Generation and utility of a single-chain fragment variable monoclonal antibody platform against a baculovirus expressed recombinant receptor binding domain of SARS-CoV-2 spike protein.

As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.