Investigation of CD8+ T cell priming by two commonly used Vaccinia virus strains

Abstract

CD8+ T cells help to control virus infections and tumours. Two main pathways, namely direct and cross presentation, are involved in generating and presenting antigenic peptides to induce CD8+ T cell responses. In general, antigens that require minimal processing or are rapidly degraded allow better direct presentation while only stable antigens can be cross presented. Currently, several vaccinia virus (VACV) recombinants are being tested in clinical trials as vaccines to induce protective immunity, including CD8+ T cell responses, against heterologous pathogens and cancers. The current paradigm, based on work with the virulent VACV strain Western Reserve (WR), suggests an important role of direct presentation for CD8+ T cell priming. However, a study has reported that cross presentation is the major pathway for priming CD8+ T cells against the highly attenuated modified VACV Ankara (MVA), a candidate vaccine vector. Understanding this disparity between WR and MVA may improve the design of VACV-based recombinant vaccines. The priming pathway of native antigens expressed from WR and MVA was first examined in mice pre-treated with Toll-like receptor 9 ligand or cytochrome c. These methods have been reported to selectively inhibit cross presentation. However, results in this thesis demonstrate that these treatments also suppress direct presentation. Further investigations show that the extent of inhibition on CD8+ T cell priming by CpG is dependent on the immunisation route, the viral replicative ability and, more crucially, the immunisation dose. Similarly, the replicative ability of VACV influences the inhibition effect of cytochrome c treatment. Overall, these two strategies do not reveal how VACV antigens are presented. The immunogenicity of recombinant antigens expressed as minigenes from WR and MVA was then examined. The magnitude of CD8+ T cell responses induced by the minigenes, which can only be directly presented, are at least similar to, if not higher than, the full-length antigens when expressed from WR and MVA. Contrary to the published work, the model antigen ovalbumin expressed as a minigene can be more immunogenic than the full-length antigen when expressed from MVA. This suggests that MVA behaves similarly to WR to prime CD8+ T cells, i.e. by direct presentation. We further demonstrate that the location where the transgene encoding the foreign antigen is inserted in the MVA genome determines its immunogenicity. Two unexpected phenomena observed in this thesis were also explored. Firstly, a CD8+ T cell response to an immunogenic peptide of influenza A virus, known as NP-366, can only be induced if the peptide is processed from a full-length protein from WR, but not as a minigene. This contradicts our studies described above and most of the VACV literature. Several hypotheses were tested to identify the possible mechanisms responsible. Secondly, we found that completely inactivated VACV induces CD8+ T cell responses in vivo. Its application as a recombinant vaccine was examined. In summary, this thesis has expanded our understanding on how antigens expressed from VACV strains WR and MVA are presented for CD8+ T cell priming. The findings presented here provide important insights for VACV-based recombinant vaccine design. Show more