Poly-γ-d-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule ofBacillus anthracisin guinea-pigs and rabbits

Abstract

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis. The two major virulence factors of B. anthracis are exotoxin and the poly-gamma-d-glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA-PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 x LD(50) challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA-PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.

The Poly-γ- d -Glutamic Acid Capsule of Bacillus anthracis Enhances Lethal Toxin Activity

“…The capsule produced by B. licheniformis ATCC 9945a has been used as a surrogate for B. anthracis in several studies (17,18,29). However, unlike B. anthracis, which has D-glutamic acid residues exclusively, B. licheniformis 9945a contains both D-and L-enantiomers according to culture conditions (3).…”

“…However, unlike B. anthracis, which has D-glutamic acid residues exclusively, B. licheniformis 9945a contains both D-and L-enantiomers according to culture conditions (3). In our experiment, E medium, which contains 2 mM MnCl 2 ⅐ 4H 2 O, was used to stimulate the maximal production of PGA in the D isoform (18,19,33). Usually under these conditions, approximately 84% of the glutamic acid was D-enantiomer.…”

“…Usually under these conditions, approximately 84% of the glutamic acid was D-enantiomer. PGA was purified from the supernatant of B. licheniformis cultures as described previously (18,29). B. anthracis has been reported to produce a high-molecular-mass capsule of Ͼ100 kDa, which first is polymerized on the bacterial cell surface.…”

“…The weakly immunogenic and antiphagocytic PGA capsule disguises the bacilli from immune surveillance through a mechanism that is similar to that of the capsular polysaccharides that protect other pathogens, such as pneumococci and meningococci, from phagocytosis (21). The immunogenicity of PGA, like other T-cell-independent polysaccharide antigens, is enhanced when it is conjugated with other proteins, such as PA (9,18,29). A recent report showed that the capsule of B. anthracis activates caspase-1 and induces the release of interleukin-1␤ (IL-1␤) from differentiated THP-1 cells and from human monocyte-derived dendritic cells (11).…”

“…We used PGA that was purified from the culture supernatant of Bacillus licheniformis strain ATCC 9945a (18,29). PGA enhanced the cytotoxic effect of LT on J774A.1 cells in a concentration-dependent manner and also augmented the death of mice challenged with LT. Our experimental results indicate that PGA, in combination with LT, is important in anthrax pathogenesis and acts to intensify the toxemia that occurs at the terminal stage of anthrax infection.…”