Effects of LA/antiviral drug combinations on HSV and HIV replication.

<p>(A) Effects of LA combinations on their anti-HSV-2 G activity in C8166 cells. The IC₅₀s of LA (white bars) and the combined inhibitors acyclovir, PRO2000 and LabyA1 (grey bars), alone and in combination, are represented. Mean ± SEM from 3 independent experiments is shown. *<i>p</i><0.05, **<i>p</i><0.01, according to student’s T-test, compared with single-drug treatment. (B) Determination of the combination indices (CIs) of LA/acyclovir, LA/PRO2000 and LA/LabyA1 two-drug combinations. Mean CIs ± SEM from 3 independent experiments is shown. The CIs were calculated at 3 inhibitory levels (IC₅₀, IC₇₅, IC₉₀), whereby CIs<0.9 are synergistic, 0.91.1 are antagonistic. (C) Effects of LA combinations on their anti-HIV-1 R5 BaL activity in TZM-bl cells. The IC₅₀s of LA (white bars) and the combined inhibitors PRO2000, maraviroc and tenofovir (grey bars), alone and in combination, are represented. Mean ± SEM from 3–4 independent experiments is shown. *<i>p</i><0.05, according to student’s T-test, compared with single-drug treatment. (D) Calculation of CIs of LA in combination with PRO2000, maraviroc and tenofovir. Mean CIs ± SEM from 3–4 independent experiments is shown. The CIs were again calculated at 3 inhibitory levels (IC₅₀, IC₇₅, IC₉₀), whereby CIs<0.9 are synergistic, 0.91.1 are antagonistic.</p

Effects of antiviral activity on increased viral input in MT-4 cells.

<p>^a Concentrations are in μM.</p><p>^b Viral input is expressed in pg/well, with 25 pg/well as our reference viral input.</p><p>^c Fold change in IC₅₀s: highest IC₅₀/lowest IC₅₀.</p><p>Effects of antiviral activity on increased viral input in MT-4 cells.</p

LA inhibits HSV-2 DC-SIGN-related transmission to uninfected target T cells.

<p>(A) DC-SIGN⁺ cells were generated out of buffy coat and exposed to HSV-2 G for 2 h. After several washing steps, virus exposed cells were cocultivated with uninfected CD4⁺ MT-4 cells for 5 days. Viral-induced cytopathicity was first scored microscopically. Shown here are the light microscopic pictures of cocultivated HSV-2 G-exposed DC-SIGN⁺ cells and uninfected MT-4 cells 5 days post-cocultivation (a); the effects on HSV-2 G transmission and replication in the presence of 12.5 μM (b), 2.5 μM (c) and 0.5 μM LA (d). One representative experiment out of 4 individual donors is shown. (B) Bars represent the % HSV-2 G transmission and replication in MT-4 cells after 5 days of cocultivation, relative to the positive control. Dying MT-4 infected cells were detected by flow cytometry side-scatter analyses. Mean ± SEM out of 4 individual donors is shown. (C) MDDCs were generated from buffy coat and exposed to HSV-2 G for 2h. After several washing steps, virus exposed MDDCs were co-cultivated with uninfected CD4⁺ MT-4 cells for 5 days. Viral-induced cytopathicity was scored light microscopically and supernatant was retained for further analysis of viral content. Bars represent the percentage of viral replication after 5 days of co-cultivation, relative to the positive untreated control. Mean ± SEM of 3 independent experiments is shown.</p

No toxic effects of LA on vaginal <i>Lactobacilli</i>.

<p>Growth of the gastro-intestinal <i>L</i>. <i>rhamnosus GG</i> and different vaginal <i>Lactobacilli</i> strains in the presence of various concentrations of LA for 24 h. The results are expressed in % of untreated bacterial cells. The mean ± sem of 3 independent experiments is shown.</p

Cross-resistance pattern of the HIV-1 NL4.3^LAresistant virus.

<p>(A) Fold-increase in IC₅₀ of the HIV-1 NL4.3^LAresistant virus against the entry inhibitors HHA, FGM, T20, PRO2000, AMD3100 and b12 mAb. Mean ± SEM of 3–4 independent experiments is shown. (B) Dose-dependent effects of LA on the replication of the <i>in vitro</i> generated HIV-1 entry inhibitor resistant strains in MT-4 cells. Mean ± SEM of 3 independent experiments is shown.</p

LA has a potent broad-spectrum anti-HIV activity with low, if any, cytotoxicity.

