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The Pennsylvania State University The Graduate School College of Medicine AN INVESTIGATION OF THE VACCINE GENERATED CELL MEDIATED IMMUNE RESPONSES TO HLA-A2.1 RESTRICTED HPV16E7 EPITOPES IN VIVO USING TWO PRECLINICAL ANIMAL MODELS A Dissertation in Microbiology and Immunology by Callie E. Bounds © 2010 Callie E. Bounds Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2010 The dissertation of Callie E. Bounds was reviewed and approved* by the following: Neil D. Christensen Professor of Pathology and Microbiology and Immunology Dissertation Advisor Chair of Committee Craig Meyers Professor of Microbiology and Immunology David J. Spector Professor of Microbiology and Immunology Todd D. Schell Associate Professor of Microbiology and Immunology Laura Carrel Associate Professor of Biochemistry and Molecular Biology Richard Courtney Professor of Microbiology and Immunology Department Chair *Signatures are on file in the Graduate School ii Abstract Human papillomaviruses (HPVs) are small DNA tumor viruses and “high risk” types have been recognized as the etiological agents of cervical cancer. Two prophylactic virus-like particle (VLP) vaccines that protect against the two most common “high risk” types, HPV16 and HPV18, are currently commercially available. However, the protection provided by each vaccine is type specific and neither vaccine can induce clearance of pre-existing HPV infections or established HPV disease. Moreover, approximately 30% of all cervical cancers are caused by other HPV types and at least 5 other cancers have been linked to HPV infection. Consequently, additional protective and therapeutic vaccine strategies are needed. The focus of this thesis was to investigate the protective vaccine generated immunity to HLA-A2.1 restricted HPV16 E7 epitopes using two preclinical animal models. The protective immunity generated after DNA vaccination against the well-known HLA- A2.1 restricted HPV16 E7 82-90 epitope was first examined using the CRPV/HLA-A2.1 transgenic rabbit model. Infectious CRPV genomes were developed by embedding the epitope within the E7 gene or the L2 gene using two alternative strategies. Protective vaccination studies carried out with these two genomes indicated that this epitope was processed and presented from its position within either the E7 protein or the L2 protein, as epitope vaccinated HLA-A2.1 transgenic rabbits were protected against viral DNA challenge. These studies also revealed that the CRPV genome contains areas of plasticity within both the E7 and the L2 genes that are amenable to PCR induced modification and suggested that while an epitope expressed during a late time point of a natural papillomavirus infection could be targeted by cell mediated immunity, early expressed epitopes are more readily targeted by cellular immunity. iii It has long been known that route of delivery can have an impact on the immune- stimulating capacity of vaccines. In head to head experiments comparing two vaccination strategies, rabbit groups were vaccinated three times at three-week intervals using the tattoo gun or gene gun followed by challenge with the wild type CRPV genome or an epitope-modified CRPV genome. These protective vaccination studies indicated that DNA vaccination through tattooing or with a gene gun yielded similar levels of protection. Thus the tattoo gun is a simple, useful, and cost-effective alternative to the gene gun and produces comparable results in the CRPV/HLA-A2.1 transgenic rabbit model. The focus of the third data chapter was the validation of new HLA-A2.1 restricted HPV16 E7 epitopes identified by bioinformatics. To examine the binding affinity and stability of the peptide/MHC complex, various in vitro assays were performed. The immunogenicity of these potential HPV16E7 epitopes was determined in vivo through peptide and DNA vaccination of HHD mice. HLA-A2-restricted HPV16 E7 epitopes that stimulated epitope-specific CTLs in the HHD mice after peptide vaccination were considered potential epitopes for continued testing. Of the seven candidate epitopes tested, four were immunogenic in vivo. Additional studies to examine the vaccine- induced epitope-specific protective immune responses generated to two of these epitopes were performed using the CRPV/HLA-A2.1 transgenic rabbit model. DNA vaccination was followed by challenge with modified CRPV genomes containing each epitope embedded in the E6 or E7 genes. The data collected from these studies suggested that the C-terminus region of the E7 gene has plasticity and is more amenable to PCR modification than the tested regions within the E6 gene. Additionally, HLA-A2.1 transgenic rabbits vaccinated against a newly discovered HPV16 E7 epitope were partially protected from challenge with the epitope-modified CRPV genome containing this epitope embedded in the E7 gene. iv Supplementary projects demonstrated that both the CRPV E6 and CRPV E7 genes are permissive for epitope-modification and that genome