Vibro cholerae O139 : Identification, characterization and vaccin strategies

Sammanfattning: Cholera is an acute dehydrating diarrheal disease, and epidemics as well as pandemics of cholera are a major health problem in developing countries and cause many deaths each year, especially in children. Cholera is caused by Vibrio cholerae serogroup, 01 and since 1992 also by serogroup 0139. The emergence of V. cholerae 0139 marked a turning point in the history of cholera - before it was believed that serogroups other than 01 were not able to cause epidemics, only isolated cases of diarrhea and extraintestinal infections. A V. cholerae 0139 specific DNA region was isolated and characterized by arbitrary PCR. The fragment contains open reading frames encoding two potential glycosyltransferases possibly involved in capsular polysaccharide and/or lipopolysaccharide biosynthesis. In order to evaluate the possibility that this region could be used for the specific detection of V. cholerae 0139, a PCR system was established. The specificity and sensitivity of the assay was investigated by analyzing 240 strains within the family Vibrionaceae and 178 strains of other Gram-negative bacteria. All V. cholerae 0139 strains tested were positive, and none of the 384 control strains were amplified. The sensitivity of the assay was 102 CFU ml-1. PCR with the same primer pair was used to screen 180 diarrheal stool specimens. All the 67 V. cholerae 0139 culture-positive stool specimens were positive by PCR, and the remaining specimens, which contained either other recognized enteric pathogens or no pathogens, were all negative by PCR. V. cholerae 0139 arose from a V. cholerae 01 strain by deletion of the chromosomal region encoding 01 specificity and acquisition of a novel 35-kb region encoding the 0139 specificity. Previous studies indicated significant phenotypic and genotypic changes in 0139 isolates over the years since its first appearance. This prompted us to study possible polymorphism in the 35-kb novel region encoding the 0139 specificity. A total of 17 V. cholerae 0139 isolates originating from different countries and years in South Asia and China, and a single unrelated V. cholerae 0139 isolate from Argentina were studied. The 35-kb chromosomal region was amplified as two fragments of 12- and 23-kb regions in an extended PCR from all isolates. The amplified products were digested with restriction enzymes and the fragment patterns were compared to detect polymorphism. The results indicated that the portion of the chromosome encoding the 0139 antigen specificity is highly conserved. This implies that alterations in this region might affect the serogroup specificity of the strain. The V cholerae 0139 has the potential of being responsible for the eighth pandemic of cholera and a vaccine against this serogroup would be of great importance. Prior exposure to V. cholerae 01 does not protect against infection with V. cholerae 0139, thus the 0139 strain is expected to have a selective advantage in regions where 01 infections predominate. The capsular polysaccharide of the 0139 serogroup is a T cell independent antigen and does not give rise to immunologic memory. To circumvent this problem, our approach was to select peptides mimicking the V. cholerae 0139 capsular polysaccharide. Monoclonal antibodies against the capsular polysaccharide of V. cholerae 0139 were used to screen different phage-displayed random peptide libraries. Eight different phage clones were selected, characterized using EIA with the mAbs, and then tested for specificity by competition with V. cholerae 0139 capsular polysaccharide. Selected peptides have been sequenced, synthesized, conjugated to Keyhole Limpet Hemocyanin (KLH) and used to immunize mice. The immune response in the mice showed to be specific against V. cholerae 0139 capsular polysaccharide and the anti-peptide antibodies showed to be protective in the suckling mouse model. The protective efficacy is both specific and dose-dependent. The results clearly show that the peptides induce a protective immune response in mice. It can therefore, be surmised that these peptides could serve as potential lead candidates for developing a safe and effective vaccine against the V. cholerae 0139.