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Antimicrobial Agents and Chemotherapy, May 2008, p. 1751-1758, Vol. 52, No. 5
0066-4804/08/$08.00+0     doi:10.1128/AAC.00707-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Inhibitory Effect of PRO 2000, a Candidate Microbicide, on Dendritic Cell-Mediated Human Immunodeficiency Virus Transfer

Natalia Teleshova,¹ Theresa Chang,¹ Albert Profy,² and Mary E. Klotman^1*

Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, New York, New York,¹ Indevus Pharmaceuticals, Inc., Lexington, Massachusetts²

Received 30 May 2007/ Returned for modification 25 July 2007/ Accepted 27 February 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Without an effective vaccine against human immunodeficiency virus (HIV) infection, topical microbicide development has become a priority. The sulfonated polyanion PRO 2000, a candidate topical microbicide now in phase II/III clinical trials, blocks HIV infection of cervical tissue in vitro. Dendritic cells (DC) are among the first cell types to contact HIV in the genital tract and facilitate the spread of the virus. Thus, interfering with virus-DC interactions is a desirable characteristic of topical microbicides as long as that does not interfere with the normal function of DC. PRO 2000 present during capture of the replication-defective HIV_JRFL reporter virus or replication-competent HIV_BaL by monocyte-derived DC (MDDC) inhibited subsequent HIV transfer to target cells. Continuous exposure to PRO 2000 during MDDC-target cell coculture effectively inhibited HIV infection of target cells. PRO 2000 inhibited HIV capture by MDDC. In addition, the compound blocked R5 and X4 HIV envelope-mediated cell-cell fusion. Interestingly, simultaneous exposure to PRO 2000 and lipopolysaccharide attenuated the cytokine production in response to stimulation, suggesting that the compound altered DC function. While efficient blocking of MDDC-mediated virus transfer and infection in the highly permissive MDDC-T-cell environment reinforces the potential value of PRO 2000 as a topical microbicide against HIV, the impact of PRO 2000 on immune cell functions warrants careful evaluation.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Heterosexual transmission is the leading mode of human immunodeficiency virus (HIV) transmission worldwide. Almost half of the people living with HIV/AIDS are women, and the vulnerability of women to acquiring HIV infection requires the development of effective and acceptable female-controlled methods, such as microbicides, to reduce HIV transmission (NIH fiscal year 2005 plan for HIV-related research).

Observations derived from the macaque model using either simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus have provided important insights into local events that occur at the time of infection (1, 30, 31, 42). In particular, the tracking of vaginal SIV infection in macaques has shown that dendritic cells (DC) get infected with SIV and then migrate to draining lymph nodes, where they can efficiently transmit virus to T cells, within hours (20).

DC represent immune system sentinels that engulf pathogens in the peripheral tissues, including the genital tract, and process them for antigen presentation on major histocompatibility complex class I and II molecules (25, 26, 35, 43). DC express CD4, dendritic-cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and other C-type lectin receptors that may facilitate the capture and transfer of HIV (13, 14, 45). In addition to facilitating the transfer of virus to permissive T cells (trans-infection) without de novo infection, the C-type lectin receptors can enhance CD4/CCR5-mediated de novo infection of DC, or "cis"-infection (45). The importance of antigen-presenting cells in the initial infection of the cervical epithelium has also been demonstrated in the human cervical tissue explant model developed by Greenhead et al. (16, 21). The authors demonstrated that DC migrated out of the tissue and were able to transmit HIV to susceptible target cells. The rapid transfer of the virus to lymphoid tissue by DC underscores the need to develop strategies to interfere with HIV-DC interactions early during infection. Thus, interfering with virus-DC interactions represents a unique task for topical microbicides.

The largest class of topical microbicide candidates that has moved forward to clinical trials is negatively charged sulfated or sulfonated agents, including PRO 2000 (6), polystyrene sulfonate (6, 19), dextrin 2-sulfate (22), cellulose sulfate (CS) (2, 6), and carrageenan (47). These compounds block HIV and other sexually transmitted infections (STIs), including herpes simplex virus (HSV), Chlamydia trachomatis, and Neisseria gonorrhoeae infections, to various degrees in vitro. PRO 2000, CS, and carrageenan have been formulated and are undergoing testing in phase II/III clinical trials (reviewed in reference 8). However, a recent phase III trial of CS was prematurely stopped due to safety concerns. Preliminary results at some trial sites indicated that the use of CS was associated with an increased risk of HIV infection (www.aidsinfo.nih.gov); however, the final analysis of that trial has not been completed. These compounds inhibit HIV and HSV entry by interacting directly with viral glycoproteins (6, 40).

