During the past year, this laboratory has continued studies on the mechanism of HIV entry, and on the development of novel protective and treatment strategies based on the molecules involved in entry. 1) Molecular mechanisms of gp120 interactions with coreceptors (CCR5 and CXCR4). Studies on the critical domains of gp120 and CCR5 involved in molecular interactions were extended by studying the effects of synthetic peptides and specific mutations (deletions) on functional interactions, as assessed by assays of Env-mediated cell fusion and entry of HIV Env pseudotyped retroviral particles. Inhibition by peptides derived from the CCR5 sulfated N-terminus was greatly enhanced in the presence of soluble CD4 (sCD4), suggesting that the corresponding binding site on gp120 is masked prior to CD4 binding. The effects of these peptides on inhibition with wild type CCR5 versus stimulation with delta-NT CCR5 are being examined, along with assessment of the role of specific regions of gp120 (V1/V2 and V3 loops). These studies are also being extended to synthetic peptides corresponding to the predicted extracellular loop of CCR5. It is hoped that these studies will guide the design of a soluble form of CCR5 (perhaps in the context of a protein scaffold) capable of specific interaction with the relevant sites on gp120 (collaboration with K. Ridge, U. Texas Med. Sch.). Binding studies of the CCR5-based peptides to gp120 constructs (wild type and loop-deleted) are also being performed (collaboration with C. Bewley, NIDDK). 2) Possible role of neutralizing antibodies in selection against CXCR4-using HIV-1 variants during acute transmission. We have described a novel murine monoclonal antibody (D19) that recognizes a conserved epitope within the V3 loop of gp120 that is fully exposed on trimers of Envs capable of using CXCR4 (X4 and R5X4) but masked on trimers of CCR5-specific (R5) Envs until CD4 binding; consistent with this, D19 potently neutralizes the corresponding CXCR4-using viruses, but has weak activity against CCR5-specific viruses unless sub-neutralizing amounts of sCD4 are added. These findings have led us to begin investigations of whether antibodies with such selectivity (neutralizing CXCR4-using but not CCR5-specific variants) may play a role in the selective transmission of R5 strains during acute infection. Collaborative studies of acute seroconverters (with G. Shaw) as well as SHIV-infected monkeys (W. Narayan) are under discussion. 3) Novel anti-HIV agents based on HIV Env/receptor interactions. a) Topical microbicide to protect against sexual transmission of HIV. We have extended studies on a sCD4-17b, a genetically engineered bifunctional protein with strong HIV-1 neutralization activity that shows promise as a topical microbicide to protect against sexual transmission. We have developed expression systems (baculovirus and mammalian cell) that enable production of mg quantities of sCD4-17b. Initial studies have confirmed the potent neutralizing activity of the original construct (35 aa linker), and shown similar activity for a construct with a longer linker (42 aa). Experiments are underway to test whether extending the linker broadens the activity of sCD4-17b against genetically and phenotypically diverse HIV-1 strains. In collaboration with OSEL, Inc., we have extended efforts to engineer vaginal strains of Lactobacillus to secrete HIV-neutralizing proteins. By testing various promoters, we have improved expression of sCD4-17b. We have also engineered lactobacillus to secrete the T-20 peptide (D. Hamer, collaborator). b) Immunotoxin to deplete reservoirs of HIV-infected cells. We have extended studies on 3B3-PE38, an immunotoxin targeting Env expressed on the surface of productively infected cells (I. Pastan, collaborator). The agent was found to be active against diverse HIV-1 primary strains replicating in primary cells of biological significance (PBMCs, macrophages). Most importantly, 3B3-PE38 showed greatly enhanced potency and reduced hepatotoxicity in monkeys compared to an earlier Env-targeted toxin CD4-PE40. We hope to employ 3B3-PE38 to deplete infected cell reservoirs that persist in spite of suppressive HAART.