Production and Purification of LTB-RBD: A Potential Antigen for Mucosal Vaccine Development against SARS-CoV-2
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Most of the current SARS-CoV-2 vaccines are based on parenteral immunization targeting the S protein. Although protective, such vaccines could be optimized by inducing effective immune responses (neutralizing IgA responses) at the mucosal surfaces, allowing them to block the virus at the earliest stage of the infectious cycle. Herein a recombinant chimeric antigen called LTB-RBD is described, which comprises the B subunit of the heat-labile enterotoxin from E. coli and a segment of the RBD from SARS-CoV-2 (aa 439-504, carrying B and T cell epitopes) from the Wuhan sequence and the variant of concern (VOC)—delta. Since LTB is a mucosal adjuvant, targeting the GM1 receptor at the surface and facilitating antigen translocation to the submucosa, this candidate will help in designing mucosal vaccines (i.e., oral or intranasal formulations). LTB-RBD was produced in E. coli and purified to homogeneity by IMAC and IMAC-anionic exchange chromatography. The yields in terms of pure LTB-RBD were 1.2 mg per liter of culture for the Wuhan sequence and 3.5 mg per liter for the delta variant. The E. coli-made LTB-RBD induced seric IgG responses and IgA responses in the mouth and feces of mice when subcutaneously administered and intestinal and mouth IgA responses when administered nasally. The expression and purification protocols developed for LTB-RBD constitute a robust system to produce vaccine candidates against SARS-CoV-2 and its variants, offering a low-cost production system with no tags and with ease of adaptation to new variants. The E. coli-made LTB-RBD will be the basis for developing mucosal vaccine candidates capable of inducing sterilizing immunity against SARS-CoV-2. © 2022 by the authors.
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chimeric antigen; COVID-19; humoral response; mucosal adjuvant; mucosal vaccine adjuvant; ampicillin; chimeric protein; chloramphenicol; cholera toxin B subunit; enterotoxin; epitope; Freund adjuvant; immunoglobulin A; immunoglobulin G; ltb rbd; neutralizing antibody; recombinant vaccine; unclassified drug; vaccine; virus antigen; vitronectin; amino acid sequence; animal experiment; anion exchange chromatography; antibody titer; Article; bacterium culture; biomass; Bradford assay; cell disruption; chemoluminescence; chromatography; controlled study; coronavirus disease 2019; dialysis; enterotoxigenic Escherichia coli; enzyme linked immunosorbent assay; Escherichia coli; feces; fermentation; gene translocation; heat shock; humoral immunity; immune response; immunization; immunogenicity; mouse; mucosa; nonhuman; oxygen saturation; polyacrylamide gel electrophoresis; protein analysis; protein purification; protein secondary structure; Severe acute respiratory syndrome coronavirus 2; sterilizing immunity; submucosa; ultrafiltration; vaccination; vaccine development; variant of concern; Western blotting
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