position, as well as epitope sequence, affect the stimulating capacity of individual epitopes. Moreover, the CRPV/HLA-A2.1 transgenic rabbit model is a useful and versatile tool for exploring the vaccine generated immunity in a model of natural papillomavirus infection and the use of both HHD mice and HLA-A2.1 transgenic rabbits to evaluate predicted epitopes overcomes the individual limitations of each HPV preclinical animal model. v TABLE OF CONTENTS LIST OF FIGURES xiv LIST OF TABLES xix LIST OF ABBREVIATIONS xxi ACKNOWLEDGEMENTS xxiv CHAPTER I: Literature Review 1 A. Papillomaviruses 2 1. Introduction 2 2. Human Papillomaviruses 2 a. Types and Tissues 2 i. Cutaneous and mucosal HPVs 2 ii. HPV and cancer progression 3 iii. Infections in immunocompromised patients 6 iv. Current treatments 7 b. Genome Organization 8 c. Life Cycle and Function of Proteins 10 3. Immune Responses to HPV Infections 15 a. Cellular Immunity 15 b. Humoral Immunity 17 c. Protein Localization and Immunity 18 4. An Illustration of Immune Evasion 18 a. The Infectious Cycle 18 b. Inhibition of Host’s Innate Immunity During Infection 20 c. Rare Codon Usage 23 d. Other Mechanisms of Immune Escape 24 vi 5. Commercially available VLP vaccines 24 a. Gardasil and Cervarix 24 b. Deficiencies of First Generation VLP Vaccines 25 B. Animal Model Systems for the Study of Papillomaviruses 26 1. Models of Natural Infection 26 a. Introduction 26 b. BPV 26 c. COPV 27 d. ROPV 27 e. CRPV 28 e. CRPV/HLA-A2.1 30 2. Mouse Models 31 a. Introduction 31 b. C57Bl/6 Mice 31 c. HLA-A2.1 Transgenic Models 32 i. HHD Mice 32 C. HPV Therapeutic Vaccines 33 1. Challenges of Vaccine Development 33 2. Considerations for Therapeutic HPV Vaccine Designs 34 3. Current Therapeutic HPV Vaccine Strategies 35 a. DNA Vaccines 35 b. Peptide/protein Vaccines 36 c. Viral/bacterial Vectors 37 d. Dendritic Cell Vaccines 38 e. Combination Vaccines 39 f. Non-HPV-Specific Therapies 40 vii 4. The First Human T Cell Vaccine 41 CHAPTER II: Introduction to the thesis 42 CHAPTER III: Relocation of an HPV16 E7 HLA-A2.1 Restricted CD8+ T Cell Epitope into the Cottontail Rabbit Papillomavirus (CRPV) Genome Increases the Protective Immunity Elicited in the HLA-A2.1 Transgenic Rabbit Model 47 A. Abstract 48 B. Introduction 49 C. Materials and Methods 51 1. DNA vaccines 51 2. Viral DNA challenge constructs 51 3. Rabbit vaccination and DNA challenge 54 4. Papilloma volume determination and statistical analysis 55 D. Results 56 1. Epitope modified CRPV genomes produce papillomas 56 2. DNA vaccinated HLA-A2.1 transgenic rabbits are partially protected against challenge with a modified CRPV genome 60 3. DNA vaccinated HLA-A2.1 transgenic rabbits are completely protected against challenge with a modified CRPV genome 60 4. DNA vaccination generates epitope-specific immunity in HLA-A2.1 transgenic rabbits 65 E. Discussion 72 F. Acknowledgements 77 CHAPTER IV: DNA Vaccination by Tattooing Induces Specific Protective Immunity to HLA-A2.1 Restricted CRPV E1 and HPV16 E7 Epitopes in HLA-A2.1 Transgenic Rabbits 78 A. Abstract 79 viii B. Introduction 81 C. Materials and Methods 84 1. DNA vaccines 84 2. DNA plasmids 84 3. Rabbit vaccination and DNA challenge 85 4. Histology and immunofluorescence detection 87 5. Statistical analysis 87 D. Results 88 1. Detection of EGFP 88 2. DNA vaccination by tattooing provides complete protection against wild type CRPV challenge 88 3. Gene gun and tattoo gun DNA vaccination provide similar levels of protection 93 4. DNA vaccination by tattooing provides complete protection against a modified CRPV genome 95 E. Discussion 103 F. Acknowledgements 105 CHAPTER V: Characterizing the Immunogenicity of a “Sequence Optimized” HPV16 E7 HLA-A2.1 Restricted Epitope Using Two HLA-A2.1 Transgenic Preclinical Animal Models 106 A. Abstract 107 B. Introduction 109 C. Materials and Methods 109 1. Bioinformatics and peptide synthesis 112 2. Antibodies, tetramer synthesis, and flow cytometry 112 3. HLA-A2.1 peptide binding assay 113 4. HLA-A2.1 stability assay 113 ix 5. Animals 114 6. HLA-A2.1 transgenic mice vaccination 114 7. Cell culture 115 8. Dendritic cell isolation and culture 115 9. Tetramer staining assay 116 10. Intracellular cytokine staining assay 116 11. DNA vaccine 117 12. Viral DNA challenge constructs 117 13. Rabbit vaccination and viral DNA challenge 119 14. Papilloma volume determination and statistical analysis 119 D. Results 120 1. Sequence modification increases binding affinity of HLA-A2.1 restricted epitope 120 2. Peptide immunization of HHD mice produces epitope- specific CTLs 123 3. An epitope-modified CRPV genomes produces papillomas 130 4. Epitope DNA vaccination is partially protective against an epitope-modified CRPV genome 133 E. Discussion 138 F. Acknowledgements 141 CHAPTER VI: Identification and Characterization of the Vaccine Generated Cellular Immune Responses to Computer-Predicted and Known HPV16 E7 HLA-A2.1 Restricted Epitopes In Vivo 142 A. Abstract 143 B. Introduction 145 C. Materials and Methods 147 1.