Study On High Functional Avidity CD8~+T Cells Induced By DNA Prime- VV Boost Vaccination Regimen

Cytotoxic T Lymphocytes (CTL) is a major effector for protection against many infectious diseases and cancer. Several attributes of CD8 cells contribute to the containment of pathogen replication, among which, functional avidity, also termed as antigen sensitivity, is one of the most critical properties that determine CTL’s efficacy. By definition, high functional avidity cells respond to low antigen doses, thus, CD8 cells with high avidity could exert rapid and readily effector functions and eliminate the viral infected cells effectively before the occurrence of mass propagation and mutation. Determined by ex vivo exposing T-cell populations induced by vaccines to different densities of cognate antigen, functional avidity is always served as an index of immune reponses elicited by vaccines in recent studies. Interestingly, although recent advances have disclosed the in vitro regulation of primary T cells to determine their functional avidity in reponse to different levels of stimulation, how functional avidity of antigen-speicific T cells was tuned in vivo by different vaccines remain pooly defined.Our previously finding showed that recombinant DNA prime-vaccinia virus boost (DNA-VV) regimen could elicit higher levels of antigen-specific T-cell response with enhanced functional avidity compare with DNA prime-DNA boost (DNA-DNA).In this study, using different stimulatory peptides (dominant, sub-dominant epitopes and peptide pools), antigens and mice models, we confirmed this observation. Then, we further asked that how vaccinia virus boost is able to tune the low functional avidity CD8+T cells primed by DNA vaccine into high avidity ones?Firstly, by evaluating the well known parameters of T-cell that might associated with functional avidity of CD8+ T cells between two vaccination regimens, including cytokine profile of CD4+ T helper cells, memory phenotypes and poly-functionality of antigen-specific CD8+ T cells, it’s indicated that functional avidity of antigen-specific T cells induced by vaccines is uniquely regulated by its own pathway since there is no significant correlation between functional avidity and these parameters in our system.Secondly, since replication-competent vaccinia virus Tiantan strain used in this study could persist in vivo for a short time, the increased functional avidity of CD8+ T cells in DNA-VV group might due to some potential effects of residual VV replication such as better antigen presentation efficacy of the autologous antigen presenting cells (APC) during the ex vivo stimulation. To exclude this possibility, functional avidity assay was performed after the mixture of sorted CD8+T cells from each group with the same sources of APC, or after interchanging CD8+ and CD8" cells between two groups. Data showed that the functional avidity of CD 8 T cells was not affected by the antigen presentation efficacy of exogenous APC, suggesting that the enhanced functional avidity boosted by VV was developed as an intrinsic attribute of CD8+ memory T cells.Thirdly, as the initiaiton of T-cell signaling, the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) is believed as one of the determinants of function avidity. Higher-affinity TCR might bind pMHC easier to initiate the signaliing earlier and lead to higher functional avidity. To explore the potential effect of TCR selection, the monoclonal TCR transgenic OT-I mice were introduced in this study. Briefly, the purified CD8+ T cells from OT-I mice were adoptively transferred into C57BL/6 mice and functional avidity were compared between two vaccination regimens. Interestingly, the difference in frequency and functional avidity still remained between two groups in spite of the TCR is monoclonal, demonstrating that the enhanced functional avidity induced by VV boost is independent of TCR selection.Fourth, the levels of activated ZAP-70 and Lck of antigen-specific CD8+T cells, which are key molecules of proximal TCR signaling pathway, were further evaluated. Data showed that the amount of basal phospho-ZAP-70 was significantly higher in DNA-VV regimen; in response to peptide stimulation, DNA-VV also exhibited statistically significant increases in Lck phosphorylation. This analysis suggests that VV boost could facilitate proximal TCR signaling, leading antigen-specific CD8 cells to be in a status of prone to activation, which is further confirmed by transcriptional profiling of antigen-specific CD8+T cells induced by two immunization strategies.Fifth, since inflammatory cytokines provide the third signal of T-cell signaling, we asked whether the increased TCR signaling by VV boost is associated with the inflammatory milieus induced by the viral vector. To begin with, concurrently administrating DNA vaccine and mock VV didn’t enhance the functional avidity, indicated the contribution of VV induced inflammatory milieus is not crucial; next, however, DNA-VV failed to enhance functional avidity in MyD88-/- mice, in which both secretion of inflammatory cytokines and functions of CD8+T cells are significantly impaired since the MyD88’s expression is blocked, suggesting either MyD88 mediated inflammation or intrinsic MyD88 expression in CD8 cells plays a crucial role in shaping functional avidity maturation induced by VV boost; finally, this hypothesis was further tested in wild type OT-I CD8 T cells adoptively transferred to MyD88 host, wherein the VV induced inflammatory cytokines production was muted and CD8+ T cell response to vaccination remained competently. Data showed that VV boost retained the capability of inducing higher functional avidity CD8+T cells in the absence of infection induced inflammation, proving that VV infection induced inflammatory milieus is dispensable for the memory formation of high functional avidity CD8+ T cells, instead, it’s indicated that MyD88 signaling in CD8+T cells is essential in shaping T-cell functional avidity in response to vaccinia vaccine.In summary, we found that vaccinia vectored vaccine boost is able to tune the low functional avidity CD8+T cell primed by DNA into high avidity ones through facilitating proximal TCR signaling, which is associated with MyD88 expression in CD8 cells and independent of TCR selection and MyD88 mediated inflammatory milieus induced by viral vector infection. Our data provides new insights into the regulation of different vaccine vectors in shaping functional avidity, which have important implications for the design of novel vaccines and immunization strategies.