Endotoxin [lipopolysaccharide (LPS)], the major antigen of the outer membrane of Gram-negative bacteria, consists of a variable-size carbohydrate chain that is covalently linked to N,O-acylated beta-1,6-D-glucosamine disaccharide 1,4'-bisphosphate (lipid A). The toxic activity of LPS resides in the lipid A structure. The structural features of synthetic peptides that bind to lipid A with high affinity, detoxify LPS in vitro, and prevent LPS-induced cytokine release and lethality in vivo were defined. The binding thermodynamics were comparable to that of an antigen-antibody reaction. Such synthetic peptides may provide a strategy for prophylaxis and treatment of LPS-mediated diseases.

Lipopolysaccharide (LPS) exerts its biological activity through the lipid A moiety. We tested the efficiency in inhibiting TNF production in sera and in tissues of mice and in the derma of rabbits challenged with LPS, of a synthetic anti-LPS peptide (SAEP-2) previously shown to specifically detoxify the lipid A region of LPS on the basis of structural similarities with the antibiotic polymyxin B (PMXB). In mice, SAEP-2 (100 μg/mouse, i.v.) injected with various schedules ('-30 to +10 min from LPS at 50 ng/mouse, i.v.) significantly inhibited serum TNF as well as liver, spleen and lung-associated TNF. In rabbits, SAEP-2 significantly inhibited TNF produced in dermal tissue and the resulting local hemorrhagic necrosis. The amount of tissue-associated TNF released by LPS challenge in the mouse was up to 6 times that present in the serum and inhibition by SAEP-2 or PMXB accounted for 75% of the total. Direct measurement of the binding kinetics by surface plasmon resonance and molecular filtration at equilibrium revealed that SAEP-2 and PMXB bind to LPS only in the presence of a significant amount of water but that they are unable to bind LPS in undiluted serum. Altogether these findings strongly suggest that inhibition of LPS-induced TNF by SAEP-2 and PMXB may occur in tissues.

We describe a model of vaccine based on detoxified endotoxin (LPS) conserving the supramolecular structure of micelles. Detoxification of LPS from Neisseria meningitidis group A, strain A1 (LPS A1), has been achieved by complex formation with a synthetic anti-endotoxin peptide (SAEP 2) binding to the lipid A moiety of LPS A1 with high affinity. Following subcutaneous injection in SW mice, LPS A1/SAEP 2 complex induced high titers of boostable IgG antibodies against the immunotype determinants of LPS A1, cross-reactive with group B LPS in either purified or cellassociated form. These antibodies were able to functionally fix and activate homologous and heterologous species of complement after binding to LPS A1-coated sheep erythrocytes. None of the IgG antibodies induced were specific for lipid A or SAEP 2 and none of the IgG antibodies cross-reacted with heterologous LPS. The purified IgG polyclonal antibodies significantly inhibited serum TNF production in CD1 mice intravenously challenged by homologous but not heterologous LPS. The immunogenic properties of LPS A1/SAEP 2 complex, investigated by the kinetic, magnitude and sub-isotype composition of the polyclonal antibodies induced, were comparable to those of a glycoconjugate obtained by covalent binding of LPS A1, detoxified by SAEP 2, to BSA working as a T-cell dependent carrier protein.The results obtained suggest that LPS behaves in vivo as a T-cell dependent antigen. The strategy of properly delivering to the immune system of mammalians, non-toxic LPS fully expressing its supramolecular antigenic structure, represents a novel approach for development of a new generation of R-and S-LPS/SAEP complex-based vaccines for prophylaxis of specific Gram-negative infections leading to sepsis and endotoxemia.