Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.

Genomic analysis of SARS-CoV-2 variants: diagnosis and vaccination challenges.

SARS-CoV-2 put a heavy financial burden on the healthcare system, with millions of laboratory-confirmed cases and deaths worldwide in the last 2 years. During the seventh wave of this pandemic, the continuously evolving nature of SARS-CoV-2 resulted in the emergence of new variants that harbor different mutations. Mutations are associated with changes in the virus behavior, including increased transmissibility, increased virulence, and evasion of neutralizing antibodies. Currently, we need detailed and comprehensive genomic information on all SARS-CoV-2 variants. One of the key points in this study was the genome survey of mutation profiles across variants as a genomic data source, to determine the efficiency of RT-qPCR assays. We also used the source to calculate the binding affinity changes of neutralizing antibodies-mutant receptor binding domain (RBD) complexes and determine vaccine efficacy. Our result revealed that the number of nucleotide mismatches is variable in the WHO-recommended primer-probe sets. Mismatches located at the 3' ends of the oligonucleotide, may lead to false-negative results. Only the primer-probe sets designed by the Ministry of Public Health of Thailand were exclusive and cannot detect the omicron variant reliably. Binding affinity changes showed that E484K was more deleterious than other mutations and decreased stability between the mutant RBD protein and neutralizing antibodies. The Omicrons show the highest change in binding affinity which may lead to immune escape and increase transmissibility. Additionally, the 7D6 monoclonal antibody in the 7eam complex could neutralize all variants of SARS-CoV-2. We strongly recommend creating and improving a matrix accuracy by processing a large number of SARS-CoV-2 sequences to update RT-qPCR assays and identified immunogenic residues among conserved RBD. Also, a detail computational analysis is needed to investigate distinctive amino acid substitution patterns which may be foundational in the vaccines. Finally, designing in-vitro studies can help confirm the present study and manage COVID-19 patients.Communicated by Ramaswamy H. Sarma.