The SF162 sequence was threaded over the V1V2 template coordinates using Rosetta 3.4 [32]. seven human monoclonal antibodies (mAbs) derived from HIV-infected individuals that are directed against conformational epitopes in the V1V2 domain. In this study, using lysates of SF162 pseudoviruses transporting V1V2 mutations, we mapped the epitopes of these seven mAbs. All tested mAbs demonstrated a similar binding pattern in which three mutations (F176A, Y177T, and D180L) abrogated binding of at least six of the seven mAbs to 15% of SF162 wildtype PLX8394 binding. Binding of six or all of the mAbs was reduced to 50% of wildtype by single substitutions at seven positions (168, 180, 181, 183, 184, 191, and 193), while one switch, V181I, increased the binding of all mAbs. When mapped onto a model of V2, our results suggest that the epitope of the conformational V2 mAbs is located mostly in the disordered region of the available crystal structure of V1V2, overlapping and surrounding the 47 binding site on V2. Introduction In 2009 2009, the RV144 clinical vaccine trial provided the first success in the development of an effective HIV vaccine, demonstrating a 31.2% efficacy at preventing HIV-1 infection in vaccine recipients [1]. In the recently published case-control study, levels of IgG antibodies specific for the first and second variable regions (V1V2) of gp120 were shown to inversely correlate with the reduced rate of HIV-1 contamination [2]. The hypothesis based on these data, that V1V2 antibodies are protective, is also supported by a recent publication reporting an increased RV144 vaccine efficacy against viruses with genetic signatures at two positions in V2 [3]. These data generated the hypothesis that this V2 region of gp120 is likely to be a encouraging target for vaccine development and recent HIV-1 vaccine research has increasingly focused on antibodies specific for V2. The V1V2 region can form four anti-parallel -strands (A, B, C, and D) linked via disulfide bonds [4]; these are located at the apex of the envelope trimer [5]. Comparison of all V2 sequences in the LANL database revealed that whilst V2 loops can vary in length, the majority of amino acids are highly conserved or permit only conservative substitutions [6]. This, and the broad immunologic cross-reactivity of many human V2 mAbs with gp120s PLX8394 from PLX8394 diverse clades, suggest that the V2 region DNM1 contains structurally and antigenically conserved elements [7]C[10]. V2 can bind to 47, an integrin that is expressed strongly on activated CD4+ T cells [11]. 47, in concert with chemokine receptors, is essential for homing of CD4+ T cells to the gut mucosa [12]C[14]. On activated gut CD4+ T cells, 47 has been shown to co-localize with CD4 and CCR5 in the cell membrane [14] and, whilst not required for viral access or replication, 47 interacts with gp120 proteins from diverse HIV-1 subtypes, suggesting that this integrin may play a key role PLX8394 in mucosal HIV-1 transmission [14], [15]. Binding to 47 is usually mediated by a conserved tri-pepetide in the V2 loop, the LDV/I binding motif [11], which mimics the tri-peptide binding sites encoded by the natural ligands of 47 [16]. However, in recent studies, antibodies specific for 47 failed to block contamination of activated CD4+ T cells by full-length infectious molecular clones of subtype C viruses, suggesting that differences in binding may exist between gp120 proteins and PLX8394 the trimeric envelopes present on virions [17]. Antibodies targeting the V2 region are found in approximately 20C45% of HIV-1 infected individuals [8], [18]C[20]. Monoclonal antibodies (mAbs) that identify epitopes in V2 have been shown to target linear, quaternary, or conformational.
