Neutralizing activities of caprine antibodies towards conserved regions of the HCV envelope glycoprotein E2

Abstract

Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection.

Antiviral Activity of Virocidal Peptide Derived from NS5A against Two Different HCV Genotypes: Anin vitroStudy

“…Huh7.5 cells were cultured according to previously described protocol where Huh7.5 cells were cultured till adherent cells became semi-confluent, cells were subcultured and counted. [17] Then cells were infected with positive HCV serum according to protocols described by Seipp et al [28] The total cellular RNA was extracted using acid guanidinium-phenol-chloroform according to the protocol of Chomczynski and Sacchi [29] and Goergen et al [30] The successful viral infection in Huh7.5 cells throughout the culture duration was confirmed at the transcriptional level qualitatively by Reverse Transcription Polymerase Chain Reaction (RT-PCR) amplification of HCV RNA and quatitatively by real-time RT-PCR. [17,31] Moreover, the HCV viral proteins in transfected cells were detected using immunostaining assay as previously described to confirm that direct infection of Huh7.5 cells resulted in viral replication and de novo synthesis of non structural proteins.…”

“…[17,31] Moreover, the HCV viral proteins in transfected cells were detected using immunostaining assay as previously described to confirm that direct infection of Huh7.5 cells resulted in viral replication and de novo synthesis of non structural proteins. [17,32] Transfection of Huh7.5 Cell Line with HCV cc J6/JFH…”

“…Several HCV-derived synthetic peptides that inhibit HCV infection in the cell culture infection system were reported. [6,7,17] One of those inhibitory peptides, a virocidal peptide derived from the membrane anchor domain of the HCV nonstructural protein NS5A (C5A). Virocidal peptide C5A contains amino acids 3-20 of the amphipathic α-helical N-terminal membrane anchor domain of the genotype 1a HCV NS5A protein.…”

Progress in Vaccine Development for HCV Infection

“…Previous studies focused on certain peptide sequences from envelope regions 1 and 2 of HCV as a candidate vaccine. They found that peptide region E1 (aa 315-323) and peptide regions from HCV E2 (aa 412-219) and HCV E2 (aa 517-531) had capability to introduce neutralizing antibodies in mice, rabbits, and goats, while peptide sequence from HCV E2 (aa 430-447) produced nonneutralizing antibodies, which are known interference antibodies [15][16][17][18][19][20][21][22]. Another strategy is to use viral vectors inducing T-cell responses against HCV-infected cells, e.g., adenoviral vectors that have big areas of the HCV genome itself.…”

Synthetic Peptide Vaccines

“…Several highly conserved sites were determined in HCV envelope proteins E1 and E2 (Sobolev et al, 2000); however, most these sites did not elicit specific antibodies because of insufficient Tlymphocyte help resulting from the absence of T-helper epitopes in the vicinity. Conjugation of putative B-epitopes, derived from HCV envelope proteins, to promiscuous T-helper epitopes from other sources (Torresi et al, 2007) or to the carrier protein, keyhole limpet hemocyanin (El Awady et al, 2010;El Abd et al, 2011), resulted in the formation of immunogens capable of producing antibodies specific for the whole HCV envelope proteins and viral particles. However, the use of foreign T-epitopes and carrier proteins leads to the formation of T helper memory cells that are non-specific for HCV and hence, will not be activated upon HCV infection.…”