Alain Boutonnier

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

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.

Regulation of Staphylococcus aureus CapsularPolysaccharideType 5: CO2 InhibitionIn Vitro andIn Vivo

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats, and in vitro using ELISA and IFA. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1%-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1%-5% of CP5-positive cells were detectable in pouch exudates. CF and pouch isolates, however, reexpressed CP5 (70%-90% of cells) when grown in vitro with air. Addition of > or = 1% CO2 to air or to O2/N2 gas mixtures reduced CP5 expression significantly (P < .001) in a dose-dependent manner (6%-1% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal that regulates CP5 expression.

Immunochemical characterization of an Ogawa-Inaba common antigenic determinant of Vibrio cholerae O1

Cholera remains an important public health problem in many parts of the world and the availability of an effective cholera vaccine is important for the prevention of cholera in the countries affected by this disease. Despite the appearance in 1992 of a new serogroup, 0139, of Vibrio cholerae, most of the cholera outbreaks are still caused by V. cholerae O1 biotype El Tor. Vaccine trials in Asia from 1968 to 1971, and studies of the production of serotype-specific antiserum in rabbits and of the protective activity of monoclonal antibodies against diarrhoeal disease in neonatal mice, have led to the conclusion that the Ogawa serotype contains a specific antigenic determinant whereas the Inaba serotype contains a different antigenic determinant that cross-reacts with the Ogawa serotype. By studying the binding of anti-Ogawa monoclonal antibodies to synthetic oligosaccharide fragments mimicking the Ogawa O-specific polysaccharide, it has been shown that the terminal monosaccharide, bearing the 2-O-methyl group in the O-specific polysaccharide, is most probably the serotype-specific determinant for the Ogawa strain. However, study of the binding of a monoclonal antibody recognizing both Ogawa and Inaba serotypes suggested partial recognition of the core as well as of the O-specific polysaccharide of the LPS of V. cholerae O1. To further characterize this antigenic determinant that is common to the Ogawa and Inaba serotypes, the core and the O-specific polysaccharide linked to the core of V. cholerae O1 LPS were purified by preparative electrophoresis. The O-specific polysaccharide linked to the core was subjected to periodate oxidation to destroy sugars from the core. Binding studies of these purified saccharide fragments to a monoclonal antibody which is protective in mice and specific to the antigenic determinant common to Ogawa and Inaba serotypes showed that both the core and the O-specific polysaccharide are involved in this common antigenic determinant. This explains how the presence or the absence of the Ogawa-specific antigenic determinant would lead to the expression of two independent antigenic determinants of V. cholerae O1, one specific to the Ogawa serotype and the other common to both Ogawa and Inaba serotypes.

Low West Nile virus circulation in wild birds in an area of recurring outbreaks in Southern France.

West Nile virus (WNV) has a history of irregular but recurrent epizootics in countries of Mediterranean and of Central and Eastern Europe. We have investigated the temporal enzootic activity of WNV in free-ranging birds over a 3-year period in an area with sporadic occurrences of WNV outbreaks in Southern France. We conducted an intensive serologic survey on several wild bird populations (>4000 serum samples collected from 3300 birds) selected as potential indicators of the WNV circulation. WNV antibodies were detected by seroneutralization and/or plaque reduction neutralization in house sparrows, black-billed magpies, and scops owls, but these species appeared to be insufficient indicators of WNV circulation. Overall seroprevalence was low (<1%), including in birds that had been potentially exposed to the virus during recent outbreaks. However, the detection of a seroconversion in one bird, as well as the detection of seropositive birds in all years of our monitoring, including juveniles, indicate a constant annual circulation of WNV at a low level, including in years without any detectable emergence of WN fever in horses or humans.

Investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of Vibrio cholerae O1, serotype Inaba

A free amino group present on the acid-detoxified lipopolysaccharide (pmLPS) of V. cholerae O1 serotype Inaba was investigated for site-specific conjugation. Chemoselective pmLPS biotinylation afforded the corresponding mono-functionalized derivative, which retained antigenicity. Thus, pmLPS was bound to carrier proteins using thioether conjugation chemistry. Induction of an anti-LPS antibody (Ab) response in BALB/c mice was observed for all conjugates. Interestingly, the sera had vibriocidal activity against both Ogawa and Inaba strains opening the way to a possible bivalent vaccine. However, the level of this Ab response was strongly affected by both the nature of the linker and of the carrier. Furthermore, no switch from IgM to IgG, i.e. from a T cell-independent to a T cell-dependent immune response was detected, a result tentatively explained by the possible presence of free polysaccharide in the formulation. Taken together, these results encourage further investigation towards the development of potent pmLPS-based neoglycoconjugate immunogens, fully aware of the challenge faced in the development of a cholera vaccine that will provide efficient serogroup coverage.

Crystal structure of a monoclonal antibody directed against an antigenic determinant common to Ogawa and Inaba serotypes of Vibrio cholerae O1

Crystal structure of a monoclonal antibody directed against an antigenic determinant common to Ogawa and Inaba serotypes of Vibrio cholerae O1 Firoz Ahmed, Gwénaëlle André-Leroux, Ahmed Haouz, Alain Boutonnier, Muriel Delepierre, Firdausi Qadri, Farida Nato, Jean-Michel Fournier, and Pedro M. Alzari* 1 Institut Pasteur and CNRS URA 2185, 25/28 rue du Dr. Roux, Paris, France 2 International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh