Poly( -D-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: A potential addition to the anthrax vaccine (original) (raw)

2003, Proceedings of the National Academy of Sciences

Both the protective antigen (PA) and the poly(␥-D-glutamic acid) capsule (␥DPGA) are essential for the virulence of Bacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-␥DPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic ␥DPGA or corresponding synthetic peptides were bound to BSA, recombinant B. anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA). To identify the optimal construct, conjugates of B. anthracis ␥DPGA, Bacillus pumilus ␥DLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the D or L configuration with active groups at the N or C termini, were bound at 5-32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5-to 6-week-old mice. IgG anti-␥DPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-␥DPGA were elicited by decamers of ␥DPGA at 10-20 mol per protein bound to the Nor C-terminal end. High IgG anti-␥DPGA levels were elicited by two injections of 2.5 g of ␥DPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies. rPA was the most effective carrier. Anti-␥DPGA induced opsonophagocytic killing of B. anthracis tox؊, cap؉. ␥DPGA conjugates may enhance the protection conferred by PA alone. ␥DPGA-rPA conjugates induced both anti-PA and anti-␥DPGA. A nthrax probably caused the ''festering boils'' of the people and cattle of Egypt described in the sixth plague of the Old Testament. After the discovery of Bacillus anthracis by Robert Koch in 1880 (1), Pasteur (2) developed a vaccine for sheep composed of chemically treated attenuated strains. Routine use of a noncapsulated strain has virtually eliminated anthrax among domesticated animals (3). In the only controlled study of an anthrax vaccine in humans, culture-supernatant from a capϪ nonproteolytic strain that produced protective antigen (PA), conferred 92% efficacy among woolsorters (4). The Centers for Disease Control monitored the anthrax vaccine adsorbed (AVA) in industrial settings between 1962 and 1974: none of 34 cases occurred in fully vaccinated individuals. A similar vaccine is used in the U.K. (5). This and other evidence indicate that serum IgG anti-PA confers immunity to cutaneous and inhalational anthrax in humans (6, 7). The structure and expression of the essential virulence factors of B. anthracis are controlled by two plasmids. pX01 encodes anthrax toxin (AT) composed of the PA (binding subunit of AT), and two enzymes known as lethal factor and edema factor (8, 9). Administration of AT to primates mimics the symptoms of anthrax (9). pX02 encodes the poly(␥-D-glutamic acid) (␥DPGA) capsule of B. anthracis (10, 11). Other bacilli produce poly(␥glutamic acid) (␥PGA) but only B. anthracis synthesizes it entirely in the D conformation (12). ␥DPGA is a surface structure (13), inhibits in vitro phagocytosis and, when injected, is a poor immunogen even as a bacterial component (14-18); the protective effect of anti-␥DPGA has not been reported. The capsule shields the vegetative form of B. anthracis from agglutination by monoclonal antibodies to its cell wall polysaccharide (19). Systemic infection with B. anthracis induces ␥DPGA antibodies (20). Antibodies to D-amino acid polymers may be induced in animals by injection of ␥DPGA methylated BSA complexes along with Freund's adjuvant, i.v. injections of a formalin-treated capsulated B. anthracis, or by peptidyl proteins (16, 21). We report the synthesis and evaluation of conjugates that induce ␥DPGA antibodies under conditions suitable for clinical use. Experimental Procedures Bacterial Strains. Bacillus pumilus strain Sh18 and B. anthracis strain A34, a pX01Ϫ, pX0 2ϩ variant derived from the Ames strain by repeated passage at 43°C, have been described (10, 22). Analytic. Amino acid analyses were done by GLC-MS after hydrolysis with 6 M HCl, 150°C, 1 h, derivatization to heptafluorobutyryl R-(Ϫ)isobutyl esters, and assayed with a Hewlett-Packard apparatus (model HP 6890) with a HP-5 0.32 ϫ 30 mm glass capillary column, temperature programming at 8°C per min, from 125°C to 250°C in the electron ionization (106 eV) mode (24). Under these conditions, we could separate Dglutamic acid from the L-enantiomer. The amount of each was calculated based on the ratio of D-glutamic acid relative to L-glutamic acid residues in the protein (Fig. 1). The number of peptide chains in L-peptide conjugates was calculated by the increase of total L-glutamic acid relative to aspartic acid. Protein concentration was measured by the method of Lowry (25), free amino groups were measured by Fields' assay (26), thiolation was measured by release of 2-pyridylthio groups (A 343) (27), and hydrazide was measured as reported (28). SDS͞PAGE used 14% gels according to the manufacturer's instructions. Double immunodiffusion was performed in 1.0% agarose gel in PBS. Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF). Mass spectra were obtained with a PerSeptive BioSystems Voyager Elite DE-STR MALDI-TOF instrument (Applied Biosystems) operated in the linear mode, 25-kV accelerating voltage, and a 300-nsec ion extraction delay time. Samples for analysis were prepared by a ''sandwich'' of matrix and analyte. First, 1 l of matrix (saturated solution of sinnapinic acid made