Immunogenicity and protective efficacy of Bacillus anthracis poly-gamma-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 gamma-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.
Joyce
Joseph
J
Department of Vaccine and Biologics Research, Laboratory of Science and Investigative Toxicology and Bioprocess and Bioanalytical Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. joseph.joyce@merck.com
Cook
James
J
Chabot
Donald
D
Hepler
Robert
R
Shoop
Wesley
W
Xu
Qiuwei
Q
Stambaugh
Thomas
T
Aste-Amezaga
Miguel
M
Wang
Su
S
Indrawati
Lani
L
Bruner
Mark
M
Friedlander
Arthur
A
Keller
Paul
P
Caulfield
Michael
M
D000818:Animals; D022122:Anthrax Vaccines; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000378:metabolism*; D016667:Bacterial Capsules; D055592:Biophysical Phenomena; D001703:Biophysics; D002234:Carbodiimides / Q000737:chemistry; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C; D009025:Morpholines / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011099:Polyglutamic Acid / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012542:Scattering, Radiation; D013171:Spores, Bacterial; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014227:Triazines / Q000494:pharmacology*
D022122:Anthrax Vaccines; D000942:Antigens, Bacterial; D002234:Carbodiimides; D009025:Morpholines; D010455:Peptides; D011108:Polymers; D000644:Quaternary Ammonium Compounds; D013438:Sulfhydryl Compounds; D014227:Triazines; C083125:Stymulen; D011099:Polyglutamic Acid; D018698:Glutamic Acid