The proposed research aims to produce a highly potent and broadly neutralizing cocktail of "evolved" human monoclonal antibodies for clinical evaluation in HIV-1 infections. Passive immunization is proposed for the treatment of individuals accidentally infected perhaps due to occupational exposure and in the treatment of infected mothers to reduce transmission rates and severity of disease in the infant. Treatment of long-term infected individuals with a cocktail of antibodies could be of value to reduce viral load in combination with other anti-retroviral agents. In vitro approaches will be developed using the phage display approach to evolve antibodies with the affinity and specificity required for this application. In preliminary experiments we have improved the affinity of an HIV-1 neutralizing antibody 100-fold to a final affinity of 10(-11)M. Neutralization of both laboratory and primary clinical isolates was improved. Human antibodies directed against the CD4-binding site and the V3 loop of the envelope glycoprotein gp120 and a conserved epitope on gp41 will be evolved to exceptional affinity and neutralizing ability. Antibodies will be characterized with respect to sequence, affinity for antigen, epitope recognized, and neutralizing ability as assessed with laboratory and primary clinical isolates of HIV-1. The properties of evolved antibodies will be far superior to those obtained by any other means and provide the best opportunity for testing passive immunotherapy in HIV-1 infection. The methods developed will be of general applicability to the modification of the specificity and affinity of any antibody. Mimetopes will be selected from constrained peptide libraries to bind optimized antibodies to explore their use as templates in the design of an HlV-1 vaccine. Mice will be immunized with mimetopes and the polyclonal response will be characterized as described for monoclonal antibodies. Crystallographic studies of evolved antibodies in complex with mimetopes or gp120, coupled with detailed characterization of the binding kinetics of these antibodies should accelerate the understanding of macromolecular recognition involving antibody/antigen interactions. Furthermore the solved ligand structures have potential as templates for vaccine design and inhibitors of HIV-1 infection. The generation of antibodies with exceptional affinities and specificities should both increase biological potency and decrease the cost of antibodies as therapeutics.