Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Ted Hansen

Abstract

CD8+ T cells play a major role in controlling infection and disease progression in many infectious diseases. Upon infection, antigen-specific CD8+ T cells are generated and mainly through their cytotoxic activity remove infected cells, therefore, pathogens. Ongoing research has identified antigenic epitopes in a vast number of pathogens and, using the identified epitopes, the induction of CD8+ T cell immune responses has been an important strategy for successful vaccines. However, most immunization approaches with class I binding peptides have failed to induce CD8+ T cell responses strong enough to prevent disease. This failure has been attributed to the lack of CD4+ T cell help and difficulty in maintaining a sufficient level of antigen presentation required for CD8+ T cell activation. To circumvent these limitations, we have developed fully assembled MHC molecules that can be expressed as membrane-bound proteins on the cell surface, termed single chain trimers: SCTs). SCTs are composed of an immunodominant peptide, β2m, and MHC I heavy chain covalently linked by 15-20 amino acid flexible linkers. Because SCTs are expressed as a single polypeptide chain, they do not require peptide processing, or chaperone-assisted peptide loading in the ER. Furthermore, antigen presentation by the SCT bypasses the need to compete with an extensive pool of endogenous peptides for peptide loading. SCTs are folded properly and T cells see SCTs comparably to native peptide/MHC I complexes. Various human and mouse class Ia and Ib MHC molecules have been engineered with epitope peptides into SCTs and proven as useful tools to monitor and modulate immune responses. Thus, SCT engineering offers a great potential as a platform for antigen-specific DNA vaccines. Although there have been several reports of SCT-based DNA vaccines generating antigen-specific CTL responses, there have been no reports of pathogen protection after DNA vaccine expression of SCTs. In this thesis, I examined the efficacy of SCT DNA vaccines for the first time in pathogen infection models. First, we developed a clinically relevant human HLA-A2 transgenic mouse model of West Nile virus: WNV) infection and demonstrated protective efficacy of a HLA-A2 SCT DNA vaccine against lethal viral infection. Second, I validated the potency of a SCT DNA vaccine using the BALB/c model of Listeria monocytogenes infection, which indicated that SCT DNA vaccines also provide protective immunity against bacterial infection. Lastly, I demonstrated that further engineering of SCTs: dtSCT) using a disulfide trap to better accommodate epitopes can potentiate the capacity of SCTs to stimulate CD8+ T cells, suggesting broad application of SCTs even with low immunogenic peptides. I also used disulfide trap- or chimeric SCTs to test the mechanism of antigen presentation after DNA vaccination. My studies showed that SCTs are presented to T cells as intact molecules after DNA immunization, suggesting direct presentation by transfected DCs or cross-dressing as a major mechanism of the antigen presentation by DNA vaccine. In summary, these dissertation studies demonstrated that SCT-based DNA vaccines can provide pathogen protection and SCTs are effective probes for dissecting mechanisms of antigen presentation.

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Permanent URL: http://dx.doi.org/10.7936/K79G5JT4

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