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BACKGROUND AIMS: An effective vaccine against hepatitis C virus (HCV) infection is required to achieve viral eradication. A previous viral vectored vaccine encoding a genotype-1b T cell antigen suppressed peak viral RNA but failed to prevent chronic infection. Previous studies showed dominant vaccine-induced T cell responses were not cross-reactive with common HCV strains, possibly contributing to vaccine failure. To address this, we evaluated two novel HCV vaccine strategies designed to elicit T cells targeting multiple HCV genotypes.APPROACH RESULTS: HCV genetic segments highly conserved between genotypes 1-6 were encoded in chimpanzee adenoviral (ChAd-Gt1-6) and Modified Vaccinia virus Ankara (MVA-Gt1-6) vectors and tested in prime-boost regimens. This was compared to vaccinating with an ancestral genotype-1a non-structural antigen encoded in ChAd (ChAd-Bole1a-NS) boosted with a genotype-3a non-structural antigen encoded in MVA (MVA-Gt3a-NS). Immunogenicity was evaluated in C57BL/6 and transgenic HLA-A*02:01 mice. Splenocytes were stimulated with genotype-1a, -1b, or -3a peptide pools in ex-vivo IFNγ ELISpot and intracellular cytokine assays. Priming with ChAd-Gt1-6 elicited broad T cell responses towards all genotypes, whereas ChAd-Bole1a-NS generated a focused response to genotype-1a. Boosting ChAd-Bole1a-NS with MVA-Gt3a-NS generated cross-reactive T cells targeting multiple genotypes, though some responses were genotype-specific. In contrast, ChAd-Gt1-6 and MVA-Gt1-6 prime-boost generated high magnitude responses that were all cross-reactive between genotypes.CONCLUSIONS: Vaccinating with conserved regions of genotypes 1-6 or sequentially vaccinating with genotype-1a and genotype-3a immunogens are two novel approaches to generate cross-reactive T cells. The proportion of intergenotypic cross-reactive T cells generated was higher using the conserved region antigen.