The polyanion PRO 2000 was reported to block the binding of HIV gp120 to its primary receptor, CD4 (38). Polyanions have been reported to bind to gp120 with high affinity and inhibit the binding of gp120 V3-directed monoclonal antibody (MAb) (4, 17, 36, 40). Importantly, cervical and vaginal lavage fluids from women who received a single application of PRO 2000 had significant HIV and HSV inhibitory activity in vitro with no apparent acute inflammatory response (23, 28). In the cervical tissue explant model, PRO 2000 was effective against HIV type 1 (HIV-1) X4 and R5 viruses (11, 16) and inhibited the dissemination of virus by migratory DC (11). Partial protection (50 to 75%) in macaques treated with PRO 2000 and challenged with simian-human immunodeficiency virus has been reported as well (46).

In order to understand the potential limitations of these compounds, it is important to evaluate their interactions with mucosal cells involved in HIV transfer. In this study, we tested the effect of PRO 2000 on HIV transfer using monocyte-derived DC (MDDC), which are phenotypically similar (HLA-DR⁺ MR⁺ DC-SIGN⁺) to DC mediating HIV transfer in the cervical explant model. We also assessed the effect of PRO 2000 exposure on MDDC cytokine production.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents. Recombinant interleukin-2 (IL-2) and IL-4 were purchased from R&D Systems, Inc. (Minneapolis, MN), and granulocyte-macrophage colony-stimulating factor was purchased from Berlex Laboratories, Inc. (Montville, NJ). Reagents for tissue culture were purchased from GIBCO Life Technologies (Grand Island, NY). AB human serum was from MP Biomedicals (Solon, OH). Lipopolysaccharide (LPS) and phytohemagglutinin (PHA) were from Sigma (St. Louis, MO). PRO 2000 (sulfonated polymer) was obtained from Indevus Pharmaceuticals, Inc. (Lexington, MA). Fusion inhibitor T20 was obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP).

Cell preparation and culture. Human immature MDDC were generated from CD14⁺ monocytes in the presence of IL-4 (100 U/ml) and granulocyte-macrophage colony-stimulating factor (1,000 U/ml). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of anonymous donors using Ficoll-Hypaque density gradient centrifugation. CD14⁺ monocytes were isolated by using CD14 magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol, with modifications, and cultured as previously described (12) in the presence of 1% AB human serum. To characterize the MDDC phenotype, cells were stained with fluorescein isothiocyanate-conjugated MAbs against HLA-DR (G46-6) used in combination with phycoerythrin-conjugated MAbs against DC-SIGN (120507; R&D Systems) and MR (19.2), CD80 (L307.4), CD83 (HB15e), and CD86 (IT2.2) (all from BD PharMingen). Fluorescence was detected by using a FACScan flow cytometer (BD) and analyzed using FlowJo software. Background fluorescence was defined with irrelevant mouse immunoglobulin Gs. To generate mature MDDC (mMDDC), LPS (100 ng/ml) was added to the cells on day 6 of culture. After 48 h of LPS stimulation, mMDDC had an intermediate/mature phenotype with increased CD80, CD83, and CD86 levels and decreased DC-SIGN and MR expression (not shown).

CD4⁺ T cells were isolated from PBMCs (which were first depleted of CD14⁺ to generate MDDC) by using a CD4⁺ T-cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec) and were cultured in RPMI medium supplemented with 10% fetal bovine serum, antibiotics, and IL-2 (50 U/ml). CD4⁺ T cells were stimulated with PHA (5 µg/ml) for 1 to 2 days at 37°C prior to use.

HeLa-CD4⁺ CCR5⁺ cells expressing endogenous CXCR4 and stably transfected with CD4 and CCR5 were a gift from Shawn E. Kuhmann (34). Cells were maintained in Dulbecco's minimal essential medium containing 10% fetal bovine serum and glutamine.

