Conjugation of protein antigen to microparticulate beta-glucan from Saccharomyces cerevisiae: a new adjuvant for intradermal and oral immunizations.

Immunostimulatory glucose polymers known as beta-glucans have been studied for many years. Our laboratory has prepared and characterized a novel microparticulate beta-glucan (MG) from the budding yeast Saccharomyces cerevisiae. Because MG particles are rapidly phagocytized by murine peritoneal macrophages and induce the expression of B7 costimulatory molecules, we hypothesized that MG could serve as a vaccine adjuvant to enhance specific immune responses. Here, we describe a procedure for conjugating the test vaccine antigen bovine serum albumin (BSA) to MG via water-soluble carbodiimide linkage. Conjugates with up to 0.4 mg of BSA/mg MG were prepared. MG/BSA conjugates were still actively phagocytized by mouse peritoneal macrophages. When used to immunize mice by the intradermal route, these conjugates enhanced the primary IgG antibody response to BSA in a manner comparable to the prototypic complete Freund's adjuvant. Although primary oral immunization with MG/BSA caused no increase in serum anti-BSA antibody titers, booster immunization elicited a significant anti-BSA antibody response. These results suggest that protein antigens can be conjugated to MG via a carbodiimide linkage and that these conjugates provide an adjuvant effect for stimulating the antibody response to the protein antigens.

IFN-gamma primes macrophages for enhanced TNF-alpha expression in response to stimulatory and non-stimulatory amounts of microparticulate beta-glucan.

beta-(1-->3)-D-Glucan is an integral cell wall component of a variety of fungi, plants, and bacteria. Like the prototypic inflammatory mediator lipopolysaccharide (LPS), some beta-(1--> 3)-D-glucan-containing preparations have been shown to induce the production of proinflammatory cytokines by macrophages. In the present study, we have tested a new microparticulate form of beta-(1--> 3)-D-glucan (MG) from Saccharomyces cerevisiae for its ability to induce proinflammatory cytokine secretion in mouse peritoneal macrophages in vitro, and we have examined the effect of IFN-gamma. MG was rapidly phagocytized by peritoneal macrophages, and these MG-treated macrophages upregulated TNF-alpha, IL-6, and IL-1beta mRNAs and secreted these proinflammatory cytokines. IFN-gamma treatment alone did not induce unstimulated macrophages to produce TNF-alpha. However, a 4 h IFN-gamma pretreatment augmented TNF-alpha secretion by peritoneal macrophages subsequently treated with an optimally stimulatory dose of MG. IFN-gamma pretreatment for 2 h followed by thorough washing and a further 2 h incubation without IFN-gamma still resulted in enhanced TNF-alpha production in response to MG, suggesting that IFN-gamma can prime macrophages for a subsequent proinflammatory response. Most interestingly, we found that IFN-gamma pretreatment of peritoneal macrophages enhanced the TNF-alpha response to amounts of MG that were poorly stimulatory or non-stimulatory in the absence of IFN-gamma priming. These data suggest that a synergy between IFN-gamma and beta-glucan may have evolved to lower the threshold of sensitivity of the innate immune response to fungal pathogens.

Enhanced immunoglobulin A response and protection against Salmonella enterica serovar typhimurium in the absence of the substance P receptor.

The development of the neurokinin-1 receptor-deficient (NK1R(-/-)) mouse permitted inquiry into the regulation of secretory immunoglobulin A (S-IgA) responses by substance P (SP) after oral immunization with a Salmonella enterica serovar Typhimurium vector expressing colonization factor antigen I (CFA/I) from enterotoxigenic Escherichia coli. In NK1R(-/-) mice, mucosal and serum IgA anti-CFA/I fimbrial responses were augmented, while secreted IgG anti-CFA/I fimbrial responses remained unaffected compared to those of BALB/c (NK1R(+/+)) mice. Supportive antibody-forming cells were present in the small intestinal lamina propria and spleen. To gain insight as to why the augmented S-IgA responses occurred, minimally, the responses were not attributed to differences in vaccine colonization of Peyer's patch (PP) and spleen or in their respective tissue weights. However, these S-IgA responses were supported by increased numbers of PP CD4(+) T helper (Th) cells secreting interleukin-5 (IL-5) and IL-6 and splenic CD4(+) Th cells secreting IL-6 compared to NK1R(+/+) mice. Challenge of naive NK1R(-/-) mice with wild-type Salmonella showed improved median survival compared to naive NK1R(+/+) mice. Data from peritoneal macrophage infection studies suggest that this survival is in part contributed by increased IL-10 production. Oral vaccination with Salmonella CFA/I or Salmonella vector showed no significant differences in conferred protection against wild-type challenge for either NK1R(-/-) or NK1R(+/+) mice. Thus, these studies suggest that SP mediation contributes to proinflammatory responses to Salmonella infections.