During FY2008, this laboratory has expanded studies on the molecules involved in HIV entry, and on the development of related HIV treatment and prevention strategies using recombinant bifunctional proteins. 1) sCD4-17b. We designed this recombinant bifunctional protein and showed previously that it had broad HIV-1 neutralizing activity, using the single cycle TZM-bl reporter gene assay. In FY2008 we greatly extended the number of isolates examined to include 12 clade B and 12 clade C isolates representing the standardized panels selected for vaccine analyses, plus 8 isolates from clades A and F. Strong potency was observed throughout, with the IC50 values generally ranging around 0.1 0.2 microgm/ml (2 4 nM) for laboratory-adapted strains and 0.5 4 microgm/ml (10 80 nM) for most primary isolates. Particularly dramatic was the efficacy of sCD4-17b on many isolates that are highly resistant (IC50 much greater that 50 microgm/ml) to one or more of the well characterized broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5 and 4E10. sCD4-17b also showed potent neutralization of spreading infection in PBMCs. The varying sCD4-17b sensitivities of different isolates was found to correlate with the reactivities of the functional Env trimers with CD4 rather than with the exposed 17b epitope. These results support the potential use of sCD4-17b for protection against HIV-1 infection. To this end, we have found modifications of the linker composition that give much better yields of functional sCD4-17b in Lactobacillus, supporting the use of this engineered commensal organism as a live microbicide to protect against sexual transmission. We have also initiated a collaboration with Dr. Kenneth Palmer (U. Louisville) who has developed recombinant tobacco mosaic virus as a vector to express large quantities of foreign proteins in Nicotinia plants (e.g. 60 grams of another candidate microbicide protein, 99% purity in 2 weeks). 2) 3B3-PE38 immunotoxin. We are continuing to develop this bifunctional protein for depletion of HIV-1 reservoirs that persist after HAART. Extending our collaboration with Dr. Ira Pastan (NCI), we have examined a variant of 3B3-PE38 with several mutations in known B cell epitopes, in the hope of reducing the immunogenicity of this protein for therapeutic application. The effect of the mutations on specific killing of HIV Env-expressing cells was found to be dependent on the cell type. Thus, the mutations had no effect when tested against a CHO-Env transfectant cell lines, but reduced the activity against an HIV-1 chronically infected cell line. These results suggest that only a subset of the mutations tested may be suitable for clinical use of this agent. Collaborative experiments with Dr. Tae-Wook Chun are being conducted to test whether 3B3-PE38 can deplete cells responsible for the residual viremia still detectable under highly suppressive HAART. With support from the NIAID Scientific Director, we are collaborating with Dr. Clifford Lane and Dr. Richard Davey (NIAID) to formulate a pre-IND with the goal of obtaining FDA approval for a phase 1 clinical trial of 3B3-PE38 in infected people with strong HAART suppression of viral load. 3) We are continuing studies of the functional features of the HIV-1 Env trimer. In an extension of previous work, we have observed functional complementation upon co-expression of a diversity of Envs containing mutations in distinct determinants involved in fusion. The results indicate that fusion does not require every member of the Env trimer to contain binding sites for CD4 or coreceptor; similar, every member need not contain an active fusion peptide or N-helix or normal transmembrane domain. These findings indicate functional cross-talk between different protomers within the Env trimer. In collaboration with Dr. Susan Zolla-Pazner, we are examining the molecular basis for masking of specific epitopes on the V3 loop. Our sCD4-activated cell fusion assay enables us to assess the potential neutralizing activities of antibodies whose epitopes become accessible only after receptor binding.