AN INVESTIGATION OF THE VACCINE GENERATED CELL MEDIATED IMMUNE RESPONSES TO HLA-A2.1 RESTRICTED HPV16E7 EPITOPES IN VIVO USING TWO PRECLINICAL ANIMAL MODELS

Open Access
Author:
Bounds, Callie E
Graduate Program:
Microbiology and Immunology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
September 08, 2010
Committee Members:
  • Neil David Christensen, Dissertation Advisor
  • Neil David Christensen, Committee Chair
  • Todd Schell, Committee Member
  • Craig Matthew Meyers, Committee Member
  • David Joseph Spector, Committee Member
  • Laura Carrel, Committee Member
Keywords:
  • Gene Gun
  • HLA-A2.1 Rabbits
  • DNA Vaccines
  • CRPV
  • HPV
  • HHD mice
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. 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. 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.