Additional conjugation methods and immunogenicity of Bacillus anthracis poly-gamma-D-glutamic acid-protein conjugates

The capsule of Bacillus anthracis, composed of poly-gamma-d-glutamic acid (gammaDPGA), is an essential virulence factor of B. anthracis. The capsule inhibits innate host defense through its antiphagocytic action. gammaDPGA is a poor immunogen, but when covalently bound to a carrier protein, it elicits serum antibodies. To identify the optimal construct for clinical use, synthetic gammaDPGAs of different lengths were bound to carrier proteins at different densities. The advantages of the synthetic over the natural polypeptide are the homogeneous chain length and end groups, allowing conjugates to be accurately characterized and standardized and their chemical compositions to be related to their immunogenicities. In the present study, we evaluated, in addition to methods reported by us, hydrazone, oxime, and thioether linkages between gammaDPGA and several proteins, including bovine serum albumin, recombinant Pseudomonas aeruginosa exotoxin A, recombinant B. anthracis protective antigen (rPA), and tetanus toxoid (TT). The effects of the dosage and formulation on the immunogenicities of the conjugates were evaluated in mice. All conjugates were immunogenic. The optimal gammaDPGA chain length of 10 to 15 amino acids and the density, an average of 15 mol gammaDPGA per mol of protein, were confirmed. The thioether bond was the optimal linkage type, and TT and rPA were the best carriers. The optimal dosage was 1.2 to 2.5 microg of gammaDPGA per mouse, and adsorption of the conjugates onto aluminum hydroxide significantly increased the antibody response to the protein with a lesser effect on anti-gammaDPGA levels.