 mM L- glutamic acid pH7.4; (g) diluted in water; (h) 100 mM glycine buffer pH 2. These samples were o incubated at 4 C overnight; spun at 13,000 rpm for 25 minutes and supernatant fractions were analysed using SDS-PAGE. CMV polyepitope protein was dialysed against 25 mM MES buffer at pH 5.6. The CMV polyepitope protein was concentrated using Ultracel-10K spin columns (Millipore, County Cork, Ireland) followed by sterile filtration using 0.22μm membrane filter, total protein was estimated using BIO-RAD Bradford protein assay kit and various concentrations of CMV polyepitope protein was analysed using SDS-PAGE to determine the final purity of polyepitope protein. o The purified protein was stored in 1 ml aliquots at -70 C. 4.3.4 In vitro stimulation and expansion of CMV specific T-cells from healthy donors using polyepitope proteins PBMC from healthy virus carriers were incubated with 25 μg of purified o polyepitope protein at 37 C, 6.5% CO2 for 2 hours. After incubation, these PBMC 121 were mixed with un-pulsed PBMC and resuspended in RPMI 1640 medium supplemented with 10% FBS (referred to as growth medium). These cells were o cultured in a 24 well plate for 10 days at 37 C, 6.5% CO2. On days 3 and 6, cultures were supplemented with 1 mL of growth medium containing 100 U of recombinant IL-2. The T cell specificity of these in vitro expanded cells was assessed using a standard ICS assay (see detailed method for ICS assays described in Chapter 2). In addition, T cells in these cultures were also assessed for polyfunctional capacity using multi-parameter flow cytometery (detailed method described in Chapter 2). + 4.3.5 Analysis of processing and presentation of CD8 T cell epitopes from CMV polyepitope protein by human cells Epstein-Barr virus (EBV) transformed Lymphoblastoid cell lines (LCLs) and HEK 293 cells were used as antigen presenting cells in these assays. These cells were o pulsed with 25-100 µg of CMV polyepitope protein for two hours at 37 C, 6.5% CO2 and then washed twice with RPMI 1640 medium, resuspended in growth medium and o incubated overnight at 37 C, 6.5% CO2. After overnight incubation, antigen presenting cells were exposed to CMV-specific T cells at a responder to stimulator ratio of 4:1 o for four hours at 37 C, 6.5% CO2 and T cells assessed for cytokine expression using ICS assays (the detailed method for ICS assays is described in Chapter 2). 4.3.6 Enzyme inhibition assays To assess the role of various proteases involved in the processing of CMV polyepitope protein, LCLs were pre-treated with different inhibitors and then used as antigen presenting cells. These inhibitors were specifically targeted to inhibit lysosomes/endosome acidification (80 µM chloroquine and 10 mM Bafilomycin A1), 122 the recycling pathway (200 µM primaquine), cysteine proteases (100 µM leupeptin and 100 µM E64), acid proteases (10 µM pepstatin A), autophagy mediators (10 mM 3-methyladenine(3-MA), the proteasome complex (10 µM lactacystine, 1 µM epoxomicin and MG132), golgi transport (1 µg/mL brefeldin A and 0.7 µg/mL monensin) or aminopeptidase enzymes (30 µM leucinethiol with 0.5mM dithiothreitol (DTT)). Following pre-treatment with these inhibitors, cells were incubated with 25 o µg of CMV polyepitope protein for two hours at 37 C, 6.5% CO2, washed twice with o R-medium, resuspended in growth medium and incubated overnight at 37 C, 6.5% CO2. After overnight incubation, cells were exposed to CMV-specific T cells at a o responder to stimulator ratio of 4:1 for four hours at 37 C, 6.5% CO2 and T cells assessed for cytokine expression using ICS assays (the detailed method for ICS assays is described in Chapter 2). 4.3.7 Silencing of ATG12 or Sec61 with short hairpin RNA (shRNA) Lentivirus‐based vectors encoding ATG12 shRNA (clone ID NM_004707.2‐485s1c1, (CCGGTGTTGCAGCTTCCTACTTCAACTCGAGTTGAAGTAGGAAGCTGCAA CATTTTT) or Sec61 shRNA (clone ID NM_006808.2-410s1c1, CCGGCCCAACATTTCTTGGACCAAACTCGAGTTTGGTCCAAGAAATGTTGG GTTTTTTG) were obtained from Sigma‐Aldrich in an E.coli host. Plasmid encoding shRNA was purified using the large scale plasmid purification kit (Qiagen, Hilden, Germany). Lentivirus was produced in HEK293T cells by co‐transfecting the shRNA vector or control vector (pLKO.1‐puro) with a packaging vector, pHR8.2ΔR, and an envelope vector, pCMV‐VSV‐G (vesicular stomatitis virus glycoprotein G). Following 123 48 and 72 hours of transfection, lentivirus‐containing supernatant was harvested, 0.45μm filtered, and stored at ‐80°C. Transduction was performed by resuspending 5 3x10 CEM.T1, CEM.T2 cells in 1mL of lentivirus‐containing supernatant and centrifuging for 30 minutes at 800g, 32°C. Puromycin (1μg/mL) was added 48 hours after transduction. To generate complete knock down cells were reinfected with the identical lentivirus vector on day 10 and cells were used for downstream assays after 5‐7 days of second transduction. 4.3.8 Western blotting Western blot analysis was performed as previously described (Ausubel, 1995). Briefly, lentivirus shRNA infected cells were washed in PBS and lysed with RIPA buffer (Thermo Scientific, Rockford, IL, USA) on ice according to the manufacturer’s instructions. Protein was quantified using a DC protein assay kit (Bio-Rad laboratories, Hercules, CA, USA). Lysate was mixed with SDS-PAGE loading buffer and resolved on 12-15% SDS-PAGE gels, then transferred to a nitrocellulose membrane (using a Mini Trans-Blot apparatus (Bio-Rad, CA, USA) in pre-chilled transfer buffer (1X Tris-glycine buffer containing 20% methanol) at 100V for 1 hour. Following transfer the nitrocellulose membrane was washed three times in wash buffer (PBS containing 0.05% V/V Tween-20), then incubated in blocking buffer (PBS containing 5% skim milk) for 1 hour at room temperature on a shaker. The membrane was incubated in rabbit anti-Sec61 (Thermo Scientific, Australia) or rabbit anti‐ATG12 (Cell Signaling Technology,Danvers, MA) primary antibody solution o (diluted in blocking buffer) overnight at 4 C on a shaker. The membrane was washed 6 times with wash buffer for 10 minutes each wash, then incubated with sheep anti‐rabbit conjugated to horseradish peroxidase (Chemicon, Australia) secondary 124 antibody (diluted in blocking buffer) for 1 hour at room temperature. The nitrocellulose membrane was washed in wash buffer, incubated with ECL reagent (Merck, Darmstadt, Germany) and protein was visualised on an X-ray film. 4.3.9 Statistical analysis Statistical analyses were carried out using Graph Pad software or Microsoft + Office Excel 2007. For CD8 T cell responses, the means ± SD were calculated and p values were determined using the Student’s t-test. Error bars represent S.E.M. Where indicated with *, ** and *** represents statistically significant with p<0.05, p<0.01 and p<0.001 respectively when compared to the controls. 125 4.4 Results 4.4.1 Purification and characterisation of CMV polyepitope protein + CMV polyepitope inserts encoding 13, 14, 15 or 20 minimal CD8 T cell epitopes were designed as outlined in Figure 4.1. A comprehensive list of CMV epitopes included in FPTKDVALADRIWGPISGHVLKADNQYQYDPVAALFADRQWQEFFWDANDI YADRIWTPRVTGGGAMRNIWQIKVRVDMVRNQYIPSINVHHYRNQYKLGG each of these ALQAKADRIWRPHERNGFTVLRNIWELRRKMMYMADNIWVTEHDTLLYKR QWNLVPMVATVKRQWVLEETSVMLKNIWAYAQKIFKILADRIWTRATKMQ VIADRQWARVYEIKCRRNQYCPSQEPMSIYVYKRQWCEDVPSGKLRNIWYAY polyepitope IYTTYL KRQWQAIRETVELKRQWHHHHHH sequences are Nde I BamH I presented in Table pJexpress 404 4.1. These CMV polyepitope Transformation into E. coli constructs were transformed into E. coli, protein Induction of protein expression expression conditions were optimised and Purification of recombinant CMV analysed on SDS- polyepitope PAGE. Results Figure 4.1: Illustration of the design of the CMV polyepitope encoding sequence and downstream processing. The design obtained from these of the CMV polyepitope 20mer encoding sequence is shown as an example. Individual epitope amino acid sequence are experiments showed shown in bold; grey letters following the epitope sequence represent the amino acid residues for processing of the CMV that CMV polyepitope protein by the proteasome and the underlined amino acid sequences represent the motifs for TAP. The DNA polyepitope protein sequence encoding the CMV polyepitope protein was synthetically made, cloned into an E. coli inducible plasmid, (13, 14, 15 and pJexpress 404, and transformed into E. coli to carry out protein expression and purification. 20mer) can be successfully expressed using a bacterial expression system under an IPTG inducible 126 o promoter at 37 C (Fig. 4.2A & B). Because of the hydrophobic nature of the linear + CD8 T cell epitopes, the CMV polyepitope protein was aggregated in the form of inclusion bodies (IBs, data not shown). These IBs were solubilised and CMV polyepitope proteins from constructs encoding 13, 14 or 15 epitopes were purified using Ni-NTA matrix. This one step purification process allowed us to purify these CMV polyepitope proteins to homogeneity (Fig. 4.3A-C). 1 2 3 4 5 6 7 1 2 3 A B 37KDa 37KDa 20mer 25KDa 25KDa 20KDa 20KDa * * 13, 14 * & 15mer Figure 4.2: Expression and purification of CMV polyepitope proteins. The pJexpress 404 plasmids expressing the CMV polyepitope 13, 14, 15 or 20mer were transformed into E.coli BL21 (DE3) pLysS. Protein expression was induced with IPTG and pre and post-induction samples were analysed using SDS-PAGE. (A) Expression of CMV polyepitope 13, 14 and 15mer proteins in E. coli. Lane 1, molecular weight marker (kDa). Lanes 2 & 3, 13mer un- induced and induced E. coli cell lysate. Lanes 4 & 5, 14mer un-induced and induced E. coli cell lysate. Lanes 6 & 7, 15mer un-induced and induced E. coli cell lysate. (B) Expression of CMV polyepitope 20mer protein in E. coli. Lane 1, molecular weight marker; (2 & 3) 20mer un-induced and induced cell lysate. * indicates the polyepitope protein. However, purification of the CMV polyepitope 20mer was not successful, despite using two different denaturing agents, 8M urea and 6M guanadine hydrochloride, to solubilise the IBs. Following solubilisation the 20mer protein remained in the pellet fraction; and no protein was detected in the elution fractions (Fig. 4.3D). The data obtained from the solubility test to identify a compatible buffer system, indicated that CMV polyepitope proteins require MES or glycine buffers at an acidic pH to remain soluble (Fig. 4.4A). Following CMV polyepitope purification, various concentrations of protein were analysed on SDS-PAGE to check integrity. Data presented in Figure 127 4.4B-D shows minimal impurities and the molecular weights of the recombinant polyepitope proteins were approximately 19, 21 and 25 kDa which matched with the theoretically calculated molecular weight of the 13, 14 and 15mer polyepitope respectively. This one step purification step allowed us to obtain 80 mg of the 13mer, 4 mg of the 14mer and 15 mg of the 15mer protein from 2 L of culture. 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 A B 37KDa 37KDa 25KDa 25KDa 20KDa 20KDa 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 C D 37KDa 25KDa 20KDa 37KDa * 25KDa Figure 4.3: SDS-PAGE analysis of purified CMV polyepitope proteins. Following CMV polyepitope purification on Ni-NTA column, samples from various stages of purification were analysed by SDS- PAGE. Panels A, B & C represent the purification of the CMV polyepitope protein 13mer, 14mer and 15mer. For all the SDS-PAGE gels Lane 1, molecular weight marker. Lane 2, solubilised protein prior to loading. Lane 3, flow through. Lane 4, wash. Lanes 5, 6, 7 & 8, elution fractions. Panel D represent purification of CMV polyepitope 20mer. Lane 1 pellet fraction after solubilisation. Lane 2 molecular weight marker. Lane 3-8, solubilised protein, flow through, wash and elution fractions. * indicates polyepitope protein + 4.4.2 Ex vivo expansion of CMV epitope specific CD8 T cells from PBMC following stimulation with polyepitope protein To evaluate the immunogenicity of the CMV polyepitope proteins, we performed several in vitro experiments using various HLA typed CMV-seropositive 128 + donor PBMC to expand CMV specific CD8 T cells. PBMC from healthy donors were stimulated ex vivo with purified CMV polyepitope proteins and then assessed for antigen specificity by ICS assay and compared with ex vivo responses. The data obtained from these experiments showed that the 13, 14 and 15mer CMV polyepitope + proteins induced a rapid expansion of CMV specific CD8 T cell specific for the + epitopes included in the polyepitope (Fig. 4.5 & 4.6). In most cases dominant CD8 T cell responses were against multiple epitopes included in the CMV 1 2 3 4 5 6 7 8 9 10 A 37KDa 25KDa 20KDa Mw 2µg 4µg 6µg 8µg Mw 2µg 4µg 6µg 8µg Mw 2µg 4µg 6µg 8µg B C D 37KDa 25KDa 20KDa Figure 4.4: CMV polyepitope protein solubility test and characterisation. To determine a compatible buffer system for CMV polyepitope storage as a soluble protein, purified protein was diluted with various buffer compositions at different pH ranges, incubated at 4o C O/N, centrifuged and supernatant fractions were analysed on SDS-PAGE. (A) Lane 1, molecular weight marker. Lane 2, diluted with 25 mM MES buffer pH 5.6. Lane 3, diluted with 25 mM MES buffer pH 3.2. Lane 4, diluted with 25 mM MES pH 4.5. Lane 5, diluted with 25 mM MES pH 4.5 and 400 mM L-arginine. Lane 6, diluted with 10 mM Tris and 100 mM NaH2Po4 pH 4.3. Lane 7, diluted with 10 mM Tris, 100 mM NaH2Po4 and 400 mM L- arginine pH 4.3. Lane 8, diluted with PBS, 50 mM L-arginine and 50 mM L- glutamic acid pH7.4. Lane 9, diluted with water. Lane 10, diluted with 100 mM glycine buffer pH 2. (B, C & D) CMV polyepitope protein purity check. Following dialysis of the CMV polyepitope protein 13mer, 14mer and 15mer against MES buffer pH 5.6, different concentrations of each protein was analysed on SDS-PAGE to observe the final purity and degradation products. polyepitope. For example, data presented in Figures 4.5 & 4.6 shows that a + considerable increase in the percentage of CMV specific CD8 T cells against multiple 129 epitopes from individual donor PBMCs. These results clearly demonstrate that sensitisation of human PBMC with polyepitope protein induces the rapid expansion of CMV-specific T cells. + 4.4.3 CD8 T cells expanded following stimulation with polyepitope proteins display polyfunctional profile + A large body of documented evidence suggest that polyfunctional CD4 and + CD8 T cell responses are crucial in providing protection against a range of viral and A NP TPR QIK RPH ELR VTE KLG 5 0.084 5 510 10 2.31 10 1.96 510 2.11 510 8.78 5 510 2.12 10 0.24 4 4 4 4 4 4 4 10 10 10 10 10 10 10 Ex vivo 10 3 10 3 10 3 10 3 10 3 10 3 103 2 2 2 2 2 2 2 10 10 10 10 10 10 10 0 0 0 0 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 5 5 5 5 5 5 5 10 0.3 10 43.5 10 4.76 10 8.87 10 14 10 26.2 10 2.79 Expanded 4 4 4 4 4 4 4 10 10 10 10 10 10 10 with CMV 103 103 103 103 103 103 103 polyepitope 2 2 2 2 2 2 2 10 10 10 10 10 10 10 13mer 0 0 0 0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD8+ T cells 100 proliferated peptide specific CD8+ T cells ex vivo B 10 1 B35 A11 A24 B44 B7 B8 B35 A3 B7 B8 A1 A2 A2 FPT GPI QYD QEF TPR QIK IPS KLG RPH ELR VTE NLV VLE Figure 4.5: Stimulation of CMV-seropositive donor PBMC with the CMV polyepitope protein 13mer. PBMC from various healthy CMV-seropositive donors were stimulated ex vivo with recombinant CMV polyepitope protein 13mer and cultured for 10 days in the presence of recombinant IL-2. The percentage of expanded peptide specific CD8+ T cells producing IFN- γ was determined using an ICS assay and results were analysed using FlowJo. (A) The data shown in the FACS plots represents the percentage of CMV specific CD8+ T cells producing IFN-γ against TPR, QIK, RPH, ELR, VTE and KLG peptides following stimulation of PBMC from an individual donor with or without the CMV polyepitope protein 13mer. (B) The bar chart represents the percentage of expanded CMV specific CD8+ T cells producing IFN- γ following stimulation of different donor PBMC with (black bars) or without the CMV polyepitope (empty bars). microbial pathogens (Betts et al., 2006; Darrah et al., 2007; Millington et al., 2007). In + addition, in the context of CMV, polyfunctional CD8 T cells protect against high levels of viral replication after liver transplant (Nebbia et al., 2008). These + observations clearly highlight that polyfunctional CD8 T cell responses are a 130 IFN-γ % of IFN-γ producing CD8+ T cells prerequisite for the development of a potent CMV vaccine. In our subsequent + experiments, we analysed effector functions of CMV specific CD8 T cells expanded by polyepitope proteins. These analyses were designed to assess the ability of these effector cells to perform cytolytic function (CD107a mobilization) and express multiple cytokines (IFN-γ, TNF and MIP-1β). Representative data from one of these + analyses is presented in Figure 4.7. The majority of the CMV-specific CD8 T cells expanded with the polyepitope displayed strong cytolytic function (as indicated by + + + CD107a mobilization) and expressed multiple cytokines (IFNγ , TNF and MIP1β ). A NLV VLE FPT TRA IPS AYA TTV 10.1 13.8 41.9 5.15 21 39.3 28.4 CMV polyepitope 14mer 11.9 12.1 31.6 17.3 27.4 29.9 22.5 CMV polyepitope 15mer CD8+ T cells Donor 1 Donor 2 100 CMVpolyepitope 14mer CMVpolyepitope 15mer ex vivo B 10 1 B35 A24 B44 B7 B8 B35 A3 B7 B8 A1 A2 A2 A23/24 cw6 FPT QYD QEF TPR QIK IPS TTV RPH ELR VTE NLV VLE AYA TRA Figure 4.6: Stimulation of CMV-seropositive donor PBMC with the CMV polyepitope protein 14 and 15mer. PBMC from different donors were stimulated with or without the CMV polyepitope protein 14mer or 15mer, cultured in the presence of IL-2 then tested for antigen specificity using ICS assays. (A) The data presented in the FACS plots shows the percentage of CMV peptide specific CD8+ T cells producing IFN-γ against NLV, VLE, FPT and TRA peptides from donor 1 PBMC and IPS, AYA and TTV peptides from donor 2 PBMC following stimulation with the CMV polyepitope 14mer or 15mer or ex vivo. (B) The bar chart represents the percentage of expanded CMV specific CD8+ T cells from various healthy donor PBMC producing IFN- γ after stimulation with CMV polyepitope 14mer (empty bars), 15mer (black bars) or ex vivo (grey bars). 