<p>(A) Concentrations up to 62.5 μM (500 μg/ml) of LA were given to MT-4 cells, HEK293T cells and PBMCs. Cytotoxicity levels and CC₅₀s were determined spectrophotometrically using MTS/PES method. (B) Dose-dependent anti-HIV activity of LA in the CD4⁺ T-lymphoma cell line MT-4 against 3 laboratory HIV-1 strains (NL4.3, IIIB and HE) and 1 HIV-2 strain (ROD) with respectively the following IC₅₀s: 0.20 ± 0.03 μM (NL4.3), 0.22 ± 0.06 μM (IIIB), 0.22 ± 0.00 μM (HE) and 0.34 ± 0.04 μM (ROD). Mean IC₅₀ ± SEM up to 6 independent experiments is shown. (C). Evaluation of the IC₅₀s of LA against various clinical isolates representing different subtypes (A-H), irrespective of HIV-1 coreceptor tropism in primary cells (PBMCs and monocyte/macrophages [MDM]). Viral replication was measured using HIV-1 p24 Ag ELISA with exception of p27 HIV Ag ELISA for BCF-06 (group O). The IC₅₀s for viral inhibition were as follows: 0.10 ± 0.03 μM (UG273); 0.12 ± 0.06 μM (US2); 0.20 ± 0.05 μM (BK132); 0.15 ± 0.04 μM (ETH2220); 0.19 ± 0.01 μM (DJ259); 0.19 ± 0.04 μM (SE365); 0.16 ± 0.04 μM (BZ163); 0.13 ± 0.08 μM (BCF-DIOUM); 0.17 ± 0.08 μM (HH8793); 0.24 ± 0.07 μM (BCF-KITA); 0.07 ± 0.05 μM (BCF-06) and 0.11 ± 0.02 μM (BaL). Mean IC₅₀s ± SEM from 2–6 independent donor experiments is shown. (D) Dose-dependent effect of LA on the giant cell (syncytia) formation between persistently HIV-1 IIIB-infected T cells (HUT-78/IIIB) and non-infected CD4⁺ target SupT1 T cells. Percentage syncytia inhibition was measured by flow cytometry. Mean ± SEM of 3 independent experiments is shown.</p

LA inhibits HIV-1 DC-SIGN-related transmission to uninfected CD4⁺ target T cells.

<p>(A) Raji.DC-SIGN⁺ cells were pretreated with or without various concentrations of LA for 30 min. The binding of PE-conjugated anti-DC-SIGN mAb (clone DCN46) was measured by flow cytometry. The bars represent the % of anti-DC-SIGN binding relative to the untreated conditions. Mean ± SEM of 3 independent experiments is shown. (B) Effect of LA, HHA and PRO2000 on the capture of HIV-1 strain HE (R5/X4) by DC-SIGN on Raji.DC-SIGN⁺ cells. HIV-1 virions were pre-incubated for 30 min. with the test compounds before being exposed for 1h to Raji.DC-SIGN⁺ cells. After washing the cells, p24 HIV-1 Ag ELISA was used to quantify the amount of cell-associated virus. Bars represent the % virus binding relative to untreated conditions. Mean ± SEM of 3 independent experiments is shown with *<i>p</i><0.05, according to Student’s T-test. (C) HIV-1 HE (R5/X4) virions were exposed to Raji.DC-SIGN⁺ cells for 1 h. After extensive washing, Raji.DC-SIGN/HE cells were cocultivated with or without compound pretreated uninfected CD4⁺ target C8166 T cells for 48h. Cocultures were stained with PE-conjugated anti-DC-SIGN and FITC-conjugated anti-CD4. The % of positive gated cells in the quadrants of the dot plots is given. One representative experiment out of 3 is shown.</p

Broad-spectrum anti-HSV-2 activity of LA in epithelial HEL cells, MDDCs and CD4⁺ HIV-susceptible T cells.

