Abstract:The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~ 300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the… Show more
“…Figure 1 is the 1D gel of purified S-614D and S-614G along with the results of an affinity pulldown of the two spike variants with ACE2 and with monoclonal antibodies. The 1D SDS-PAGE gels show the purity of both S-614D and S-614G and their ability to bind to ACE2 receptor and the two monoclonal antibodies 34 against the SARS-CoV-2 spike receptor binding domain, indicating these two recombinant proteins are functionally active with proper structures suitable for this study. Figure 1 SDS gel analysis of the SARS-CoV-2 S proteins interacting with other proteins.…”
Section: Resultsmentioning
confidence: 91%
“…Biotinylated ACE2 was purchased from Sino Biological Inc (Wayne, PA). Mouse SARS-CoV2 monoclonal antibodies (3G7 and 3A2) against the RBD of the spike were prepared by an accelerated immunization and hydridoma screening process 34 .…”
N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.
“…Figure 1 is the 1D gel of purified S-614D and S-614G along with the results of an affinity pulldown of the two spike variants with ACE2 and with monoclonal antibodies. The 1D SDS-PAGE gels show the purity of both S-614D and S-614G and their ability to bind to ACE2 receptor and the two monoclonal antibodies 34 against the SARS-CoV-2 spike receptor binding domain, indicating these two recombinant proteins are functionally active with proper structures suitable for this study. Figure 1 SDS gel analysis of the SARS-CoV-2 S proteins interacting with other proteins.…”
Section: Resultsmentioning
confidence: 91%
“…Biotinylated ACE2 was purchased from Sino Biological Inc (Wayne, PA). Mouse SARS-CoV2 monoclonal antibodies (3G7 and 3A2) against the RBD of the spike were prepared by an accelerated immunization and hydridoma screening process 34 .…”
N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.
“…COVID-19 is still a public health threat to societies since the complexity of mass vaccination programs, the lack of effective drugs to specifically treat SARS-CoV-2 infected individuals ( 30 – 32 ) and the emergence of VOC predict that virus circulation may last for years ( 33 ). A more detailed and broad understanding of the molecular mechanisms underlying the pathogenesis of COVID-19 will have significant implications for developing countermeasures against the virus with particular emphasis to drug discovery, diagnosis and more effective and safe vaccine design ( 34 ). The SARS-CoV-2 spike protein is a high glycosylated transmembrane protein assembled as a homotrimer on the virus surface, with three S1 subunits containing the RBD sitting on the top of a trimeric membrane fusion envelope-anchored S2 subunits ( 35 ).…”
Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.
“…As the world continues to grapple with the coronavirus COVID-19 pandemic (Zhu et al, 2020; Sun et al, 2020 ), monoclonal antibodies (mAbs) have become increasingly used in applications such as basic research, diagnosis, therapeutics for SARS-CoV-2 infection ( Hwang et al, 2022 , Chapman et al, 2021 ). Fortunately, the hybridoma technology developed by Georges Kohler and Cesar Milstein in 1975 has made it possible to obtain large numbers of mAbs for these purposes (Köhler et al, 1975).…”
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