Immuno-proteomic interrogation of antigen presentation during Dengue infection reveals novel and HLA haplotype-specific MHC-I antigens

Dengue (DEN) virus infection is a leading cause of hospitalization and death in the developing world and broadly effective vaccines and therapies remain elusive. Identifying infection-specific peptide antigens would open new avenues for developing T cell based interventions. Past efforts towards mapping viral antigens used computational predictions that only partially reflect actual antigens presented by the major histocompatibility complex (MHC). To identify DEN-specific antigens without relying on error prone predictions, we developed an immuno-proteomics approach for interrogating antigen presentation in DEN infected B-lymphocytes. This approach enabled three fundamental findings; First, we identified 86 viral MHC-I antigens (59 novel), and mapped them to presentation hotspots in the DEN genome. Second, we discovered post-translationally modified viral and host antigens and antigens derived from alternate reading frames. Predicting these antigens using computational methods alone would be infeasible. Third, we found antigens responsible for HLA-haplotype dependent immune responses against DEN infection: we genetically engineered B-lymphocytes to express HLA alleles associated with either strong or weak DEN-immune responses and identified corresponding allele specific antigens. Together these discoveries let us assay the immunogenic potentials of an unprecedented range of DEN-specific antigens. Ex-vivo assays including ELISPOT and HLA tetramer staining supported our identification of immunogenic antigens in DEN-specific CD8+ T cells. These antigens have potential as both diagnostic tools to characterize DEN-specific T cell immunity, and serve as candidates for designing effective DEN vaccines.