search efforts focus on vaccine and therapeutic development through both basic and applied research projects. Studies are designed to increase our understanding of pathogenic mechanisms associated with microbial infections and devise novel vaccine and therapeutics strategies to protect humans from the sever effects of infectious diseases. In recent research studies, our laboratory determined the molecular mechanism responsible for antibiotic resistance in several clinical isolates of Streptococcus pneumoniae from patients at Children's Hospital in Washington, DC . In one study, we demonstrated that a specific single amino acid mutation in the bacterial chromosomal gene for dihydrofolate reductase was responsible for high level trimethoprim resistance seen in these pneumococcal clinical isolates. In a similar study, we have isolated and characterized the molecular determinant responsible for optochin resistance in S. pneumoniae. This is the first reported cases of infection with S. pneumoniae in the United States that can not be detected by the optochin resistance/sensitive diagnosis test. We have isolated the gene and the mutation responsible for failure of this strain to be detected by the clinical laboratory test. In addition to this research, the laboratory continues to work on the development of genetically detoxified pertussis toxin for acellular whooping cough vaccines. Whooping cough is an upper respiratory tract infection caused by Bordetella pertussis. resulting in mortality rates estimated at about 500,000 deaths per year. This disease has been effectively controlled by the current vaccine which consists of killed whole B. pertussis cells. Although efficacious, the present whole cell vaccine produces unacceptable side effects. The major protective antigen in whooping cough vaccines is pertussis toxin. Chemically inactivated pertussis toxin vaccines have been produced with reduced side effects and reasonable efficacy, however, these product suffer from reduced antigenicity and difficulties in vaccine manufacture processing. In addition, residual activity may exist from reversion or incomplete chemical inactivation. Using site-specific DNA mutagenesis, we modified E. coli subclones of pertussis toxin and used these constructs to replace the chromosomal copy of the toxin gene in B. pertussis vaccine strain 3779. The resulting new strain produces a fully genetically detoxified form of pertussis toxin which is strongly immunoprotective and can be used as a vaccine antigen without chemical inactivation. In a recently competed NIAID-supported clinical trial in Sweden and Italy, pertussis toxin emerged as an essential component of any new whooping cough vaccine. One of the most successful acellular pertussis vaccines used in this clinical trail contained a genetically altered version of pertussis toxin that was developed from basic research generated through this intramural research project. Molecular studies are currently underway in our laboratory to develop higher yield bacterial strains to enhance expression of pertussis toxin for use in acellular and conjugate vaccine manufacture. In addition, an avirulent, live attenuated B. pertussis vaccine is being developed which is capable of delivering other immunoprotective antigens such as proteins from HIV, TB, and hepatitis C virus and well as the protective pertussis antigens.""""""""
