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.
Highlights
An outbreak of severe transmittable pneumonia, later referred to as COVID-19 (COronaVIrus Disease 19), was first described in China in late 2019 [1]
RRBD was well detected in both the cell lysate and the supernatant of transiently transfected HEK293T cells as a band of approximatively 35 kDa. This result contrasts with the predicted molecular mass of 29.1 kDa calculated on the basis of recombinant RBD (rRBD) amino acid sequence but could be explained by post-translational modifications that occur in mammalian cells
COVID-19 is still a public health threat to societies since the complexity of mass vaccination programs, the lack of effective drugs to treat SARS-CoV-2 infected individuals [30,31,32] and the emergence of variants of concern (VOC) predict that virus circulation may last for years [33]
Summary
An outbreak of severe transmittable pneumonia, later referred to as COVID-19 (COronaVIrus Disease 19), was first described in China in late 2019 [1]. The disease resulted in high occurrences of fatal pneumonia with clinical symptoms resembling those of severe acute respiratory syndrome coronavirus (SARS-CoV) infections observed during the 2002-2004 SARS epidemic. The causative pathogen was identified as a novel coronavirus, initially designated 2019-nCoV and subsequently SARS-CoV-2 [3]. SARS-CoV-2 has been able to spread rapidly worldwide since it is transmitted efficiently from human to human, even prior to the onset of symptoms, via droplets/aerosol from coughing or sneezing, or direct contact [4]. In March of 2020, the World Health Organization officially declared COVID-19 as a pandemic. The pandemic is having a devastating impact on the global economy and public health systems worldwide. In addition to safe and highly protective vaccines against SARS-CoV-2 and its VOC, monoclonal antibodies (mAbs), able to recognize and neutralize SARS-CoV-2 to be employed as new diagnostic tools and efficacious therapeutic approaches are still urgently needed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
