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J. Biol. Chem., Vol. 281, Issue 8, 4831-4843, February 24, 2006

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

Joseph Joyce¹, James Cook, Donald Chabot, Robert Hepler, Wesley Shoop^¶, Qiuwei Xu, Thomas Stambaugh^||, Miguel Aste-Amezaga, Su Wang, Lani Indrawati, Mark Bruner, Arthur Friedlander, Paul Keller, and Michael Caulfield

From the Departments of Vaccine and Biologics Research, Laboratory of Science and Investigative Toxicology and Bioprocess and Bioanalytical Research, Merck Research Laboratories, West Point, Pennsylvania 19486, United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21701, the ^¶Department of Human and Animal Infectious Disease Research, Merck Research Laboratories, Rahway, New Jersey 07065, and ^||Lockheed Martin, Edison, New Jersey 08837

The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by -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 -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--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 -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 -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.

Received for publication, August 25, 2005 , and in revised form, October 20, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Vaccine and Biologics Research, Merck Research Laboratories, P. O. Box 4, West Point, PA 19486. Tel.: 215-652-5617; Fax: 215-652-2142; E-mail: joseph_joyce{at}merck.com.

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