Effect of presence of PRO 2000 during HIV capture on MDDC-mediated HIV transfer. Replication-defective HIV pseudotyped with JRFL envelope (RD HIV_JRFL) was used in a single-cycle infection assay. RD HIV_JRFL was prepared in 293T cells by using a previously described three-plasmid cotransfection system consisting of the packaging construct pCMV delta R8.2 (32), the HIV reporter construct pNL4-3.Luc.R-E (obtained from Nathaniel Landau through the ARRRP) (7, 18), and the envelope-expressing construct pJRFL that expresses an R5 envelope (gift from D. Littman, Skirball Institute, New York University, New York, NY). Transfections were done using Lipofectamine 2000 (Invitrogen).

Replication-competent R5 strain HIV_BaL (Advanced Biotechnologies, Inc., Columbia, MD) was used in a multiple-round infection assay. For purposes of comparison, both viruses were titrated in CD4⁺ CCR5⁺ CXCR4⁺ TZM-bl cells (from ARRRP; gift of J. C. Kappes and X. Wu) and comparable doses (63 and 100 50% tissue culture infective doses [TCID₅₀]/1 x 10⁶ cells, respectively) were used in the study.

To determine the effect of PRO 2000 on HIV transfer by MDDC, the compound was present only during HIV capture. In all experiments, viruses were preincubated for 1 h at 37°C with PRO 2000 or with medium before being added to the cells. MDDC were incubated with RD HIV_JRFL or HIV_BaL for 2 h at 37°C in the presence or absence of PRO 2000. Then, cells were washed four times and plated with target cells (5 x 10⁵ MDDC:5 x 10⁴ HeLa CD4⁺ CCR5⁺ cells or 1.25 x 10⁵ to 2.5 x 10⁵ MDDC:1 x 10⁶ PHA-activated autologous CD4⁺ T cells). Unbound HIV was efficiently removed before coculture, as cell-free final washes from virus-exposed MDDC failed to infect target cells (not shown). However, we cannot exclude the presence of bound residual PRO 2000 after washes. As a control, MDDC were cultured in the absence of target cells after exposure to the virus. To investigate the effect of PRO 2000 on direct infection of target cells, HeLa CD4⁺ CCR5⁺ and CD4⁺ T cells were infected with cell-free HIV in the presence or absence of PRO 2000 for 2 h at 37°C, washed, and cultured with or without the compound.

The luciferase activity of the lysates in the single-cycle infection assay was quantitated after 72 h of culture by using a luciferase assay system (Promega) according to the manufacturer's instructions. The luciferase activity (in relative light units) was measured on an EG&G Berthold MiniLumat LB 9506 luminometer (Berthold Technologies, Bad Wildbad, Germany).

In the multiple-round infection assay, supernatants (100 µl) were collected at the beginning of the culture (day 0) and on days 5, 7, 9, and 14 for p24 enzyme-linked immunosorbent assay (ELISA).

Effect of continuous exposure to PRO 2000 on MDDC-target cell infection. To determine the effect of continuous exposure to PRO 2000 on HIV infection in a single-cycle infection assay, MDDC were mixed with RD HIV_JRFL in the presence or absence of PRO 2000 and added to HeLa CD4⁺ CCR5⁺ cells. After 2 h of incubation, the medium was carefully changed, and cells were cultured with or without PRO 2000. The luciferase activity of the lysates was measured as described above after 72 h of culture. In a multiple-round infection assay, MDDC were mixed with HIV_BaL in the presence or absence of PRO 2000 and then added to autologous PHA-activated CD4⁺ T cells. After 2 h of incubation, the MDDC and T-cell mixtures were washed four times and cultured with or without PRO 2000 for 14 days. Supernatants (100 µl) for p24 ELISA were collected at the beginning of the culture (day 0) and on days 5, 7, 9, and 14.

p24 ELISA. p24 antigen was measured by ELISA (NCI, Frederick, MD) according to the manufacturer's protocol. The detection limit of the assay was 80 pg/ml of p24.

Toxicity assay. The toxicity of PRO 2000 was determined for CD4⁺ T cells and MDDC. The cells were exposed to PRO 2000 for 4 days. The viability of the cells was analyzed by using a tetrazolium compound assay (CellTiter96; Promega, Madison, WI).

