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 has the unique property of recognizing DNA/RNA hybrids in a sequence-independent manner. Previously, we popularized this antibody and made it available to hundreds of researchers. We 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, but ones 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 papilloma virus (HPV) DNA in vaginal swabs. It follows that the S9.6 antibody offers a generic approach to detection and quantitation of any nucleic acid of known sequence. Development of this diagnostic platform could lead to methods for detection of any pathogen of interest. During FY2022 we continued to improve methods for production of the S9.6 immunoglobulin (IgG) antibody in other formats including single-chain variable fragment (scFv), fragment antigen-binding region (Fab), and molecularly well-defined Fab-reporter conjugates. Thus, we prepared the S9.6 Fab as a recombinant protein equipped with a C-terminal sequence recognized by the sortase transpeptidase. After also producing secreted embryonic alkaline phosphatase (SEAP) with a Gly3 N-terminus, the two proteins were conjugated by sortase in excellent yield to produce a purified and fully defined S9.6 Fab-SEAP conjugate. Once this conjugate proved useful in various diagnostic assay formats (described below), we extended this work to produce the S9.6 Fab (heavy chain)-SEAP protein as a genetic fusion. Because SEAP exists only as a dimer, this genetic fusion protein is effectively bivalent, with an S9.6 Fab attached to each of the SEAP monomers in the dimer. All these S9.6 proteins were produced in Chinese hamster ovary (CHO) cells grown in suspension culture, an efficient system that can yield > 50 milligrams protein per liter. The purified S9.6 antibody variants and conjugates were employed during FY2022 in a variety of assay formats for detection of RNA of Bacillus anthracis and Toxoplasma gondii, as models for future application to COVID RNA detection. In one successful format, hybrids produced from poly-biotinylated single-stranded DNA probes and complementary B. anthracis mRNA were captured on a polystreptavidin-coated assay plate and detected using S9.6 and HRP-conjugated anti-mouse IgG. We also 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. 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. Further optimization of this format is continuing. As previously described, work with S9.6 as a diagnostic tool would be aided by understanding the basis for the specificity of the antibody. Our collaborators Charles Bou-Nader and Jinwei Zhang of NIDDK had successfully produced crystals of the S9.6 Fab in complex with a small DNA/RNA hybrid. This work has now been completed by publication of the structure of the complex. Having a structure will facilitate rational approaches to optimizing the selectivity of the antibody.