SARS CoV2 Studies in the Microbial Pathogenesis Section/LPD

NIH Pandemic-Era Grants

Pandemic Era Grants

2021

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Principal Investigator: Stephen  Leppla
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2021
Award: $20,392
Funding agency: National Institute of Allergy and Infectious Diseases

The monoclonal S9.6 antibody has the unique property of recognizing DNA/RNA hybrids in a sequence-independent manner. A polyclonal antibody having this specificity 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 FY2021 we have analyzed a variety of formats for use of S9.6 in detection of DNA of B. anthracis and Toxoplasma gondi, as models for future application to COVID RNA detection. In one successful format, poly-biotinylated single-stranded DNA probes complementary to B. anthracis mRNA were captured on an S9.6-coated assay plate and detected using enzyme-linked streptavidin. 

Optimization of S9.6 as a diagnostic tool would be aided by production of the antibody as a recombinant protein. This would facilitate its production as well as structure-function analysis and protein engineering. To achieve this, we used amino acid sequence data from several experimental approaches to deduce the S9.6 Fab sequence, had the heavy and light chain genes synthesized, and produced the Fab in expiCHO cells in amounts sufficient for structural analysis. Jinwei Zhang of NIDDK obtained a crystal structure of the S9.6 Fab in complex with a small DNA/RNA hybrid. The structure helps to explain how the antibody discriminates against RNA:RNA duplexes to achieve specificity for DNA/RNA duplexes. 

Optimal sensitivity of hybrid capture type assays with S9.6 requires high quality reporter enzyme conjugates. One of our S9.6 Fab constructs is equipped with a C-terminal amino acid sequence that is the substrate for sortase-mediated conjugation to proteins having a tri-glycine N-terminal signal. After producing secreted embryonic alkaline phosphatase (SEAP) with the required tri-glycine sequence (again using the expiCHO cells), these two proteins were conjugated by sortase in excellent yield to produce a purified and fully defined S9.6 Fab-SEAP conjugate. This was shown in several assay formats to be effective in detecting DNA/RNA hybrids.  Subsequently, we also produced in the expiCHO cells a genetic fusion of the S9.6 Fab to SEAP and confirmed its activity.

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