HIV-1 capture assay. An HIV_BaL capture assay was performed as described elsewhere, with modifications (21). MDDC were seeded at 2 x 10⁵ per well in 96-well flat-bottomed plates. HIV_BaL alone (420 TCID₅₀/2 x 10⁵ MDDC) or in the presence of PRO 2000 was preincubated for 1 h at 37°C. This higher dose of HIV was chosen based on the limits of detection in this system. To test the effect of PRO 2000 on virus binding, plated MDDC were incubated for 30 min on ice, exposed to HIV for 2 h on ice in the presence or absence of PRO 2000, washed four times, and lysed with 1% Triton X-100. To test the effect of PRO 2000 on binding/internalization, cells were incubated with virus for 2 h at 37°C, washed four times, and lysed. The amount of cell-associated virus was determined by quantitating HIV p24 by ELISA.

HIV glycoprotein-mediated cell-cell fusion. HeLa cells expressing Tat proteins (HeLa-Tat; from ARRRP) were transfected with the HIV_JRFL envelope plasmid (gift of D. Littman, New York University) for 48 h. Transfected HeLa-Tat cells and HL2/3 cells containing stably integrated copies of the HIV-1 molecular clone HXB2/3gpt (from ARRRP) expressing HIV glycoproteins were incubated with PRO 2000 or T20 at 37°C for 1 h before being added to TZM-bl indicator cells, expressing the CD4, CXCR4, and CCR5 coreceptors, at 5 x 10⁵ per well in a 48-well plate. TZM-bl cells contain HIV long terminal repeat-driven beta galactosidase and luciferase reporter genes which are activated by Tat proteins when cell-cell fusion occurs. After incubation for 8 to 24 h at 37°C, TZM-bl cells were treated with lysis buffer (Promega Corp.) and luciferase activity was measured by using a Perkin-Elmer 1420 luminometer.

Effect of PRO 2000 on MDDC cytokine production. A series of experiments were performed to determine the effects of short- and long-term exposure of PRO 2000 on MDDC baseline cytokine production and response to LPS. LPS stimulation was done either in the presence of PRO 2000 or following washout.

MDDC were exposed to PRO 2000 for 1 h or 48 h, washed four times, and recultured without the compound for 48 h. LPS (100 ng/ml) was added to the cells to test if prior exposure to PRO 2000 changes the cytokine response induced by LPS stimulation. To determine cytokine production in response to LPS stimulation in the presence of PRO 2000, both the compound and LPS were added to MDDC simultaneously for 48 h. Supernatants were collected and stored at –20°C until use.

Luminex fluorescent-bead assay. Cytokine measurement was done by using a Beadlyte human multicytokine detection system 2 (IL-1β, IL-6, IL-8, IL-10, IL-12p70, and tumor necrosis factor alpha [TNF-]) (Upstate Biotechnology, Lake Placid, NY).

Statistical analysis. Statistical analysis was performed with the two-tailed Student's t test, with significance set at a P value of <0.05.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Presence of PRO 2000 during RD HIV_JRFL capture by MDDC inhibits virus transfer. We first determined whether PRO 2000 interferes with the MDDC-mediated transfer of RD HIV_JRFL reporter virus in a synchronized system using a single-cycle infection assay. MDDC were exposed to RD HIV_JRFL in the presence of PRO 2000 for 2 h and washed before being added to HeLa cells expressing CD4 and CCR5, which provided a better luciferase readout than primary CD4⁺ T cells. As expected, MDDC efficiently transferred RD HIV_JRFL to target cells in the absence of the compound (Fig. 1A). Consistent with the results of a published report (29), there was no infection in MDDC cultured in the absence of target cells (not shown). The presence of PRO 2000 during the exposure of MDDC to RD HIV_JRFL inhibited the subsequent infection of HeLa CD4⁺ CCR5⁺ cells in a dose-dependent manner (Fig. 1B). Exposure to PRO 2000 at 100 µg/ml, a concentration similar to the one detected after the application of a 0.5% formulation into a human vagina (23), resulted in 55% inhibition of virus transfer (Fig. 1A and B). This inhibitory effect, present even when MDDC were extensively washed to eliminate cell-free virus and residual compound prior to coculture with HeLa CD4⁺ CCR5⁺ cells, likely reflects a decreased amount of virus sequestered by MDDC in the presence of PRO 2000, although we cannot exclude the inhibition of transfer by residual compound.

