Neutralization and immunoperoxidase to estimate the immunogenicity of recombinant human rotavirus VP6 structural protein in vitro after insertion of the antibodies into cells using electroporation

Abstract

Rotaviruses (RVs) represent the principal causative agent of severe gastroenteritis leading to high mortality rates, especially in children < 5 years in both developed and developing countries. Although, the first-generation of live attenuated RV vaccines such as RotaTeq and Rotarix achieved partial success in reducing the number of RV deaths worldwide, several concerns, such as low efficacy especially in developing countries, safety, and cost imply a dire need to develop these vaccines. Also, sensitive methods to estimate the immunogenicity of the candidate recombinant subunit VP6 vaccines in vitro are of great need. In the present study, 1232 bp of the most frequent full length VP6 in clinical and environmental isolates in Egypt with 98% nucleotides identity and 98% amino acid identity in comparison to human RoV Wa reference strain was expressed in E.coli. The examination of the sensitivity of the antibodies produced in the male rabbits which were immunized intramuscularly with 20 µg of the purified VP6 proteins, indicated a sensitivity up to 1/24000 dilution of antibodies against the expressed protein using ELISA. Introduce antibodies into MA104 cell line was performed using electroporation to neutralize the human rotavirus Wa strain VP6 when exposed after viral uncoating. Higher sensitivity of neutralization in relation to immunoperoxidase was observed for the estimation of the antibodies which act intracellularly against high and low infectious units of human rotavirus Wa strain in vitro. Promising sensitivity of the produced antibodies against the infectious human RV Wa strain was observed.