<p>(A) Dose-depedent anti-HSV activity of LA in HEL fibroblasts against wild-type HSV-1 strains [KOS (reference) and RV-174 (clinical isolate)], TK^- HSV-1 strains RV-179 and RV-117 (clinical isolates)], wild-type HSV-2 strains [G (reference), RV-124 and RV-24 (clinical isolates)] and TK^- HSV-2 strains (RV-129 and BA19026589 (clinical isolates)]. The following IC₅₀s were obtained: 0.53 ± 0.08 μM (KOS strain); 0.59 ± 0.03 μM (RV-174); 0.62 ± 0.06 μM (RV-179); 0.51 ± 0.05 μM (RV-117); 0.42 ± 0.09 μM (strain G); 0.51 ± 0.05 μM (RV-124); 0.56 ± 0.00 μM (RV-24); 0.54 ± 0.02 μM (RV-129) and 0.42 ± 0.09 μM (BA19026589). Mean ± SEM out of 2 independent experiments is shown. (B) HEL cells were infected with HSV-2 G and the compounds [LA (0.5 μM), LabyA1 (9.6 μM) and Acyclovir (10 μM)] were added at the time of infection (0 h) or 2 h post-infection. After 3 days, virus-induced CPE was scored microscopically. Mean ± SEM up to 3 experiments is shown. (C) Infection of HSV-2 G in CD4⁺ HIV-1 susceptible T cell lines. MT-4-, C8166 T-lymphoma cells were infected with HSV-2 G. Percentage of infected cells is indicated after 3 days (MT-4 cells) or 4 days (C8166 cells) post-infection as detected using the mAb [10.B.7] directed to the HSV-2 envelope glycoprotein B (gB) by flow cytometry. Grey histograms represent the background fluorescence, while the black histograms show virus binding. The mean fluoresence intensity (MFI) values are indicated in each histogram. (D) Dose-dependent anti-HSV-2 G activity of LA in the CD4⁺ T-lymphoma cell lines MT-4 and C8166 as measured spectrophotometrically. Mean IC₅₀ ± SEM from 5–6 independent experiments is shown. (E) Infection of HSV-2 G in MDDCs. Viral replication was measured 5 days post-infection, relative to the postive control, based on flow cytometry side-scatter analyses. Mean ± SEM of 4 individual donors is shown.</p

LA inhibits efficiently single and dual HIV-1/HSV-2 infection in T cells.

<p>MT-4 cells were infected with HIV-1 NL4.3, HSV-2 G and HIV-1 NL4.3 + HSV-2 G for 3 days in the presence of various concentrations of LA (A), AMD3100 (B) and acyclovir (C). Cytopathicity was measured spectrophotometrically using MTS/PES (left panels). The results are normalized to untreated infected cells. Viral replication at dual infection (shown in blue) was also further investigated by flow cytometry (right panels). HIV-1 NL4.3 replication was detected with b12 mAb + RaH-FITC (white bars) and HSV-2 strain G with agB + GaM-PE (grey bars). Bars indicate the % viral replication in MT-4 cells, compared to untreated conditions. The results shown are the mean ± SEM of 3–5 independent experiments.</p

Lack of stimulatory effects of LA on PBMCs.

<p>(A) LA does not activate HIV-1 susceptible CD4⁺ T cells. Freshly isolated PBMCs were cultured in the presence of LA, tenofovir or PHA for 3 days at 37°C. The cells were then analyzed for their expression of the activation markers CD69 (early), CD25 (late) and HLA-DR (very late) by the mAbs PE-conjugated anti-CD69 (left), anti-CD25 (middle) and anti-HLA-DR (right) together with PerCP-conjugated anti-CD4. Mean ± SEM of 6 independent experiments is shown. ***<i>p</i><0.001; ****<i>p</i><0.0001, according to student’s T-test for comparison with untreated PBMCs. (B) Freshly isolated PBMCs were pretreated with LA, tenofovir, PRO2000, acyclovir or PHA for 24h, then thoroughly washed and infected with HIV-1 R5 BaL for 7 days in the abscence of compounds. Viral replication in the supernatant was measured using HIV-1 p24 Ag ELISA. Mean of 3 independent donor experiments is shown.</p