131 IFN-γ % of IFN-γ producing CD8+ T cells NLV QEF QYD IPS TPR QIK ELR FPT RPH VTE CD107a IFN-γ MIP-1β TNF Figure 4.7: The magnitude and quality of expanded CMV specific CD8+ T cells following stimulation with CMV polyepitope protein. Following PBMC stimulation with the CMV polyepitope protein, cells were analysed to assess their effector functions by multi-parameter flow cytometry. The frequency of CD8+ T cells demonstrating cytolytic function (CD107a degranulation marker) and intracellular cytokine production (IFN-γ, TNF and MIP-1β) were analysed on FlowJo and multifunctional cytokine producers were plotted using the SPICE program. Data in the pie chart is shown for an individual epitope and each slice of the pie chart represents each possible combination of functions. + 4.4.4 CD8 T cell epitopes from the polyepitope protein are cross-presented through a TAP-independent pathway but involves proteasome and the autophagy dependent pathway + To delineate the precise pathway for the presentation of CD8 T cell epitopes from the polyepitope protein, we first assessed the processing of the polyepitope protein in human B cells and epithelial cells. Representative data presented in Figure 4.8A shows that both B cells (EBV-transformed LCLs) and epithelial cells (HEK293) efficiently processed and presented the HLA A2-restricted NLV epitope and induced strong stimulation of antigen-specific T cells. Further testing of the polyepitope 132 protein showed that other HLA class I- restricted CMV epitopes (FPT, TPR, VTE and NP NLV A 5 5 B NP peptide pulsed LCL + T cells polyepitope pulsed LCL+ T cells10 0.1 10 7.5 90 104 104 80 103 103 LCL 70 102 102 0 0 60 0 102 103 104 105 0 102 103 104 105 50 40 30 105 1.34 105 17.6 20 104 104 10 103 103 HEK 293 0 2 FPT QEF TPR QIK ELR VTE NLV VLE10 102 0 0 0 102 103 104 105 0 102 103 104 105 CD8+ T cells Figure 4.8: Analysis of the processing and presentation of CD8+ T cell epitopes from the CMV polyepitope protein by LCL and HEK 293 cells. To determine the ability of LCL and HEK 293 to cross-present the CMV polyepitope protein, cells were incubated with and without the CMV polyepitope protein for two hours, washed, incubated overnight and then co-cultured with CMV specific CD8+ T cells in the presence of Brefeldin A. CD8+ T cells were then assessed for IFN-γ production by ICS. (A) Representative flow cytometry analysis of NLV specific CD8+ T cells producing IFN-γ following co-culture with CMV polyepitope pulsed LCL or HEK 293 cells. (B) Data represents the mean ± SEM of IFN-γ producing CMV epitope specific CD8+ T cells following co-culture with LCL pulsed with no peptide (grey bars), cognate peptide (empty bars) or polyepitope (black bars). VLE) were also efficiently processed and presented by human B cells. However, we noticed that the HLA B44-restricted, QEF and the HLA B8-restricted, ELR and QIK epitopes were not efficiently presented by human B cells. In the next set of experiments we attempted to delineate the precise pathway for the processing of exogenously loaded polyepitope protein. First we pulsed + - polyepitope protein in TAP (CEM.T1) and TAP (CEM.T2 and CEM.T2-HLA B7) LCLs and then exposed these cells to CMV-specific T cells. Data presented in Figure + - + 4.9 shows that both TAP and TAP B cells can efficiently present CD8 T cell epitopes from the polyepitope protein. To delineate the mechanisms of polyepitope presentation we used CEM.T1 and CEM.T2 cells as antigen presenting cells to + stimulate HLA A2 restricted NLV-specific CD8 T cells. These antigen presenting cells were first pre-treated with inhibitors for lysosome/endosomal acidification (chloroquine and bafilomycin A1), the recycling pathway (primaquine), cysteine 133 IFN-γ % of IFN-γ producing CD8+ T cells proteases (leupeptin and E64), and acid proteases (pepstatin A) and autophagy A NP NLV peptide Figure 4.9: Analysis of the processing 0.099 25.4 and presentation of the CMV polyepitope protein by TAP+ and TAP- cells. To identify the role of TAP in the CEM.T1 cells presentation of CMV polyepitope protein, TAP+ cells (CEM.T1) and TAP- cells (CEM.T2 or CEM.T2 HLA-B7) were pulsed with CMV polyepitope B NP NLV protein for two hours, washed, 0.075 27.8 incubated overnight and exposed to NLV or TPR peptide specific CD8+ T cells. (A & B) The data shows the CEM.T2 cells percentage of NLV peptide specific CD8+ T cells producing IFN-γ following co culture with CMV Polyepitope protein pulsed CEM.T1 cells (TAP+) C NP TPR and CEM.T2 cells (TAP-). (C) The percentage of TPR specific CD8+ T 0.026 17.8 cells producing IFN-γ following co culture with CMV polyepitope pulsed CEM.T2-HLA B7 cells CEM.T2 HLA B7 cells. The data shown in panels A, B & C is one representative experiment from two independent experiments. CD8+ T cells (3-MA) and then pulsed with polyepitope protein. Data presented in Figures 4.10A shows that rather than blocking the presentation of polyepitope proteins, lysosome, recycling pathway and cysteine protease inhibitors significantly increased the T cell recognition of CEM.T1 and/or CEM.T2 cells pulsed with the polyepitope protein. These observations suggest that the pre-treatment with leupeptin, E64 or + pepstatin A may protect the CD8 T cell epitopes within the polyepitope protein from degradation by cysteine and acid proteases. Unexpectedly, chloroquine and bafilomycin A1 showed opposing effects on the cross-presentation of the polyepitope protein. While chloroquine enhanced the antigen presentation in CEM.T2 cells, pre- treatment with bafilomycin A1 significantly reduced the T cell recognition of polyepitope pulsed antigen presenting cells (Fig. 4.10A). Previous studies have shown 134 IFN-γ that bafilomycin A1 is also a potent and specific inhibitor of vacuolar H+ ATPase and A no drug Figure 4.10: The effect of T2 cells different chemical inhibitors on 3-MA * the processing and presentation T1 cells of the polyepitope protein. bafilomycin A1 **** CEM.T1 and CEM.T2 cells were ** either untreated or pre-treatedchloroquine with inhibitors for autophagy (3- leupeptin ** MA), lysosomes/endosome (chloroquine or bafilomycin A1), E64 ** the recycling pathway (primaquine), cysteine proteases pepstatin (leupeptin or E64) or acid proteases (pepstatin A) (Panel primaquine A), proteasomal inhibitors, lactacystin, epoxomicin and 0 25 50 75 100 125 150 MG132 (Panel B) and ER- relative % of antigen presentation B resident aminopeptidase inhibitor (leucinethiol + DTT) or its control (DDT alone) or golgi No drug inhibitors (brefeldin-A or monensin) (Panel C) prior to ** incubation with the CMVlactacystin ** polyepitope protein. Cells were T2 cells washed and cultured in the epoxomicin ** T1 cells presence of respective inhibitors ** for twelve hours and then exposed to NLV specific CD8+ T MG132 ** cells to assess the relative ** percentage of antigen presentation by ICS assay. Bars 0 20 40 60 80 100 120 represent the relative relative % of antigen presentation percentage of antigen C presentation by CEM.T1 (T1, empty bars) and CEM.T2 (T2, no drug T2 cells black bars) cells. The results are presented as the relative T1 cells percentage of antigen DTT presentation, with 100% corresponding to the number of IFN-γ producing cells following DTT+leucinethiol incubation with untreated cells. The data represents the mean of two independent experiments brefeldin A ** performed in triplicates. Error ** bars represent the ± SEM. * or ** indicates statistically monensin ** significant (p<0.05 or p<0.01), ** calculated by 2-tailed Student’s 0 50 100 150 T-test. relative % of antigen presentation prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes. To explore whether the polyepitope protein processing may involve the autophagy pathway, we pre-treated antigen presenting cells with the PI3K inhibitor, 3-methyladenine (3-MA) and then exposed to CMV- 135 specific T cells. Data presented in Fig. 4.10A shows that 3-MA treatment also effected + the presentation of CD8 T cell epitopes from the polyepitope protein. These observations suggest that it is likely that cross-presentation of the polyepitope protein is via an autophagy dependent pathway. In the next set of experiments we investigated the potential role of the proteasome complex in cross-presentation of the polyepitope protein. CEM.T1 and CEM.T2 cells were pre-treated with the proteasome inhibitors lactacystin, epoxomicin and MG132 and then pulsed with polyepitope protein. These cells were then assessed + for the presentation of CD8 T cell epitopes. Data presented in Figure 4.10B shows + that all three proteasome inhibitors completely blocked the presentation of CD8 T cell + epitopes from the polyepitope protein. It is important to note that presentation of CD8 T cell epitopes does not depend on the expression of immunoproteasomes since CEM.T2 cells, which don’t express these components of the proteolytic complex, can + efficiently process CD8 epitopes from the polyepitope protein. We next focused our attention on the potential role of the secretory pathway and ER-resident + aminopeptidases in the presentation of CD8 T cell epitopes from the polyepitope protein. Data presented in Figure 4.10C shows that pre-treatment with brefeldin-A and + monensin significantly blocked presentation to CD8 T cells, while leucinethiol treatment had minimal effect on the T cell recognition of CEM.T1 and CEM.T2 cells. These results suggest that the polyepitope protein is processed via a proteasome dependent but ER independent pathway that may involve the retrotranslocation pathway, which degrades misfolded ER proteins. To further elucidate the influence of retrotranslocation and autophagy mediated pathways in the cross-presentation of the polyepitope, CEM.T1 and CEM.T2 cells were infected with lentivirus expressing shRNAs for silencing of the Sec61β subunit and ATG12 (autophagy regulator 12) genes. The data presented in Figures 4.11A-C 136 shows that although shRNA expression dramatically reduced the expression of the A D 37kDa GAPDH 49kDa ATG12 15kDa 37kDa GAPDHSec61β CEM.T1 CEM.T2 CEM.T1 CEM.T2 B pLKO Sec61-2.4 E pLKO ATG 7393 19.3 17.4 32.4 9.81 46.1 44.4 35.8 19.4 CD8+ T cells CD8+ T cells C F PLKO PLKO T2 T1 T2 Sec61-2.4 ATG12-7393 ** T1 *** 0 50 100 0 50 100 150 relative % of antigen presentation relative % of antigen presentation Figure 4.11: Effect of Sec61 and autophagy inhibition on the processing and presentation of the polyepitope protein. CEM.T1 and CEM.T2 cells were transduced with recombinant lentivirus encoding shRNA for Sec61β subunit (referred to as Sec61-2.4) or ATG12 (referred to as ATG7393) or a control vector (pLKO), cultured for two day in R-10 medium, selected in puromycin for seven days and then used as antigen presenting cells. (A) Western blot analysis of Sec61 protein expression. GAPDH was used as a control for protein loading. (B-C) FACS plots and bar graphs represent the percentage of NLV specific CD8+ T cells producing IFN-γ following stimulation with CEM.T1 and CEM.T2 cells infected with Sec61β shRNA lentivurus or control vector. (D) Western blot analysis of ATG12 protein expression. (E-F) Data represent the percentage of NLV specific CD8+ T cells producing IFN-γ following stimulation with ATG12 knock down CEM.T1 and CEM.T2 cells. Sec61β subunit, this loss of expression had minimal effect on the presentation of T cell epitopes from the polyepitope protein. In contrast, down-regulation of ATG12 expression in both CEM.T1 and CEM.T2 cells significantly reduced the recognition of CMV polyepitope protein sensitised cells. Taken together these observations 137 IFN-γ CEM.T2 CEM.T1 IFN-γ CEM.T2 CEM.T1 demonstrate that cross-presentation of the polyepitope protein occurs through a novel pathway which involves both proteasomal and autophagy pathways. 138 4.5 Discussion + Emerging evidence suggests that CMV-specific CD8 T cell responses in healthy CMV-seropositive individuals, are directed towards multiple CMV antigens, predominantly pp65 and IE1, but also other structural, early/late antigens and immunomodulators (pp28, pp50, pp150, IE2 gH, gB, US2, US3, US6 and UL18) (Elkington et al., 2004; Elkington et al., 2003; Khan et al., 2005; Manley et al., + 2004a; Sylwester et al., 2005). These CD8 T cell responses play a critical role in immunity to CMV, controlling viral replication and preventing the clinical manifestations of progressive infection in both animal models as well as in humans (Quinnan et al., 1982; Reddehase et al., 1985; Rook et al., 1984). These observations indicate that a vaccine against CMV that can induce T cells responses against multiple antigens will likely strengthen protection against CMV-associated disease. Therefore, + to target multiple antigens, especially to induce CD8 T cell responses, in this study we have proposed a novel recombinant based polyepitope vaccine technology. Polyepitope based vaccines provide a powerful approach to induce immune responses against a variety of conserved epitopes from a number of antigens, without the use of full length antigens which may comprise unknown or pathogenic properties. A series of CMV polyepitope proteins (13mer, 14mer, 15mer and 20mer) were designed by covalently linking multiple HLA class I restricted T-cell epitopes to + potentiate CMV-specific CD8 T cell responses against a number of antigens in different ethnic populations. Selected epitopes in the CMV polyepitope constructs were derived from highly conserved multiple antigens of CMV, including pp65, pp50, pp150, DNAse, and IE-1 (Brytting et al., 1992; Retiere et al., 1998; Solache et al., + 1999). To enhance the immunogenicity of the CMV polyepitope, the selected CD8 T cell epitopes were linked together with a linker sequence consisting of a proteasome 139 liberation amino acid sequence (AD or K or R) and a TAP (transporter associated with antigen processing) recognition motif (RIW, RQW, NIW or NQY) at the carboxyl terminus of each epitope. In this regard, published data shows that the use of the amino acid residues to provide proteasomal processing of the polyepitope proteins (Ishioka et al., 1999; Kuttler et al., 2000; Livingston et al., 2001) and the motifs for TAP recognition are necessary for transporting the proteasome generated peptides into the endoplasmic reticulum (ER) (Bazhan et al., 2010; Uebel et al., 1999). The 13mer, 14mer and 15mer CMV polyepitope proteins were successfully expressed as recombinant proteins in E.coli and purified using Ni-NTA chromatography. However, our attempts to make the CMV polyepitope 20mer were unsuccessful due to its highly hydrophobic nature. The optimised protein expression conditions and purification protocol were consistent. Approximately 2 L of shaker flask culture yielded a substantial quantity of polyepitope proteins. Next we tested the CMV polyepitope proteins immunogenicity in in vitro experiments by stimulating healthy donor PBMC to augment the frequencies of CMV + epitope specific CD8 T cells. The data from these studies clearly demonstrated that these CMV polyepitope proteins are highly efficient in generating CMV-specific + CD8 T cells responses in healthy virus carriers. Interestingly, our results showed