Specificity of anti-sTLR2 antibodies.

<p>The specificity of anti-sTLR2 antibodies was confirmed using a peptide competition assay and CBA. (A) N-17 was preincubated without (-P) or with (+P) 5× molar excess of peptide (N-17P) prior to immunoblotting. Pre-incubation with excess peptide markedly reduced both the ∼83 kDa and ∼38 kDa isoforms of sTLR2. Results representative of breast milk (BM) samples from different donors tested. (B) Schematic of cytometric bead array shows that beads coated with T2.5 mAb pulled natural sTLR2 out of milk and were detected with phycoerythrin (PE) labeled N-17 detection antibody. (C) CBA analysis of commercial rsTLR2 and BM dilution clearly shows system can detect natural sTLR2 but cannot detect commercial rsTLR2. A representative data set from triplicate experiments is shown.</p

A Conserved Glycan in the C2 Domain of HIV-1 Envelope Acts as a Molecular Switch to Control X4 Utilization by Clonal Variants with Identical V3 Loops

<div><p>Nearly all persons newly infected with HIV-1 harbor exclusively CCR5-using virus. CXCR4-using variants eventually arise in up to 50% of patients infected with subtypes B or D. This transition to efficient CXCR4 utilization is often co-incident with progression to AIDS. The basis for HIV-1’s initial dependence on CCR5, the selective force(s) that drive CXCR4-utilization, and the evolutionary pathways by which it occurs are incompletely understood. Greater knowledge of these processes will inform interventions at all stages, from vaccination to cure. The determinants of co-receptor use map primarily, though not exclusively, to the V3 loop of gp120. In this study, we describe five clonal variants with identical V3 loops but divergent CXCR4 use. Mutagenesis revealed two residues controlling this phenotypic switch: a rare polymorphism in C1 and a highly conserved N-glycan in C2. To our knowledge, this is the first description of co-receptor usage regulated by the N-glycan at position 262.</p></div

Variation in sTLR2 polypeptides between different women.

<p>Breast milk samples (10 µg total protein) from HIV-uninfected women were evaluated using a cocktail of anti-sTLR2 antibodies (N-17 pAb and T2.5 mAb) using Western blot analysis. Results show dramatic variation in predominant sTLR2 polypeptide (∼38 kDa and ∼26 kDa) expression in milk from different women. (A) Samples taken within one week postpartum. (B) Samples taken within one month postpartum.</p

Partial alignment and phenotypic data for all parental and mutants envelopes from subject 1010.

<p>Mutant sequences are identified by the parental clone (i.e. 542) and the residues present at positions 123 and 264 (i.e. 543 I+G indicates a mutant derived from parental clone 543 with an I at position 123 and a G at position 264). The presence or absence of a potential N-linked glycosylation site (PNG) at position 262 (as determined by the N-glycosite web tool from LANL) is denoted by a + or—in the column labeled PNG 262, and the relevant sequons [Nx(S/T)] are shaded dark gray in the abbreviated alignment on the right. Efficiency of entry into CXCR4-expressing (X4) GHOST cells [relative to CCR5/CXCR4-expressing (R5/X4) GHOST cells] is indicated for each clone, as are fifty-percent inhibitory concentrations (IC₅₀) in nM for the R5 inhibitors Maraviroc (MVC), TAK779, and AD101; the peptide fusion inhibitor T-20; and in μg/ml for the plant-derived lectins GNA, HHA, and UDA. Columns have been color-coded to highlight the concentration of a given compound that is required to achieve the IC50. Expressing cut-offs for green, yellow, and red coding: for MVC and AD101:0–9.9, 10–24.9, >25nM; for TAK-779: 0–24.9, 25.0–49.9, >50nM; and for GNA and HHA: 0–0.49, 0.5–0.99, and >1.0 μg/ml. T20 and UDA were not color coded, as their IC50s did not correspond to the % X4 usage of the clone. Phenotypic data for reference viruses Bori, 89.6, and pNL4-3 are shown at the bottom of the figure.</p

sTLR2 significantly inhibits HIV-1 infection in reporter assay.

