RESUMEN
AIM: Assessment of the neutralizing activity of human monoclonal antibodies against HCV and also study their safety in experimental small animals (Swiss mice). MATERIALS AND METHODS: Assessment of neutralizing activity of human monoclonal antibodies against HCV envelope regions (E1, E2) by two methods: by HCV cc infectious system 1) and by using positive HCV positive serum as source of HCV particles genotype 4a (neutralizing assay 2). Dot ELISA was used to study the activity of the generated antibodies. Safety and toxicity of the generated human antibodies were tested by assessing the changes in the biochemistry of liver function and kidney function tests, Complete blood counts (CBC) and studying the pathological changes with different concentrations of purified human antibodies were carried out.. RESULTS: Human Abs # 5 & 11 showed neutralizing activity by (neutralizing assay 2) but were not neutralizing by HCV cc assay. Human Abs # 12 & 15 showed neutralizing activity by the two methods i.e our generated human antibodies Abs# 5 &11 & 12 & 15 were neutralizing for HCV genotype 4a and Abs # 12 & 15 were neutralizing for HCV genotypes 4a and 2a. Liver and kidney functions and CBC results indicated that doses of 10 µg, 100 µg were safe. The histopathological results indicated that the dose of 10 µg of purified human monoclonal antibodies per mouse body weight was safe. CONCLUSION: The generated human monoclonal antibodies can be used to develop potent immunotherapy agents that can be administrated for the post-transplantation patients to prevent the recurrence of HCV infection. Also, the monoclonal antibodies can be used to develop a candidate vaccine against HCV.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Animales , Línea Celular , Hepatitis C/inmunología , Humanos , Ratones , Pruebas de Neutralización/métodos , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
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 â¼1000 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.