Antigenicity and Immunogenicity of Novel Chimeric Hepatitis B Surface Antigen Particles with Exposed Hepatitis C Virus Epitopes

Abstract

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.Hepatitis C virus (HCV) is now recognized as the major cause of non-A, non-B hepatitis. It has been estimated that about 170 million people worldwide are infected with HCV, of whom 70 to 80% will develop chronic liver disease, leading to cirrhosis in 10 to 20% and liver cancer (hepatocellular carcinoma) in 1 to 5% of chronically infected individuals (6). The linear, single-stranded, positive-sense HCV RNA genome of ca. 9.5 kb contains a single open reading frame (ORF) encoding a polyprotein which is cleaved into the individual mature viral proteins by host-and virus-specific proteinases. Three structural proteins have been identified, the core protein and two envelope proteins, E1 and E2.It has been reported that cellular and humoral immune responses play a pivotal role in the host defense mechanism against HCV (8, 36). Most HCV carriers have circulating antibodies to the virus envelope proteins and to a region located in the extreme amino terminus of E2, hypervariable region 1 (HVR1), which has been reported to contain neutralizing Bcell epitopes and a T-cell epitope (16,17,44). HVR1 probably represents the major site of HCV genetic drift, with amino acid substitutions leading to escape from recognition by existing anti-HVR1 antibodies. Due to the variability within the HVR1 region, it has been proposed that these mutations are responsible for the persistence of HCV infection through neutralizing antibody escape mutants (25,46,52). Qualitative antibody changes accompany HVR1 epi...

Hepatitis B Surface Antigen-based Vaccines for Human Neoplastic Disease

“…VLPs are highly immunogenic and easy to produce for vaccination (Netter et al, 2001;de Jong et al, 2002;Villa et al, 2005). These vaccines rely on a structural protein which can self assemble into viral capsid structures (VLPs).…”

“…HBsAg-based vaccines frequently illustrate broad and robust CTL immunogenicity (Schirmbeck et al, 1995a;Loirat et al, 2000;Netter et al, 2001;Schirmbeck et al, 2002;Marsac et al, 2005;Woo et al, 2006;Schirmbeck et al, 2008).…”

“…The efficacy of this vaccine was attributable to the retained ability of recombinant HBsAg to form immunogenic VLPs in which fused proteins were displayed in the correct conformation within a highly immunogenic array. Molecules of HBsAg have demonstrated retained ability to form and secrete VLPs when 36 (Netter et al, 2001), and up to 113 (Lee et al, 1996) amino acids are inserted into the α-loop. However conflicting results reporting abrogated VLP formation and secretion after insertion of 24 and 33 amino acid sequences (Delpeyroux et al, 1987) sugests that intrinsic properties of inserted sequences affect HBsAg-VLP formation and secretion (Netter et al, 2001).…”

“…HBsAgencoding sequences have been modified to incorporate foreign sequences into the backbone (Woo et al, 2006;Haigh et al, 2007), or the surface-exposed central α-loop region (von Brunn et al, 1991;Netter et al, 2001) of HBsAg. The fusion of circumsporozite protein of P.…”

“…pCDNA3-HBsAg (Netter et al, 2001) was the source of pCDNA3 vector and was also used as a control DNA vaccine. Transcription from pCDNA3 is controlled by a CMV promoter which allows for expression in mammalian cells.…”