Cholera is an epidemic diarrheal disease caused by a bacterium, Vibrio cholerae. We are now in the seventh pandemic, due to Vibrio cholerae O1, which began in India in 1960, invaded Africa in 1970 and spread to Latin America in 1991. In 1999, cholera appeared on the island of Madagascar for the first time since the beginnining of the seventh pandemic. A new Vibrio cholerae serogroup, O139, which appeared in India in 1992 could be responsible for an eighth cholera pandemic. Cholera remains a major individual and public health problem. According to the WHO, the number of persons now vulnerable to cholera due to socioeconomic problems has dramatically increased worldwide, creating conditions favorable for a global cholera crisis.
1. Bacteriological diagnosis of cholera and non cholera vibrios (Jean-Michel FOURNIER, Marie-Laure QUILICI, Alain GUENOLE)
As most of the developed countries having high level of hygien, France is not directly concerned by cholera epidemics. However, imported cases of cholera are regularly identified in travellers. Such imported cholera cases are notified to the WHO.
Cholera epidemics do not recognize national borders. We therefore collaborate with biologists from other countries reporting cholera outbreaks, with institutes from the International Network of Institut Pasteur and with nongovernmental humanitarian organizations. These collaborations make it possible for us to study new cholera vibrio strains that might be imported into France.
The occurrence of several cholera cases in the Mayotte island during the first semester of year 2000, has leaded the National Reference Center to alert the health authorities against a risk of the development of an outbreak of cholera in this french territory in the Indian Ocean. The french gouvernement decided to immunize all the population of the island against cholera. The director of the Reference Center spent one week in Mayotte, in October 2000, in order to prepare the population to this vaccination campaign. This campaign was successful since, among estimated total of 150 000 persons, 115 000 have been vaccinated.
Clinical cases of non cholera infections that have occurred in France have been identified by the National Reference Center. These are mainly due to Vibrio cholerae non-O1/non-O139 and to Vibrio parahaemolyticus. Most of these cases are linked to direct contact with the sea or to consumption of seafood.
2. Molecular epidemiology of cholera (Marie-Laure QUILICI)
The spreading of cholera vibrio strains responsible for cholera outbreaks in Africa since 1970, year of the start of the 7th pandemic in this continent, is done by using two molecular typing methods (ribotyping and pulsotyping). Results of the study of 347 strains isolated from 34 countries are being studied.
3. New chemically defined glycoconjugate vaccine against cholera (Alain BOUTONNIER, Bruno DASSY, Jean-Michel FOURNIER)
The long-term control of cholera depends on improving hygiene by providing safe drinking water and appropriate sewage disposal, and by changing personal hygiene behavior. However, these control measures are expensive and it is difficult to change people's hygiene and water-use behavior. Hence, research to develop an effective cholera vaccine, especially in children, necessarily plays a key role in the prevention of the disease. Thus, it remains imperative to develop a new vaccine against cholera.
The starting point of our research was to determine which antibodies protect against cholera. We have shown that IgG monoclonal antibodies directed to the polysaccharide moiety of the lipopolysaccharide of Vibrio cholerae O1, are immunoprotective in an experimental model of cholera in neonatal mice. We have also shown that IgG antibodies injected intravenously in adult mice are transferred from blood vessels to the lumen of the intestine. These results explain the correlation, observed in human, between the level of serum vibriocidal antibodies and protection against cholera. These results encourage the development of vaccines based on polysaccharide-protein glycoconjugate to induce systemic IgG antibodies directed against the O-antigens of Vibrio cholerae O1 and Vibrio cholerae O139.
We purified the polysaccharide moieties of the lipopolysaccharide of Vibrio cholerae O1 and of the lipopolysaccharide and capsular polysaccharide of Vibrio cholerae O139. We then characterized the antigenic determinants expressed by these polysaccharides, using immunochemical and physicochemical methods. We also prepared glycoconjugates by coupling a carrier protein (tetanus toxoid) to the purified polysaccharide moiety of the two Vibrio cholerae strains. The immunogenicity of these conjugates was assessed in mice.
A positive result is the characterization of the antigenic determinant of serotype Ogawa of Vibrio cholerae O1. For the first time, the crystal structure of the Fab from a protective anti-cholera monoclonal antibody specific for the lipopolysaccharide antigen of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide. This work was done in collaboration with 3 laboratories from Institut Pasteur (Unité de Biochimie Structurale/CNRS URA 2185, Unité d'Immunologie Structurale, Laboratoire d'Ingénierie des Anticorps) and one laboratory from NIH (Bethesda, United States). This work has been published in PNAS in July 2000.
Another critical step is the preparation and the demonstration of immunoprotective efficacy in a murine model of a glycoconjugate vaccine composed of the polysaccharide moiety of the lippolysaccharide of Vibrio cholerae O139 bound to tetanus toxoid. These results, published in Infection and Immunity in May 2001, justify clinical evaluation of this glycoconjugate.
4. Molecular characterization of non cholera vibrios (Annick ROBERT- PILLOT, Marie-Laure QUILICI, Jean-Michel FOURNIER)
Molecular methods, such as PCR and colony hybridization, were developed to improve identification and characterization of Vibrio species pathogenic for humans. The warming of waters in certain coastal regions, due to global climate change or local industrial activities, favors the proliferation of these bacteria which may be involved in the contamination of humans, either by direct contact with seawater or following the consumption of seafood. An ecological study of vibrios isolated from the Baie de la Rance is currently done in collaboration with the Ecole Nationale de la Santé Publique in Rennes. These molecular methods are also applied to the specific detection of genes coding for pathogenicity factors in two Vibrio species of medical interest, Vibrio cholerae and Vibrio parahaemolyticus. These molecular methods are used to identify pathogenic Vibrio strains isolated from imported seafood.
Three-dimensional structure, determined in cristallography, of the complex formed by a protective monoclonal antibody (left) and the polysaccharidic moiety of the lipopolysaccharide (right) of the agent of cholera, Vibrio cholerae O1. The 2-O-methyl group (yellow sphere), at the center of the interface, plays a determining role in this binding site. This is the first time that a monoclonal antibody protective against a bacterial infection is crystallized in complex with ligands.