the + feasibility of simultaneously amplifying multiple CMV peptide-specific CD8 T cell + responses, and these expanded CD8 T cells demonstrated strong expression of IFN-γ, + TNF, MIP-1β and CD107a by CMV-specific CD8 T cells following stimulation with polyepitope protein. These functional characteristics of the T cells are highly important for predicting the efficacy of T cell mediated immune responses and virus clearance [reviewed in (Seder et al., 2008)]. In addition to expanding virus-specific + CD8 T cells from healthy donors, we also tested the immunogenicity of the polyepitope protein using human B cells (LCLs) and epithelial cells (HEK293). In this 140 context, the majority of the HLA restricted epitopes encoded by the CMV polyepitope were processed and presented efficiently to antigen-specific T cells, confirming the propensity of the polyepitope protein to deliver epitopes for presentation via the MHC class I pathway. Although many studies have shown how exogenous proteins are internalised, processed and presented by MHC class I molecules on antigen presenting cells, exogenously loaded polyepitope protein processing and presentation by antigen presenting cells has never been reported. In general, cross-presentation of exogenous antigens by dendritic cells has been shown to operate using three different pathways. The first proposed model uses an indirect pathway of transferring exogenous antigens from phago-endosomes to the cytosol for proteasome dependent processing. Processed peptides are then loaded in the endoplasmic reticulum by the classical MHC class I machinery (Huang et al., 1996). The second model is a direct, proteasome independent pathway whereby antigens are processed and loaded on MHC class I entirely in endosomal compartments (Shen et al., 2004). The third proposed model utilises the delivery of endoplasmic reticulum components to endocytic organelles or the transport of incoming antigen to the endoplasmic reticulum (Guermonprez et al., 2003; Houde et al., 2003). Indeed, in the development of effective vaccines, immunotherapies against cancers as well as in immune tolerance to self antigens to prevent + autoimmunity, cross-presentation of exogenous antigens to naïve CD8 T cells is the prerequisite for the induction of cytotoxic T cell responses (Rock and Shen, 2005). We therefore elucidated the pathway by which CMV polyepitope was processed and cross-presented by CEM.T1 and CEM.T2 cells in the presence of various chemical inhibitors, involved in different stages of antigen presentation. Our results clearly demonstrate that the polyepitope is degraded into peptides in a TAP-independent, proteasome and autophagy-dependent pathway. Both CEM.T1 and CEM.T2 cells 141 treated with proteasome inhibitors and autophagy inhibitors prevented effective + presentation of CD8 T cells epitopes, while presentation was enhanced with lysosome, recycling pathway, cysteine proteases, acid proteases and ER-resident amino peptidases inhibitors. In addition, we also observed reduced presentation of + CD8 T cell epitopes by CEM.T1 and CEM.T2 following treatment with brefeldin A + and monensin. This effect could be an indirect effect on presentation of CD8 T cell epitopes because these inhibitors are known to block the transport of newly synthesised MHC I molecules to the cell surface. Because processing and presentation + of CD8 T cell epitopes was blocked by proteasome but not ER inhibitors, we + hypothesised that CD8 T cell epitope presentation was mediated via a retrotranslocation pathway whereby exogenously antigens are internalised into phagosomes, then delivered into the cytosol through a Sec61 channel, then degraded by proteasomes into oligopeptides before being transferred to MHC class I molecules in the ER (Ackerman et al., 2006; Rock, 2006). However, knock down of the Sec61β + subunit protein in CEM.T1 and CEM.T2 had no effect on presentation of CD8 T cells epitopes, indicating that the retrotranslocation pathway may not involve the processing + and presentation of the polyepitope encoded CD8 T cell epitopes. Although we found no evidence for the retrotranslocation pathway in the processing of the polyepitope proteins, we did find evidence for a role for the autophagy pathway following knockdown of ATG12. ATG12 is an ubiquitin-like modifier and its covalent conjugation with another autophagy regulator, ATG5 plays an essential role in autophagy formation and elongation (Mizushima et al., 1998a; Mizushima et al., + 1998b). Therefore, we conclude that CD8 T cell epitopes from the polyepitope protein are processed and presented by CEM.T1 and CEM.T2 cells through a novel TAP-independent, proteasome and autophagy dependent pathway. This pathway is difficult to reconcile with the previously proposed cross-presentation models. 142 However, documented evidence suggests that the collaboration between the proteasome and autophagy pathways is essential for protein quality control in the cell (Ding et al., 2007). In addition, although a proteasome and autophagy dependent pathway has never been reported in the context of cross-presentation it has been show to be involved in the degradation of endogenously over-expressed proteins (Webb et al., 2003). Thus, based on these observations we speculate that the polyepitope protein is processed and presented through a novel proteasome and autophagy dependent pathway. In summary, polyepitope proteins can be expressed as recombinant proteins using a prokaryotic expression system in a stable form. These polyepitope proteins are highly immunogenic, and may have preferential access to a proteasome and autophagosome dependent pathway for cross-presentation by antigen presenting cells. 143 144 Chapter 5: Generation of robust CMV-specific cellular and humoral immunity following immunisation with recombinant viral antigens in combination with TLR4 and TLR9 agonists 5.1 Abstract There is now convincing evidence that the successful development of an effective CMV vaccine will require improved formulation and adjuvant selection that is capable of inducing both humoral and cellular immune responses. Here, we have designed a novel subunit vaccine formulation based on CMV-encoded glycoprotein B (gB) and polyepitope proteins in combination with human compatible TLR agonists. + The polyepitope protein includes multiple minimal HLA class I-restricted CD8 T cell epitopes from different antigens of CMV. This subunit vaccine generated durable anti- + + viral antibody, Th1 CD4 and CD8 T cell responses. The humoral immune response induced by the vaccine displayed strong neutralisation capacity and the antigen- specific T cells expressed multiple cytokines with long-term memory maintenace. Furthermore, this subunit CMV vaccine, through the activation of TLR4 and TLR9, activated different dendritic cell (DC) subsets expressing IL12p70, IFN-α, IL-6 and TNF-α, which play a crucial role in the activation of antigen-specific T cells. These observations argue that this novel vaccine strategy, if applied to humans, could facilitate the generation of robust humoral and cellular immune responses which may be more effective in preventing CMV-associated complications in various clinical settings. 145 5.2 Introduction Empirical vaccines that have been developed using live/attenuated pathogens can successfully generate protective immune responses against various infectious diseases including smallpox, polio and diphtheria; however, for a large numbers of intracellular pathogens this strategy has not been reproducibly successful. The development of effective vaccine formulations against such intracellular pathogens + + requires Th1 CD4 T cell and/or CD8 T cell responses (Plotkin, 2008b). Development of vaccine formulations which are capable of generating potent and durable T cell responses remains a significant challenge. A number of vaccine delivery platforms have been considered to specifically generate T cell responses. These include attenuated or killed pathogens, recombinant viral vectors and DNA vaccines, but these delivery platforms are associated with a number of safety concerns. Therefore, a major trend in vaccine development is a move towards the use of recombinant protein-based vaccines. Optimising the formulation of such vaccines with an appropriate adjuvant to generate protective T cell responses is critical. Several + + factors influence the generation of CD4 and CD8 T cell responses using protein- based vaccines, including the dose and physical form of the antigen, (Gamvrellis et al., 2004) the duration of antigen exposure (Obst et al., 2005), the cytokine milieu (Mattei et al., 2010; Trinchieri, 2003) and the type and differentiation state of APCs (Pulendran and Ahmed, 2006; Steinman and Banchereau, 2007). Adjuvants that induce IL-12 and type I IFN by dendritic cells (Akira et al., 2001; Iwasaki and + Medzhitov, 2010) are crucial in the activation/differentiation of Th1 CD4 T cells (Trinchieri, 2003) and the cross-presentation of protein antigens (Le Bon et al., 2006; Le Bon et al., 2003). The function of these adjuvants is primarily dependent upon their capacity to signal via TLRs, and recent evidence suggests that activation of multiple 146 TLRs may be more effective than activation of a single pathway to elicit broad spectrum innate and adaptive immune responses including the enhanced production of + IFN-γ and IL-2-producing CD8 T cells (Grossmann et al., 2009; Querec et al., 2006). In order to produce an effective antigen formulation to generate T cell responses, various strategies have been followed in preclinical and clinical trials. Some of these strategies include the formulation of free antigen and adjuvant, conjugate vaccines or antigen encapsulated in synthetic nanoparticles. These studies have shown that delivery of the vaccine formulation to specific dendritic cell subsets + capable of cross-presenting the antigens and activating CD4 Th1 cells is critical for the induction of a robust immune response. Taking into consideration these findings and other previously published data, the immunogenicity and protective efficacy of novel CMV vaccine formulations, which include recombinant viral protein antigens in combination with different human compatible TLR agonists has been assessed. It has been shown that a subunit vaccine formulation based on recombinant glycoprotein B and CMV polyepitope proteins formulated with TLR4 and TLR9 agonists activates various subsets of DCs and induces a potent durable humoral and cellular immune response. This polyfunctional immune response is likely to be more effective in resisting viral challenge and preventing active replication and persistence of the virus in vaccinated individuals. 147 5.3 Materials and methods 5.3.1 Vaccine formulations All vaccine formulations were prepared using CMV polyepitope 13mer protein because for consistency with the in vitro experiments described in Chapter 4. For in vivo experiments, the gB-CMVpoly protein vaccine was formulated by mixing 5 μg of ® gB and 20 μg of CMV polyepitope with (1)13 μg of AbISCO 100 (ISCOMs) and 50 μg of CpG ODN1826 (TLR9 agonist), (2) 25 μg of MPL (TLR4 agonist) and 50 μg of CpG ODN1826, (3) 25 μg of MPL and 20 μg of Gardiquimod (TLR7 agonist) or (4) 50 μg of CpG ODN1826 and 20 μg of Gardiquimod per dose in a 100 μL volume. TLR agonists were purchased from InvivoGen (San Diego, CA, USA). 5.3.2 Mice immunisations HHD I mice containing human HLA-A*0201 with a disrupted murine MHC class I (Pascolo et al., 1997) were bred and maintained under specific pathogen-free conditions at the QIMR. All protocols were followed in compliance with the QIMR animal ethics committee. In each group at least 5, six-to-eight week old mice, were immunised subcutaneously (s.c.) at the base of the tail with the gB and CMV polyepitope vaccine formulated with the various combinations of TLR agonists. Mice were bled regularly after prime and boost immunisation from the lateral tail vein and + + serum and PBMCs were isolated to assess the gB-specific antibody, CD4 and CD8 T + cell responses, and the CMV polyepitope-specific CD8 T cell responses using ELISA and ICS assays as described in Chapter 2: Materials and Methods. 148 5.3.3 Isolation of murine dendritic cells DCs were purified from the spleens of HHD mice. Spleens were placed in 7 mL of enzymatic digestion mix (R-medium with 2% FBS, 1 mg/mL collagenase and 20 µg/mL DNase I) in a Petri dish and tissue was cut into small fragments and transferred into a 10 mL polypropylene tube. The tissue was digested by frequently o mixing with a wide bore pasture pipette for 25 minutes at 25 C before 600 µL of 0.1 M EDTA was added to disrupt any DC-T cell complexes and mixing was continued for another 5 minutes. Undigested tissue fragments were removed by filtration through a 70 µM cell strainer. Cells were washed twice with R-2% FBS to recover the cells from the digest, counted and resuspended in 400 µL of buffer (PBS, 0.5% FBS and 2 8 mM EDTA) per 10 total cells. To isolate CD11c positive DCs, 100 µL of CD11c 8 MicroBeads (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) per 10 total o cells were added and incubated at 4 C for 15 minutes. Cells were washed by adding 1- 7 2 mL of buffer per 10 cells, centrifuged at 200 xg for 10 minutes and resuspended up 8 + to 10 cells in 500 µL of buffer. CD11c DCs were separated by positive selection using an autoMACS (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany), washed and resuspended in DC growth medium (R-5% FBS, penicillin, streptomycin, non- essential amino acids, sodium pyruvate and β-mercaptoethanol). The purity of + enriched CD11c DCs was determined by surface staining with AF700-conjugated anti-CD11c antibody. Surface stained cells were acquired on a BD FACSCanto II and analysed using FlowJo software. 149 + 5.3.4 In vitro stimulation of CD11c DCs with various combinations of adjuvant formulations 6 Approximately 1x10 enriched DCs were stimulated with PBS, MPL (25 µg), CpG ODN1826 (50 µg), MPL and CpG ODN1826, MPL and Gardiquimod (20 µg), and CpG ODN1826 and Gardiquimod. Cells were cultured in 800 µL of DC growth medium in 48-well plates for 20 hours. Following stimulation, 700 µL of the supernatants were collected and a cytokine ELISA (IL-6, IL-12p70, TNF, IFN-α) was performed to quantify the amount of innate immune stimulation mediated by various adjuvant formulations. Production of IL-12p70 cytokines was also analysed by intracellular staining following stimulation with the various adjuvant combinations. After harvesting supernatants, cells were resuspended in the remaining 100 µL of medium, transferred to 96 well ‘V’ bottom plates and brefeldin A was added in medium at a concentration of 1 µg/mL. The cells were incubated for 6 hours, washed twice, and then incubated with AF700-conjugated anti-CD11c and FITC conjugated anti-PDCA-1 monoclonal antibodies. After washing, fixing and permeabilising, cells were stained intracellularly with PE-conjugated anti-IL-12p70 antibody. Cells were acquired on a FACSCanto II and analysed using FlowJo software. 