Membrane separation was verified with SDS-PAGE. == Enzyme linked immunosorbent assay (ELISA). findings nor-NOHA acetate support the importance of OmpA at the host-pathogen interface and begin to explore the implications and utility ofE. coli-specific antibodies in human hosts. KEYWORDS:Escherichia coli, aggregation, monoclonal antibodies, outer membrane proteins, phagocytosis == INTRODUCTION == Escherichia coliis a versatile Gram-negative bacterial organism that can be a human enteric commensal, a widely used laboratory reagent, and in some settings a dangerous human pathogen. The latter of these is especially concerning because pathogenicE. colifrequently exhibits antimicrobial resistance (AMR), even to the most advanced antibiotics in clinical use. Carbapenem-resistant or extended-spectrum beta lactamase (ESBL)-producingE. colicauses over 200,000 infections and more than 10,000 deaths annually in the United States (1). These infections lead to extensive morbidity, mortality, and more than 1 billion USD of excess health care costs annually (2). TreatingE. coliinfections and stopping the spread of AMR bacteria require a multipronged approach involving public health containment, clinical care improvement, and novel therapeutic strategies. The utility of monoclonal antibodies (MAbs) as an antibacterial strategy has only recently begun to be systematically evaluated, and yet, numerous groups have espoused the promise of this class of therapeutics (37). Although interest in antibacterial MAbs has recently increased, several MAbs have been tested in clinical trials with various degrees of success, including MEDI4983 (8), 514G3 (9), and AR301 (10) againstStaphylococcus aureusand MEDI3902 (11,12) againstPseudomonas aeruginosa. MAbs toE. colihave not been evaluated extensively, although previous work identified an outer membrane porin, likely outer membrane protein A (OmpA), as a dominant antibody target followingE. coliinfection (1317). More recently, a murine MAb, 49.4-15, was identified that is specific forE. coliOmpA (18). Multiple studies of OmpA protein variants as vaccine candidates have also demonstrated the development of OmpA-specific humoral immunity in immunized animals, although the usefulness of OmpA as a vaccine may be limited by the high level of conservation ofompAacross commensal bacteria (1922). It is evident that antibodies toE. coliare induced in humans after infection, and the murine antibody studies suggest these human antibodies may impact the virulence or pathogenesis ofE. coliinfection by targeting OmpA. In this study, we identified and evaluated naturally occurring human MAbs that specifically bind to OmpA. We investigated the binding of these antibodies to protein and intact bacteria under various growth conditions and determined functional properties of MAb-OmpA binding uponE. colipathogenesis. We assessed the characteristics of this MAb against K-12 MG1655 and the well-characterized urinary tract Mouse monoclonal to CER1 isolate UTI89. Urinary tract infections represent a large burden of disease caused byE. coli, and OmpA has been reported previously as important forE. coliuropathogenesis (23,24). This work provides a foundation for experiments to obtain a better understanding of the interaction ofE. coliouter membrane proteins and the human immune system. == RESULTS == == Isolation of human MAbs that bind to OmpA. == Peripheral blood samples were collected after written informed consent was obtained from healthy laboratory personnel working in microbiology research laboratories. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient fractionation and used to make human B cell hybridoma cell lines secreting naturally occurring MAbs that bound toE. coliouter membrane fractions by ELISA. Most MAbs were found to be of the IgG1 isotype, and the antibody variable gene sequences nor-NOHA acetate were obtained and used to produce recombinant IgG1 proteins. If the native isotype was not IgG1, we recombinantly produced and tested both the native isotype IgG as secreted by the hybridoma cell line and the IgG that was isotype nor-NOHA acetate switched to IgG1. We also produced recombinant LALA-PG Fc region-variant IgG proteins (which do not bind to mouse nor human FcR [25]), fragment antigen binding (Fab) variants (which only contain a single binding site instead of the paired binding arms of IgG MAbs), and rSTAU-228 IgG1 (a negative-control antibody specific forStaphylococcus aureusIsdA [26]). To determine the antigen specificity of these MAbs, they.
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