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

ABSTRACT 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 capsulatedV. 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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Immunomodulative properties of conjugates composed of detoxified lipopolysaccharide and capsular polysaccharide of Vibrio cholerae O135 bound to BSA-protein carrier

Biologia ◽

10.2478/s11756-010-0092-9 ◽

2010 ◽

Vol 65 (5) ◽

Cited By ~ 6

Author(s):

Jana Korcová ◽

Eva Machová ◽

Pavol Farkaš ◽

Slavomír Bystrický

Keyword(s):

Vibrio Cholerae ◽

Capsular Polysaccharide ◽

Size Exclusion ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibodies ◽

Polymer Carrier ◽

Protein Carrier ◽

Exclusion Chromatography ◽

Polysaccharide Antigens ◽

Surface Polysaccharide

AbstractO135 serotype Vibrio cholerae isolated from Slovak river was used as a source of surface polysaccharide antigens. Following detoxification procedure, fractions of polysaccharides were separated by size exclusion chromatography. Two resultant fractions were the capsular polysaccharide (M w ∼ 197,000 Da) and the lipopolysaccharide fragment (M w ∼ 13,300 Da). These materials were used for preparation of four novel glycoconjugates. Two of them containing detoxified lipopolysaccharide as antigen were prepared by original chemical method using the new biocompatible polymer as carrier of antigen. Additionally, other two conjugates were prepared by direct linking of capsular and detoxified lipopolysaccharide antigens to the protein carrier using adipic acid dihydrazide spacer. The immunogenicities (induced IgM, IgG, IgA antibodies) of all conjugates were determined by enzyme-linked immunosorbent assay. Polymer containing conjugates elicited higher levels of specific anti-lipopolysaccharide IgM and IgG antibodies in comparison with other conjugates without polymer carrier. Enhanced IgM vibriocidal activity of mice antisera was also evident here.

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Preparation, Immunogenicity, and Protective Efficacy, in a Murine Model, of a Conjugate Vaccine Composed of the Polysaccharide Moiety of the Lipopolysaccharide of Vibrio cholerae O139 Bound to Tetanus Toxoid

Infection and Immunity ◽

10.1128/iai.69.5.3488-3493.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3488-3493 ◽

Cited By ~ 51

Author(s):

Alain Boutonnier ◽

Sylvain Villeneuve ◽

Farida Nato ◽

Bruno Dassy ◽

Jean-Michel Fournier

Keyword(s):

Vibrio Cholerae ◽

Tetanus Toxoid ◽

Capsular Polysaccharide ◽

Protective Efficacy ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibodies ◽

Vibrio Cholerae O139 ◽

Specific Polysaccharide ◽

Antibody Levels

ABSTRACT The epidemic and pandemic potential of Vibrio choleraeO139 is such that a vaccine against this newly emerged serogroup ofV. cholerae is required. A conjugate made of the polysaccharide moiety (O-specific polysaccharide plus core) of the lipopolysaccharide (LPS) of V. cholerae O139 (pmLPS) was prepared by derivatization of the pmLPS with adipic acid dihydrazide and coupling to tetanus toxoid (TT) by carbodiimide-mediated condensation. The immunologic properties of the conjugate were tested using BALB/c mice injected subcutaneously three times at 2 weeks interval and then a fourth time 4 weeks later. Mice were bled 7 days after each injection and then once each month for the following 6 months. LPS and TT antibody levels were determined by enzyme-linked immunosorbent assay using immunoplates coated with either O139 LPS or TT. Both pmLPS and pmLPS-TT conjugate elicited low levels of immunoglobulin M (IgM), peaking 5 weeks after the first immunization. The conjugate elicited high levels of IgG antibodies, peaking 3 months after the first immunization and declining slowly during the following 5 months. TT alone, or as a component of conjugate, induced mostly IgG antibodies. Antibodies elicited by the conjugate recognized both capsular polysaccharide and LPS from V. cholerae O139 and were vibriocidal. They were also protective in the neonatal mouse model of cholera infection. The conjugation of the O139 pmLPS, therefore, enhanced its immunogenicity and conferred T-dependent properties to this polysaccharide.

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Reduction in Capsular Content and Enhanced Bacterial Susceptibility to Serum Killing of Vibrio cholerae O139 Associated with the 2002 Cholera Epidemic in Bangladesh

Infection and Immunity ◽

10.1128/iai.73.10.6577-6583.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6577-6583 ◽

Cited By ~ 17

Author(s):

Firdausi Qadri ◽

Ann-Mari Svennerholm ◽

Sohel Shamsuzzaman ◽

Taufiqur Rahman Bhuiyan ◽

Jason B. Harris ◽

...

Keyword(s):

Vibrio Cholerae ◽

Capsular Polysaccharide ◽

Bactericidal Effect ◽

Electron Microscopic ◽

Recent Infection ◽

Phenotypic Properties ◽

Immunological Responses ◽

Serum Killing ◽

Vibrio Cholerae O139 ◽

Microscopic Studies

ABSTRACT Vibrio cholerae O139 emerged in 1992 as a major cause of epidemic cholera. However, the incidence of disease due to this new serogroup subsequently decreased for almost a decade. In April 2002, there was a dramatic resurgence of V. cholerae O139 in Bangladesh. We compared the phenotypic properties of the bacterial isolates and the immunological responses in patients with disease due to V. cholerae O139 during the 2002 epidemic with those dating to the emergence of this disease in 1993 to 1995. Strains isolated from patients in the two time periods were compared with respect to capsular polysaccharide, their resistance to the bactericidal effect of serum, and their capacity to be used as target strains in complement-mediated vibriocidal assays. Phase-contrast microscopy showed that strains isolated in 2002 had less capsular material than those isolated from 1993 to 1995 (P = <0.001), a finding confirmed by electron microscopic studies. Strains isolated in 2002 were more susceptible to the bactericidal activity of serum compared to strains from 1993 to 1995 (P = 0.013). Compared to results using a standard O139 strain, a modified vibriocidal assay utilizing a 2002 strain, CIRS 134, as the target organism detected higher vibriocidal responses in both O139-infected cholera patients as well as O139 vaccine recipients. The vibriocidal assay utilizing the less encapsulated 2002 strain, CIRS 134, is a more sensitive indicator of adaptive immune responses to recent infection with V. cholerae O139. Consequently, this assay may be useful in studies of both O139-infected patients and recipients of O139 vaccines.

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