Gram-negative bacteria are common pathogens of man. Gram-negative sepsis is the most serious consequence of infections caused by these bacteria. Lipopolysaccharides (LPS), or endotoxins, are an essential component of the outer cell membrane of all Gram-negative bacteria and play a central pathogenic role in Gram-negative sepsis. The core region of endotoxin, which contains the lipid A moiety, is structurally and antigenically conserved among many Gram-negative bacteria. Lipid A mediates most biological activities important in pathogenesis. The goal of this proposal is to test the hypothesis that antibodies directed toward shared LPS core determinants cross-react broadly among heterologous LPS's, exhibit anti-endotoxic activity, and provide a protective function in septicemic infections due to diverse Gram-negative pathogens. Specific murine and human monoclonal antibodies (mAbs) will be prepared against the exposed core glycolipids of rough mutant Gram-negative bacteria. These mAbs will be evaluated in binding and immunoblotting assays for cross-reactivity with heterologous LPS's. Specific core epitopes recognized by mAbs will be identified in direct binding and competition studies employing LPS subcomponents, lipid A precursors, and synthetic lipid A analogs. The avidity with which mAbs bind to these ipitopes will be measured by solid-phase radioimmunoassay. The ability of core-specific mAbs to recognize antigenic target sites on the bacterial surface will be evaluated using whole bacterial cell assays, and the possible antibacterial activities mediated by such interactions will be determined. The blocking or neutralizing effect of core-specific mAbs on in vitro functional properties of homologous and heterologous LPS will be assessed. The in vivo inhibitory action of core-specific mAbs on LPS-induced shock and death will be evaluated, and therapeutic activity tested in animal models of Gram-negative sepsis. It is anticipated that these studies will: 1) Further elucidate the concept of cross-protective, anti-endotoxic immunity, 2) Define the therapeutic role of core-specific mAbs in Gram-negative sepsis, and 3) Provide fundamental new insights concerning the immunopathogenic role of LPS at the molecular level.
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