Project 3: Determination of the minimal MHC-E-restricted SIV epitope targeting required for RhCMV/SIV vaccine-mediated SIV replication arrest efficacy

PROJECT 3 - PROJECT SUMMARY Immune responses elicited by strain 68-1 Rhesus Cytomegalovirus vaccine vectors expressing simian immunodeficiency virus (SIV) inserts (RhCMV/SIV) stringently control SIV (via “replication arrest”) in 59% of vaccinees, with subsequent viral clearance over time. The ability of this vaccine to mediate replication arrest has been strongly linked to its ability to elicit SIV-specific CD8+ T cells that recognize viral peptides in the context of functionally non-polymorphic major histocompatibility complex (MHC)-E rather than classical highly polymorphic MHC-Ia. These MHC-E-restricted CD8+ T cell responses intercept primary SIV infection before the massive viral replication and sequence diversification that leads to mutational escape, and thus they may only require effective overall SIV recognition and not necessarily epitope breadth. Preliminary work by our group has found that MHC- E-restricted TCRs and T cell clonotypes are fundamentally different from those of classical MHC-Ia-restricted CD8+ T cell responses. Whereas conventional MHC-Ia-restricted epitope-targeted responses are mediated by independent TCRs, MHC-E-restricted SIV-specific responses (even when directed at numerous SIV epitopes) are largely comprised of only a handful (<10) of independent TCRs that are relatively low avidity and highly cross-reactive – with each TCR recognizing one or more universal epitopes called supertopes (epitopes that are recognized by all 68-1 RhCMV/SIV vaccinated RMs) and a variable number of diverse non-universal epitopes. In this project we seek to define the minimal MHC-E-restricted CD8+ T cell TCR recognition unit capable of mediating SIV replication arrest-type efficacy, asking the questions of whether efficacy requires recognition of one or multiple supertopes, and whether this efficacious recognition requires one or multiple TCR clonotypes (effectively asking whether cross-reactive TCR recognition of multiple SIV epitopes is an intrinsic requirement for effective overall SIV-infected cell recognition and thus for efficacy). Also, given the lack of MHC-E polymorphism, we ask whether development of a universal MHC-E-restricted supertope-targeted HIV vaccine is possible – a vaccine that has similar specificity in all vaccinees and is broadly targeted enough to ensure efficacy- sufficient recognition across global HIV sequence variability. These questions will be answered by addressing the following Specific Aims: S.A.1 - to design and validate SIV inserts for miR-126-restricted (MHC-E-only) 68-1 RhCMV vectors that will (exclusively) elicit SIV MHC-E-restricted SIV-specific CD8+ T cell responses limited to 1, 2, and 10 SIV supertopes; S.A.2 - to compare immunogenicity (including response specificity, TCR clonotypic hierarchies, and TCR cross-reactivity) and replication arrest-type efficacy of single, dual and 10 MHC-E supertope-only vectors; and S.A.3 - to develop ex vivo engineered autologous T cells expressing validated MHC- E-restricted SIV supertope-specific TCRs for in vivo infusion at the time of SIV challenge to determine whether CD8+ T cells expressing protective MHC-E-restricted TCRs can mediate replication arrest efficacy in the absence of RhCMV/SIV vaccination.