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

¹ Laboratory of Food Microbiology and Immunology, Research Center for Animal Hygiene and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido 080-8555, Japan
² Department of Pathobiological Science, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido 080-8555, Japan
³ Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka 4, Toyohira-Ku, Sapporo, Hokkaido 062-0045, Japan

Correspondence
Keiko Kawamoto
kkeiko{at}obihiro.ac.jp

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.

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