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FIG. 1. PRO 2000 inhibits MDDC-mediated RD HIVJRFL transfer to HeLa CD4+ CCR5+ cells and infection in MDDC-HeLa CD4+ CCR5+ cell cocultures. (A and B) In transfer experiments, MDDC or mMDDC were mixed with RD HIVJRFL preincubated with PRO 2000 or medium for 1 h at 37°C. After 2 h of incubation, MDDC were washed and plated with HeLa CD4+ CCR5+ cells. The results of a representative experiment (mean ± standard deviation [SD] of the results of duplicate wells) (A) and the summary of three to eight experiments (mean ± SD) (B) are shown. Percent inhibition of HeLa CD4+ CCR5+ cell infection by PRO 2000 in transfer experiments and significant differences are shown relative to infection in the absence of the compound (0% inhibition). (C) In coculture experiments, MDDC were mixed with RD HIVJRFL preincubated with PRO 2000, immediately added to target cells, and then incubated in the presence or absence of the compound for 2 h. Then, medium containing the virus and the compound was aspirated and cells were cocultured in fresh medium with or without PRO 2000 for 72 h. HeLa CD4+ CCR5+ cells were infected with cell-free RD HIVJRFL in the presence or absence of PRO 2000, washed, and incubated with or without the compound. Percent inhibition of infection by PRO 2000 and significant differences are shown relative to level of infection in the absence of the compound (0% inhibition). A summary of the results of three to eight experiments (mean ± SD) is shown. R.L.U., relative light units; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

We also examined the effect of PRO 2000 on mMDDC-mediated HIV transfer, as encounter with sexually transmitted pathogens in the setting of coexisting STI can induce DC maturation (15, 39). Similar to the observed effect on MDDC-mediated HIV transfer, PRO 2000 inhibited mMDDC-mediated HIV transfer (Fig. 1B).

Continuous exposure to PRO 2000 inhibits RD HIV_JRFL infection in MDDC-target cell cocultures. A microbicide could be present during both DC HIV uptake and subsequent transfer to T cells via DC-T-cell conjugates in the mucosa and submucosa. To test the efficacy of PRO 2000 in this setting, MDDC and HeLa CD4⁺ CCR5⁺ mixtures were infected with RD HIV_JRFL in the presence of PRO 2000 and cultured in the presence of the compound. Continuous exposure to PRO 2000 at 10 and 100 µg/ml inhibited HeLa CD4⁺ CCR5⁺ infection more effectively than the presence of compound only during capture (Fig. 1C). However, this experimental approach does not discriminate which step(s) during RD HIV_JRFL uptake or transfer is affected. For comparison, the infection of HeLa CD4⁺ CCR5⁺ cells with cell-free virus was effectively inhibited in the presence of PRO 2000 (Fig. 1C).

Presence of PRO 2000 during HIV_BaL capture by MDDC inhibits virus transfer. To assess the extent of PRO 2000 inhibition on replication-competent virus transfer, MDDC were exposed to HIV_BaL in the presence of PRO 2000 for 2 h and washed before coculture with autologous CD4⁺ T cells. PRO 2000 at 100 µg/ml inhibited the MDDC-mediated HIV_BaL infection of T cells by up to 87 to 100% (days 5 to 14 of culture) (Fig. 2A and B).

