Mapping of the HD5-binding site in HIV-1 gp120.

<p>Competition of HD5 with a panel of anti-gp120 mAbs with various epitope specificity for binding to plastic-immobilized recombinant gp120 in ELISA. The plates were coated with gp120_BaL at 2 µg/mL; HD5 at 5.6 µM was added prior to the anti-gp120 mAbs, all used at 5 µg/ml. CD4 binding site (CD4-BS), CD4-induced coreceptor binding site (Coreceptor-BS). The data represent mean values (±SD) from three experiments.</p

Effect of HD5 on cell-surface HIV-1 coreceptor expression: induction of CXCR4 downmodulation.

<p>PM1 cells and primary peripheral blood lymphocytes activated with PHA and IL-2 were preincubated for 2 hours with or without HNP1 or HD5 (each at 14.7 µM) in RPMI-ITS without serum; the cells were then stained with 12G5 or 44717.11, two_conformation-dependent mAbs that recognize antigenically distinct populations of CXCR4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045208#pone.0045208-Baribaud1" target="_blank">[39]</a> and analyzed by flow cytometry. The level of expression was determined by differences between the level of staining with specific mAbs and with the isotype control. The results are expressed as percent reduction of mean fluorescent intensity (MFI) compared to untreated controls. Data are representative of 4 to 6 independent experiments performed with similar results. Error bars show standard deviations from the mean. *, p = 0.032 and **, p = 0.0084. Wilcoxon Mann-Whitney rank-sum test, 2-tailed.</p

HD5 inhibits HIV-1 envelope-mediated fusion induced by clinical and laboratory HIV-1 isolates with different coreceptor usage.

<p>Fusion assays were performed using the human T-cell line (PM1) chronically infected with two prototypic laboratory-adapted strains (HIV-1_IIIB and HIV-1_BaL) and four primary clinical isolates (R5: HIV-1_J6366 and HIV-1_J2615; X4: HIV-1_J57 and HIV-1_J287) cocultured with NIH-3T3 cells expressing membrane-bound CD4 and either CCR5 or CXCR4 in the presence or absence of increasing concentrations of HD5 (filled circles) for 2 hrs at 37°C in medium without FCS. For comparison, inhibition by HNP1 (open squares) used at the same concentrations is shown. The data are normalized with respect to fusion observed in the absence of inhibitors. The data represent mean values (±SD) from 3 experiments.</p

Effect of HD5 on HIV-1 replication in purified CD4⁺ T lymphocytes.

<p>(A) Effect of recombinant HD5 on infection by primary isolate HIV-1_J176 in cell-free infection assays performed using the spinoculation method. HIV-1 virions were preincubated for 1 hour at 37°C with HD5 at the indicated concentrations in either RPMI-10 (10%FBS), RPMI-0.3-ITS (no serum) or 10 mM phosphate buffer 0.3-ITS (low salt). Gag p24 concentrations in the extracellular supernatant were measured on day 6 post-infection. Neutralizing mAbs directed to gp120 (2G12) or CD4 (Sim4) were used as positive controls to exclude non-specific infection. The data represent mean values (±SD) from 3 experiments each performed in triplicate. (B) Broad-spectrum inhibition of different HIV-1 strains by HD5. Purified CD4⁺ T lymphocytes were infected with two laboratory-adapted strains (HIV-1_IIIB and HIV-1_BaL) or 4 primary clinical isolates (HIV-1_J1005, HIV-1_p36, HIV-1_J6195 and HIV-1_J176) in the presence of HD5 at 1.4 µM in RPMI-0.3-ITS. Gray bars indicate R5 HIV-1 isolates; solid bars X4 isolates; the striped bar a dualtropic isolate. The data represent mean values (±SD) of three separate experiments, each performed in triplicate.</p

HD5 binds to both gp120 and CD4, and blocks the interaction of CD4 with gp120.

<p>(A) Effect of HD5 on HIV-1 envelope-mediated fusion after pre-incubation with effector cells (expressing the viral envelope) or target cells (expressing CD4 and the coreceptor). Prior to the fusion reaction, HD5 was pre-incubated for 20 minutes with effector cells or target cells followed by washing to remove the unbound defensin. Control fusion was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045208#pone-0045208-g002" target="_blank">Figure 2</a> with addition of HD5 at the time of culture between effectors and targets. (B) Binding of HD5 to HIV-1 gp120. Competition of synthetic HD5 with the anti-gp120 mAb IgG1b12 (used at 5 µg/mL) for binding to plastic-immobilized recombinant gp120_BaL in ELISA. (C) Binding of HD5 to human CD4. Competition of synthetic HD5 with the anti-CD4 mAb Leu3a for binding to plastic-immobilized recombinant sCD4 in ELISA. The plates were coated with sCD4 at 5 µg/mL; HD5 was added prior to Leu3a and kept in the wells throughout the reaction period. (D) Inhibition of gp120/CD4 binding by HD5. Competition of HD5 with recombinant HIV-1 gp120_BaL for binding to plastic-immobilized sCD4 in ELISA. The plates were coated with sCD4 at 5 µg/mL; HD5 was added prior to Leu3a or gp120_BaL and kept in the wells throughout the reaction period. Error bars indicate SD of mean values obtained from 3 repeated assays.</p

