Synthetic peptide-based immunoassay as a supplemental test for HCV infection.

INTRODUCTION: Hepatitis C virus (HCV) is a single strand RNA hepatotrophic virus infecting 170 millions around the world and 20% of Egyptian blood donors. Although there has been significant improvement in the enzyme immunoassays (EIAs) in population screening of HCV infection, the development of a low variability, easy to automate and inexpensive supplemental test to support the current immunoassays was of a major concern to several laboratories. OBJECTIVES: In the current study, we embarked on a systematic study to analyze by DNA sequencing several HCV isolates to identify conserved core protein sequences and perform explorative analysis of five synthetic peptides from the core/E1 region in anti-HCV antibody assays. METHODS: We designed four synthetic-core specific peptides and an E1-specific peptide. These peptides were used to screen HCV antibodies in sera of 100 HCV positive patients and 100 HCV negative subjects and compared the results with those obtained by the commercial systems based on second and third generation enzyme-linked immunosorbent assays. RESULTS: Our results showed that all peptides detect HCV antibodies in infected sera to varying degrees. The synthetic peptide (a.a. 21-40) of the core protein had 99% sensitivity, 100% specificity and was highly reproducible. CONCLUSION: The above findings make this core peptide a candidate product for developing a supplemental test for chronic HCV infection in the Egyptian population.

Murine neutralizing antibody response and toxicity to synthetic peptides derived from E1 and E2 proteins of hepatitis C virus.

INTRODUCTION: The highest estimated prevalence of HCV infection has been reported in Egypt, nearly 12% mostly type 4. Currently, a commercial vaccine to protect this high risk population as well as global HCV infected patients is not available. OBJECTIVES: In the present study, we aim at: (1) examining the viral binding capacities of purified monospecific polyclonal murine antibodies raised against genetically conserved viral protein sequences, i.e. synthetic peptides derived from those sequences located within envelope proteins and (2) assessment of immunogenic properties and safety parameters of those peptides individually and in a vaccine format in mice. METHODS: Purified IgG Abs from immunized mice were used in immunocapture RT-PCR experiments to test viral neutralization by Abs raised against each of 4 peptides termed p35 (E1), p36 (E2), p37 (E2) and p38 (E2). Swiss mice were immunized with each of the 3 peptides (p35, p37 and p38) which generated neutralizing antibodies in immunocapture experiments. Antibody responses to corresponding peptides were determined using different routes of administration, different adjuvants, different doses and at different time points post-injection. To explore the dose range for future pharmacological studies, three doses namely 50 ng, 10 µg and 50 µg/25 gm mouse body weight were tested for biochemical and histopathological changes in several organs. RESULTS: Murine Abs against p35, p37 and p38 but not p36 showed HCV neutralization in immunocapture experiments. Subcutaneous injection of peptides elicited higher responses than i.m. and i.p. Immunization with Multiple Antigenic Peptide (MAP) form or coupled to Al PO4 elicited the highest Ab responses. Peptide doses of 50 ng/25 gm body weight or less were effective and safe, however dose assessment still requires further study. Histopathological changes were observed in animals that received doses ∼1000 times higher than the potential therapeutic dose. CONCLUSION: Exploration of humoral immunogenicity, neutralization capacity and safety suggested that the peptides presented herein are candidate vaccine components for further preclinical assessment.

Establishment of hybrid cell lines producing monoclonal antibodies to a synthetic peptide from the E1 region of the hepatitis C virus.

We aimed at establishing hybridoma cells secreting monoclonal antibodies (mAbs) against E1 synthetic peptide of HCV. BALB/c mice were immunized with HCV E1-synthetic peptide (GHRMAWDMM) and its spleenocytes were fused with the P3NS1 myeloma cell line. Two highly reactive and specific mAbs (10C7 IgG2b mAb, and 10B2 IgG1 mAb) were generated. The target HCV E1 antigen was identified at approximately 38 kDa in serum of infected individuals. A newly developed ELISA detected the target antigen in 90% of sera from HCV RNA infected individuals with a specificity of 84%. So, the generated mAbs may provide promising probes for serodiagnosis of HCV infection.