5.3.5 Statistical analysis Statistical analyses were carried out using Microsoft Office Excel 2007. For + + ELISA antibody titres, CD4 and CD8 T cell responses, the mean ± SD were calculated and p values were determined using the Student’s t-test. Error bars represent SEM. Where indicated *, ** and *** represents statistically significant with p<0.05, 0.01 and 0.001, respectively 150 5.4 Results 5.4.1 Evaluation of the immunogenicity of the recombinant gB-CMVpoly proteins ® in combination with CpG ODN1826 and AbISCO 100 We previously reported that gB protein formulated with CpG ODN1826 and ® AbISCO 100 induced strong gB-specific antibody and cellular immune responses (Dasari et al., 2011). Here we have extended these studies to include recombinant CMV polyepitope 1. gB + CMV poly + AbISCO 100 + CpG ODN1826 Figure 5.1: Schematic 2. gB + CMV poly representation of the gB- protein in this CMVpoly proteins 3. AbISCO® 100 + CpG ODN1826 vaccination in combination with or vaccine formulation. without CpG ODN1826 and AbISCO® 100. (a) To The polyepitope evaluate the CpG ODN1826 and AbISCO® HHD-I 100 combination, three protein includes groups (n=5) of HHD mice were immunised multiple HLA class subcutaneously with gB and CMV polyepitope + Day 21 proteins formulated with I-restricted CD8 T Tail bleed and then AbISCO® 100 and CpG boost ODN 1826 or proteins cell epitopes from alone or adjuvants alone. On day 21, a booster dose was given. Mice were CMV-encoded Day 30 sacrificed on day 30 to Assess gB-specific antibody and CD4+ assess the gB and CMV antigens and covers and CD8+ T cell responses and CMV polyepitope-specific polyepitope specific CD8+ T cell immune responses. >96% of the world- response wide population. To determine the immunogenicity of this new formulation, HLA-A2 transgenic mice (referred to as HHD-1) were immunised subcutaneously with gB- ® CMVpoly proteins formulated with or without CpG ODN1826 and AbISCO 100 and boosted with the same vaccine formulation on day 21 (See Fig. 5.1). Mice immunised with adjuvant alone were included as a negative control. Glycoprotein B-specific + + antibody and CD4 T cell responses and CMV polyepitope-specific CD8 T cell responses against the HLA A2-restricted epitopes, 151 A B 100 gB + CMV poly + 4.5 gB + CMV poly + 90 AbISCO ®100 + CpG 4 AbISCO®100 + CpG 80 gB + poly 3.5 gB + poly 70 3 60 AbISCO ®100 + CpG 2.5 AbISCO® 100 + CpG 50 2 40 1.5 30 1 20 0.5 10 0 0 serum dilutions serum dilutions C D gB + CMV poly + gB + CMV poly + AbISCO®100 + CpG ** AbISCO®100 + CpG gB + CMV poly gB + CMV poly AbISCO®100 + CpG AbISCO®100 + CpG 0 0.5 1 0 0.2 0.4 + % of CD8 + T cells producing IFN-γ % of CD4 T cells producing IFN-γ Figure 5.2: Assessment of gB-specific and CMV polyepitope-specific immune responses following immunisation with AbISCO® 100 and CpG ODN 1826. (A) gB-specific antibody titres in sera from mice immunised with gB-CMVpoly proteins in combination with or without AbISCO® 100 and CpG ODN 1826 or adjuvants alone. Error bars represent ± S.E.M. **p<0.01 calculated by 2-tailed Student’s t test. (B) CMV-specific neutralising antibody titres induced following immunisation with gB- CMVpoly proteins in combination with or without AbISCO® 100 and CpG ODN 1826 or adjuvants alone. On day 30 sera was pooled from individual immunised groups, serially diluted and incubated with the CMV AD169 strain prior to MRC-5 cell infection. Virus infectivity was determined using an IE-1/IE-2 micro-neutralisation assay. The percentage of neutralisation of viral infectivity was calculated using the following formula: [(number of IE1+ nuclei of HCMV infected cells – number of IE1+ nuclei of serum treated HCMV infected cells/ number of IE1+ nuclei of HCMV infected cells) ×100]. (C) On day 30, splenocytes were stimulated with gB protein overnight in the presence of brefeldin A and then assessed for IFN-γ production using an ICS assay. Bars represent the magnitude of gB-specific IFN-γ producing CD4+ T cell responses following immunisation. Error bars represent means ± S.E.M. **p<0.01 calculated by 2-tailed Student’s t test. (D) On day 30, splenocytes from immunised mice were stimulated with NLV & VLE peptides in the presence of brefeldin A and then assessed for IFN-γ production. Bars represent the magnitude of CMV polyepitope-specific CD8+ T cell responses. Error bars represent means ± S.E.M. NLVPMVATV (referred to as NLV) and VLEETSVML (referred to as VLE) were then analysed on day 30. The data presented in Fig. 5.2A shows that immunisation of HHD-1 mice with gB-CMVpoly proteins in combination with CpG ODN1826 and ® AbISCO 100 induced significantly higher gB-specific antibody titres when compared 152 OD at 450nm ** ** ** ** ** % of neutralisation to mice immunised with recombinant proteins alone or adjuvants alone. More importantly, these antibodies displayed strong neutralising capacity against the CMV AD169 strain (Fig. 5.2B). Although this vaccine formulation was highly effective in + inducing gB-specific CD4 T cell responses, very low to undetectable CMV-specific + CD8 T cell responses were observed against the HLA A2-restricted NLV and VLE epitopes. (Fig. 5.2C & D). Taken together, these experiments showed that although ® + CpG ODN1826 and AbISCO 100 is capable of inducing strong antibody and CD4 T cell responses, this adjuvant combination may not be effective at inducing CMV + polyepitope specific CD8 T cell responses. 5.4.2 Assessment of immunogenicity of gB-CMVpoly vaccine with alternative immunoadjuvants ® Since the CpG ODN1826 and AbISCO 100 adjuvant combination failed to + induce CMV polyepitope-specific CD8 T cell responses, we next formulated the gB- CMVpoly vaccine with a series of human compatible experimental TLR agonists including monophosphoryl lipid A (MPL), Gardiquimod and CpG ODN1826. These adjuvants have been shown to activate TLR4 (MPL), TLR7/8 (Gardiquimod) and TLR9 (CpG ODN1826) and induce strong T cell responses in both experimental animals and humans (Ahmed et al., 2011; Coffman et al., 2010; Coler et al., 2011; Garcon and Van Mechelen, 2011). Multiple groups of HHD-1 mice were immunised with the gB-CMVpoly vaccine in combination with MPL, Gardiquimod and/or CpG ODN1826 or with adjuvants alone (negative control), and boosted with the same formulations on day 21. These mice were scarified on day 30 to assess gB-specific + + CD4 T cell and antibody responses and CMV polyepitope-specific CD8 T cell responses using ELISA and ICS assays (Fig 5.3). 153 1. gB + CMV poly + G ardiquimod + C pG ODN1826 Figure 5 . 3 : Illustration of the 2. g B + CMV poly + MPL + Gardiquimod gB -C MVpoly proteins 3. gB + CMV poly + MPL + CpG ODN1826 vaccination study in 4. Gardiquimod + CpG ODN1826 5. MPL + Gardiquimod combination with various 6. MPL + CpG ODN1826 combinations of TLR agonists. To identify a compatible adjuvant system for gB and CMV polyepitope proteins to induce the appropriate immune HHD- I responses, six groups (n=5 ) of HHD mice were immunised subcutaneously with gB - CMVpoly proteins formulated Day 21 with Gardiquimod and CpG Tail bleed and then ODN 1826 , MPL and boost Gardiquimod , MPL and CpG ODN 1826 or adjuvants alone. These mice were boosted with Day 3 0 the same formulations on day 21 , Assess gB- specific antibody and CD4+ and and then sacrificed on day 30 to CD8+ T cell responses and CMV assess gB and CMV polyepitope- polyepitope specific CD8 + T cell response specific immune responses Data presented in Fig 5.4A shows that HHD-1 mice vaccinated with gB- CMVpoly vaccine formulated with MPL, Gardiquimod and/or CpG ODN1826 showed significantly higher levels of gB-specific antibody titres when compared to the control animals immunised with adjuvants alone. Furthermore these gB-specific antibodies displayed strong neutralising capacity against CMV (Fig. 5.4B). It is important to mention here that animals immunised with CMV vaccine formulated with MPL and CpG ODN1826 induced higher levels of neutralising antibody titres when compared to animals immunised with the MPL and Gardiquimod or CpG ODN1826 and Gardiquimod formulations (Fig. 5.4B). Ex vivo analysis of antigen-specific T cell responses revealed that although all TLR + agonists efficiently induced gB-specific CD4 T cell responses (Fig. 5.4C), CMV + polyepitope-specific CD8 T cell responses, were only detected following immunisation with the CMV vaccine formulated with MPL and CpG ODN1826 or 154 CpG ODN1826 and Gardiquimod (Fig. 5.4D). To further confirm the activation of + these antigen-specific CD8 T cell responses, splenocytes were stimulated with the HLA A2-restricted NLV and VLE peptide epitopes and cultured for 10 days in growth A B gB + CMV poly + 100 gB + CMV poly + 4.5 Gardiquimod + CpG Gardiquimod + CpG 90 4 gB + CMV poly + MPL + gB + CMV poly + MPL + Gardiquimod 80 Gardiquimod 3.5 gB + CMV poly + MPL + 70 gB + CMV poly + MPL + 3 CpG CpG 60 2.5 Gardiquimod + CpG Gardiquimod + CpG 50 2 MPL + Gardiquimod 40 MPL + Gardiquimod 1.5 30 MPL + CpG MPL + CpG 1 20 0.5 10 0 0 D serum dilutionsC Serum dilutions gB + CMV poly + gB + CMV poly + Gardiquimod + CpG Gardiquimod + CpG gB + CMV poly + MPL + gB + CMV poly + MPL + Gardiquimod Gardiquimod gB + CMV poly + MPL + CpG gB + CMV poly + MPL + CpG Gardiquimod + CpG Gardiquimod + CpG MPL + Gardiquimod MPL + Gardiquimod MPL + CpG MPL + CpG 0 0.2 0.4 0.6 0.8 0 0.2 0.4 % of CD4+ T ccells producing IFN-γ % of CD8+ T cells producing IFN-γ Figure 5.4: Evaluation of gB and CMV polyepitope-specific immune responses following vaccination with various combinations of TLR agonists. (A) gB-specific antibody titres in mice vaccinated with the gB-CMVpoly proteins formulated with Gardiquimod and CpG ODN1826, MPL and Gardiquimod, MPL and CpG ODN1826 or adjuvants alone. Error bars represent means ± S.E.M. * or ** represent p<0.05 or 0.01 calculated by 2-tailed Student’s t test. (B) Neutralising antibody titers induced following immunisation with gB-CMVpoly proteins in combination with CpG ODN1826, MPL and Gardiquimod, MPL and CpG ODN1826 or adjuvants alone. On day 30 sera was pooled from individual immunised groups, serially diluted, incubated with CMV AD169 and used to infect MRC-5 cells. Virus infectivity was determined using an IE- 1/IE-2 micro-neutralisation assay. (C) Splenocytes from vaccinated mice were stimulated with gB protein overnight in the presence of brefeldin A and then assessed for IFN-γ production using an ICS assay. The bar graph represents the frequencies of gB-specific CD4+ T cells producing IFN- γ. (D) To assess the CMV polyepitope-specific CD8+ T cell responses on day 30, splenocytes were stimulated with NLV and VLE peptides in the presence of brefeldin A and then measured for IFN-γ production. Bars indicate the frequencies of CMV polyepitope-specific CD8+ T cells producing IFN-γ following stimulation with NLV and VLE peptides. Error bars represent means ± S.E.M. * represent p<0.05 calculated by 2-tailed Student’s t test. media supplemented with IL-2. Functional analysis of these in vitro cultured T cells showed a massive expansion of CMV epitope-specific T cells from mice immunised with the gB-CMVpoly vaccine formulated with MPL and CpG ODN1826 and to a lesser extent in mice vaccinated with the CpG ODN1826 and Gardiquimod formulation (Fig. 5.5A & B). Collectively these observations suggest that the gB- CMVpoly proteins formulated with MPL and CpG ODN1826 adjuvants were the most 155 OD at 450nm ** ** ** ** * % of neutralisation * * + efficient at inducing gB-specific CD4 T cell and neutralising antibody responses, and + CMV polyepitope-specific CD8 T cell responses. gB + CMV poly + gB + CMV poly + A CpG + MPL + gB + CMV poly + Gardiquimod Gardiquimod MPL + CpG 28.8 2.84 47.1 CD8+ T cells B gB + poly + Gardiquimod + CpG gB + poly + MPL + Gardiquimod gB + poly + MPL + CpG 0 20 40 60 % of IFN-γ producing CD8+ T cells Figure 5.5: Assessment of CMV polyepitope-specific CD8+ T cell responses following stimulation with NLV and VLE peptides (A) Splenocytes from mice immunised with gB-CMVpoly proteins in combination with CpG ODN1826 and Gardiquimod, MPL and Gardiquimod, MPL and CpG ODN1826 were stimulated in vitro with NLV and VLE peptides for 10 days in the presence of IL-2. These cells were assessed for CMV polyepitope-specific reactivity using ICS. Each FACS plot is representative of one of five mice per group. (B) Bars represent the frequencies of expanded CMV polyepitope-specific CD8+ T cells from each group. Error bars represent the mean ± S.E.M. ** represent or 0.01 calculated by 2-tailed Student’s t test. 156 IFN-γ ** 5.4.3 Effect of single and combined TLR4 & 9 agonists on the immunogenicity of gB-CMVpoly protein vaccine Having established that a combination of TLR4 and TLR9 agonists could efficiently induce humoral and cellular immune responses, the next set of experiments were designed to determine whether these adjuvants have complementary, synergistic 1. gB + CMV poly + MPL + CpG Figure 5.6: Schematic 2. gB + CMV poly + CpG representation of the gB- 3. gB + CMV poly + MPL CMVpoly protein vaccination 4. MPL + CpG study with single and double TLR agonists. To study the synergistic or antagonistic effect of MPL and CpG ODN1826, four groups (n=5/group) of HHD HHD-I mice were immunised subcutaneously with gB- CMVpoly proteins formulated with MPL alone, CpG ODN1826 Day 21 alone, MPL and CpG ODN1826 Tail bleed and then or adjuvants alone. On day 21 boost these mice were tail bled and a booster dose was given. Mice were tail bled again on day 28 and 35 and sacrificed on day 50 Day 28, 35 & 50 to assess gB and CMV Assess gB-specific antibody and CD4+ polyepitope specific immune and CD8+ T cell responses and CMV responses. polyepitope specific CD8+ T cell response or antagonistic effects. To address this issue, multiple groups of HHD-1 mice were immunised with the gB-CMVpoly vaccine formulated with MPL and CpG ODN1826, MPL alone, or CpG alone and then boosted with the same formulation on day 21 (Fig. 5.6). In addition, mice immunised with MPL and CpG alone were included as a negative control. Following vaccination, peripheral blood samples were collected through the lateral tail bleed at different time points (days 21, 28 and 35) to analyse 157 + gB-specific CD4 T cell and humoral immune responses and CMV polyepitope A B 4 4 gB + CMV poly + MPLgB + CMV poly + MPL 3.5 gB + CMV poly + CpG 3.5 gB + CMV poly + CpG 3 3 gB + CMV poly + MPL + CpG gB + CMV poly + MPL + CpG 2.5 2.5 MPL + CpG MPL + CpG 2 2 1.5 1.5 1 1 0.5 0.5 0 0 C Serum dilutions D Serum dilutions 4 gB + CMV poly + MPL 100 gB + CMV poly + MPL 3.5 90gB + CMV poly + CpG gB + CMV poly + CpG 80 3 gB + CMV poly + MPL + CpG 70 gB + CMV poly + MPL + CpG 2.5 MPL + CpG 60 MPL + CpG 2 50 40 1.5 ** 30 1 20 0.5 10 0 0 serum dilutions Serum dilutions Figure 5.7: gB-specific antibody responses induced following vaccination with gB-CMVpoly proteins in combination with MPL, CpG ODN1826, MPL & CpG ODN1826 or adjuvants alone. (A-C) Data represents the gB-specific antibody titres determined by ELISA on day 21 and 35 in pooled sera from individual groups and on day 50 in individual mouse serum. Error bars represent the mean ± S.E.M. (D) CMV-specific neutralising antibody titers induced following immunisation with above indicated adjuvants. On day 50, sera from individual groups were pooled, serially diluted and pre- incubated with HCMV AD169 strain. MRC-5 cells were infected with serum-treated virus and virus infectivity was determined using an IE-1/IE-2 micro-neutralisation assay. + specific CD8 T cell responses. Animals were sacrificed on day 50 to determine the long term gB-specific humoral and cellular memory responses (Fig. 5.6). Data presented in Figure 5.7A-C shows the level of gB-specific humoral immune responses in animals immunised with different formulations of the CMV vaccine on days 21, 35 and 50 respectively. These observations revealed a synergistic effect of MPL & CPG ODN1826. The CMV vaccine formulated with MPL & CpG ODN1826 consistently induced higher levels of gB-specific antibody responses, which were sustained at high levels until at least day 50 post-immunisation, when compared to the vaccine formulated with CpG ODN1826 or MPL alone. Furthermore, the combination of MPL 158 OD at 450 nm OD at 450nm ** ** ** ** OD at 450nm % of neutralisation and CpG ODN1826 adjuvants induced very high levels of anti-viral neutralising responses although these levels were comparable to those induced with the vaccine formulated with the single adjuvants alone (Fig. 5.7D). A gB + CMV poly gB + CMV gB + CMV + MPL + CpG poly + CpG poly + MPL MPL + CpG 1.77 0.24 0.017 0.035 0.88 0.15 0.048 0.034 2.64 0.87 0.48 0.13 CD4+ T cells B C gB + CMV polyepitope gB + CMV polyepitope + MPL + CpG + MPL + CpG ** gB + CMV polyepitope 50D gB + CMV polyepitope + CpG 28D + CpG gB + CMV polyepitope gB + CMV polyepitope + MPL + MPL MPL + CpG MPL + CpG 0 0.5 1 1.5 0 0.5 1 1.5 + % of CD4+ T cells producing IFN-γ % of CD4 T cells producing IFN-γ Figure 5.8: Evaluation of gB-specific effector and memory CD4+ T cell responses following immunisation with the gB-CMVpoly vaccine formulated with MPL, CpG ODN1826, MPL & CpG ODN1826 or adjuvants alone. PBMC (on day 28 and 50) or splenocytes (on day 50) from immunised mice were stimulated in vitro with gB protein overnight in the presence of brefeldin A and then IFN-γ secretion was measured using an ICS assay. Each FACS plot is representative of one of five mice per group. (A) FACS plots represent the frequencies of gB-specific, IFN-γ producing CD4+ T cells following immunisation with gB-CMVpoly proteins in combination with MPL and CpG ODN1826, CpG ODN1826 alone, MPL alone or adjuvants alone, on day 28 and 50 in peripheral blood and on day 50 in the spleen post-immunisation (PI). (B & C) Bars represent the frequencies of gB-specific IFN-γ producing CD4+ T in peripheral blood on day 28 and 50 and in spleen on day 50. Error bars represent the mean ± S.E.M. To assess the synergistic effects of MPL & CPG ODN1826 on the gB-specific cellular + + immune response, gB-specific CD4 and CD8 T cell responses were evaluated using 159 IFN-γ Day 50 PI (spleen) Day 50 PI (blood) Day 28 PI (blood) ICS assays. A detailed analysis of these responses is presented in Figures 5.8-5.10. + The induction of an optimal gB-specific CD4 T cell response (Fig. A gB + CMV poly + gB + CMV poly + gB + CMV poly + MPL + CpG