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

Both the protective antigen (PA) and the poly(gamma-d-glutamic acid) capsule (gamma 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-gamma 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 gamma 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 gamma dPGA, Bacillus pumilus gamma 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-gamma dPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-gamma dPGA were elicited by decamers of gamma dPGA at 10 -20 mol per protein bound to the N- or C-terminal end. High IgG anti-gamma dPGA levels were elicited by two injections of 2.5 microg of gamma dPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies. rPA was the most effective carrier. Anti-gamma dPGA induced opsonophagocytic killing of B. anthracis tox-, cap+. gamma dPGA conjugates may enhance the protection conferred by PA alone. gamma dPGA-rPA conjugates induced both anti-PA and anti-gamma dPGA.