Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency

Abstract

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.

Development and characterization of a multimeric recombinant protein based on the spike protein receptor binding domain of SARS-CoV-2 that can neutralize virus infection

“…For example, antibodies generated by the Alpha, Beta, Gamma, and Delta variants are not efficient against Omicron, even though they share some mutations [56]. In contrast, it has been shown that monoclonal antibodies made from a recombinant RBD (Wuhan sequence) neutralized major variants [57]. However, in our case a full C-terminal domain of the S1 protein along the RBD domain was introduced and might affect the production of new neutralizing antibodies.…”

Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles

“…Expression plasmids encoding wild type and variants of concern (VoC) Spike ORFs used in the pseudovirus neutralization assay were previously described ( 45 , 46 ) and are depicted in Supplementary Figure 1B . Briefly, plasmid pSpike-C3 encodes the wild type (Wuhan-Hu-1) codon optimized SARS-CoV-2 delta21 Spike protein open reading frame (ORF) ( 40 ); plasmids pSpike-UKC3, pSpike-SAC3 and pSpike-BRC3 encode the delta21 codon optimized B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) Spike ORFs, respectively, while plasmid pSpike-INC3 encodes the B.1.617.2 (Delta) Spike ORF with a 19 aa deletion in the cytoplasmic tail ( 45 , 46 ).…”

Persistent immunogenicity of integrase defective lentiviral vectors delivering membrane-tethered native-like HIV-1 envelope trimers

“…Integrase competent Lentiviral Vector expressing Luciferase (LV-Luc) and pseudotyped either with Indiana or Cocal VSV.G were tittered in VERO E6 cells and used in the neutralization assays 66 , 67 . Briefly, 5-fold serial dilutions starting from 1/100 of heat inactivated sera from immunized monkeys were incubated with 200.000 relative luminescence units (RLU) of LV-Luc/VSV.G In or LV-Luc/VSV.G Co in 96 deep well plate plates (Resnova, Genzano di Roma, Italy) at 37 °C for 30 minutes at 37 °C.…”

“…Results are expressed as inhibitory dilution (ID) 50 corresponding to the serum dilution giving 50% inhibition of infection (neutralization) compared to the virus-only control wells after background subtraction in cell control wells. ID50 was calculated with a linear interpolation method 66 , 67 .…”