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FIG. 2. PRO 2000 inhibits MDDC-mediated transfer of HIVBaL to CD4+ T cells and infection in MDDC-CD4+ T-cell cocultures. (A) For transfer experiments, MDDC were mixed with HIVBaL preincubated with PRO 2000 (100 µg/ml) or medium for 1 h at 37°C. After 2 h of incubation, MDDC were washed and added to PHA-activated CD4+ T cells. As a control, MDDC were cultured without T cells after exposure to the virus. Alternatively, T cells were infected directly with cell-free HIVBaL in the presence or absence of PRO 2000, washed, and cultured with or without the compound. Supernatants were collected at the beginning of the culture (day 0) and on days 5, 7, 9, and 14. Results of a representative experiment (mean ± standard deviation [SD] of the results of duplicate wells) are shown. (B) Summary of the results of three to five transfer experiments (mean ± SD) done as described for panel A is shown. Percent inhibition of CD4+ T-cell infection by PRO 2000 in transfer experiments and significant differences relative to the level of infection in the absence of the compound (control; 0% inhibition) are shown. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (C) For coculture experiments, MDDC were mixed with virus in the presence or absence of PRO 2000 (100 µg/ml) and immediately added to PHA-activated CD4+ T cells. After 2 h of incubation, MDDC and T cells were washed and plated with or without PRO 2000. Supernatants were collected as described for panel A. Results representative of four experiments (mean ± SD of the results of duplicate wells) are shown.

However, in one out of a total of seven experiments, PRO 2000 at 100 µg/ml did not have a significant inhibitory effect (not shown), suggesting that donor cell variability influences the efficacy of the compound. Exposure to the compound at 10 µg/ml inhibited CD4⁺ T-cell infection up to day 9, with breakthrough virus replication at day 14 of the culture (Fig. 2B). The exposure of MDDC to a higher dose of HIV_BaL (1,900 TCID₅₀) resulted in less inhibition of infection in the transfer experiments (not shown). MDDC were not infected with HIV_BaL (Fig. 2A). PRO 2000 at 100 µg/ml effectively (up to 100%) inhibited direct cell-free virus infection of T cells (Fig. 2A).

Continuous exposure to PRO 2000 inhibits HIV_BaL infection in MDDC-target cell cocultures. PRO 2000 efficiently inhibited HIV_BaL replication in MDDC-T-cell cocultures when DC-T-cell mixtures were exposed to the virus in the presence of PRO 2000, washed, and then cultured in the presence of the compound (Fig. 2C).

PRO 2000 is not cytotoxic to MDDC and CD4⁺ T cells. To ensure that the inhibitory effect of PRO 2000 on MDDC-mediated HIV transfer and infection in the cocultures was not due to cytotoxicity, we examined the viability of MDDC and CD4⁺ T cells in the presence of PRO 2000 by MTS assay (Promega). PRO 2000 at 10 or 100 µg/ml did not decrease the viability of T cells (0 to 7% in two experiments) and MDDC (0 to 3% in two experiments) (Fig. 3).

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FIG. 3. PRO 2000 does not cause cytotoxicity in MDDC and CD4+ T-cell cultures. MDDC or PHA-activated CD4+ T cells were cultured in the presence of PRO 2000 for 4 days at 37°C. Cytotoxicity was determined by MTS assay (Promega). Data are means ± standard deviations of the results of triplicate wells and represent two experiments each with MDDC and T cells.

PRO 2000 inhibits HIV_BaL capture. To test the effect of PRO 2000 on HIV binding and internalization by MDDC, cells were incubated with HIV_BaL in the presence or absence of PRO 2000 at 4°C or 37°C, carefully washed, and lysed. We observed an inhibitory effect of PRO 2000 (100 µg/ml) on HIV_BaL binding (4°C) and binding/internalization (37°C) by MDDC (Fig. 4A and B). The inhibition of binding and binding/internalization in the presence of PRO 2000 relative to those in the no treatment control were 51 to 73% (two experiments) and 63 to 72% (three experiments), respectively. Of note is that in one out of a total of four experiments, exposure to PRO 2000 increased virus binding (42%) (not shown). This suggests that the efficacy of the compound can be influenced by donor cell variability.

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FIG. 4. HIVBaL capture by MDDC is inhibited in the presence of PRO 2000. HIVBaL binding at 4°C (A) and binding/internalization by MDDC at 37°C (B) in the presence of PRO 2000 (100 µg/ml; means ± standard deviations of the results of triplicate wells) are shown. The minimum levels of p24 detected in binding and binding/internalization experiments were 1.1 ng/ml and 0.82 ng/ml, respectively. Mean background values were subtracted. Results representative of two binding and three binding/internalization experiments are shown.

