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Title: Studies on cross presentation of antigen by human dendritic cells
Author: Pillay, Sirika
ISNI:       0000 0004 2716 4443
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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It is widely acknowledged that an HIV-1 vaccine requires a robust CD8+ T-cell response to effectively combat HIV-1. However, past vaccine candidates have failed to stimulate adequate cellular responses in humans, highlighting the need to explore alternative ways to induce strong cellular immunity. Animal studies have shown that if exogenous antigen can be targeted to dendritic cells (DCs), strong CD8+ T-cell responses can be generated via the cross-presentation pathway. In this study we evaluated the ability of four DC surface receptors in two different human DC subsets (monocyte-derived DCs and blood myeloid CDlc+ DCs) to efficiently target model antigens to the cross-presentation pathway to induce enhanced CD8+ T-cell responses. These receptors included DEC-205, MMR, DC-SIGN and/or CD1a. Four receptor-targeting strategies were explored, each exploiting receptor-specific antibodies in various ways to direct antigen towards the respective receptors. Initially, selected model antigens (CMV pp65, MART-1, and streptavidin-fusion proteins) were produced in large quantities for in vitro experiments. Two expression systems were evaluated to produce antigens, namely, the generation of a stably-expressing cell line and a recombinant adenovirus expression system (AES). The AES emerged as a better expression system, and was used to produce the proteins of interest. These proteins were then purified using a Nickel-NTA/anti-His tag column, and yield and purity of recombinant proteins were determined. The immunogenicity of the purified antigens was assessed to confirm the integrity of the bulk-produced proteins. Additionally, TLR agonists were evaluated for their ability to augment T-cell responses. LPS (TLR4 agonist) was determined to be the favoured agonist. The strategy utilising chemically-conjugated antigen-antibodies yielded the most significant enhancements in CD8+ T-cell proliferative responses, specifically for targeting of DEC-205, as well as MMR. Overall, this study details the successful design and production of various DC receptor targeting reagents, and goes on to assess the efficacy of novel receptor targeting strategies to enhance CD8+ T-cell responses for the eventual purpose of improving current HIV-1 vaccine strategies.
Supervisor: Patterson, Steven Sponsor: Commonwealth Scholarship Commission ; SSRC-Mellon Foundation ; British Society of Immunology ; Chelsea and Westminster Charity Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral