Novel Immunization Strategies Against Multi-drug-resistant Gram-negative Bacteria

Abstract

BackgroundHealthcare-related infections due to multi-drug resistant (MDR) Gram-negative bacteria (GNB) such as Acinetobacter baumannii (AB) and carbapenemase producing Klebsiella pneumoniae (KPC) are associated with high mortality rates. New methods to prevent or treat these infections are needed. Candidaantigen Hyr1p is predicted to share structural and sequence homology with the hemagglutinin/hemolysin protein (FhaB) and siderophore-binding protein of GNB including AB and KPC, respectively. Indeed, active and passive immunization using Hyr1p as a target protect against AB infections in mice. Thus, we attempted to develop protective monoclonal antibodies (mAb) and test their efficacy against AB and KPC in vitro and in vivo.MethodsMurine hybridomas were generated from Balb/c mice after vaccination with recombinant Hyr1p. The concentration and identification of the collected mAbs were determined using Bradford and SDS-PAGE. Binding ability of mAb was tested against AB and KPC using flow cytometry. In-vitro studies on the ability of these mAbs to kill KPC and AB were tested by quantitative culturing. The ability of these mAbs to protect from AB- or KPC-mediated alveolar epithelial cell (A549) damage was studied with 51Cr-release assay. The efficacy of mAb in protecting against AB- or KPC-induced pneumonia was studied in neutropenic or immunocompetent CD1 mice by administering 30 µg of mAb (i.p.) on Day +1 and +4, relative to infection, respectively. Survival of mice served as an endpoint.ResultsFour different mAb-producing hybridoma cells generated IgM that bound to AB and KPC. 40–80 µg/mL of mAb resulted in 100% killing effect of AB or KPC in vitro. Two of mAb (25 µg/mL each) resulted in protecting A549 cells from AB- or KPC-induced damage by ~80% vs. cells incubated with isotype-matching Ab (P < 0.05). Finally, one of the mAb resulted in 70% or 100% long-term survival of mice infected with lethal doses of KPC or AB, respectively (P < 0.05).ConclusionWe used Candida Hyr1p to generate cross-protective mAb against MDR AB and KPC. Our results warrant the further development of these mAb as novel immunotherapeutics against MDR GNB.Disclosures All authors: No reported disclosures.