Effect of inoculation route on priming pathway and CD8+ T cell immunodominance during Vaccinia virus infection

Abstract

The CD8+ T cell response is essential for defence of mammals against pathogens and tumours, but its initiation and modulation are not fully understood. This thesis focuses on two aspects of CD8+ T cell responses. First, antigen priming pathways, which dictate the initiation of epitope-specific responses. Second, immunodominance representing the breadth of immune responses. Additionally, we explored the influence of inoculation route on antigen-priming pathways and immunodominance to delineate the role of vaccination route in shaping CD8+ T cell responses during infection. The antigen-priming pathways of VACV epitopes were investigated by employing the recombinant virus MVACPX2, expressing CPXV12 and CPXV203, which interfere with epitope presentation on MHC-I. Of eight epitopes that give substantial CD8+ T cell responses to MVA, priming of four was significantly inhibited by CPXV12/203 following i.p. infection, whereas the remaining four were unaffected. For the epitopes inhibited by CPXV12/203 we can conclude that direct priming must be important. By further examining the induction of CD8+ T cell responses to the direct-priming dependent OVA mini-gene (MSIINFEKL) co-expressed with CPXV12/203 in the recombinant virus, we demonstrated that 'leaky' direct-presentation as well as cross-presentation could explain why some native MVA epitopes were able to prime normal CD8+ T cells under the inhibition of CPXV12/203. To link the extent of direct-presentation to immunogenicity of native MVA epitopes in the face of CPXV12/203, we used mass spectrometry to quantify peptide presentation on MVACPX2-infected cells. Among the four epitopes with priming that was unaffected by CPXV12/203, small amounts of three of these were detected, indicating that incomplete inhibition of direct-presentation could not be ruled out. By contrast, presentation of the fourth was undetectable on MVACPX2-infected cells, suggesting that cross-priming explains the continued immunogenicity of this epitope. Surprisingly and in contrast to the epitope-specific inhibition of priming by CPXV12/203 in i.p.- and i.v.- infected mice, no suppression of CD8+ T cell responses was found following i.d. inoculation. As SIINFEKL-specific responses were induced after i.d. infection with X2-miniOVA, we suggested that direct-priming remained operable and cross-priming is not solely responsible for eliciting CD8+ T cells in this route. Immunodominance, which is the unequal responses elicited across a range of different epitopes, is a fundamental property of the cellular immunity to infection and vaccination. We re-examined the effect of vaccination route on immunodominance using a panel of 15 VACV epitopes and four routes. CD8+ T cell immunodominance was found to be sharpened following peripheral infection (i.d. and s.c.) compared with the systemic routes (i.p. and i.v.) which had a greater focus on the subdominant epitopes. With experiments examining the virus spread and CD8+ T cell activation sites, we showed that strong immunodominance is associated with restricting priming to local draining lymph nodes. Moreover, we demonstrated that immunodomination occurs more readily following i.d. infection, and it can be reduced by over-expressing costimulators B7-1/2, suggesting competition for these molecules as a mechanism for sharpened immunodominance following peripheral infection. Together, this thesis expanded our understanding of antigen priming and immunodominance during primary infection, providing insight into the immune system and vaccine development.