<p>Development of Anti-<em>Yersinia pestis</em> Human Antibodies with Features Required for Diagnostic and Therapeutic Applications</p>

Abstract

Background: Yersinia pestis is a category A infective agent that causes bubonic, septicemic, and pneumonic plague. Notably, the acquisition of antimicrobial or multidrug resistance through natural or purposed means qualifies Y. pestis as a potential biothreat agent. Therefore, high-quality antibodies designed for accurate and sensitive Y. pestis diagnostics, and therapeutics potentiating or replacing traditional antibiotics are of utmost need for national security and public health preparedness. Methods: Here, we describe a set of human monoclonal immunoglobulins (IgG1s) targeting Y. pestis fraction 1 (F1) antigen, previously derived from in vitro evolution of a phagedisplay library of single-chain antibodies (scFv). We extensively characterized these antibodies and their effect on bacterial and mammalian cells via: ELISA, flow cytometry, mass spectrometry, spectroscopy, and various metabolic assays. Results: Two of our anti-F1 IgG (αF1Ig 2 and αF1Ig 8) stood out for high production yield, specificity, and stability. These two antibodies were additionally attractive in that they displayed picomolar affinity, did not compete when binding Y. pestis, and retained immunoreactivity upon chemical derivatization. Most importantly, these antibodies detected <1,000 Y. pestis cells in sandwich ELISA, did not harm respiratory epithelial cells, induced Y. pestis agglutination at low concentration (350 nM), and caused apparent reduction in cell growth when radiolabeled at a nonagglutinating concentration (34 nM). Conclusion: These antibodies are amenable to the development of accurate and sensitive diagnostics and immuno/radioimmunotherapeutics.

Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera

“…Therefore, eventual competition between the two antibody formats is strong and easy to detect. Our ongoing work includes development of an LFA-based detection system for plague using antibodies YP2 and YP8, identified as orthogonal binder pairs using the scTGP displacement assay described here ( Lillo et al ., 2020 ).…”

“…FLISA with gradient protein concentration (2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\times$\end{document} ) starting at 20 000 ng in 100 μl volume/well was used to determine the binding curve of scTGP YP2, YP3 and YP8 (these antibodies were determined to be the most functional antibodies in this suite). The corresponding anti-YP IgGs were synthesized at ATUM Inc. and the F1V recognition by these IgGs was evaluated by ELISA using anti-human HRP (97165, Abcam) as the secondary antibody ( Lillo et al ., 2020 ). The epitope binning assay was performed similarly to FLISA.…”

“…Finally, we use scTGP and IgGs to develop a novel epitope binning method for multimeric proteins. We demonstrate this method using three antibodies against Yersinia pestis , the causative agent of plague ( Lillo et al ., 2011 ; Lillo et al ., 2020 ). Epitope binning uses competitive pairwise binding assays to sort antibodies into bins recognizing the same or different epitopes.…”

Plague Prevention and Therapy: Perspectives on Current and Future Strategies

“…Additional anti-Y. pestis human antibodies have recently been produced and are currently being evaluated for therapeutic application [204].…”

Optimised Heterologous Expression and Functional Analysis of the Yersinia pestis F1-Capsular Antigen Regulator Caf1R

“…The F1 capsule also has substantial applied value. Due to its surface location, high level of expression and the fact that the F1 polymer is unique to Y. pestis, F1 remains a primary target for plague diagnostics [11][12][13][14] and a key component of anti-plague vaccines [6,[15][16][17].…”