Diverse and representative peptide set.

<p>The sequences of the four peptides selected to represent the V2 loop region for a variety of HIV-1 viruses. Peptide 1 represents the most polar V2 peptide, Peptide 2 the most commonly occurring peptide, Peptide 3 the most polar peptide of the most common length, and Peptide 4 the published consensus V2 peptide.</p><p>Diverse and representative peptide set.</p

gp120 trimer showing linked functional sites.

<p>A representation of the QRV^170–172 functional site in relation to the LDV^179–181 α4β7 binding site. Residues shown in sphere representation and colored by residue type (glutamine = orange; arginine = cyan; valine = green; leucine = olive; aspartate = red). Protein shown in ribbon representation. This model was built using PDB 4nco as a template.</p

Protection maps to positions 166–179 of the V2 loop.

<p>Of the approximate 270 assays performed in the RV144 immune correlates analysis, only Abs binding three reagents showed an OR of 0.5 or lower. These reagents were the gp70-V1V2 glycoprotein (sequence for a portion of the V1V2 domain of gp70 shown in blue heptagons with glycosylation sites indicated by black stars), the MN peptide (sequence shown as green heptagons from position 161–183), and the V2 Hotspot (shown as purple heptagons spanning positions 166–179). All three of these reagents include an unglycosylated portion of the V2 domain spanning positions 166–179.</p

Alignment of V2 sequences.

<p>A logo indicating the conservation pattern of the amino acids comprising the V2 loop from position 158 to position 192. 464 V2 sequences from subtype AE viruses were aligned, with larger letters indicating higher conservation. Hydrophobic amino acids are indicated by black letters, charged amino acids by blue (+) and red (-) letters and polar amino acids are colored pink or green. Valine at position 172 is highly conserved in this population (numbering is according the Hxbc2 reference sequence by convention <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108446#pone.0108446-Korber1" target="_blank">[21]</a>).</p

gp120 and gp145 bind α4β7 expressing cells.

<p>The blue histograms show binding of biotin labeled variants of gp120 and biotin labeled gp145 to activated primary CD4⁺ T cells, CD8⁺ T cells and the RPMI 8866 cell line expressing α4β7. Each row represents a different cell type: row 1) RPMI 8866 cells; row 2) CD4⁺ T cells; row 3) CD8⁺ T cells. Each column represents a different ligand: column 1) gp120 of clade A; column 2) gp145 of clade C; column 3) gp120 (CM235) Clade A/E; column 4) gp120 (RV254.006) clade A/E. The green histogram shows background binding to the NeutraAvidin negative control, LFI = Log Fluorescence Intensity.</p

Specific monoclonal antibodies inhibit gp120 binding at protein or integrin level.

<p>CD4⁺ cells (left) and CD8⁺ cells (right) were incubated with biotinylated gp120 from clade A either without antibody pretreatment, red line (control) or after pretreatment with 2 µg of anti-integrin α4 mAb, HP2/1, (blue line), anti-V2 mAb 697D (orange line) or anti-V2 mAb 2158 (aqua). Binding was detected with NeutrAvadin PE and analyzed by FACS. The binding of clade A gp120 (red line) is inhibited by the addition of 2 µg anti-α4 mAb (blue line), anti-V2 mAb 697D (orange line) and anti-V2 mAb 2158 (aqua line). Green histogram is NeutrAvidin control, LFI = Log Fluorescence Intensity.</p

<i>ab initio</i> peptide folding.

<p>Peptide 1 (the sequence used in strain A244 of the RV144 vaccine) is predicted to consistently fold into a beta hairpin (upper panels). However, with the addition of QRV to the N-terminus of the peptide, the peptide is predicted to fold into more variable conformations including beta-like and alpha helical-like folds (lower panels). Conformations 1 and 6 of the 50 conformations generated by our software are shown. The peptide is shown in ribbon representation with the α4β7 binding domain (LDI^179–181) shown in ball-and stick and colored according to residue.</p

α4β7 diverse peptide binding assay.

<p>The binding of the representative set of 4 diverse peptides listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108446#pone-0108446-t001" target="_blank">Table 1</a> to α4β7 on CD4⁺ T cells (top row) and α4β7 on CD8⁺ T cells (bottom row). All peptides were tested at both 2 µg (blue line) and 5 µg (red line). In both cell lines, Peptide 1, the most polar and soluble peptide, showed maximal binding, while Peptides 2 and 3 showed submaximal binding. Peptide 4 showed no reactivity in either primary cell type. Green histogram is NeutrAvidin control, LFI = Log Fluorescence Intensity.</p

V2 derived peptides suggest a second determinant of integrin α4β7 binding.

<p>Biotinylated peptides representing the region around the canonical α4β7 tripeptide binding site (LD[I/V]^179–181) in HIV strains A244 subtype AE (¹⁷³HALFYKLDIVPIE¹⁸⁵; red line, left panel) and MN subtype B (¹⁷³YALLYKLDIEPID¹⁸⁵;red line, right panel) were incubated with RPMI 8866 cells and detected with NeutrAvidin PE. The overlap with the control (green histogram) indicates no α4β7-binding. The aqua lines in both panels show the signal obtained when the QRV tripeptide is added to the N terminus of each of the original peptides. The blue line (both panels) indicates the effect of adding EDTA during incubation. Green histogram is NeutrAvidin control, LFI = Log Fluorescence Intensity.</p

Env-A244 peptides generated by CAT and proteasomal cleavage of CAT degradation products.

a<p>The number of Env-A244 peptides identified by mass spectrometry.</p>b<p>As reported in the Los Alamos database.</p