Development and Validation of a Detection System for Wild-type Vibrio cholerae in Genetically Modified Cholera Vaccine

• Published in: Biologicals 2000-09, Vol.28 (3), p.149-154
• Main Authors: Studer, Edgar, Candrian, Urs
• Format: Article
• Language: English
• Subjects: Cholera Toxin - genetics; Cholera Toxin - immunology; Cholera Vaccines - standards; Confidence Intervals; ctxA gene; DNA Primers; Drug Contamination; mer operon; Oligodeoxyribonucleotides, Antisense; Polymerase Chain Reaction - methods; Quality Control; Reproducibility of Results; Sensitivity and Specificity; Sequence Deletion; Vaccines, DNA - standards; Vibrio cholerae; Vibrio cholerae - genetics; Vibrio cholerae - isolation & purification
• ISSN: 1045-1056; 1095-8320
• DOI: 10.1006/biol.2000.0252

Orochol®, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR. This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance. In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches. A multiplex PCR system that targets the intactctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed. To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V. cholerae 569B cells and tested by PCR. The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit). This number is below the infective dose of wild-type Vibrio cholerae. In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol®.