Significant passive protective effect against anthrax by antibody to Bacillus anthracis inactivated spores that lack two virulence plasmids

Abstract

The protective-antigen (PA)-based cell-free vaccine is the only vaccine licensed for use against Bacillus anthracis infection in humans. Although the PA shows strong immunogenicity, the capsule or spore-associated somatic antigens may be important as additional vaccine targets for full protection against anthrax. In this study, the protective effect of spore-associated antigens against B. anthracis infection was determined. Rabbits were immunized with formalin-fixed spores of a non-toxigenic unencapsulated B. anthracis strain that lacked the two virulence plasmids pXO1 and pXO2, and the protective effects of the immune antibody were evaluated. Immunostaining and Western blot analysis revealed that the anti-B. anthracis (anti-BA)-spore IgG specifically bound to the surface of spores or endospores of B. anthracis, but not to vegetative cells, or closely related Bacillus species, such as Bacillus cereus, Bacillus subtilis and Bacillus thuringiensis. Passively transferred anti-BA-spore IgG protected mice from intraperitoneal challenge with a lethal dose of fully virulent B. anthracis spores, and increased the survival rate in a dose-dependent manner. Pre-incubation of spores with antibody also reduced their infectivity in a dose-dependent manner. The number of bacteria (c.f.u.) in spleens and livers of infected mice was significantly lower in antibody-treated mice than in untreated mice. Treatment with anti-BA-spore IgG also inhibited the germination of spores in J774.1 macrophages, suggesting that opsonization of spores promotes phagocytosis and subsequent killing by macrophages. These results indicate the usefulness of spore surface antigens as vaccine targets. In combination with major virulence factors such as the PA, spore-associated antigens may offer a safer and more effective multicomponent vaccine for B. anthracis infection.

Recombinant Exosporium Protein BclA of Bacillus anthracis Is Effective as a Booster for Mice Primed with Suboptimal Amounts of Protective Antigen

“…The importance of the latter observation cannot be overstated because spore germination within macrophages is considered to be a key step in the pathogenesis of anthrax. In support of the possible use of this spore surface-based antigen as a boost for a PA-based antianthrax vaccine for humans, we detected little or no cross-reactivity between rBclA and human collagen types I, III, and V. Our observation that anti-rBclA IgG led to a prolongation in the MTD of anthrax spore-challenged mice is consistent with data from a recent report by Enkhtuya et al (11). Those investigators demonstrated protection of mice from anthrax by passive immunization of the animals with anti-spore IgG isolated from immune sera of rabbits inoculated multiple times with formalin-fixed spores of a nonencapsulated, nontoxinogenic B. anthracis mutant strain.…”

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…Studies involving passive immunization with spore-specific antisera have produced mixed results. Protection has been reported in some studies but could not be demonstrated in others (148,(157)(158)(159). The distinct difference between the unambiguous benefit obtained with active immunization with PA and spore components and the less clear-cut results obtained via passive immunization involving anti-sporecomponent antibodies underscores the likely involvement of cellular immunity in protection (159).…”

“…Animal studies of the efficacy of the human vaccine found that protection against the most virulent strains of B. anthracis required the addition of spore antigens to the vaccination regimen (144)(145)(146)(147). Improved vaccine protection was observed when the added spore antigens included inactivated whole spores (147)(148)(149)(150), live attenuated spores (146,(151)(152)(153)(154), BclA (81,83,148,155), BxpB (68,148), and BAS5303 (68,148). Antibodies directed against whole spores or, specifically, BclA have been shown to be inhibitory to spore germination in vitro (148,156).…”

Efficacy of a Vaccine Based on Protective Antigen and Killed Spores against Experimental Inhalational Anthrax

“…For instance, PA-based vaccines confer better protection to guinea pigs, rabbits, and nonhuman primates than to mice, probably because the ␥PDGA capsule is the primary virulence factor in mice (87). Indeed, many reports suggest that capsule antigen(s) (13,47,64,67,81) and spore antigen(s) (10,16,23) might confer additional protection. An immunodominant glycoprotein antigen of the spore surface (BclA) has been identified among the various surface proteins of the exosporium and may contribute to protective immunity (72,74).…”