<p>(A) MTT assay indicates that HIV-uninfected breast milk (BM) was not toxic to TZM-bl cells. (B) HIV-uninfected BM was incubated with either N-17 pAb or T2.5 mAb (200 before R5 HIV-1 (200 TCID₅₀) and then placed on TZM-bl cells. T20, 2F5IgG, and 1∶100 BM significantly inhibited infection (<i>P<0.001)</i>. A significant increase (<i>P<0.001, 0.01,</i> respectively<i>)</i> in HIV-1 infection was shown when sTLR2-specific N-17 or T2.5 Ab were pre-incubated with BM. N-17 pAb and T2.5 mAb alone and isotype control (200 ng/mL) did not inhibit HIV-1 infection. (C) rsTLR2+/− sCD14 or pooled HIV-uninfected mock-D or sTLR2-D BM (described in materials and methods) were incubated with R5 HIV-1 (200 TCID₅₀) before addition to TZM-bl cells for 48 hours. A significant decrease (<i>P<0.001</i>) in HIV infection was observed in cells exposed to mock-D BM. However, a significant increase (<i>P<0.001</i>) in HIV infection was detected with sTLR2-depleted BM. (D) rsTLR2+/− sCD14, mock-D, or sTLR2-D was incubated with cells for 1 hour at 37°C, washed with PBS, and then exposed to R5 HIV-1 (200 TCID₅₀) did not alter HIV-1 infection. <i>** P<0.01,</i> ***<i>P<0.001</i>. Errors bars, SEM. A representative data set from four experiments is shown.</p

sTLR2-mediated augmentation of pro-inflammatory cytokines during bacterial lipoprotein exposure.

<p>(A) Immunodepletion of ∼38 kDa sTLR2 in breast milk (BM) described in materials and methods. Quantitative analysis using TLR2 ELISA indicated a significant (<i>P<0.05)</i> decrease in sTLR2-depleted (sTLR2-D) compared to mock-D breast milk (BM). Western blot analysis revealed that ∼38 kDa and ∼83 kDa sTLR2 were markedly reduced compared to mock-D BM. (B-D) 500 ng/mL Pam₃CSK₄ was incubated with media or BM that was either mock-D or sTLR2-D for 1 hr at 37°C before being placed on cells. Supernatants were collected for IL-8 ELISA after 18 hours. Results represent (B) U937, (C) HEK293-TLR2 and (D) HEK293. (E) Commercial rsTLR2 was used at varying concentration with or without sCD14 and showed no inhibition of IL-8 or (F) IL-6 production in HEK293-TLR2 cells. Significant increases in pro-inflammatory cytokine, IL-8, was observed in sTLR2-D compared to mock-D BM. *<i>P<0.05,</i> **<i>P<0.01</i>, ***<i>P<0.001</i>. Errors bars, SEM. A representative data set from triplicate experiments is shown.</p

Predominant sTLR2 polypeptides profiles in breast milk.

<p>Predominant sTLR2 polypeptide profiles found in multiple breast milk (BM) samples using Western and Native blot analysis. (<b>A</b>) BM samples (10 µg total milk protein) with commercial rsTLR2 were evaluated by Western blot analysis with N-17 pAb and 4 mAb (sc-52909, T2.5, TL2.3, TL2.1). pAb N-17 detected commercial rsTLR2 as well as multiple bands in BM; the predominant BM forms were ∼83 kDa and ∼38 kDa sTLR2 forms. In contrast, mAbs did not detect the commercial rsTLR2. In BM mAbs detected the ∼83 kD band, as well as a unique ∼26 kDa sTLR2 form, which was not detected with the N-17 pAb. (<b>B</b>) N-17 pAb and T2.5 mAb were used in Native blot analysis of two BM samples. N-17 pAb detected two large proteins (arrow 1 & 2), while T2.5 mAb detected 3 proteins (arrow 1, 2 & 3). A representative data set from three experiments is shown.</p

Characteristics of Healthy HIV-uninfected Women Participants.

<p>Characteristics of Healthy HIV-uninfected Women Participants.</p

Clinical characteristics of study subjects.

<p>¹ Some subjects were identified as ineligible for P1020 before a CD4 count was performed</p><p>² Anti-retroviral drug susceptibility was determined genotypically using the Stanford HIV Drug Resistance Database</p><p>³ Maraviroc susceptibility cut-offs reflect the ability to achieve 50% neutralization at the highest drug concentration tested (200nM)</p><p>Clinical characteristics of study subjects.</p

Correlation of R5 inhibitor susceptibility and high mannose specific (GNA/HHA) or complex glycan specific (UDA) lectin susceptibility.

<p>For each parental (n = 5) and mutant (n = 15) clone from patient 1010, the 50% inhibitory concentration (IC₅₀, in nM) of maraviroc (A-C), TAK779 (D-F), or AD101 (G-I) is plotted on the x-axis and the IC₅₀s (in μm) for the high mannose specific lectin GNA (A,D,G), the high mannose specific lectin HHA (B,E,H), and the complex glycan specific lectin UDA (C,F,I) are plotted on the y-axis. Pearson correlation coefficients (r) and associated p-values are also displayed.</p