In vitro neutralization assay for estimating immunogenicity of recombinant human Rotavirus VP6 protein via electroporation | ||
| Egyptian Journal of Botany | ||
| Articles in Press, Accepted Manuscript, Available Online from 03 October 2025 | ||
| Document Type: Regular issue (Original Article) | ||
| DOI: 10.21608/ejbo.2025.345178.3123 | ||
| Authors | ||
| Marwa A. Kamel1; Abeer Shokeer2; Usama Hegazy2; Amr E. El-Hakim2; Omar EL-Farouk Rabia Elsayed3; Ahmed B. Barakat3; Aly F. Mohamed4; Ibrahim Ahmed Hamza1; Cristina Fuentes5; Rosa M. Pinto5; Albert Bosch5; Waled Morsy El-Senousy* 1 | ||
| 1Environmental Virology Lab, Water Pollution Research Department, Environment and Climate Change Research Institute, National Research Centre, Dokki, Giza 12622, Egypt | ||
| 2Molecular Biology Department, Biotechnology Research Institute, National Research Centre, Dokki, Giza 12622, Egypt | ||
| 3Microbiology Department, Faculty of Science, Ain Shams University, Cairo, Egypt | ||
| 4The International Center for Training and Advance Researches (ICTAR- Egypt), Cairo, Egypt | ||
| 5Enteric Virus Laboratory, Department of Microbiology, School of Biology, University of Barcelona, Barcelona, Spain | ||
| Abstract | ||
| 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. | ||
| Keywords | ||
| ELISA; Human rotaviruses; Neutralization test; Recombinant protein; Subunit vaccine; VP6 whole gene | ||
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