The Regulation of the bvg-Repressed Genes in B. pertussis. Our work is directed towards a better understanding of the interaction between Bordetella pertussis and it's human host. Specifically we are addressing the mechanism of regulation of the bvg-repressed genes and their role in the infectious life cycle. In previous work we identified the bvgR locus of B. pertussis and demonstrated that it is responsible for the repression of expression of the bvg-repressed genes. The mechanism by which the expression of the bvg-repressed genes is regulated by BvgR appears to be novel in that the putative BvgR-binding site lies well downstream of the start of translation within the coding sequence of the target gene. As a first step in the examination of this mechanism, we constructed, this year, a recombinant a strain of Escherichia coli that overexpress the B. pertussis BvgR protein. We initially determined that constitutive overexpression of the bvgR gene is lethal in both B. pertussis and E. coli but overcame this lethality by placing the expression of the bvgR gene under the control of a tightly-regulated phage T7 promoter. We have successfully purified the BvgR protein to near homogeneity and have undertaken a biochemical analysis of the interaction between BvgR and nucleic acid. We have also undertaken a genetic analysis of the promoter of the BvgR-repressed gene vrg18. We constructed a detailed collection of 5' and 3' nested deletions and linker substitutions of the vrg18 promoter and have made transcriptional fusions of these constructs to a lacZ reporter. We transferred these PbvgR-lacZ fusions onto the B. pertussis chromosome in single copy and assayed the activity of the recombinant promoters. This analysis, combined with the results of biochemical studies discussed above should allow us identify the elements of the vrg18 promoter required for transcriptional activation and repression by BvgR and allow us to propose a model of the mechanism by which BvgR regulates expression of its target genes. We have undertaken an analysis of the bvgR promoter. We identified the bvgR transcript in Northern blots and identified the start site of transcription by primer extension analysis. We constructed a detailed collection of nested deletions and linker substitutions of the bvgR promoter and have made transcriptional fusions of these constructs to a lacZ reporter. We transferred these PbvgR-lacZ fusions onto the B. pertussis chromosome and measured their activities under inducing and non-inducing conditions. This analysis suggests that the mechanism by which the bvgR promoter is regulated is different from the mechanism by which other well-characterized BvgA-activated promoters are regulated. A careful examination of the similarities between this system and the other well-characterized systems will likely provide insights into the mechanism by which this important global regulator regulates the expression of genes involved in the infectious life-cycle of B. pertussis. Therapeutic Development Bordetella pertussis, the etiologic agent of whooping cough, is a highly infectious human pathogen with a strong capacity to infect the human respiratory tract. Pertussis in the United States and Canada is primarily seen in young adults with waning immunity and unprotected infants under 6 months of age. Infants with severe pertussis often require long hospitalizations. Although antibiotics and bronchodilators are often given, they do little to alter the severity or duration of the disease. The development of therapies that can reduce the severity and duration of the paroxysmal coughing is required. Compelling evidence has accumulated that suggests that anti-PT antibodies provide protection against disease. It has been demonstrated that both polyclonal and monoclonal anti-PT antibodies can reverse the severe systemic manifestations of pertussis infection in mice and two recent small, clinical studies indicated that the administration of high-titer immunoglobulin prepared from the plasma of donors immunized with purified pertussis toxin was well tolerated by infants and resulted in a decrease in the severity and duration of pyroxysmal coughing {518,519}. Because of the need for an effective therapy for severe pertussis and because of the recent encouraging clinical results with high titer anti-pertussis immunoglobulin in the treatment of pertussis in infants, we sought to develop human monoclonal antibodies and single-chain antibodies against pertussis toxin for use in pertussis therapy. The use of monoclonal antibodies ensures a homogenous preparation of high and well-characterized activity. Work conducted during the last year in our collaborator's laboratory at the University of Texas at Austin has resulted in the successful humanization of the murine monoclonal antibodies 1B7 and 11E6. These particularly potent anti-pertussis toxin antibodies have previously been shown to be protective in the mouse aerosol challenge model. In addition to producing humanized Mab 1B7 and Mab 11E6, our collaborators have derived single chain antibodies (scFv) from 1B7 and 11E6. We are undertaking a series of mouse aerosol challenge experiments designed to determine if the humanized monoclonal antibodies and single-chain antibodies derived from murine Mab 1B7 and murine Mab 11E6 can provide a therapeutic benefit to mice exposed to virulent B. pertussis in the mouse aerosol challenge model. This study will provide us with important insights into the mechanism by which anti-pertussis antibodies confer protection against B. pertussis infection and will provide the data required to support an Investigational New Drug application when these therapeutic agents move forward to clinical studies.