CpG MPL MPL + CpG 0.21 0 0 0 0.13 0.018 0 0 0.92 0.15 0.13 0.01 CD8+ T cells B C gB + CMV polyepitope gB + CMV polyepitope + + MPL + CpG MPL + CpG gB + CMV polyepitope + CpG 50D gB + CMV polyepitope + CpG gB + CMV polyepitope 28D gB + CMV polyepitope + + MPL MPL MPL + CpG MPL + CpG 0 0.2 0.4 0 0.2 0.4 % of CD8+ T cells producing IFN-γ % of CD8+ T cells producing IFN-γ Figure 5.9: Evaluation of gB-specific effector and memory CD8+ T cell responses following immunisation with gB-CMVpoly proteins formulated with MPL and CpG ODN1826, CpG ODN1826 alone, MPL alone or adjuvants alone. PBMC (on day 28 and 50) or splenocytes (on day 50) from immunised mice were stimulated in vitro with gB protein overnight in the presence of brefeldin A and then gB-specific CD8+ T cells producing IFN-γ was measured using an ICS assay. (A) FACS plots represent the percentage of gB-specific IFN-γ producing CD8+ T cells in the blood and spleen. Each plot is representative of one of five mice from each group. (B & C) Bars represent the frequencies of gB-specific IFN-γ producing CD8+ T cells in peripheral blood (day 28 and 50) and in the spleen (day 50) following stimulation with gB protein. Error bars represent mean ± S.E.M. + 5.8) and CD8 T cell response (Fig. 5.9) was dependent upon the co-delivery of both MPL and CPG ODN1826. An important outcome of these experiments was that the CMV vaccine formulated with MPL and CpG ODN1826 adjuvants not only induced strong effector T cell responses but also generated a gB-specific memory T cell response which was detectable on day 50 (Figs. 5.8 & 5.9). Furthermore, the gB- 160 IFN-γ Day 50 PI (spleen) Day 50 PI (blood) Day 28 PI (blood) + specific CD4 T cells induced following delivery with both adjuvants were polyfunctional with the majority of these effector cells expressing either triple + + + + + - (IFNγ TNF IL-2 ) or double (IFN TNF IL-2 ) cytokines (Fig. 5.10). Whilst, the gB- gB + polyepitope + gB + polyepitope + gB + polyepitope + A MPL + CpG CpG MPL IFN+ TNF+ IL2+ 1% 5% 10% 10% IFN+TNF+IL2- 3% 3% 11% 25% IFN+TNF-IL2+ 0% 5%39% 38% IFN+TNF-IL2- 8% 6% 1% 0% IFN-TNF+IL2+ 0% 51% 8% 44% IFN-TNF+IL2- 32% IFN-TNF-IL2+ B * 0.80 gB + polyepitope + MPL + CpG* gB + polyepitope + CpG 0.60 gB + polyepitope + MPL MPL + CpG 0.40 0.20 0.00 Figure 5.10: Multiple cytokine expression by gB-specific CD4+ T cells from mice immunised with the gB-CMVpoly vaccine formulated with MPL and CpG ODN1826, CpG ODN1826 alone, MPL alone or adjuvants alone. On day 50, splenocytes from immunised mice were in vitro stimulated with gB protein in the presence of brefeldin A and production of IFN-γ, TNF-α or IL-2 was determined using multiparameter ICS. Data was analysed using FlowJo software and gB- specific CD4+ T cell inducing different combinations of cytokines were plotted. (A) Pie charts indicate the proportion of gB-specific CD4+ T cells producing seven combinations of cytokines. (B) The bar graph represents the absolute percentage of gB-specific CD4+ T cells inducing multiple cytokines. + specific CD4 T cells displayed similar polyfunctionality following delivery with CpG ODN1826 alone, the response generated following delivery with MPL was primarily - + - monofunctional (IFN TNF IL-2 ). 161 % of cytokine producing CD4+ T cells Another important aspect of these studies was the induction and long-term + maintenance of CMV-specific CD8 T cell responses. Data presented in Figure 5.11 A gB + CMV poly + gB + CMV poly + gB + CMV poly + MPL + CpG CpG MPL MPL + CpG 0.11 0.039 0 0 0.1 0.19 0.02 0 0.38 0.61 0.069 0.05 CD8+ T cells B C gB + CMV gB + CMV polyepitope + MPL polyepitope + MPL * + CpG + CpG gB + CMV gB + CMV polyepitope + CpG polyepitope + CpG 50D gB + CMV gB + CMV polyepitope + MPL 28D polyepitope + MPL MPL + CpG MPL + CpG 0 0.05 0.1 0.15 0.2 0 0.1 0.2 0.3 % of CD8+ T cells producing IFN-γ % of CD8+ T cells producing IFN-γ Figure 5.11: Assessment of CMV polyepitope-specific effector and memory CD8+ T cell responses following immunisation with gB-CMVpoly proteins formulated with MPL and CpG ODN1826, CpG ODN1826 alone, MPL alone or adjuvants alone in peripheral blood and spleen. Following immunisation PBMC (day 28 & 50) and splenocytes (day 50) were stimulated with NLV and VLE peptides and IFN-γ producing CMV polyepitope-specific CD8+ T cells were determined by ICS. (A) The data in the FACS plots represent the frequencies of CMV polyepitope specific CD8+ T cells producing IFN-γ, on day 28 and 50 in peripheral blood and on day 50 in the spleen post-immunisation. (B) Bar graphs represent the frequencies of IFN-γ producing CMV polyepitope-specific CD8+ T cells in pooled blood from mice immunised with gB-CMVpoly proteins formulated with gB and CMV polyepitope vaccine formulated with MPL and CpG ODN1826, CpG alone MPL alone or adjuvants alone, on day 28 and 50 and in peripheral blood and in individual spleens on day 50 (C). Error bars represent the mean ± S.E.M. shows that CMV vaccine formulations with CpG ODN1826 alone or a combination of MPL and CpG ODN1826 adjuvants were the most efficient at generating HLA class I- 162 IFN-γ Day 50 PI (spleen) Day 50 PI (blood) Day 28 PI (blood) + restricted CD8 T cell responses when compared to the formulation with MPL alone. A gB + CMV poly + gB + CMV poly + gB + CMV poly + MPL + CpG CpG MPL MPL + CpG 30.3 30.8 0.61 0.14 CD8+ T cells B gB + CMV polyepitope + MPL + CpG gB + CMV polyepitope + CpG gB + CMV polyepitope + MPL MPL + CpG 0 5 10 15 20 % of CD8+ T cells producing IFN-γ Figure 5.12: Assessment of the CMV polyepitope-specific CD8+ T cell responses following in vitro stimulation of splenocytes with NLV and VLE peptides. On day 50 post-immunisation, splenocytes were stimulated in vitro with NLV and VLE peptides for 10 days in media supplemented with recombinant IL-2 and then these cells were assessed for CMV polyepitope- specific T-cell reactivity using an ICS assay. (A) The data in the FACS plots represent the percentage of CMV polyepitope-specific IFN-γ producing CD8+ T cells from one of five mice immunised with gB-CMVpoly proteins in combination with MPL and CpG ODN1826, CpG ODN1826 alone, MPL alone or adjuvants alone. (B) Bar graph represents frequencies of IFN-γ producing CMV polyepitope-specific CD8+ T cells. Error bars represent the mean ± S.E.M. These responses were detected in both the peripheral blood (Fig 5.11B) and spleen (Fig 5.11C), and were maintained as a robust memory response for the long-term. Most importantly, in vitro stimulation of these T cells with the HLA A2-restricted T 163 IFN-γ cell epitopes, NLV and VLE, resulted in rapid and massive A gB + polyepitope gB + polyepitope gB + polyepitope + MPL + CpG + CpG + MPL IFN+ TNF+ IL2+ 2% 0% 12% 7% 1% 8% IFN+TNF+IL2- 5% 15% 5% 0% 0% IFN+TNF-IL2+ 4% 32% IFN+TNF-IL2- 38% 25% 44% 58% IFN-TNF+IL2+ 32% 2% IFN-TNF+IL2- 5% 5% IFN-TNF-IL2+ B 16 14 * gB + polyepitope + MPL + CpG 12 gB + polyepitope + CpG * 10 gB + polyepitope + MPL 8 MPL + CpG 6 4 2 0 Figure 5.13: Multiple cytokine expression by CMV-polyepitope specific CD8+ T cells. On day 10, following in vitro expansion of splenocytes from mice immunised with gB- CMVpoly proteins in combination with MPL and CpG ODN1826, CpG ODN1826 alone, MPL alone or adjuvants alone were restimulated with NLV and VLE peptides, cultured for 10 days in the presence of IL-2 and then assessed for their capacity to produce IFN-γ, TNF-α or IL-2 by ICS. (A) Pie charts represent the percentage of CMV polyepitope- specific CD8+ T cells producing seven combinations of cytokines. (B) The bar graph represents the absolute frequencies of multiple cytokine producing CMV polyepitope- specific CD8+ T cells. Error bars represents the mean ± S.E.M. * indicates p<0.05. + expansion of antigen-specific CD8 T cell responses which expressed IFN-γ following stimulation with the relevant peptide epitopes (Fig. 5.12). Polyfunctional analysis of antigen-specific T cells revealed that the majority of these expanded cells expressed multiple cytokines (Fig. 5.13A & B). These observations clearly demonstrate that formulation of the recombinant gB and polyepitope proteins with both TLR4 and TLR9 agonists was critical for the 164 % of cytokine producing CD8+ T cells induction of a pluripotent immune response. Although the TLR9 agonist was sufficient + to activate polyepitope-specific CD8 T cell responses, the optimal induction of both + gB-specific CD4 T cell and antibody responses required the synergistic effects of the TLR4 and TLR9 agonists. 5.4.4 TLR4 and TLR9 agonists synergistically promote the activation of dendritic cells and induce pro-inflammatory cytokine expression To delineate the potential mechanism for the induction of pluripotent immune responses by a combination of MPL and CpG ODN1826 adjuvants, we next tested the effect of these adjuvants on murine DCs. Murine DCs were purified using CD11c microbeads and then cultured in vitro in the presence of the various combinations of adjuvants. Supernatants from cultures were harvested after 20 h and then assessed for the expression of the pro-inflammatory cytokines IL-6, IL-12p70, TNFα and IFN-α using ELISA. Data presented in Figure 5.14 shows that following in vitro stimulation, mouse DCs stimulated with CpG ODN1826 or MPL and CpG ODN1826 induced high levels of IL-6, IL-12p70, TNFα and IFNα when compared to DCs stimulated with MPL, MPL and Gardiquimod or Gardiquimod and CpG ODN1826 (Fig. 5.14A- D). Furthermore, IL-12p70 expression was also assessed in cultured DC using ICS assays. Consistent with the ELISA results, data presented in Figure 5.14D shows that stimulation with CpG ODN1826 or MPL and CpG ODN1826 induced strong + + expression of IL12p70 in CD11c DCs and PDCA plasmacytoid DCs (pDCs). It is important to note that the combination of MPL and CpG ODN1826 does appear to have a synergistic effect on IL-12 production by pDCs. 165 IL-6 IL-12p70 A 120500 100 B 400 80 300 60 200 40 100 200 0 TNF IFN-α 400 C 40 D 300 30 200 20 100 10 0 0 E MPL MPL CpG & & & PBS MPL CpG CpG Gardiquimod Gardiquimod 1.7 1.34 8.77 4.99 1.84 3.28 IL-12p70 0.43 1.03 1.9 3.9 2.19 2.78 IL-12p70 Figure 5.14: Assessment of proinflammatory cytokines production following DC stimulation with various adjuvant combinations. CD11c+ DCs were enriched using CD11c microBeads, based on positive selection, and stimulated with PBS, MPL, CpG ODN1826, MPL & CpG ODN1826, MPL & Gardiquimod, CpG ODN1826 and Gardiquimod or AbISCO®100 and CpG ODN1826 for 20 hours in a 48 well plate. Supernatants were collected and ELISAs were performed to quantify the pro- inflammatory cytokines. (A-D) Bar graphs represent the production of the pro-inflammatory cytokines IL-6, IL-12p70, TNF-α and IFN-α. (E) FACS plots represent the CD11c+ and pDCs producing IL-12p70 following stimulation with indicated adjuvants. 166 pg/mL pg/mL PDCA-1 CD11c+ pg/mL pg/mL 5.5 Discussion Cellular immune responses are typically initiated by activation of APCs, notably DCs and B cells through the interaction between their pathogen recognition receptors (PRRs). Amongst these PRRs, TLRs are the best characterised and they are widely expressed either on the cell surface or on endosome by a variety of cells in the blood, spleen, lung, muscle and intestine (Abreu et al., 2005; Kabelitz, 2007; Schuster and Nelson, 2000; Takeda et al., 2003; Zarember and Godowski, 2002). A number of TLR agonists in clinical and preclinical stages are emerging and various reports have shown that the use of these TLR agonists, in particular the TLR7 agonist imiquimod, TLR 7/8 agonist resiquimod, and the TLR9 agonist CpG ODN induces long-term immune responses and strong protection against related pathogens (Krieg, 2006; Wille-Reece et al., 2005b). Taking these observations into consideration, we formulated recombinant gB-CMVpoly proteins with different combinations of TLR agonists to investigate the induction of CMV-specific humoral and cellular immune responses. Although we had previously observed that the combination of CpG ® ODN1826 and AbISCO 100 lead to the efficient induction of a gB-specific cellular and humoral response, this combination did not induce a CMV polyepitope-specific + CD8 T cell response. Subsequent examination of three TLR agonists in three different combinations (MPL and Gardiquimod, MPL and CpG ODN1826 or CpG ODN1826 and Gardiquimod) revealed that a combination of the TLR4 agonist MPL and the TLR9 agonist CpG ODN1826 lead to the most efficient induction of both gB- specific humoral and cellular responses, and to the induction of a polyepitope specific + CD8 T cell response. These observations suggested that innate signalling via both TLR4 and TLR9 lead to the optimal induction of both cellular and humoral immunity following 167 immunisation with the gB-CMVpoly vaccine formulations. To delineate the role of + MPL or/and CpG ODN1826 in the enhancement of gB-specific antibody, CD4 and + + CD8 T cell responses and CMV polyepitope-specific CD8 T cell responses, we investigated the use of these adjuvants in combination or alone. Whilst immunisation in combination with CpG ODN1826 alone was sufficient to induce a polyepitope + specific CD8 T cell response, only when the gB-CMVpoly proteins were formulated with a combination of MPL and CpG ODN1826 was an optimal gB-specific antibody and polyfunctional cellular immune response generated. This confirmed the synergistic effect of the TLR4 and TLR9 agonists on the induction of a gB-CMVpoly response. The ability of a vaccine to skew the immunological response towards a specific type is highly important for the induction of pathogen-specific immune responses. Emerging evidence suggests that to manipulate the immune system to induce particular correlates of protective immunity, activation of resting DCs is a crucial factor (Banchereau et al., 2000; Kalinski et al., 1999; Moser and Murphy, 2000). Following activation, different subsets of DCs produce pro-inflammatory cytokines, migrate to lymph nodes and stimulate naïve T cells to initiate immune responses (Coffman et al., 2010; Steinman, 2008). Pro-inflammatory cytokines play an integral role in the regulation of innate and adaptive immune responses. Emerging evidence shows that IL-6 is critical for resolving innate immunity and promoting adaptive immune responses [reviewed in (Jones, 2005)], IL-12p70 and IFN-α induced from activated DCs stimulate Th1 responses [reviewed in (Janeway and Medzhitov, 2002; Medzhitov and Janeway, 2000a; Takeda et al., 2003)] and TNF influences the adaptive immune responses against viruses. In the current study, we observed that CpG alone or in combination with MPL was capable of activating inflammatory cytokine production + from CD11c dendritic cells in vitro, which correlated with the ability of CpG alone to 168 + induce a polyepitope-specific CD8 T cell response. Conversely, MPL alone was much less efficient at inducing inflammatory cytokine production, as has been shown previously, and was inefficient at promoting gB-CMVpoly specific cellular responses. Despite the enhanced immunogenicity of gB-CMVpoly in combination with both MPL and CpG, the addition of MPL appeared to enhance the overall induction of inflammatory cytokines by CpG. However, MPL did appear to function synergistically + with CpG to enhance IL-12 production by PDCA1 pDCs. Emerging evidence has demonstrated that pDCs play a critical role in the induction of both cellular and humoral immune responses (Pulendran, 2004; Takagi et al., 2011). Therefore, the synergistic effect of MPL and CpG on pDCs provides one potential mechanism via which MPL further enhanced the immunogenicity of the gB-CMVpoly vaccine. Alternatively, other undefined mechanisms may play a role in the synergistic effects of MPL and CpG. A recent study conducted in non human primates demonstrated that although MPL did not induce inflammatory cytokines and co-stimulatory molecules, it was able to induce expansion of myeloid DCs and monocytes in the draining lymph node. In contrast, CpG did not induce expansion of monocytes but was able to induce inflammatory cytokines and costimulatory molecules (Kwissa et al., 2012). This suggests that these adjuvants function synergistically by differentially stimulating components of the systemic and local innate immune response. In summary, the data obtained from this study demonstrates that formulation of the gB-CMVpoly vaccine with a combination of multiple TLR ligands is critical for the optimal induction of long-term CMV-specific humoral and cellular immune responses. 