The major accomplishments for this year are summarized below. 1) Soluble forms of the HIV-1 receptor CD4 (sCD4) have been extensively characterized for more than two decades as promising inhibitors and components of vaccine immunogens. However, they were mostly based on the first two CD4 domains (D1D2) and numerous attempts to develop functional high-affinity stable soluble one-domain sCD4 (D1) have not been successful because of the strong interactions between the two domains. We have hypothesized that combining the power of structure-based design with sequential panning of large D1 mutant libraries against different HIV-1 envelope glycoproteins (Envs) and screening for soluble mutants could not only help solve the fundamental stability problem of isolated D1 but may also allow for improvement of D1 affinity while preserving its cross-reactivity. By using this strategy we identified two stable monomeric D1 mutants, mD1.1 and mD1.2, which were significantly more soluble and bound to Env gp120s more strongly (50-fold) than D1D2, neutralized a panel of HIV-1 primary isolates from different clades relatively more potently than D1D2, induced conformational changes in gp120, and sensitized HIV-1 for neutralization by CD4-induced (CD4i) antibodies. mD1.1 and mD1.2 exhibited much lower binding to human blood cell lines than D1D2;moreover, they preserved a beta strand secondary structure and stability against thermal-induced unfolding, trypsin digestion and degradation by human serum. Because of their superior properties, mD1.1 and mD1.2 could be potentially useful as candidate therapeutics, components of vaccine immunogens, and research reagents for exploration of HIV-1 entry and immune responses. Our approach could be applied to other cases where soluble isolated protein domains are needed. 2) We continued to characterize our newly identified cross-reactive HIV-1-neutralizing human monoclonal antibody m66.6. We found that it is considerably less autoreactive than 4E10 and 2F5 and one of his possible predecessors lacks or shows very weak reactivity to some autoantigens. The knowledge gained from the analysis of the maturation pathway of those antibodies and the antibodies themselves could be used as tools to further explore the mechanisms of elicitation of broadly neutralizing antibodies targeting the 2F5 epitope. These and related mAbs could also help explore the complex maturation pathways of other broadly neutralizing antibodies, autoreactivity, design of vaccine immunogens and development of therapeutics. 3) We continued to analyze the results of our high-throughput 454 sequencing which is currently the method of choice for sequencing of antibody repertoires and libraries containing large numbers (106 to 1012) of different molecules. Because these molecules are with similar frameworks and variable regions it poses significant challenges for identifying sequencing errors. Identification and correction of sequencing errors in such mixtures is especially important for the exploration of complex maturation pathways and identification of putative germline predecessors of highly somatically mutated antibodies. To quantify and correct errors incorporated in 454 antibody sequencing, we sequenced six antibodies at different known concentrations twice over and compared them with the corresponding known sequences as determined by standard Sanger sequencing. We found that 454 antibody sequencing could lead to approximately 20% incorrect reads due to insertions that were mostly found at shorter homopolymer regions of 2-3 nucleotide length, and less so by insertions, deletions and other variants at random sites. Correction of errors might reduce this population of erroneous reads down to 5-10%. However, there are a certain number of errors accounting for 4-8% of the total reads that could not be corrected unless several repeated sequencing is performed, although this may not be possible for large diverse libraries and repertoires including complete sets of antibodies (antibodyomes). By using this knowledge we are currently analyzing our 454 sequencing data for HIV-1-infected humans and healthy individuals.
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