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Infection and Immunity, September 2000, p. 5037-5043, Vol. 68, No. 9
0019-9567/00/$04.00+0

Vibrio cholerae O139 Conjugate Vaccines: Synthesis and Immunogenicity of V. cholerae O139 Capsular Polysaccharide Conjugates with Recombinant Diphtheria Toxin Mutant in Mice

Zuzana Kossaczka,^1,* Joseph Shiloach,² Virginia Johnson,³ David N. Taylor,⁴ Richard A. Finkelstein,⁵ John B. Robbins,¹ and Shousun C. Szu¹

National Institute of Child Health and Human Development¹ and National Institute of Diabetes, Digestive and Kidney Diseases,² National Institutes of Health, and Center for Biologics Evaluation and Research,³ Bethesda, Maryland; Walter Reed Army Institute For Research, Washington, D.C.⁴; and Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri⁵

Received 25 February 2000/Returned for modification 15 May 2000/Accepted 9 June 2000

Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to 50. Treatment with 2-ME reduced the titers of 17 of 20 patients to 50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned.

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^* Corresponding author. Mailing address: National Institute of Child Health and Development, National Institutes of Health, Bldg. 6, Rm. 424, Bethesda, MD 20892-2720. Phone: (301) 496-1185. Fax: (301) 402-9108. E-mail: kossaczz{at}mail.nih.gov.

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Infection and Immunity, September 2000, p. 5037-5043, Vol. 68, No. 9
0019-9567/00/$04.00+0

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