Evaluation of the immunogenicity of liposome encapsulated HVR1 and NS3 regions of genotype 3 HCV, either singly or in combination

Abstract

Doc number: 74

Abstract

Background: Hepatitis C virus displays a high rate of mutation and exists as a quasispecies in infected patients. In the absence of an effective universal vaccine, genotype-specific vaccine development represents an alternative. We have attempted to develop a genotype 3 based, liposome encapsulated HCV vaccine with hypervariable region-1 (HVR1) and non-structural region-3 (NS3) components.

Results: HCV RNA extracted from serum samples of 49 chronically infected patients was PCR amplified to obtain HVR1 region. These amplified products were cloned to obtain 20 clones per sample in order to identify the quasispecies pattern. The HVR1 consensus sequence, along with three variants was reverse transcribed to obtain peptides. The peptides were checked for immunoreactivity individually, as a pool or as a single peptide tetramer interspersed with four glycine residues. Anti-HCV positivity varied from 42.6% (tetramer) to 92.2% (variant-4) when 115 anti-HCV positive sera representing genotypes 1, 3, 4 and 6 were screened. All the 95 anti-HCV negatives were scored negative by all antigens. Mice were immunized with different liposome encapsulated or Al(OH)₃ adjuvanted formulations of HVR1 variants and recombinant NS3 protein, and monitored for anti-HVR1 and anti-NS3 antibody titres, IgG isotypes and antigen specific cytokine levels. A balanced Th1/Th2 isotyping response with high antibody titres was observed in most of the liposome encapsulated antigen groups. The effect of liposomes and aluminium hydroxide on the expression of immune response genes was studied using Taqman Low Density Array. Both Th1 (IFN-gamma, Il18) and Th2 (Il4) genes were up regulated in the liposome encapsulated HVR1 variant pool-NS3 combination group. In-vitro binding of the virus to anti-HVR1 antibodies was demonstrated.

Conclusion: The optimum immunogen was identified to be combination of peptides of HVR1 consensus sequence and its variants along with pNS3 encapsulated in liposomes, which could generate both cellular and humoral immune responses in mice deserving further evaluation in a suitable cell culture system/non-human primate model.