The major accomplishments for this year are summarized below: 1) Neutralizing antibodies that target the membrane-proximal external region (MPER) of the HIV-1 gp41 glycoprotein fall into two classes, depending on whether they target the N-terminus of the MPER or its C-terminus. The antibody m66 and its more potent variant m66.6, as well as antibody 2F5, represent the only neutralizing antibodies known to target the N-terminus of the gp41 MPER. Our collaborators solved the crystal structure of m66 in complex with its gp41 epitope, as well as the free structures of m66 and m66.6. When bound by m66, the N-terminus of the gp41 MPER adopts a conformation composed of a helix followed by a strand. Comparison of m66 recognition of gp41 with that of 2F5 reveals similarities in the conformations of shared critical epitope residues, 664DKW666, as well as in the angles by which they approach these residues. A stretch of aromatic residues at the tip of the m66 heavy chain third complementarity determining region (CDRH3) loop is positioned along the same plane formed by functional hydrophobic residues at the tip of the 2F5 CDRH3. Mutation of these aromatic residues in the m66.6 context, to either increase or decrease hydrophobicity, dramatically altered m66.6 neutralization of HIV-1 and its recognition of the MPER in a lipid context, but had little effect on its recognition of a soluble MPER peptide. The similarities observed between antibodies m66 and 2F5 thus suggest that both antibodies have converged on common structural and mechanistic elements in recognition and neutralization of HIV-1. 2) By combining a protein cavity-filling strategy and the power of library technology, we further improved mD1.2 identifying an engineered cavity-altered single-domain sCD4 (mD1.22) with a unique combination of excellent properties including broad and potent neutralizing activity, high specificity, stability, solubility, and affinity for the HIV-1 envelope glycoprotein gp120, and small molecular size. To further improve its neutralizing potency and breadth, we generated bispecific multivalent fusion proteins of mD1.22 with another potent HIV-1 inhibitor - an antibody domain (m36.4) targeting the coreceptor-binding site on gp120. The fusion proteins neutralized all HIV-1 isolates tested with potency about 10-, 50-, and 200-fold higher than that of the broadly neutralizing antibody VRC01, the US FDA-approved peptide inhibitor T20, and the clinically tested sCD4-Fc fusion protein CD4-Ig, respectively. In addition, they exhibited higher stability and specificity, and lower aggregation propensity than CD4-Ig. Therefore, mD1.22 and related fusion proteins could be potentially useful for HIV-1 prevention and therapy including eradication of the virus. 3) We also analyzed the potency of potent bnAbs in a macaque model of HIV-1 infection. Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. Our collaborators have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian-human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4-7 days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3-5 weeks in some long-term chronically SHIV-infected animals with low CD4(+) T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. I did computational analysis of the slopes of viral decline as a predictor of the therapy efficacy and found the important result that it is the same as for humans treated with HAART which we showed previously is a strong predictor of efficacy (67). These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a therapy for chronically HIV-1-infected individuals experiencing immune dysfunction.