In vitro neutralization assay for estimating immunogenicity of recombinant human Rotavirus VP6 protein via electroporation

Rotaviruses (RVs) represent the principal causative agents of severe gastroenteritis leading to high mortality rates, especially in children < 5 years old 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% nucleotide identity and 98% amino acid identity in comparison to human RoV Wa reference strain was expressed in E. coli. Examination of the sensitivity of antibodies produced in the male rabbits which were immunized intramuscularly with 20μg of the purified VP6 protein indicated a sensitivity up to 1/24000 dilution of antibodies against the expressed protein using ELISA. Introducing antibodies into the MA104 cell line was performed using electroporation to neutralize the human rotavirus Wa strain VP6 when exposed after viral uncoating. The efficiency of the antibodies which act intracellularly to neutralize the infectious human rotavirus Wa strain used in challenge was 1.4, 1.6, and 1.8 log10 for high, moderate, and low viral titres at 1/10 dilution of the antibodies and the efficiency decreased gradually by increasing the dilution.