SARS CoV2 Studies in the Microbial Pathogenesis Section/LPD

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

The monoclonal S9.6 antibody uniquely recognizes DNA/RNA hybrids in a largely sequence-independent manner. We previously popularized this antibody and made it available to hundreds of researchers. We also worked with NIH tech transfer staff to license this antibody to several leading reagent supply companies. S9.6 has become a key tool for the study of R-loops, which are natural structures that become excessively abundant in certain pathological situations. A polyclonal antibody recognizing DNA/RNA hybrids is the basis of hybrid capture ELISA-type commercial diagnostic assays for human papillomavirus (HPV) DNA in vaginal swabs. The S9.6 antibody offers a generic approach to the detection and quantitation of any nucleic acid of known sequence. The development of this diagnostic platform could lead to methods for the detection of any pathogen of interest. Previously, we demonstrated the structural basis of DNA/RNA hybrid recognition by S9.6 monoclonal (Bou-Nader et al., Nat. Commun. 13, 1641 (2022)). This research demonstrated the high specificity of S9.6 with minimal cross-reactivity to other forms of double-stranded and single-stranded nucleic acids. Building on this foundation, our efforts in FY2023 refined production methods for the S9.6 antibody in diverse formats. These included single-chain variable fragment (scFv), fragment antigen-binding region (Fab), and innovative well-defined Fab-reporter conjugates. Consequently, we generated the S9.6 Fab as a recombinant protein equipped with a C-terminal sequence recognized by the sortase transpeptidase. Simultaneously, we produced secreted embryonic alkaline phosphatase (SEAP) with a Gly3 N-terminus. Through the efficient action of sortase, we conjugated these two proteins, yielding a purified and fully characterized S9.6 Fab-SEAP conjugate with excellent efficiency. Using both S9.6 IgG and the S9.6 Fab-SEAP conjugate in ELISA assays, we were able to specifically identify synthetic DNA/RNA hybrids. We extended application of this conjugate to various diagnostic assay formats (outlined below) and expanded our efforts by creating the S9.6 Fab (heavy chain)-SEAP protein as a genetic fusion. Since SEAP exists as a dimer, this fusion protein is effectively bivalent, with an S9.6 Fab attached to each monomer in the SEAP dimer. All these S9.6 proteins were generated in Chinese hamster ovary (CHO) cells grown in suspension culture, a highly efficient system that yields over 50 milligrams of protein per liter. During FY2023 we employed the purified S9.6 antibody variants and conjugates in a range of assay formats to detect the RNA of Bacillus anthracis and Toxoplasma gondii, serving as models for future applications in COVID RNA detection. In one successful format, we established assays like the HPV ELISA assay, where hybrids are captured on surface-bound S9.6 IgG and then detected with an S9.6 Fab-SEAP conjugate. Later, we improved this assay to detect hybrids produced from poly-biotinylated single-stranded DNA probes and complementary B. anthracis mRNA, which were captured on a polystreptavidin-coated assay plate and detected using S9.6 and HRP-conjugated anti-mouse IgG. To make S9.6-based assays more convenient for low-resource areas, we initiated work to implement a lateral flow assay. Our collaborator Marco Biamonte (DDTD, CA) developed an S9.6 IgG-gold nanoparticle conjugate, which we employed to identify SARS-CoV-2-specific DNA/RNA hybrids on a lateral flow strip. We are continuing to optimize this format.