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Induction of endotoxin tolerance with nonpyrogenic O-antigenic oligosaccharide-protein conjugates

We prepared a dodecasaccharide, specific for the O-antigenic polysaccharide chain of Salmonella typhimurium (O-antigens 4 and 12), by the partial hydrolysis of the O-polysaccharide chain, utilizing bacteriophage 28B endo-alpha-L-rhamnosidase. The dodecasaccharide was shown by chemical and spectroscopical analyses to be totally devoid of lipid A and core oligosaccharide. By coupling this dodecasaccharide to human serum albumin, a glycoconjugate (DODECA-4809-ITC-HSA) was prepared and found to be (i) nonpyrogenic, (ii) unable to gelate a Limulus amoebocyte lysate, and (iii) unable to induce early-phase pyrogenic tolerance to endotoxin. Rabbits immunized either intravenously (with the glycoconjugate suspended in saline) or intrapopliteally (with the glycoconjugate suspended in Freund complete adjuvant) developed a significant although modest pyrogenic tolerance against challenge with the O-antigenic homologous S. typhimurium lipopolysaccharide (P less than 0.025 and P less than 0.01 for immunized and control rabbits, respectively). The evoked tolerance was O-antigen specific since no pyrogenic tolerance against challenge with lipopolysaccharide from S. thompson (possessing identical lipid A and core oligosaccharide structures but differing in the O-antigen polysaccharide chain) could be seen (P greater than 0.1). These results demonstrate that a nonpyrogenic O-antigenic polysaccharide hapten, when coupled to an immunogenic carrier protein, evokes immune responses which mediate significant, although modest, late-phase tolerance and is capable of partly reducing the pyrogenic activity of the O-antigenic homologous lipopolysaccharide.