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.

FIG. 1.

Time to death of A/J mice after immunization with rBclA (alone) followed 4 weeks later by subcutaneous challenge with 10 LD₅₀s of B. anthracis Sterne strain 34F2 spores (∼10⁴ spores). The MTD for the vaccinated group was 4.8 days; the MTD for the group given PBS (mixed 1:1 with Titermax) was 4.1 days. This difference in the MTD was statistically significant (P = 0.0045) by the Kaplan-Meier test. The average anti-rBclA ELISA OD₄₅₀ response for a 1:100 serum dilution of the rBclA-immunized mouse group was 1.6 (data not shown).

FIG. 2.

Impact of administration of rabbit anti-rBclA IgG given 1 h before subcutaneous challenge of A/J mice with 10 LD₅₀s of B. anthracis Sterne strain 34F2 spores (∼10⁴ spores) on animal survival. The MTD of mice given PBS or passively administrated normal rabbit IgG before spore challenge was 4.3 days for each group, compared to 6.8 days for mice that received rabbit anti-rBclA IgG. This difference in MTD was statistically significant as assessed by the Kaplan-Meier test (P = 0.0002).

FIG. 3.

Protection of A/J mice primed with suboptimal amounts of PA (mixed 1:1 with Titermax) and boosted with rBclA (mixed 1:1 with Titermax) from subcutaneous challenge with 10 LD₅₀s of B. anthracis Sterne strain 34F2 spores (∼10⁴ spores). A/J mice were injected with 50 ng of rPA or PBS as a control (each mixed 1:1 with Titermax), which was followed 2 weeks later with PBS or 10 μg of rBclA (each mixed 1:1 with Titermax). Spores were administered 2 weeks after the rBclA or PBS boost. (A and B) Individual mouse antibody ELISA OD₄₅₀ responses against rPA (A) or rBclA (B). (C) Survival curve after subcutaneous challenge with 10 LD₅₀s of B. anthracis Sterne strain 34F2 spores (∼10⁴ spores) (P = 0.001). Each circle represents the average of triplicate OD₄₅₀ values of a 1:100 dilution of postimmune serum from which average preimmune values were subtracted for that mouse. Horizontal bars represent the average OD₄₅₀ reading of that immunization group.

FIG. 4.

Enhanced opsonophagocytosis and killing of B. anthracis Sterne strain 34F2 spores by mouse peritoneal macrophages when spores were incubated with rBclA IgG. (A) Uptake of spores treated with either anti-PA IgG, anti-rBclA IgG, complement, or nothing by macrophages after 1 h of incubation. (B) Intracellular germination of spores treated as described above after 1 h of incubation with macrophages. (C) Intramacrophage germination of treated spores over time (1-h time point shown in B is included here for estimation of rates). Data in A to C were obtained from three independent experiments, each done in duplicate. Data in A and B are shown as means ± standard deviations of values.

FIG. 5.

Absence of cross-reactivity between anti-rBclA antibodies and human collagen. (A) Anti-human-collagen (types I, III, and V) antibodies did not bind to spores, SSPE, or rBclA. (B) Antibodies made by inoculation of spores or rBclA did not cross-react with human collagen types I, III, and V. Abbreviations: RPAb, anti-rBclA rabbit polyclonal antibody; αI, anti-collagen I antibody; αIII, anti-collagen III antibody; αV, anti-collagen V antibody; 733, rabbit antispore antibody; EG4, monoclonal anti-BclA antibody. Samples were tested in triplicate. Bars shown are means ± standard deviations of values.