Beneficial Effects of cART Initiated during Primary and Chronic HIV-1 Infection on Immunoglobulin-Expression of Memory B-Cell Subsets

<div><p>Introduction</p><p>During HIV-1 infection the B-cell compartment undergoes profound changes towards terminal differentiation, which are only partially restored by antiretroviral therapy (cART).</p><p>Materials and Methods</p><p>To investigate the impact of infection as early as during primary HIV-1 infection (PHI) we assessed distribution of B-cell subsets in 19 PHI and 25 chronic HIV-1-infected (CHI) individuals before and during 48 weeks of cART as compared to healthy controls (n = 23). We also analysed Immunoglobulin-expression of memory B-cell subsets to identify alterations in Immunoglobulin-maturation.</p><p>Results</p><p>Determination of B-cell subsets at baseline showed that total and Naive B-cells were decreased whereas Activated Memory (AM), Tissue-like Memory (TLM) B-cells and Plasma cells were increased in both PHI and CHI patients. After 4 weeks of cART total B-cells increased, while AM, TLM B-cells and Plasma cells decreased, although without reaching normal levels in either group of individuals. This trend was maintained until week 48, though only total B-cells normalized in both PHI and CHI. Resting Memory (RM) B-cells were preserved since baseline. This subset remained stable in CHI, while was expanded by an early initiation of cART during PHI. Untreated CHI patients showed IgM-overexpression at the expenses of switched (IgM-IgD-) phenotypes of the memory subsets. Interestingly, in PHI patients a significant alteration of Immunoglobulin-expression was evident at BL in TLM cells, and after 4 weeks, despite treatment, in AM and RM subsets. After 48 weeks of therapy, Immunoglobulin-expression of AM and RM almost normalized, but remained perturbed in TLM cells in both groups.</p><p>Conclusions</p><p>In conclusion, aberrant activated and exhausted B-cell phenotypes rose already during PHI, while most of the alterations in Ig-expression seen in CHI appeared later, despite 4 weeks of effective cART. After 48 weeks of cART B-cell subsets distribution improved although without full normalization, while Immunoglobulin-expression normalized among AM and RM, remaining perturbed in TLM B-cells of PHI and CHI.</p></div

B-cell subsets distribution and trend of study groups.

<p>Indicated are frequencies of B-cells subsets in PHI and CHI at baseline and after 4 and 48 weeks of cART, as well as values of healthy controls (CS). Column A shows medians with IQR of each parameter; Column B shows median values of each time-point to highlight the trend over time. Dotted horizontal line represents the mean of the CS individuals for a given cell population. P values are * = 0.01–0.05, ** = 0.001–0.01, *** = <0.001, obtained with Mann-Whitney test for results of column A and with Two-sample paired sign test for results of column B.</p

Viro-immunological follow-up of study groups.

<p>CD4+ T-cell count (cells/mm³) and plasma viremia (log copies/ml) for PHI and CHI patients are here represented. Left panels show medians with IQR of each parameter; right panels show median values of each time-point to highlight the trend over time. P values are * = 0.01–0.05, ** = 0.001–0.01, *** = <0.001, obtained with Mann-Whitney test for left panels and with Two-sample paired sign test for right panels.</p

Baseline characteristics of study participants.

<p>PHI are individuals recruited at Fiebig stage III to V of HIV-1 infection before donating baseline samples. CHI are chronically HIV-1 infected individuals having been infected for at least 0.8 years (median 2.9, IQR 0.8–5.3 years,) and not receiving cART at recruitment. MSM means Men having sex with men; IVDU means intravenous drug user; WBC means White Blood Cells; AST means aspartate aminotransferase; ALT means alanine aminotransferase. Statistical analysis was performed using (a) Mann-Whitney test and (b) Chi-square test. P values <0.05 were considered significant differences.</p

List of inhibitory reagents selected.

<p>List of inhibitory reagents selected.</p