Ligand-receptor interactions and their modulation by antibodies are important for a number of biological processes including virus entry and signal transduction through membranes. Our long-term goal has been the elucidation of the molecular mechanisms of these interactions and the development of antibody-based therapeutics, vaccines and reagents for diagnosis and research. During the fiscal year 2005 we continued our research on the identification and characterization of potent broadly neutralizing human monoclonal antibodies (hmAbs) against HIV-1, emerging and biodefense-related viruses, and cancer-related proteins. In the giant struggle with the chronic HIV infection the immune system has generated unique antibodies matured to neutralize a virus which has evolved to escape them. We discovered unique features of a CD4bs (m18), and two CD4i (m12, X5) antibodies selected from immune phage libraries developed from long-term nonprogressors with high levels of broadly neutralizing antibodies: mimicry of the receptor CD4, small size by using only the heavy chain of the antibody and high flexibility of components of the antigen binding site. We have solved the Fab m18 crystal structure at 2.03 resolution. It has unique conformations of the heavy chain complementarity determining regions 2 and 3 (H2 and H3), and the interface between the heavy and the light chains. The H2 is highly bulged and lacks cross-linking interstrand hydrogen bonds formed in all four canonical structures. The H3 is 17.5 long and rigid, forming an extended ?A-sheet decorated with an ??-turn motif bearing a phenylalanine-isoleucine fork at the apex. It shows striking similarity to the Ig CDR2-like C??C???? region of the CD4 domain 1 which dominates the binding to gp120. Docking simulations suggested significant degree of similarity between the m18 epitope and the CD4 binding site on gp120. However, m18 did not enhance binding of CD4i antibodies, and did not induce CD4-independent fusion mediated by the HIV Env. Thus, vaccine immunogens based on the m18 epitope structure are unlikely to elicit antibodies that could enhance infection. The m18 structure can also serve as a basis for design of novel highly efficient inhibitors of HIV entry. We also demonstrated by three lines of evidence that the X5 H3 is highly flexible and undergoes a large conformational change to directly contact gp120; X5 is the only HIV-specific antibody known to contain a highly flexible component of the antigen binding site. The H3 of the X5-derived scFv antibody, m9, has higher predicted flexibility than the X5 H3; m9 exhibits superior inhibitory activity compared to any antibody tested. An antibody, m12, was identified that lacks light chain. The variable domain of its heavy chain, which is the smallest antigen-binding entity, exhibits cross-reactive neutralizing activity. High flexibility, small size and mimicry of receptors are unique features of antibodies matured to counteract the HIV immune response evasion strategies. It is possible that small m12-like IgGs that target CD4i epitopes and exhibit potent and broad neutralizing activity exist in vivo or/and can be developed.m18-like antibodies may exhibit energy profiles of binding similar to that of CD4 antibody which will decrease the probability of neutralization escape mutants without inducing conformational changes that could enhance infection. These results may help in the development of vaccine immunogens that can elicit such antibodies with unique features able to fight back the immune evasion strategies of HIV.We continued to characterize the novel hmAbs against the Hendra G protein which exhibited potent inhibitory activity against fusion mediated by the envelope glycoproteins of Hendra and Nipah viruses. We found that one of them, m101, is a very potent neutralizer of infectious Hendra virus and have potential for use in humans.
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