Human monoclonal antibodies with functional activity (ie: antibacterial protective activity) against group B streptococci (GBS) may be a significant addition to the therapy of newborn sepsis and offer the opportunity to elucidate the fundamental mechanisms of antibody protection in group B streptococcal sepsis. The objective of this proposal is the development of anti-group B streptococcal human monoclonal antibodies. Human-human hybridomas will be constructed from in vitro stimulated human tonsil or spleen following short term culture with GBS type III polysaccharide antigen and pokeweed mitogen. Somatic cell fusion of the stimulated lymphocytes with human HGPRT deficient human lymphoblastoid cell lines will be performed by a modification of the monolayer fusion method. Fusion products will be screened for the production of antibodies specific for GBS type III polysaccharide using ELISA and RIA. Fusion products secreting appropriate antibodies will be cloned by limiting dilution, propogated and the monoclonal antibody products tested in a standard animal model of GBS sepsis. Protective monoclonal antibodies of the major immunoglobulin classes and subclasses will be produced by a combination of pre-fusion culture manipulations using T cell derived differentiation factors and post-fusion selection of isotype variants. Post-fusion selection of antibodies with particular constant regions will be achieved by repetitive sublining or fluorescence activated cell sorting. The development of human monoclonal antibodies that confer protection in the animal model of GBS sepsis will provide useful investigational tools and potentially important therapeutic reagents. The precise role of antibody in GBS sepsis can be characterized and the mechanisms by which antibodies of different classes and subclasses act can be defined. The approach of constructing therapeutically useful human monoclonal antibodies against important bacterial pathogens may be generally useful in those situations (eg: newborn sepsis and Haemophilus influenza meningitis in infants) in which preformed antibodies are not present and life-threatening infection progresses more rapidly than the humoral immune response.
