Recombinant mammalian cell derived hepatitisC virus-like particles induce neutralizing antibody responses to hepatitisC virus

Abstract number: P1154

Johnson D.F., Chin R., Earnest-Silveira L., Zentgraf H., Bock T., Chua B., Jackson D.C., Torresi J.

Objective: Clearance of Hepatitis C virus (HCV) requires a strong and broadly cross-reactive CD4+, CD8+ T cell and neutralising antibody (Ab) responses. Virus like particles (VLPs) resemble mature parent virus inducing protective humoral and cellular immune responses against HCV and provide a viable prophylactic vaccine candidate.

Methods: Recombinant adenoviruses expressing HCVcore-E1-E2 were used to infect Huh7 (hepatoma) cells and produce HCV VLPs. These were isolated from cell lysates and purified by Iodixanol density gradient ultracentrifugation. E1 and E2 glycoproteins of the correct size in HCV VLPs were confirmed by Western immunoblot. HCV VLPs were analysed by testing for maturation of Dendritic cells (DC) and mice were immunised with HCV VLPs alone and with alum and Freunds adjuvants. The mice were assessed for (1) humoral responses against both VLPs and a recombinant E2 protein of HCV, (2) production of mouse antibody secreting cells (memory B cells) in splenocytes using B cell Elispot assays and (3) neutralizing Ab in a Huh 7 cell entry assay. A second group of mice were immunised with VLPs and 2 novel adjuvants.

Results: We have produced, purified and confirmed the presence of HCV VLPs of genotype 1a by western immunoblot, electron microscopy (EM) and immunogold EM. HCV VLPs efficiently stimulate the maturation of dendritic cells to level that are comparable to lipopolysaccharide (LPS). Mice immunised induced strong humoral responses to E2 and VLPs and mouse anti-HCV VLP serum neutralized VLP entry into Huh7 cells. B cell elispot assays, using mouse splenocytes, demonstrated production of mouse antibody secreting cells / memory B cells. Mice immunised with VLPs and 2 novel adjuvants demonstrated a greater humoral response and increased levels of mouse antibody secreting cells compared with mice immunised with HCV VLPs alone or in Alum.

Conclusion: Mammalian cell derived HCV VLPs exhibit similar morphological, biophysical and immunological properties as putative HCV virions and are a viable vaccine strategy for HCV. HCV VLPs of genotypes 1b, 3a and 4 are currently being produced. Studies of CD8 responses and methods to improve VLP immunogenicity and alternative adjuvanting are ongoing.