Inhibition of HIV glycoprotein-mediated cell-cell fusion. The effective inhibition of HIV activity in MDDC-T-cell cocultures in the presence of the compound and the fact that virus transfer via DC-T synapse is inhibited by entry and fusion inhibitors (24) led us to test the effect of PRO 2000 on HIV glycoprotein-mediated cell-cell fusion. PRO 2000 effectively inhibited HIV_JRFL (R5) envelope-mediated cell-cell fusion (Fig. 5). PRO 2000 also inhibited envelope-mediated cell-cell fusion when donor cells expressed HIV_HXB2 (X4) envelope (not shown).

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FIG. 5. PRO 2000 inhibits R5 envelope fusion. Fusion assay with R5 envelope-expressing cells was performed in the presence of PRO 2000 or T20. Data are means ± standard deviations of the results of triplicate wells. Mean background value was subtracted (results of one of two to three experiments are shown). R.L.U, relative light units.

MDDC cytokine profile after short- and long-term exposure to PRO 2000. Exposure to microbicides might have indirect effects on virus replication in vivo through changes in the local cytokine milieu (9). We tested the effect of in vitro short- and long-term exposure to PRO 2000 on cytokine production.

In the first series of experiments, cells were exposed to PRO 2000 for 1 h (Fig. 6A) or 48 h (Fig. 6B) followed by washout and then stimulation with LPS for 48 h. The latter stimulation mimics mucosal exposure to bacterial pathogens, including sexually transmitted pathogens, such as C. trachomatis and N. gonorrhoeae. Transient or prolonged exposure to PRO 2000 did not significantly alter the baseline or LPS-induced MDDC cytokine profile; however, there were trends toward increased IL-8 and IL-12p70 after 1 h of exposure to PRO 2000, as well as trends toward increased IL-1β and TNF- in response to LPS stimulation after 1 h and 48 h of exposure to PRO 2000 (Fig. 6A and B). In a modification of the protocol, cells were stimulated with LPS in the presence of PRO 2000 (Fig. 6C). In this case, although the non-LPS cytokine profile was not significantly altered by PRO 2000, the LPS-induced changes in IL-1β, -6, -8, and -10 were significantly decreased.

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FIG. 6. MDDC cytokine profile after short- and long-term exposure to PRO 2000. (A) MDDC were exposed to PRO 2000 (100 µg/ml) for 1 h, washed, and replated in the absence (PRO 2000 1h_Med 48 h) or presence of LPS (PRO 2000 1h_LPS 48 h) at 100 ng/ml for 48 h. Controls included MDDC cultured in the presence of LPS or medium alone. Cell-free supernatants were collected at the end of the culture. (B) The experiments were performed as described for panel A except that MDDC were exposed to PRO 2000 for 48 h prior to culture in the absence (PRO 2000 48h_Med 48 h) or presence of LPS (PRO 2000 48h_LPS 48 h). In the experiments whose results are shown in panel C, LPS was added simultaneously with PRO 2000 for 48 h. The indicated cytokines were measured with a Beadlyte human multicytokine detection system. Each supernatant was tested in duplicate. Of note is that the differences in baseline cytokine production in experiments described for panels A, B, and C derive from the fact that there are differences in the experimental setups, including washouts versus no washout and total duration of MDDC culture prior to the measurements. A summary of the results (mean pg/ml ± standard deviation) of three to four experiments is shown. *, P < 0.05; ***, P < 0.001.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PRO 2000, at a concentration achieved in vivo after a single vaginal administration, inhibited the MDDC-mediated HIV infection of HeLa CD4⁺ CCR5⁺ and CD4⁺ T cells when the compound was present only during HIV capture by MDDC. PRO 2000 also inhibited target cell infection when present throughout the MDDC-target cell coculture period. These observations suggest that the compound may alter DC-mediated HIV transfer and amplification of infection whether this occurs locally or at a distance.

PRO 2000 most likely interferes with a number of steps during the DC capture and transfer of HIV. Exposure to the compound led to the partial inhibition of HIV_BaL capture by MDDC, as indicated by the binding and internalization studies. This is consistent with the results of previous reports indicating that PRO 2000 interferes with DC-SIGN-mediated HIV capture (11) and inhibits HIV binding to epithelial cells (37). The increased binding seen in one of four experiments utilizing different donor sources for cells has been an important variable and warrants further investigation. The compound also inhibited HIV envelope-mediated cell-cell fusion, which may contribute to the inhibitory effect on cell-mediated HIV transfer seen when PRO 2000 was present in the coculture. Although the exact mechanism of this interference is uncertain, the process of virus transfer via DC-T synapse is inhibited by entry and fusion inhibitors (24).

