ABSTRACT – PROJECT 2 A central hypothesis of this Program is that humoral and cellular adaptive immune responses synergize to control HCV infection. The objective of Project 2 is to develop an HCV vaccine component that elicits pan-genotypic CD4+ and CD8+ T cell memory. When combined with a vaccine component designed to elicit broadly neutralizing antibodies (Project 1), we expect to generate immunity equaling or exceeding protective adaptive responses acquired naturally by spontaneous resolution of acute HCV infection. Several observations indicate that vaccine-induced T cell immunity will be essential for protection. Initial control of acute HCV replication is temporally associated with onset of a functional T cell response. Infections resolve only if this response is sustained. Durable T cell memory develops and may prevent persistence upon re- infection. Antibody-mediated depletion of CD8+ or CD4+ T cells from immune chimpanzees before re-infection caused prolonged or persistent infection. Finally, protection against heterologous HCV genotypes has been observed in immune chimpanzees, but can fail suggesting that HCV genetic diversity limits protective T cell memory. Two major challenges for vaccine development are (i) the tremendous genetic diversity of HCV genotypes and (ii) the lack of an animal challenge model to assess vaccine efficacy. To provide broad T cell immunity, a tetravalent vaccine comprised of conserved HCV non-structural proteins from HCV gt1a, 1b, gt2, and gt3 sequences will be assessed. NS antigens will be designed computationally from a large number of available HCV sequences, resulting in Mosaics that disfavor inclusion of uncommon amino acids that would otherwise elicit less productive vaccine- or virus strain-specific T cell responses. Mosaics are indistinguishable from natural HCV proteins in sequence and processing for HLA presentation to T cells. Mosaic transgenes will be delivered by two viral vectors, Ad48 (a rare human subtype) and MVA, that can be translated for human vaccination. A key unanswered question is whether vaccines generate tissue-resident memory T cells, and whether they are equivalent to those elicited by spontaneous resolution of infection. We will undertake the first characterization of liver-resident CD4+ and CD8+ T cell populations from chimpanzees that spontaneously resolved HCV infection. This analysis will use cryopreserved liver mononuclear cells collected with NIH support before the moratorium on chimpanzee use in biomedical research. Key measures of quality (breadth, frequency and function) will be established, and compared with tissue-resident T cells primed in rhesus macaques with the tetravalent Mosaic vaccine. In co-operation with Project 3, T cells will also be compared by Cite-Seq to provide integrated cellular protein and transcriptome measurements. These comparisons are expected to provide a surrogate measure of efficacy for vaccine-primed T cells in the absence of a virus challenge model.