Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by gamma-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free alpha-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to > 95% homogeneity and have demonstrated that the polymer contains > 99% poly-gamma-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the alpha-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5) triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of alpha-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the reagent-induced reduction of polymer mass. The data suggested that for vaccine development, maintenance of the high mass of the polymer may be important.