Abstract:COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful “tool” to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterizat… Show more
“…The usage of E. coli is often not recommended for challenging proteins that undergo glycosylation and require complex folding, such as the SARS-CoV-2 spike [ 64 , 65 ]. However, recent structural data from the circular dichroism and gel-filtration chromatography of the His-tagged recombinant SARS-CoV-2 spike proteins produced in E. coli and in mammalian HEK-293 cells evidenced that both of the proteins are stable and correctly folded, and capable of binding angiotensin-converting enzyme 2 (ACE2) [ 66 ]. Moreover, the functional data provided by ThermoFisher R&D also demonstrated that the E. coli recombinant SARS-CoV-2 spike (#RP-87668) efficiently recognised and bound ACE2 (proprietary data).…”
Multisystem inflammatory syndrome in children (MIS-C) is a rare hyperinflammatory disease occurring several weeks after SARS-CoV-2 infection. The clinical similarities between MIS-C and the toxic shock syndrome, together with the preferential expansion of T cells with a T-cell receptor variable β chain (TCRVβ) skewing, suggested a superantigen theory of MIS-C. For instance, recent in silico modelling evidenced the presence of a highly conserved motif within SARS-CoV-2 spike protein similar in structure to the superantigenic fragment of staphylococcal enterotoxin B (SEB). However, experimental data on the superantigenic activity of the SARS-CoV-2 spike have not yet been provided. Here, we assessed the superantigenic activity of the SARS-CoV-2 spike by analysing inflammatory cytokine production in both Jurkat cells and the peripheral blood CD4+ T cells stimulated with the SARS-CoV-2 spike or SEB as a control. We found that, unlike SEB, the SARS-CoV-2 spike does not exhibit an intrinsic superantigen-like activity.
“…The usage of E. coli is often not recommended for challenging proteins that undergo glycosylation and require complex folding, such as the SARS-CoV-2 spike [ 64 , 65 ]. However, recent structural data from the circular dichroism and gel-filtration chromatography of the His-tagged recombinant SARS-CoV-2 spike proteins produced in E. coli and in mammalian HEK-293 cells evidenced that both of the proteins are stable and correctly folded, and capable of binding angiotensin-converting enzyme 2 (ACE2) [ 66 ]. Moreover, the functional data provided by ThermoFisher R&D also demonstrated that the E. coli recombinant SARS-CoV-2 spike (#RP-87668) efficiently recognised and bound ACE2 (proprietary data).…”
Multisystem inflammatory syndrome in children (MIS-C) is a rare hyperinflammatory disease occurring several weeks after SARS-CoV-2 infection. The clinical similarities between MIS-C and the toxic shock syndrome, together with the preferential expansion of T cells with a T-cell receptor variable β chain (TCRVβ) skewing, suggested a superantigen theory of MIS-C. For instance, recent in silico modelling evidenced the presence of a highly conserved motif within SARS-CoV-2 spike protein similar in structure to the superantigenic fragment of staphylococcal enterotoxin B (SEB). However, experimental data on the superantigenic activity of the SARS-CoV-2 spike have not yet been provided. Here, we assessed the superantigenic activity of the SARS-CoV-2 spike by analysing inflammatory cytokine production in both Jurkat cells and the peripheral blood CD4+ T cells stimulated with the SARS-CoV-2 spike or SEB as a control. We found that, unlike SEB, the SARS-CoV-2 spike does not exhibit an intrinsic superantigen-like activity.
“…Spectra of the WT match those reported in the literature. [10] , [43] , [44] , [45] More importantly, none of the mutations caused significant changes in the spectra, implying that the Delta mutations do not affect the global protein structure. Figure 2 (A) SDS-PAGE of purified RBD constructs.…”
“…The findings suggest that the absence of glycosylation could partially affect ACE2 binding in vitro ( 52 ). In another study and in parallel with HEK-293 and insect cells, the E. coli -expressed RBD characterization showed the ability of the bacterial expression system in producing high-quality and antibody-recognizable proteins ( 53 ).…”
SARS-CoV-2 has caused a global pandemic, infecting millions of people. An effective preventive vaccine against this virus is urgently needed. Here, we designed and developed a novel formulated recombinant receptor-binding domain (RBD) nucleocapsid (N) recombinant vaccine candidates. The RBD and N were separately expressed in E. coli and purified using column chromatography. The female Balb/c mice were immunized subcutaneously with the combination of purified RBD and N alone or formulated with saponin adjuvant in a two-week interval in three doses. Neutralization antibody (Nabs) titers against the SARS-CoV-2 were detected by a Surrogate Virus Neutralization (sVNT) Test. Also, total IgG and IgG1, and IgG2a isotypes and the balance of cytokines in the spleen (IFN-γ, Granzyme B, IL-4, and IL-12) were measured by ELISA. The percentages of CD4+ and CD8+ T cells were quantified by flow cytometry. The lymphoproliferative activity of restimulated spleen cells was also determined. The findings showed that the combination of RBD and N proteins formulated with saponin significantly promoted specific total IgG and neutralization antibodies, elicited robust specific lymphoproliferative and T cell response responses. Moreover, marked increase in CD4+ and CD8+ T cells were observed in the adjuvanted RBD and N vaccine group compared with other groups. The results suggest that the formulations are able to elicit a specific long-lasting mixed Th1/Th2 balanced immune response. Our data indicate the significance of the saponin-adjuvanted RBD/N vaccine in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective long-lasting and strong vaccine.
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