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

Abstract

Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis. We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we first evaluated the capacity of recombinant, histidine-tagged, nonglycosylated BclA (rBclA) given with adjuvant to protect A/J mice against 10 times the 50% lethal dose of Sterne strain spores introduced subcutaneously. Although the animals elicited anti-rBclA antibodies and showed a slight but statistically significant prolongation in the mean time to death (MTD), none of the mice survived. Similarly, rabbit anti-rBclA immunoglobulin G (IgG) administered intraperitoneally to mice before spore inoculation increased the MTD statistically significantly but afforded protection to only 1 of 10 animals. However, all mice that received suboptimal amounts of recombinant PA and that then received rBclA 2 weeks later survived spore challenge. Additionally, anti-rBclA IgG, compared to anti-PA IgG, promoted a sevenfold-greater uptake of opsonized spores by mouse macrophages and markedly decreased intramacrophage spore germination. Since BclA has some sequence similarity to human collagen, we also tested the extent of binding of anti-rBclA antibodies to human collagen types I, III, and V and found no discernible cross-reactivity. Taken together, these results support the concept of rBclA as being a safe and effective boost for a PA-primed individual against anthrax and further suggest that such rBclA-enhanced protection occurs by the induction of spore-opsonizing and germination-inhibiting antibodies.Spores of Bacillus anthracis, the causative agent of anthrax, are the infectious form of the organism and can persist in soil in a dormant stage for decades (25). Although herbivores are the primary reservoir of anthrax, humans can contract anthrax, albeit rarely, if inoculated with spores cutaneously, orally, or inhalationally (8). Although anthrax is typically seen only in individuals involved in certain occupations, the potential for infection of larger numbers of people by the aerosol route is of public health concern because of the misuse of B. anthracis spores that occurred in the United States in 2001 (9).One way to protect vulnerable individuals and populations against anthrax is through a strategy of prophylactic immunization. Currently, the anthrax vaccine adsorbed (AVA) preparation is the only licensed anthrax vaccine for use in the United States. AVA is comprised of a formalin-treated, aluminum salt-adsorbed, cell-free culture filtrate from an attenuated strain of B. anthracis (3). Although AVA is considered to be safe and effective, the utility of the vaccine is limited by its availability, reactogenicity, requirement for the administration of multiple doses (3), and the generally adverse publicity that ...

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

“…BclA has also been shown to be an immunodominant protein on the B. anthracis spore surface and contributes to the overall spore hydrophobicity (33,81). This immunogenicity provides a potential benefit for anthrax vaccine development, as immunization with protective antigen (PA) and BclA protected mice and guinea pigs from challenge with B. anthracis Sterne (pXO1 ϩ / pXO2 Ϫ ) or Ames strains better than immunization with PA alone (68,(81)(82)(83).…”

“…This immunogenicity provides a potential benefit for anthrax vaccine development, as immunization with protective antigen (PA) and BclA protected mice and guinea pigs from challenge with B. anthracis Sterne (pXO1 ϩ / pXO2 Ϫ ) or Ames strains better than immunization with PA alone (68,(81)(82)(83). Loss of BclA has little, if any, effect on sporulation rates of B. anthracis.…”

“…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).…”

Immunization of Mice with Formalin-Inactivated Spores from Avirulent Bacillus cereus Strains Provides Significant Protection from Challenge with Bacillus anthracis Ames

“…Vaccination with either component by itself provided only minor protection from s.c. challenge with B. anthracis 9602 in guinea pigs and no protection in mice (32). Studies of the efficacy of spore proteins as vaccine candidates showed that immunization with PA and BclA, BxpB/ ExsFA, or p5303 protected mice from challenge with attenuated B. anthracis Sterne (pXO1 ϩ /pXO2 Ϫ ) better than did immunization with PA alone (31,34). In a similar vaccine study conducted in mice and guinea pigs challenged with fully virulent B. anthracis Ames (pXO1 ϩ /pXO2 ϩ ), the addition of BclA, BxpB/ExsFA, and p5303 to the PA vaccine regimen enhanced protection compared to vaccination with PA alone (33).…”

“…While most of the newer vaccine candidates are still based on PA, additional research on other components of B. anthracis, such as the spore and spore components, has yielded promising results in animals (27,(30)(31)(32)(33)(34)(35). Live, attenuated spore vaccines are used safely and effectively in humans in Russia and China and in animals worldwide (36,37).…”