169 170 Chapter 6: Preclinical assessment of the immunogenicity of a glycoprotein B and polyepitope protein based cytomegalovirus vaccine in combination with human compatible adjuvants 6.1 Abstract This chapter focuses on the evaluation of the immunogenicity of the CMV vaccine based on the combination of the glycoprotein B and polyepitope proteins (referred to as gB-CMVpoly) complemented with the human compatible adjuvants ® ® IC31 , AS01 or AS15. The IC31 adjuvant includes an antibacterial peptide (KLKL(5)KLK) and a TLR9 agonist, while the AS01 and AS15 adjuvants are liposome formulations containing QS21, and a agonist, TLR4 with or without a TLR9 agonist. These adjuvants have been successfully used in various human vaccine formulations with minimal side effects. Transgenic mice expressing the human HLA A2 MHC class I allele were immunised twice with the gB-CMVpoly proteins ® formulated with IC31 , AS01 or AS15. All three adjuvants consistently induced a strong gB-specific antibody response that showed highly efficient anti-viral neutralising capacity. Furthermore, these vaccine formulations induced a long-lived + gB-specific CD4 T cell response characterised by the production of multiple + cytokines. CMV-specific CD8 T cell responses were detectable following ® immunisation with vaccines formulated with the IC31 or AS01 adjuvants, while no + CD8 T cell responses were detected in animals immunised with the gB-CMVpoly proteins adjuvanted with AS15. Based on these results, we propose that a CMV 171 ® vaccine formulation based on gB-CMVpoly proteins in combination with IC31 or AS01 should be assessed in human clinical trials. 172 6.2 Introduction One of the key aspects in the development of vaccine formulations against intracellular pathogens is the induction of long-lived humoral and cellular immune responses. Although various platform technologies including viral vectors and plasmid DNA have been successfully tested, protein-in-adjuvant formulations remain the preferred option due to the ease of manufacturing, storage and safety profile. Most of the adjuvants currently approved for human vaccines, including alum, MF59, AS03 and AS04 predominantly induce humoral immune responses, and have limited use against intracellular pathogens which require both humoral and cellular immune responses for optimal protection against disease (Mbow et al., 2010). To potentiate protective immune responses a number of experimental adjuvants have been developed and tested in preclinical and clinical trials [reviewed in (Coffman et al., 2010; Dey and Srivastava, 2011; Mutwiri et al., 2011)]. In spite of the successful outcome of these studies, most of these adjuvants have failed to achieve formal approval for human use due to underlying safety concerns (McCartney et al., 2009; McKee et al., 2010; McKee et al., 2007). To overcome the limitations of experimental adjuvants, a series of next generation adjuvants have been developed by combining classical adjuvants and novel immunomodulators, which allow the induction of both humoral and cellular immune responses. These new generation adjuvants include ® IC31 (Intercell), and AS01, AS02 and AS15 (all from GSK Biologicals). One of the unique features of these adjuvants is that they all contain one or more TLR agonists. ® IC31 contains the TLR9 agonist ODN1a without a CpG motif, while AS01 and AS15 adjuvants include a TLR4 agonist (MPL) with or without a TLR9 agonist (CpG ODN7909). These adjuvants activate the innate immune system through TLR- dependent pathways and have been shown to induce potent T cell responses against 173 various intracellular pathogens (Bernardo et al., 2011; Kamath et al., 2008; Kester et al., 2009; Olafsdottir et al., 2009; Riedl et al., 2008; Schellack et al., 2006). In addition these adjuvants have also demonstrated acceptable safety profiles in human clinical trials (Bejon et al., 2008; Kamath et al., 2008; Van Braeckel et al., 2011; van Dissel et al., 2011). Taking into consideration the findings from these studies, a series of experiments were designed to assess the immunogenicity of the gB-CMVpoly vaccine ® with IC31 , AS01 and AS15. These studies showed that these adjuvants were highly effective in inducing a gB-specific antibody response that showed strong anti-viral neutralising capacity. Furthermore, these vaccine formulations also induced long-lived + polyfunctional gB-specific CD4 T cells. Analysis of CMV-specific cytotoxic T cell ® responses showed that the CMV vaccine formulated with the IC31 or AS01 adjuvants + + induced a polyfunctional antigen-specific CD8 T cell response, while no CD8 T cell responses were detected in animals immunised with the gB and CMVpoly proteins adjuvanted with AS15. 174 6.3 Materials and Methods ® 6.3.1 Preparation of vaccine formulations with IC31 adjuvant ® The IC31 adjuvant consisting of the KLK peptide and ODN1a was manufactured by Intercell AG., Austria. Vaccines were formulated by adsorbing gB (5 ® µg) and CMV polyepitope (20 µg) proteins with IC31 (100 nmol KLK peptide and 4 ® nmol of ODN1a), or IC31 and Alum (0.15%; Pierce/ThermoScientific, Rockford, USA) in a 100 µL volume/dose. 6.3.2 Preparation of vaccine formulations with AS01 and AS15 adjuvants The adjuvant system, AS01 or AS15 (GSK, Rixensart, Belgium) used in this study contained 5 µg of MPL and 5 µg of QS-21 in 50 µL of liposomes. In addition the AS15 formulation also included CpG 7909. All the vaccine formulations were prepared just before vaccination by mixing gB (5 µg) and CMV polyepitope (20 µg) proteins (in 25 µL volume) with 25 µL of AS01 or AS15 adjuvant per dose. After mixing vaccine formulations were agitated on an orbital shaker for 10 min mice were then immunised within one hour of preparation. 6.3.3 Mice immunisation HHD 1 (HLA A2 transgenic) mice were bred and maintained in a pathogen free facility at QIMR. All protocols were followed in compliance with the requirements of the QIMR animal ethics committee. Six-to-eight week old HHD 1 mice were immunised subcutaneously at the base of tail with the above mentioned 175 formulations or with MPL and CpG as a positive control, or with proteins alone or adjuvants alone as a negative control. On day 21 these mice were boosted with the same vaccine formulation. Mice were bled via the lateral tail vein at regular intervals following primary and secondary immunisations. PBMC and sera were collected for analysis of cellular immune responses by ICS and antibody responses by ELISA as described in Chapter 2: Material and Methods. 6.3.4 Statistical analysis Statistical analyses were carried out using Graph Pad software or Microsoft + + Office Excel 2007. For ELISA antibody titers, CD4 and CD8 T cell responses, the means ± SD were calculated and p values were determined using the Student’s t-test. Error bars represent S.E.M. Where indicated with *, ** and *** represents statistically significant with p<0.05, p<0.01 and p<0.001 respectively when compared to the control groups. 176 6.4 Results 6.4.1 Immunisation of HHD mice with the gB-CMVpoly proteins in combination with human compatible adjuvants To evaluate the immunogenicity of the gB and CMV polyepitope proteins in combination with human compatible adjuvants two different sets of experiments were 1. gB + CMV poly 1. gB + CMV poly 2. gB + CMV poly + IC31 2. gB + CMV poly + AS01 3. gB + CMV poly + IC31 + Alum 3. gB + CMV poly + AS15 4. gB + CMV poly + MPL + CpG 4. AS01 ODN1826 5. AS15 5. IC31 6. IC31 + Alum HHD-I Day 21 Tail bleed and then boost Day 28, 35 & 50 Assess gB-specific antibody and CD4+ and CD8+ T cell responses and CMV polyepitope specific CD8+ T cell response Figure 6.1: Schematic representation of the gB and CMV polyepitope vaccination study in combination with IC31, AS01 or AS15 adjuvants. To evaluate the IC31 adjuvant, six groups (n=5) of HHD mice were immunised subcutaneously with unadjuvanted gB-CMVpoly proteins or formulated with IC31®, IC31® and alum or MPL and CpG ODN1826, or with adjuvants alone. To evaluate AS01 and AS15 adjuvants, five groups (n=5) of mice were immunised with gB and CMV polyepitope alone or formulated with AS01, AS15 or adjuvants alone. Mice were tail bled and then a booster dose was given on day 21. On day 28 and 35 these mice were again tail bled to assess cellular immune responses and antibody responses and sacrificed on day 50 to assess gB and CMV polyepitope-specific immune responses. performed. In the first set of experiments six groups of HLA A2 transgenic mice (HHD-1) were immunised with unadjuvanted gB-CMVpoly proteins in combination ® ® with IC31 alone, IC31 and Alum, MPL and CpG ODN1826, or with adjuvants alone as outlined in Figure 6.1. In the second set of experiments five groups of mice were 177 immunised with unadjuvanted gB-CMVpoly proteins, or formulated with AS01, AS15 or adjuvants alone as outlined in Figure 6.1. On day 21 mice were bled via the lateral tail vein, then boosted with an identical formulation. Mice were tail bled after secondary immunisation (on day 28 and 35) to assess gB and CMV polyepitope- specific effector cellular immune responses using an ICS assay and antibody responses using a gB ELISA. These mice were sacrificed on day 50 to evaluate memory responses in the peripheral blood and the spleen (Fig. 6.1). 6.4.2 Evaluation of the gB-specific antibody response following immunisation with the gB-CMVpoly proteins in combination with human compatible adjuvants The data presented in Figures 6.2A & B shows that on day 21 mice immunised ® ® with gB-CMVpoly proteins in combination with IC31 alone, IC31 and Alum, MPL and CpG ODN1826, and AS15 showed higher gB-specific antibody titers when compared to animals immunised with the unadjuvanted formulation or with adjuvants alone. While an increase in the gB-specific antibody titre was not evident following primary immunisation with the AS01 formulation, it was evident on day 35 following the boost immunisation (Fig 6.2B & D). A similar increase in titre was also observed following the boost immunisation with the other adjuvant formulations (Fig 6.2C & D). More importantly these antibody titers were maintained at high levels until day 50 (Fig. 6.2E & F). The neutralising capacity of the CMV vaccine induced antibody responses were next assessed against the HCMV AD169 strain in MRC-5 cells using a micro-neutralisation assay. Neutralising titres were defined as the dilution of serum capable of inducing a 50% reduction of CMV infected nuclei. The data presented in Figures 6.3A & B shows that neutralising antibody titres in serum samples from mice ® immunised with gB-CMVpoly vaccine formulated without adjuvants or with IC31 178 ® alone, IC31 and Alum, AS01, AS15 and MPL and CpG ODN1826. These analyses showed that the CMV proteins formulated with MPL and CpG or with AS15 adjuvants showed the highest level of neutralizing antibodies, while the antibodies generated ® following immunisation with IC31 and AS01 adjuvanted vaccines were comparably A 3.5 BgB + CMV poly 4 gB + CMV poly 3 gB + CMV poly + IC31 3.5 gB + CMV poly + AS01 2.5 gB + CMV poly + IC31 + Alum 3 gB + CMV poly + AS15 2 gB + CMV poly + MPL + CpG 2.5 AS01 2 1.5 IC31 AS15 1.5 Day 21 IC31 + Alum 1 1 0.5 0.5 0 0 serum dilutions D serum dilutionsC 3.5 gB + CMV poly 4 gB + CMV poly 3 gB + CMV poly + IC31 3.5 3 gB + CMV poly + AS01 2.5 gB + CMV poly + IC31 + Alum 2.5 gB + CMV poly + AS15 2 gB + CMV poly + MPL + CpG 2 AS01 1.5 IC31 1.5 AS15 1 IC31 + Alum 1 Day 35 0.5 0.5 0 0 E serum dilutions F serum dilutions 3.5 gB + CMV poly 4 gB + CMV poly 3 gB + CMV poly + IC31 3.5 gB + CMV poly + AS01 2.5 3gB + CMV poly + IC31 + Alum gB + CMV poly + AS15 2.5 2 gB + CMV poly + MPL + CpG AS01 2 1.5 IC31 AS15 1.5 1 IC31 + Alum 1 Day 50 0.5 0.5 0 0 serum dilutions serum dilutions Figure 6.2: Anti gB antibody titers induced following immunisation with gB and CMV polyepitope vaccine in combination with IC31, AS01 or AS15. gB-specific antibody titres in mice immunised with the gB-CMVpoly vaccine formulated without adjuvant or with IC31®, IC31® and alum, MPL and CpG or with AS01, AS15 or adjuvants alone on day 21 (A & B), 35 (C & D) and 50 (E & F). The gB-specific antibody titers were measured in pooled sera on day 21 and 35 and in individual mice on day 50 using an ELISA. The data represent the means ± S.E.M. from five mice in each group. 179 OD at 450nm OD at 450nm OD at 450nm ** ** ** ** OD at 450nm OD at 450nm OD at 450nm ** ** ** ** ** ® less efficient at neutralising the virus. The addition of Alum to IC31 had no effect on the neutralising titres (Fig. 6.3A). 6.4.3 Assessment of the gB-specific cellular immune response following immunisation with the CMV vaccine formulations To explore the A gB-specific cellular 100 gB + CMV poly 90 immune response gB + CMV poly + IC31 80 70 gB + CMV poly + IC31 + Alum following immunisation 60 gB + CMCV poly + MPL + CpG 50 with gB-CMVpoly 40 IC31 30 IC31 + Alum proteins in combination 20 10 ® ® with IC31 alone, IC31 0 and Alum, AS01, AS15 serum dilutions B gB + CMV poly and MPL and CpG 100 90 gB + CMV poly + AS01 80 ODN1826, ex vivo gB- gB + CMV poly + AS15 70 60 AS01 specific effector and 50 AS15 40 + memory CD4 T cell 30 20 10 responses were assessed 0 on days 28 and 50 in the serum dilutions peripheral blood and on Figure 6.3: Assessment of the virus-neutralising capacity of antibodies induced following immunisation. (A) CMV-specific day 50 in the spleen neutralising antibody titres induced following immunisation with the gB-CMVpoly vaccine formulated without adjuvant, with IC31®, using ICS assays. IC31® and Alum, MPL and CpG or adjuvants alone. (B) Neutralising antibody titres induced following immunisation with the gB- CMVpoly vaccine in combination without adjuvant, with AS01 or Animals immunised with AS15, or following immunisation with adjuvants alone. On day 50 serum samples from individual groups were pooled, serially diluted gB-CMVpoly proteins in and pre-incubated with the HCMV AD169 strain. MRC-5 cells were infected with serum-treated virus preparations and virus infectivity ® combination with IC31 was determined using an IE-1/IE-2 micro-neutralisation assay. The percentage of neutralisation of viral infectivity was calculated using the following formula: [(number of IE1+ nuclei of HCMV infected or MPL and CpG cells – number of IE1+ nuclei of serum treated HCMV infected cells/ number of IE1+ nuclei of HCMV infected cells) ×100]. 180 % of neutralisation % of neutralisation A ODN1826 showed gB + CMV poly + IC31 enhanced frequencies gB + CMV poly + IC31 + Alum 50D of IFN-γ producing gB + CMV poly + MPL + CpG 28D + gB + CMV poly gB-specific CD4 T IC31 cell responses in blood IC31 + Alum when compared to 0 0.05 0.1 0.15 0.2 0.25 ®IC31 and alum, % of IFN-γ producing CD4+ T cells B unadjuvanted or gB + CMV poly + IC31 * adjuvant alone groups gB + CMV poly + IC31 + Alum on day 28, however, gB + CMV poly+ MPL + CpG by day 50 these gB + CMV poly responses were IC31 significantly reduced IC31 + Alum (Fig. 6.4A ). In 0 0.2 0.4 0.6 0.8 % of IFN-γ producing CD4+ T cells contrast to the T cell Figure 6.4: Representative data of gB-specific effector and responses observed in memory CD4+ T cell responses following immunisation with IC31, IC31 & alum or MPL & CpG formulations in peripheral the peripheral blood blood and in the spleen. (A) Bars represent the frequencies of gB-specific IFN-γ producing CD4+ T cells in pooled blood from on day 50, higher mice immunised with the gB and CMV polyepitope vaccine formulated with IC31, IC31 and alum, MPL and CpG or frequencies of gB- without adjuvants, or with adjuvants alone, on day 28 and 50 and in individual spleens on day 50 (B). Error bars represent + specific CD4 T cell the mean ± S.E.M. responses were maintained in the spleen (Fig. 6.4B). Interestingly, mice immunized with gB-CMVpoly proteins formulated with AS01 or AS15 adjuvants showed higher gB-specific T cell responses in the peripheral blood on day 50 when compared to day + 28 (Fig. 6.5A). Furthermore, high frequencies of CD4 T cell responses were also detected in the spleen on day 50 (Fig. 6.5B). 