SAMMA, a mandelic acid condensation polyanion that is being developed as a topical microbicide, has recently been reported to have similar inhibitory effects on DC-mediated T-cell infection (5).

Langerhans cells, langerin-negative lamina propria DC, macrophages, and T cells are the first cells to be infected in the vaginal epithelium following atraumatic intravaginal challenge of macaques with SIV (20). In our study, MDDC were chosen as a model based on their phenotypic similarity to lamina propria DC. The latter have been shown to mediate HIV transfer to susceptible target cells in the human cervical explant tissue model (21). Lamina propria DC may have a central role in early HIV infection in the setting of an inflamed or traumatized squamous epithelium (41, 45).

Our data are in agreement with those of a recent report describing >90% inhibition of HIV transmission by cervical tissue-derived DC upon tissue exposure to PRO 2000 at 100 µg/ml, with an IC₅₀ of 29.1 µg/ml (11). A similar effect of PRO 2000 (50% effective concentration, 32 µg/ml) on MDDC-mediated transmission was reported recently as well; however, it was less pronounced than the effects of the other carbohydrate-binding agents tested in the study (3).

A critical function of DC is to trigger a potent immune response through the production of cytokines. Proinflammatory cytokines may play opposing roles in HIV, as they may induce protective immune responses as well as directly enhance viral replication (9). It has been shown that genital mucosal fluids from women with bacterial vaginosis have high levels of TNF- and IL-1β, cytokines known to up-regulate HIV replication through activation of the long terminal repeat promoter (33). Bacterial vaginosis and STIs have been associated with increased acquisition of HIV (27, 44). Changes in proinflammatory cytokines (increased IL-1, IL-6, and IL-8) are also considered surrogate markers of microbicide toxicity (10). A single application of 0.5% PRO 2000 gel was not associated with any significant change in the levels of proinflammatory cytokines in cervicovaginal lavage samples from women given the compound compared to those in samples from women given a placebo gel (23). PRO 2000 at 1 mg/ml has been shown to enhance the production of IL-1β and RANTES in cervical tissue in vitro (11); this concentration is higher than the level of PRO 2000 detected following the in vivo application of the 0.5% gel (23).

We investigated if exposure to PRO 2000 influenced MDDC cytokine production. Although PRO 2000 did not induce significant proinflammatory cytokine production, the compound blocked the cytokine response induced by LPS when it was present during LPS exposure. This could lead to less of an inflammatory response during exposure to HIV and/or coexisting STIs and could have a protective effect. On the other hand, an inadequate cytokine response may lead to an impaired immune response against HIV and other mucosal pathogens. It is difficult to predict overall whether these effects of PRO 2000 on DC function would have a beneficial or deleterious outcome relative to HIV transmission. The MDDC cytokine response to LPS stimulation after long-term exposure to PRO 2000 (followed by washout) was comparable to the response to LPS stimulation of nonexposed cells, indicating that PRO 2000 did not induce any persistent changes in DC function at the level of cytokine production.

In summary, PRO 2000 effectively blocked HIV transfer and infection in DC-T-cell cocultures, suggesting that this compound may be effective at inhibiting viral infection and spread within the mucosa as well as spread to local lymph nodes via DC. However, it is important to consider that PRO 2000 interferes with a host immune response and may alter the balance between virus dissemination and a local antimicrobial immune response.

 
This work was supported by grant U19HD48733 from the NIH/NIAID.

We thank members of M. Klotman's and V. Simon's laboratories for helpful discussions.

 
* Corresponding author. Mailing address: Division of Infectious Diseases, Mt. Sinai School of Medicine, New York, NY 10029. Phone: (212) 241-2950. Fax: (212) 534-3240. E-mail: mary.klotman{at}mssm.edu

Published ahead of print on 10 March 2008.

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Antimicrobial Agents and Chemotherapy, May 2008, p. 1751-1758, Vol. 52, No. 5
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