The generation of a simian adenoviral vectored HCV vaccine encoding genetically conserved gene segments to target multiple HCV genotypes

Abstract

Background: Hepatitis C virus (HCV) genomic variability is a major challenge to the generation of a prophylactic vaccine. We have previously shown that HCV specific T-cell responses induced by a potent T-cell vaccine encoding a single strain subtype-1b immunogen target epitopes dominant in natural infection. However, corresponding viral regions are highly variable at a population level, with a reduction in T-cell reactivity to these variants. We therefore designed and manufactured second generation simian adenovirus vaccines encoding genomic segments, conserved between viral genotypes and assessed these for immunogenicity.

Methods: We developed a computer algorithm to identify HCV genomic regions that were conserved between viral subtypes. Conserved segments below a pre-defined diversity threshold spanning the entire HCV genome were combined to create novel immunogens (1000-1500 amino-acids), covering variation in HCV subtypes 1a and 1b, genotypes 1 and 3, and genotypes 1-6 inclusive. Simian adenoviral vaccine vectors (ChAdOx) encoding HCV conserved immunogens were constructed. Immunogenicity was evaluated in C57BL6 mice using panels of genotype-specific peptide pools in ex-vivo IFN-ϒ ELISpot and intracellular cytokine assays.

Results: ChAdOx1 conserved segment HCV vaccines primed high-magnitude, broad, cross-reactive T-cell responses; the mean magnitude of total HCV specific T-cell responses was 1174 SFU/10⁶ splenocytes for ChAdOx1-GT1-6 in C57BL6 mice targeting multiple genomic regions, with mean responses of 935, 1474 and 1112 SFU/10⁶ against genotype 1a, 1b and 3a peptide panels, respectively. Functional assays demonstrated IFNg and TNFa production by vaccine-induced CD4 and CD8 T-cells. In silico analysis shows that conserved immunogens contain multiple epitopes, with many described in natural HCV infection, predicting immunogenicity in humans.

Conclusions: Simian adenoviral vectored vaccines encoding genetic segments that are conserved between all major HCV genotypes contain multiple T-cell epitopes and are highly immunogenic in pre-clinical models. These studies pave the way for the assessment of multi-genotypic HCV T-cell vaccines in humans.

Keywords: Conserved segments; Cross-reactivity; HCV vaccine; Simian adenovirus; T cell.

Fig. 1

Sequence diversity plot of the full HCV genome with defined conserved HCV segments. (A) Sequence diversity (normalized raw diversity, NDR) for an example sequence dataset is shown for the full HCV genome (sequence dataset HCV GT1/3a, containing 72 sequences) using a window size of W = 20. For vaccine design, segments with variability below 25% of the overall diversity distribution (first quartile Θ, marked blue) were defined as conserved and selected for conserved immunogens. For visualization purposes, diversity obtained from hamming distance measures (NDR) is normalized to 1 using the maximum found (black full line). Gaps in the dataset were ignored in the calculation of NDR, and we therefore present the sequence coverage used for the NDR measurement at each window (orange full line). Boundaries of genome regions are marked by vertical dashed lines, with gene nomenclature on the top. (B) Schematics of long and (C) short versions of conserved HCV immunogens for HCV GT1 (red), GT1/3 (orange) and GT1-6 (blue) are depicted.

Fig. 2

Patient sequence selection for final immunogen design. (A) Similarity of subtype consensus sequences (depicted as coloured spots) to overall consensus sequences at each conserved segment, shown for analyses HCV GT1 (a, left), HCV GT1/3 (b, middle) and HCV GT1-6 (c, right) immunogens. Conserved segment ID numbers (as defined in Supplementary Fig. S2) are marked on the y-axis. (B) Overall similarity between subtype consensus sequences and overall consensus sequences for all conserved segments of analyses HCV GT1 (left), HCV GT1/3 (middle) and HCV GT1-6 (right). Cons: consensus.

Fig. 3

Epitopes defined in natural HCV genotype-1 and genotype-3 infection populate HCV conserved and variable regions. HCV sequence heterogeneity (top panel, normalized raw diversity [NDR]) as defined for HCV GT1-6 with conserved regions marked blue and HCV epitopes described in natural HCV infection (bottom panels) are shown. T-cell epitopes described in natural HCV infection are depicted for HCV genotype-1 CD8 (green) and CD4 (red) epitopes, and HCV genotype-3 CD8 (purple) and CD4 (blue) epitopes.

Fig. 4

Production of HCV conserved immunogens and pre-clinical assessment in mice. (A) Core expression analysis from ChAdOx1 based vaccines encoding 6 different conserved HCV immunogens in HEK293A cells at a MOI of 100 after an infection duration of 24 h. Controls included a ChAdOx1 vector and a pENTR4-HCV-GT1-6-long-TPA construct. M – Marker Precision Plus Dual Color (BioRad). (B) Magnitude of murine splenocyte-derived T-cell responses measured in IFNγ-ELISpot assays using HCV genotype-1a, genotype-1b and genotype-3a peptide pools in C57BL6 mice vaccinated with the viral vector ChAdOx1 containing the long construct of conserved HCV GT1-6 immunogen (ChAdOx1-gt1-6L-TPA) 2 weeks after immunization at dose 1 × 10⁸ I.U. given intramuscularly. Spot-forming units (SFU) were normalized to 10⁶ cells. Results were defined as significant when p < .05 when compared with the DMSO background (Mann-Whitney test). (C and D) Total magnitude of C57BL6 splenocyte-derived CD4 (C) and CD8 (D) T-cell responses measured in intracellular staining assays and flow cytometry analysis. IFNγ⁺ and TNFα⁺,cytokine production following stimulation with core-E1-E2, NS3-4, and NS5 peptide pools for genotype-1a and -3a was assessed. Mice were sacrificed 2-weeks after immunization with ChAdOx1-gt1-6L-TPA at dose 1 × 10⁸ I.U. given intramuscularly (I.M.).