Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen

Abstract

Inactivated Bacillus anthracis spores given with protective antigen (PA) contribute to immunity against anthrax in several animal models. Antiserum raised against whole irradiated B. anthracis spores has been shown to have anti-germination and opsonic activities in vitro. Based on these observations, we hypothesized that surface-exposed spore proteins might serve as supplemental components of a PA-based anthrax vaccine. The protective anti-spore serum was tested for reactivity with recombinant forms of 30 proteins known, or believed to be, present within the B. anthracis exosporium. Eleven of those proteins were reactive with this antiserum, and, subsequently a subset of this group was used to generate rabbit polyclonal antibodies. These sera were evaluated for recognition of the immunogens on intact spores generated from Sterne strain, as well as from an isogenic mutant lacking the spore surface protein Bacillus collagen-like antigen (BclA). The data were consistent with the notion that the antigens in question were located beneath BclA on the basal surface of the exosporium. A/J mice immunized with either the here-to-for hypothetical protein p5303 or the structural protein BxpB, each in combination with subprotective levels of PA, showed enhanced protection against subcutaneous spore challenge. While neither anti-BxpB or anti-p5303 antibodies reduced the rate of spore germination in vitro, both caused increased uptake and lead to a higher rate of destruction by phagocytic cells. We conclude that by facilitating more efficient phagocytic clearance of spores, antibodies against individual exosporium components can contribute to protection against B. anthracis infection.

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

“…Therefore, my colleagues and I have proposed a model whereby BxpB is delivered to the spore by virtue of forming a complex with BclA and possibly, to a lesser extent, with other exosporium-targeting motif sequences (58). In keeping with this, we observed a reduction in the amount of BxpB extractable from spores of a BclA-negative strain, a finding similar to that observed by others (65,66,68). However, this model is disputed by Rodenburg et al (39), based on their analysis of B. anthracis spore structure by high-resolution electron microscopy.…”

“…Inclusion of this 133-residue protein of unknown function enhanced the efficacy of an anti-protective antigen (anti-PA) vaccine in a mouse model of infection, and anti-BAS5303 antibodies were found to increase phagocytic uptake of spores (68). Although the location of this protein has not been mapped precisely, it was reported to likely lie below the BclA nap layer on the exosporium, based on increased exposure to antibodies in the absence of BclA (68). The bas5303 determinant is monocistronic and expressed at a very late stage of sporulation (73).…”

Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model

“…Recent studies exposed BclA as not being the only relevant immunogen of the spore and further defined its role rather as means of masking these other, possibly much more relevant spore antigens (Cybulski et al, 2008, 4927-4939, Cote et al, 2012, 1380-1392. Taking the lack of reactivity against BclA in goats into account, this might be a much more pressing issue in this animal model and should be considered in future live and acellular vaccine trials.…”

Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins

“…Although the isolated sdAbs were specific for B. anthracis, EA1 has been shown to be a spore contaminant and not an integral component of the bacterial spore [17]. The two llamas were subjected to a second series of immunizations with a combination of recombinant spore-specific proteins: BclA, gerQ, SODA1, SOD15, BxpB, and p5303 [18,19]. Centavo received a mix of all six proteins; while Whisper was immunized with a cocktail that did not include the BclA protein in an attempt to allow a stronger immune response toward the other spore proteins that may not be as immunodominant as the BclA antigen.…”