The major accomplishments for this year are summarized below. 1) m36 is the first member of a novel class of potent HIV-1 entry inhibitors based on human engineered antibody domains (eAds) which we discovered several years ago. It exhibits broad inhibitory activity suggesting that its CD4-induced epitope is highly conserved. Fine mapping of its epitope was performed by using three approaches. First, a panel of mimotopes was affinity-selected from a random peptide library and potential m36-binding residues were computationally predicted. Second, homology modeling of m36 and molecular docking of m36 onto gp120 revealed potentially important residues in gp120-m36 interactions. Third, the predicted contact residues were verified by site-directed mutagenesis. It was found that the m36 epitope includes three discontinuous sites including six key gp120 residues (Site C1: Thr123 and Pro124;Site C3: Glu370 and Ile371;Site C4: Met426 and Trp427). In the 3D structure of gp120, the sites C1 and C4 are located in the bridging sheet and the site C3 is within the beta15-alfa3 excursion, which play essential roles for the receptor- and coreceptor-binding and are major targets of neutralizing antibodies. Based on these results we proposed a precise localization of the m36 epitope and suggested a mechanism of its broad inhibitory activity which could help in the development of novel HIV-1 therapeutics based on eAds. 2) The extensive carbohydrate coat, the variability of protein structural features on HIV-1 envelope glycoproteins (Env), and the steric constraints of the virus-cell interface during infection, present challenges to the elicitation of effective full-length (150 kDa), neutralizing antibodies against HIV. These hurdles have motivated the engineering of smaller antibody derivatives that can bind Env and neutralize the virus. To further understand the mechanisms by which these proteins neutralize HIV-1, cryoelectron tomography of native HIV-1 BaL virions complexed separately to two small (15 kDa) HIV-neutralizing proteins: A12, which binds the CD4-binding site on Env, and m36, whose binding to Env is enhanced by CD4 binding, was carried out. We show that despite their small size, the presence of these proteins and their effects on the quaternary conformation of trimeric Env can be visualized in molecular structures derived by cryoelectron tomography combined with subvolume averaging. Binding of Env to A12 results in a conformational change that is comparable to changes observed upon its binding to the CD4-binding site antibody, b12. In contrast, binding of Env to m36 results in an """"""""open"""""""" quaternary conformation similar to that seen with binding of soluble CD4 or the CD4i antibody, 17b. Because these small neutralizing proteins are less sterically hindered than full-length antibodies at zones of virus-cell contact, the finding that their binding has the same structural consequences as that of other broadly neutralizing antibodies highlights their potential for use in therapeutic applications. 3) All known broadly neutralizing antibodies are highly somatically mutated and therefore significantly differ from their germline predecessors. Thus although the mature bnAbs bind to conserved epitopes of the HIV-1 envelope glycoprotein (Env) with high affinity their germline predecessors do not or weakly bind Envs failing to initiate an effective immune response. The identification of less somatically mutated bnAbs and/or antibody maturation intermediates that are clonally related to bnAbs may be useful to circumvent the major problem of initiating immune responses leading to elicitation of bnAbs. We attempted to identify IgGs from an acutely HIV-1-infected patient using a combination of phage display and high-throughput sequencing. We found two antibodies with only a single point mutation in the V region of their heavy chain variable domains compared to their putative germline predecessors which bound with high affinity to several Envs. They targeted the Env gp41 and did not neutralize HIV-1. Using high-throughput sequencing, we identified several highly abundant CDR3s, germline-like as well as somatically mutated V genes in the VH/VL repertoires of the patient which may provide antibody intermediates corresponding to known bnAbs as templates for design of novel HIV-1 vaccine immunogens.
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