A small, well-defined group of organisms account for nearly 95% of all rapidly progressive toxin-mediated necrotizing soft tissue infections. This group includes beta hemolytic streptococci including group A streptococcus, several histotoxic clostridial species, Vibrio species, and Staphylococcus aureus, including hypervirulent methicillin-resistant strains (MRSA). Irrespective of the etiologic agent, it is essential that treatment of these infections be initiated early. However, obtaining definitive clinical laboratory identification of the causative agent and its antimicrobial sensitivities can cost valuable time. Empiric antibiotic treatment at this stage of illness can prove disastrous given the rise of emerging resistance and the fact that some antimicrobials can increase bacterial toxin production. As proof of principle, we will exploit cutting-edge B-cell isolation and enrichment technologies for the discovery, sequence and recombinant expression of human monoclonal antibodies to the model cholesterol-dependent cytolysin toxin, streptolysin O (SLO), from individuals naturally immunized by group A streptococcus infections. Hypothesis: Our hypothesis is that a) administration of a fully human immunoglobulin cocktail containing high-titer neutralizing antibodies against the principal exotoxins of the primary pathogens associated with devastating Gram positive infections will provide a vital, early therapeutic edge to current treatment strategies; b) B-cells elicited by the immune response to natural infection will provide key, toxin-specific, neutralizing antibody genes for the in vitro generation of fully-human monoclonal antibodies.
Specific Aims : For transient infections with no vaccination, we must rely on the capture and cloning of antibody genes from highly abundant, short-lived, plasmablasts (Aim 1) or low frequency, long lasting, memory B-cells (Aim 2), from the peripheral blood of naturally immunized individuals. In both Aims 1 and 2, immunoglobulin gamma genes from isolated cells will sequenced, cloned and recombinantly expressed in a human cell line to generate full-length, toxin-specific human monoclonal antibodies.
In Aim 3, we will verify the efficacy of the fully-human monoclonal antibodies by their ability to neutralize toxin in vitro and asses the degree of protection in animal models of infection.
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