181 A There is now emerging gB + CMV poly + AS01 evidence which indicates gB + CMV poly + AS15 that measuring the gB + CMV poly magnitude of IFN-γ 50D AS15 28D producing T cells is not AS01 sufficient to predict 0 0.5 1 1.5 2 2.5 protection, and the % of IFN-γ producing CD4+ T cells B evaluation of additional T gB + CMV poly + cell functions is required AS01 gB + CMV poly + to define a protective T AS15 cell response. Therefore, gB + CMV poly + gB-specific CD4 T cell AS15 responses were further AS01 characterised by 0 0.5 1 1.5 2 2.5 % of IFN-γ producing CD4+ T cells determining their capacity Figure 6.5: Representative data of gB-specific effector to induce multiple and memory CD4+ T cell responses following immunisation with AS01 or AS15 formulations in cytokines, including IFN- peripheral blood and in the spleen. (A) Bars represent the frequencies of gB-specific IFN-γ producing CD4+ T γ, TNF and IL-2, using cells in pooled blood from mice immunised with gB and CMV polyepitope in combination with AS01 or multiparametric ICS. The AS15, or without adjuvants, or with adjuvants alone, on day 28 and 50 and in individual spleens on day 50 data presented in Figures (B). Error bars represent the mean ± S.E.M. 6.6 & 6.7 shows that the + + + + majority of the gB-specific memory CD4 T cells were triple (IFNγ TNF IL-2 ) and + + double (IFNγ TNF IL2-) cytokine producers in mice immunised with gB-CMVpoly ® ® proteins in combination with IC31 alone, IC31 and Alum, AS01, AS15 and MPL and CpG ODN1826, while gB-specific T cells generated in mice 182 gB + CMV poly gB + CMV poly + A + IC31 IC31 + Alum 0% 0% 8% 13% 13% 19% 44% 6% 51% IFN+ TNF+ IL2+ 10% 6% IFN+TNF+IL2- 12% 4% IFN+TNF- IL2+ 14% IFN+TNF- IL2- gB + CMV poly + IFN -TNF+IL2+ MPL + CpG gB + CMV poly IFN -TNF+IL2- 7% 2% IFN -TNF -IL2+ 6% 0% 22% 10% 41% 3% 5% 1% 51% 33% 18% 1% B 0.45 * 0.4 gB + CMV poly + IC31 0.35 gB + CMV poly + IC31 + Alum 0.3 gB + CMV poly + MPL + CpG 0.25 gB + CMV poly 0.2 0.15 0.1 0.05 0 Figure 6.6: Multiple cytokine expression by gB-specific CD4+ T cells from mice vaccinated with gB and CMV polyepitope vaccine formulated with IC31®, IC31® and alum, MPL and CpG or without adjuvants. On day 50, splenocytes from vaccinated mice were stimulated in vitro with gB protein overnight in the presence of brefeldin A and the production of IFN-γ, TNF-α or IL-2 was determined by ICS. Data was analysed using FlowJo software and gB-specific CD4+ T cells producing various combinations of cytokines were plotted. (A) Pie charts indicate the proportion of gB-specific CD4+ T cells producing seven combinations of cytokines. (B) The bar graph represents the absolute percentage of gB-specific CD4+ T cells producing multiple cytokines. immunised with the unadjuvanted gB and polyCMV proteins were primarily single - + - (IFN TNF IL-2 ) cytokine producers. 183 % of cytokine producing CD4+ T cells gB + CMV poly + gB + CMV poly + A AS01 AS15 IFN+ TNF+ IL2+ 1% 2% IFN+TNF+IL2- 23% 19% IFN+TNF- IL2+ 0% IFN+TNF- IL2- 1% 5% 2% 1%8% IFN -TNF+IL2+ 0% 2% 65% 71% IFN -TNF+IL2 - IFN -TNF -IL2+ B 0.7 0.6 gB + CMV poly + AS01 0.5 gB + CMV poly + AS15 0.4 gB + CMV poly 0.3 0.2 0.1 0 Figure 6.7: Multiple cytokine expression by gB-specific CD4+ T cells from mice vaccinated with gB and CMV polyepitope vaccine formulated with AS01, AS15 or without adjuvants. On day 50, splenocytes from vaccinated mice were stimulated in vitro with gB protein overnight in the presence of brefeldin A and the production of IFN-γ, TNF-α or IL-2 was determined by ICS. Data was analysed using Flowjo software (A) Pie charts represent the proportion of gB-specific CD4+ T cells producing seven combinations of cytokines. (B) The bar graph represents the absolute percentage of gB-specific CD4+ T cells producing multiple cytokines. 6.4.4 Evaluation of CMV polyepitope-specific responses following immunisation with the CMV vaccine formulated with human compatible adjuvants + In the final set of experiments, we assessed the antigen-specific CD8 T cell responses following immunisation with the CMV vaccine formulations. Ex vivo + analysis using ICS assays demonstrated low to undetectable CMV-specific CD8 T 184 % of cytokine producing CD4+ T cells ** ** cell responses (data not shown). Therefore, to expand the frequency of antigen- + specific CD8 T cells, splenocytes were stimulated in vitro with the HLA A2-restricted + CD8 T cell epitopes, NLV and VLE, and cultured for 10 days in growth medium supplemented with IL-2. On day 10, expanded splenocytes were assessed for CMV- + specific CD8 T cell responses using ICS assays. The data presented in Figures 6.8A + & B shows an expansion of CMV polyepitope specific, IFN-γ producing CD8 T cells from mice immunised with the gB and CMV polyepitope proteins in combination with ® ® IC31 , IC31 and alum or MPL and CpG. A gB + CMV poly gB + CMV poly + gB + CMV poly + IC31 IC31 + Alum + MPL + CpG gB + CMV poly IC31 IC31 + Alum 3.15 3.16 5.22 0.058 0.036 0.038 CD8+ T cells B gB + CMV poly + IC31 gB + CMV poly + IC31 + Alum gB + CMV poly + MPL + CpG gB + CMV poly IC31 IC31 + Alum 0.5 2.5 4.5 6.5 8.5 10.5 12.5 14.5 % of IFN-γ producing CD8+ T cells Figure 6.8: Assessment of the CMV polyepitope-specific CD8+ T cell responses following immunisation with the gB and CMV polyepitope vaccine in combination with IC31®, IC31® and alum MPL and CpG, without adjuvants, or with adjuvants alone. On day 50 post immunisation, splenocytes were stimulated in vitro with HLA A2 peptides for 10 days in media supplemented with recombinant IL-2. Cultured cells were assessed for CMV polyepitope-specific T-cell reactivity using an ICS assay. (A) The data in the FACS plots represent the percentage of CMV polyepitope-specific IFN-γ producing CD8+ T cells. A representative plot from one of five mice per group is shown. (B) Graphs represent the frequency of IFN-γ producing CD8+ T cells (mean ± S.E.M). It is important to note that higher frequencies of CMV polyepitope-specific IFN-γ 185 IFN-γ gB + CMV poly gB + CMV poly + + IC31 IC31 + Alum A 0% 8% 1% 0%7% 0% 0% 0% 47% 34% IFN+ TNF+ IL2+ 44% 59% IFN+TNF+IL2- 0% 0% IFN+TNF -IL2+ gB + CMV poly + IFN+TNF -IL2- MPL + CpG gB + CMV poly IFN -TNF+IL2+ 0% 1% 0% 6% 6% 14%1% 15% IFN -TNF+IL2- 0% 1% IFN -TNF -IL2+ 15% 2% 53% 86% B gB + CMV poly + IC31 10 gB + CMV poly + IC31 + Alum gB + CMV poly + MPL + CpG 1 gB + CMV poly 0.1 0.01 Figure 6.9: Multiple cytokine expression by CMV polyepitope-specific CD8+ T cells. In vitro expanded CMV polyepitope-specific CD8+ T cells from mice immunised with the gB and CMV polyepitope vaccine in combination with IC31, IC31 and alum, MPL and CpG or without adjuvant were restimulated with HLA A2 peptides and cultured for 10 days. Following restimulation, expanded CMV polyepitope-specific CD8+ were stimulated with HLA A2 peptides and then assessed for their capacity to produce IFN-γ, TNF-α or IL-2 using an ICS assay. (A) Pie charts represent the percentage of CMV polyepitope-specific CD8+ T cells producing seven combinations of cytokines. (B) Bar graph represent the absolute frequency of multiple cytokine producing CMV polyepitope-specific CD8+ T cells (mean ± S.E.M). 186 % of cytokine producing CD8+T cells A gB + CMV poly gB + CMV + AS01 poly + AS15 gB + CMV poly AS15 AS01 17.5 0.26 0.5 0 0 CD8+ T cells B gB + CMV poly + AS01 gB + CMV poly + AS15 gB + CMV poly AS15 AS01 0 1 2 3 4 5 6 7 8 % of IFN-γ producing CD8+ T cells Figure 6.10: Assessment of CMV polyepitope-specific CD8+ T cell responses following immunisation with the gB and CMV polyepitope vaccine in combination with AS01, AS15 or without adjuvants, or with adjuvants alone. Splenocytes were stimulated in vitro with HLA A2 peptides for 10 days in media supplemented with recombinant IL-2 and then these cells were assessed for CMV polyepitope-specific T-cell reactivity using an ICS assay. (A) The data in the FACS plots represent the percentage of CMV polyepitope-specific IFN-γ producing CD8+ T cells. A representative plot from one of five mice per group is shown. (B) Data represents the frequency of IFN-γ producing CD8+ T cells (mean ± S.E.M). + producing CD8 T cells were observed in mice immunised with gB-CMVpoly vaccine ® ® with MPL and CpG adjuvants when compared to IC31 or IC31 and alum adjuvanted + vaccines. Furthermore, these CMV-specific CD8 T cells were polyfunctional, with + + the majority of these effector cells producing two or more cytokines (IFNγ TNFα IL- +/- + 2 ) (Fig. 6.9A & B). CD8 T cell responses in mice immunised with CMV vaccine complemented with AS01 and AS15 adjuvants showed discrepant and unexpected + patterns. The data presented in Figures 6.10A & B shows that CD8 T cell responses 187 IFN-γ could only be detected in animals immunised with the CMV vaccine formulated with ® the AS01 adjuvant. In accordance with the results obtained with the IC31 adjuvant, + CMV-specific CD8 T cells from mice immunised with CMV vaccine supplemented with the AS01 adjuvant demonstrated polyfunctionality with the majority of these + + - effector cells producing multiple cytokine (IFNγ TNFα IL-2 ) (Fig. 6.11A & B). gB + CMV poly + AS01 A IFN+ TNF+ IL2+ 14% 0% IFN+TNF+IL2 - 3% 0% IFN+TNF - IL2+ IFN+TNF - IL2- 23% 60% IFN -TNF+IL2+ 0% IFN -TNF+IL2 - IFN -TNF - IL2+ B 3.5 3 2.5 gB + CMV poly + AS01 2 1.5 1 0.5 0 Figure 6.11: Multiple cytokine expression by CMV polyepitope-specific CD8+ T cells. Following secondary stimulation, expanded CMV polyepitope-specific CD8+ T cells were stimulated with HLA A2 peptides, then assessed for their capacity to produce IFN-γ, TNF-α and IL-2. (A) Pie charts represent the percentage of CMV polyepitope-specific CD8+ T cells producing seven combinations of cytokines. (B) Bar graph represents the absolute frequencies of multiple cytokine producing CMV polyepitope- specific CD8+ T cells (mean ± S.E.M). 188 % of cytokine producing CD8+ T cells 6.5 Discussion In this study we have shown that a CMV vaccine based on gB and the polyepitope protein can be successfully formulated with human compatible adjuvants. Since protection from CMV infection requires both humoral and cellular immune response, the data presented in this study provides a potential strategy for the ® prevention of CMV disease. Two of the human compatible adjuvants, IC31 and AS01, were the most efficient in activating the relevant arms of the immune system when compared to our experimental adjuvant formulation based on MPL and CpG. ® IC31 is a synthetic bi-component adjuvant consisting of a cationic poly-amino acid ® KLK, and ODN1a, mixed at the molar ratio of 25:1. IC31 has been shown to induce potent and sustained humoral and cellular immune responses against a range of vaccine antigens in murine models by exerting its immunostimulatory effects via the TLR9/MyD88-signaling pathway (Agger et al., 2006; Cheng et al., 2011; Riedl et al., 2008; Schellack et al., 2006) and in humans (van Dissel et al., 2010; van Dissel et al., ® 2011). These observations clearly imply that IC31 may possess the requirements as an adjuvant to induce antigen-specific humoral and cellular immune responses. Based ® on these observations in this study we evaluated the suitability of the IC31 adjuvant system to potentiate the gB and CMV polyepitope-specific immune responses in the HHD transgenic mouse model. Similar to previous published observations, the present study also evidently shows that the gB-CMVpoly vaccine formulation in combination ® with IC31 significantly enhanced the gB-specific neutralising antibody titre, the gB- + + specific CD4 T cell response and the CMV polyepitope-specific CD8 T cell response. Following the booster dose on day 21, gB-specific antibody titres increased significantly, persisted until day 50 without any significant decrease and demonstrated neutralising capacity against the HCMV AD169 strain. In addition, the gB-CMVpoly ® vaccine in combination with IC31 induced higher frequencies of polyfunctional gB- 189 + specific CD4 effector and memory T cell responses. Most importantly, delivery with ® IC31 lead to the establishment of long-lasting memory gB and CMV polyepitope- specific responses. Although the precise mechanism for this long-lasting memory responses is not known, previous studies have shown that the KLK cationic peptide in ® the IC31 adjuvant promotes antigen association with the APC and induces depot formation at the site of injection (Fritz et al., 2004; Schijns, 2003). This depot formation also helps in retaining the ODN1a and protects the vaccine components against enzymatic degradation. Besides enhancing the immunogenicity of vaccine ® antigens, IC31 in various toxicology studies as well as in a human clinical trial demonstrated no systemic toxic effects and was locally very well tolerated (Schellack et al., 2006; van Dissel et al., 2011). Consistent with these observations, we also did not observe any adverse reactions in vaccinated mice. Taken together, these results ® demonstrated that IC31 is safe and can induce a robust antibody and cellular immune + response against the gB protein and a CD8 T cell response against the CMV polyepitope protein. In this study we also tested two additional adjuvants from GSK Biologicals, AS01 and AS15. AS01 and AS15 are specially designed to improve the cell mediated immunity response against recombinant antigens for prophylactic and therapeutic vaccines (Garcon et al., 2007). AS01 consists of MPL, QS-21 and liposomes and AS15 is a modified version of AS01 and contains CpG ODN 7909. Immunogenicity studies performed in different animal models and humans have shown that activation of TLR 4 by MPL induces innate immune responses, which then lead to the induction of humoral and cellular immune responses (Garcon et al., 2011; Tomai et al., 1987; Ulrich and Myers, 1995). In addition, QS-21 has also been shown to enhance humoral and cellular immune responses against recombinant antigens or peptides in human clinical trials (Evans et al., 2001; Gahery-Segard et al., 2003; Kashala et al., 2002). 190 Although no clinical data is available for AS15, the AS01 adjuvant formulation has been tested extensively in various clinical trials in combination with HIV-1 vaccine candidates (Leroux-Roels et al., 2010; Van Braeckel et al., 2011), recombinant hepatitis B surface antigen (Vandepapeliere et al., 2008), Plasmodium falciparum RTS,S (Kester et al., 2009) and Mycobacterium tuberculosis antigens (Reed and Lobet, 2005; Skeiky et al., 2004). In these clinical trials AS01 had an acceptable safety profile and elicited strong cellular immune responses [reviewed in (Garcon and Van Mechelen, 2011)]. These observations suggested that AS01 or AS15 might have the potential to enhance the desired immune responses against the gB and CMV polyepitope proteins. Animals immunised with the gB-CMVpoly vaccine formulated with AS01 or AS15 induced modest levels of antibody responses on day 21 and following a booster dose antibody titres peaked significantly and persisted until day 50 without a significant decrease. These antibodies demonstrated strong neutralising capacity against the HCMV AD169 strain. Besides the demonstration of strong gB-specific antibody titers, the CMV vaccine formulated with AS01 and AS15 also induced strong + gB-specific CD4 T cell responses in the peripheral blood as well as in spleen. Further + characterisation of gB-specific CD4 T cell indicated that these cells were able to + + + produce multiple cytokines (IFN-γ , TNF and IL-2 ). Surprisingly, undetectable or + low frequencies of IFN-γ producing CMV-specific CD8 T cells were detected in mice immunised with the gB-CMVpoly vaccine formulated with AS01 or AS15 adjuvants. However, following in vitro stimulation of splenocytes with the HLA A2- restricted peptide epitopes NLV and VLE there was a dramatic expansion of CMV- + specific CD8 T cells in mice immunised with the CMV vaccine formulated with + AS01, while no CD8 T cell expansion was observed with the AS15 formulation. The + lack of CD8 T cell response in the vaccine formulated with AS15 adjuvant was 191 + unexpected as this adjuvant has been shown to induce CD8 T cell responses in both animal and human studies. Although the precise reason for this discrepancy is not know, it is possible that the polyepitope protein may be poorly presented with the + AS15 adjuvant, preventing the induction of CD8 T cell responses. Further studies will need to be carried out to clarify this issue. ® In summary, the data obtained from the IC31 and AS01 adjuvant formulated CMV vaccine shows that both humoral and cellular immune responses are efficiently generated and this response is sustained long-term, suggesting that both effector and memory immune responses are induced by these formulations. The persistence of these immune responses is regarded as highly important against pathogens which cause latent infection. In naturally infected individuals, CMV infection is controlled by a combination of antibody and cellular immune responses, and high levels of neutralising antibodies are crucial in limiting the viral load both at systemic and mucosal sites. It will be important to extend these studies in humans to test the safety and efficacy of these novel CMV vaccine formulations. 