Polyclonal antisera directed to vaccines made from rough mutant bacteria that display the endotoxin core structure on their surface have been reported to substantially decrease mortality when passively infused into animals or humans with endotoxic shock. However, the protective element(s) in these antisera remain controversial and the mechanism of protection is not known. In addition, there is no in vitro test that correlates with the protection provided in vivo. The overall objective of the proposed work is to provide a fundamental understanding of how these antisera interact with bacterial endotoxins (LPS). The ability of the antisera to neutralize LPS will be studied using in vitro assays of LPS bioactivity including the production of tumor necrosis factor and interleukin 1 by macrophages, the mitogenic effect of LPS on splenocytes, the adherence and superoxide production of granulocytes, and the coagulation of limulus lysate. A recently developed radioimmunoassay will be utilized to study the fluid phase binding of several physicochemical forms of LPS to different elements in the antisera. The biological assays and radioimmunoassay will then be employed to evaluate the role of immunoglobulin and inflammatory proteins in the neutralization and binding of LPS. If highly purified immunoglobulin preparations fail to neutralize and/or bind to a panel of different LPS, the assay systems will be used to isolate and characterize the induced components that are active. Since LPS bound to lipoprotein is less toxic than unbound LPS, the binding of radiolabeled LPS to lipoprotein in the antisera will be studied in detail. In a different series of experiments, complexes of radiolabeled LPS that are formed in the antisera will be characterized and analyzed for the presence of immunoglobulins, proteins, and lipoproteins using electrophoresis, western blotting, and high pressure liquid chromatography. It is hoped that the proposed work will (1) resolve the ongoing controversy regarding the protective element in these antisera, (2) provide a means of testing current and future candidate antibodies and proteins for the ability to bind and neutralize LPS, and (3) provide the knowledge necessary to develop a therapeutic agent for endotoxemia based upon the protective activity of these antisera.