192 Chapter 7: General Discussion and Future Directions Human cytomegalovirus (CMV) is a ubiquitous β human herpes virus that establishes lifelong infection. Primary CMV infection in immunocompetent individuals is generally asymptomatic or in some cases causes mononucleosis-like illness, but in congenitally infected children and in transplant patients CMV causes significant morbidity and mortality. In addition, CMV has been linked with well- known clinical disorders, such as cancers (Baryawno et al., 2011; Samanta et al., 2003), cardiovascular disease (Liu et al., 2006; Sorlie et al., 2000) and functional impairment and immunosenescence in the elderly (Aiello et al., 2008; Pawelec et al., 2009). Therefore there is an emerging need for the development of an effective vaccine formulation capable of inducing highly protective immune responses against CMV. It is very clear from many studies that CMV infection is maintained under strict control by the immune system by a combination of virus specific humoral and cellular + immune responses, particularly CD8 T cells [reviewed in (Crough and Khanna, + + 2009)]. Documented evidence also shows that these CD8 T cells likely require CD4 T cell help to achieve effective viral control (Casazza et al., 2006). These observations clearly suggest that an effective CMV vaccine should be designed to induce virus- + + specific antibody, and CD4 and CD8 T cell responses. In spite of extensive efforts over the last 30 years, a clinically licensed vaccine formulation with convincing clinical efficacy remains elusive. The main objective of vaccine development against CMV is to reduce the risk of CMV associated injury to the developing fetus and in immunologically compromised individuals such as recipients of solid organ and hematopoietic stem cell transplants. Considering these observations, the aims of this PhD were to (1) developing a new formulation strategy for CMV encoded gB vaccine which induces both humoral and cellular immunity, (2) to express multiple T cell 193 epitopes from CMV as a polyepitope protein using a prokaryotic expression system and assess its immunogenicity in vitro, (3) to develop and test a prophylactic vaccine formulation based on a combination of CMV-encoded gB and polyepitope proteins with various combinations of TLR agonists, and (4) to perform preclinical assessment of the immunogenicity of a glycoprotein B and polyepitope protein based cytomegalovirus vaccine in combination with human compatible adjuvants. Recently, a recombinant gB protein vaccine formulated with MF59 adjuvant produced by Sanofi Pasteur was evaluated in seronegative women and kidney or liver transplant patients (before transplantation) in two different phase 2 double-blind randomised placebo-controlled trials. The gB/MF59 vaccine protected 50% of seronegative mothers from acquiring primary CMV infection and reduced viral load successfully following post-transplantation in kidney and liver transplantation patients (Griffiths et al., 2011; Pass et al., 2009). The primary objective of the gB/MF59 vaccine study was to target humoral immune responses. However, emerging evidence suggests that to improve the efficacy of the gB vaccine both humoral and cellular immune responses are essential. Previous studies carried out in pregnant women and transplant recipients have established the importance of both virus neutralising + antibodies and CD8 T cell responses in limiting virus dissemination and re-infection with heterologous strains (Boppana et al., 2001; Hansen et al., 2010; Ishibashi et al., 2007; Ross et al., 2010). Considering these observations, in chapter 3 we explored the capacity of different formulations of the CMV gB vaccine to induce humoral and cellular immune responses. Our results showed that a CMV gB vaccine formulation complimented with immune stimulating complexes and a TLR9 agonist was highly effective at inducing strong CMV-specific humoral and cellular immune responses following a prime-boost vaccination. More importantly, neutralising antibodies induced following immunisation were able to demonstrate cross-neutralising activity 194 against heterologous strains of CMV expressing different genotypes of gB. In addition, immune responses induced following gB vaccination was effective in providing strong resistance against viral infection as demonstrated following challenge with recombinant vaccinia virus expressing gB protein. We also showed that this vaccine formulation can dramatically enhance the effector and long-term memory gB- + + specific CD4 and CD8 T cells responses. One of the interesting features of the cellular immune response induced by this gB vaccine was the qualitative enhancement + + of gB-specific CD4 T cells. A large proportion of these gB-specific CD4 T cells + + + demonstrated the ability to produce triple (IFN-γ , TNF and IL-2 ) and double (IFN- + + - γ , TNF and IL-2 ) cytokines. Although virus-specific neutralising antibodies and + CD8 T cells are believed to be the primary effector arms of the adaptive immune + system, both responses are critically depend on CD4 T cell help. In addition, human + studies have been shown that CD4 T cells may also play a direct role in the control of CMV infection (Gamadia et al., 2003; Tu et al., 2004). The most likely explanation for the enhancement of gB-specific humoral and cellular immune responses in this study could be because of delivery of gB protein with a combination of immunostimulating complex and TLR9 agonist. In this regard recent evidence suggest that the generation of protective immune responses likely requires the use of multiple adjuvants in a vaccine formulation (Mutwiri et al., 2011). In contrast to MF59 which induce strong antibody responses, immunostimulating complex and TLR9 agonist adjuvants have been shown to enhance antibody as well as cellular immune responses. These observations therefore provided a platform to improve gB-specific immunity following vaccination through enhanced innate stimulation via the inclusion of + multiple adjuvants. CMV-specific CD8 T cell responses play a critical role in controlling viral replication and preventing the clinical manifestations of progressive infection (Quinnan et al., 1982; Reddehase et al., 1985; Rook et al., 1984). Emerging 195 + evidence suggests that CMV-specific CD8 T cell responses in healthy CMV- seropositive individuals, are directed towards multiple CMV antigens, predominantly pp65 and IE1, but also other structural, early/late antigens and immunomodulators (pp28, pp50, pp150, IE2 gH, gB, US2, US3, US6 and UL18) (Elkington et al., 2004; Elkington et al., 2003; Khan et al., 2005; Manley et al., 2004a; Sylwester et al., 2005). Indeed, the magnitude of CTL responses observed in natural infection can be used in vaccine design to induce CTL responses, but targeting the multiple potential antigens to induce CTL responses through any vaccine delivery platform is practically not feasible. Thus in chapter 4 we employed a novel platform technology that allows the + activation of CMV-specific CD8 T cell responses directed towards multiple epitopes restricted through a range of HLA class I molecules. Previous studies from our laboratory have shown that a replication deficient adenovirus-based polyepitope + vaccine encoding multiple CMV CD8 T cell epitopes activates strong virus-specific + CD8 T cell responses from humans in vitro and in HLA A2 transgenic mice (Zhong et al., 2008). However, a replication deficient adenovirus-based vaccine delivery system posses potential safety concerns. To overcome these safety concerns we developed a novel recombinant-based polyepitope vaccine platform. Multiple minimal + HLA class I-restricted CD8 T cell epitopes were linked covalently as a string of beads and expressed as a polyepitope protein using a prokaryotic expression system. These + CD8 T cell epitopes are directed against multiple antigens (pp65, pp50, pp150, IE-1, DNAse and gB) of CMV which are expressed during various stages of CMV replication and are crucial for virus attachment, replication, assembly and reactivation. To maximise the coverage of CMV antigens and HLA restriction a series of polyepitope proteins were designed. To enhance the immunogenicity of the + polyepitope protein the selected CD8 T cell epitopes were linked together with a linker sequence consisting of a proteasome liberation amino acid sequence and a TAP 196 recognition motif. While we successfully expressed 13mer, 14mer and 15mer polyepitope proteins, our attempts to make polyepitope 20mer were unsuccessful due to its highly hydrophobic nature. Although much speculation is possible on the number of antigens and HLA restriction targeted in the CMV polyepitope proteins 13, 14 and 15mer proteins, we certainly conclude that proof of principle is demonstrated in this study and it is feasible that in order to extend HLA and epitope coverage a series of 15mers polyepitope proteins could be generated. The data from these studies clearly showed that the CMV polyepitope proteins are highly effective at expanding + CMV-specific CD8 T cells from healthy virus carriers. In addition, we also showed that the polyepitope protein was processed and presented through a novel cross- presentation pathway which is independent of TAP, but proteasome and autophagy dependent pathway. Although published reports suggest that the processing and presentation of epitopes included in the polyepitope protein require TAP recognition motifs, surprisingly, in our study we did not see a role for the TAP recognition motifs because TAP deficient cells were able to efficiently process and present polyepitope protein. Conformation of this novel cross-presentation pathway will require additional studies involving microscopic techniques to monitor localisation of CMV polyepitope protein in intracellular organelles. Recently a DNA based vaccine expressing CMV gB and pp65 proteins has been evaluated in phase two clinical trial in CMV seropositive HSCT recipients (Kharfan-Dabaja et al., 2012). This vaccine significantly reduced viraemia and improved the time-to-event for viraemia episodes in vaccine recipients compared to a placebo group. Although this vaccine was developed to induce antibody and cellular immune responses, results obtained from this study showed that while the number of pp65 IFN-γ producing T cells was significantly increased, there was no significant difference in the number of gB IFN-γ producing T cells in vaccine recipients 197 compared to the placebo group. These results suggest that this vaccine formulation may not be optimal at inducing CMV-specific immunity. To improve the efficacy of CMV vaccination approaches we have designed a novel vaccine based on gB and CMV polyepitope proteins in combination with various human compatible TLR agonists. This vaccine formulation was designed especially to target gB-specific + humoral and cellular immune responses and polyepitope-specific CD8 T cell responses. A large body of documented evidence shows that targeted manipulation of TLR signalling promotes differentiation and maturation of DCs then lead to antigen uptake and cell-surface presentation, co-stimulatory molecule expression and cytokine production. These processes trigger expansion and differentiation of naïve T cells towards a T helper 1(Th1) phenotype (Dabbagh and Lewis, 2003; Pasare and Medzhitov, 2005). A range of TLR agonists have been evaluated clinically to enhance vaccine efficacy and promote a robust T helper 1-predominant immune responses against intracellular pathogens. Among these TLR agonists, TLR4, TLR7/8 and TLR9 agonists have been evaluated extensively in various preclinical and clinical trial settings and these TLR agonists predominantly induce cellular immune responses. Therefore, to enhance the gB-CMVpoly vaccine durable immune responses we formulated these proteins in three different combinations (MPL and Gardiquimod, MPL and CpG ODN1826 or CpG ODN1826 and Gardiquimod). Interestingly, the data presented in chapter 5 showed that the bivalent gB-CMVpoly vaccine in combination + with most combinations of TLR agonists induce gB-specific antibody and CD4 T + cell responses, but strong gB and polyepitope-specific CD8 T cell responses were induced only with the MPL and CpG ODN1826 combination. MPL and CpG ODN1826 appeared to function synergistically to induce production of the inflammatory cytokine IL-12 by PDCA1+ pDCs, which play a critical role in the induction of both cellular and humoral immune responses. However, when the TLR7/8 198 agonist Gardiquimod was combined with MPL or CpG ODN1826 only a modest immune responses against gB and CMV polyepitope proteins was generated. Consistent with documented evidence, our results also showed that a vaccine formulation of simply combining antigen with the TLR7/8 agonist did not induce strong immune responses (Weeratna et al., 2005; Wille-Reece et al., 2005a; Wille- Reece et al., 2005b). This is likely due to the water soluble nature of TLR7/8 agonist, which is a small molecule and upon injection distributes throughout the body rather than forming a depot at the injection site (Tomai and Vasilakos, 2011). In contrast, conjugation of antigens with the TLR7/8 agonist or encapsulation of antigens and TLR7/8 in PLGA nanoparticle induces robust immune responses (Kastenmuller et al., 2011; Kasturi et al., 2011). These results further emphasize that in addition to the selection of adjuvant(s), an appropriate formulation strategy play a critical role in a rational approach for generating robust humoral and cellular immune responses against an antigen. Although over the last few decades various CMV vaccine development strategies have been tested in preclinical and clinical trials, the majority of these strategies have never been translated to the clinic due to their limited efficacy or safety concerns. Thus, there is an emerging need for the development of a highly efficacious vaccine which can be translated into the clinic with a short transition period. To accomplish this goal in chapter 6 we evaluated various new generation human ® compatible adjuvant systems which include IC31 , AS01 and AS15 to potentiate the immune responses of the gB-CMV poly vaccine. These adjuvants are specially designed to improve the cell mediated immune response against recombinant antigens for prophylactic and therapeutic vaccines, and have been extensively investigated in preclinical and clinical trials with different vaccine antigens. In addition these adjuvants also demonstrated acceptable safety profiles in human clinical trials. Results 199 presented in chapter 6 showed that gB and CMV polyepitope proteins formulated with ® IC31 , AS01 or AS15 consistently induced strong gB-specific neutralising antibody + and cellular responses in the HHD mice model. CMV polyepitope-specific CD8 T cells responses were detectable following immunisation with gB-CMVpoly formulated ® + with the IC31 or AS01, while AS15 induced no detectable CD8 T cell responses. + + Interestingly, CD4 and CD8 T cell responses generated against gB and CMC polyepitope were highly polyfunctional and long-lived. Since protection from CMV infection requires both humoral and cellular immune responses, the data presented in this chapter provides a potential strategy for the prevention of CMV disease. A summary of immunogenicity analysis of various CMV vaccine formulations is shown in Table 7.1. Unfortunately, although antibody and cellular immune responses were efficiently generated following gB-CMVpoly vaccination in combination with MPL and CpG ODN1826, IC31 or AS01, we are unable to co-relate these results with actual protection due to the species specificity of CMV viruses. The data presented in this thesis provides significant insight into the design and formulation of a CMV vaccine capable of generating strong humoral and cellular immunity. The targeted delivery of the gB and CMV polyepitope proteins in combination with TLR agonists and human compatible adjuvants successfully induced + + neutralising antibody and CD4 and CD8 T cell responses. Overall, the preclinical data presented in this thesis provides the rationale for future testing of the gB and ® CMV polyepitope vaccine in combination with IC31 or AS01 in human clinical trials. 200 Table 7.1: summary of immune responses generated with gB and gB-CMVpoly vaccines in combination with experimental and human compatible adjuvants Immune responses + + + CD4 T cells + CD8 T cells Adjuvant Neutralising CD4 T cells(IFN- CD8 T cells(IFN- Antigen Antibody producing producing combination antibody responses γ) γ) multiple cytokines multiple cytokines AbISCO®100 + NT + NT - NT gB CpG ODN1826 + NT + NT - NT AbISCO®100 & +++ +++ +++ +++ ++ NT CpG ODN1826 AbISCO®100 & +++ +++ ++ NT - NT CpG ODN1826 Gardiquimod & ++ + ++ NT - NT MPL Gardiquimod & ++ +/- + NT + NT CpG ODN1826 MPL & CpG +++ +++ +++ +++ +++ +++ ODN1826 MPL + ++ - - - - gB & CMV polyepitope CpG ODN1826 ++ ++ + + +++ ++ IC31® ++ + ++ ++ + + IC31® & Alum ++ + + + + + AS01 ++ ++ ++ ++ + + AS15 +++ +++ +++ +++ - - + NT- not tested. 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