HIV mucosal transmission represents the principal route of virus acquisition, particularly of R5-tropic virus.1,2 In the initial phase of infection, HIV crosses the mucosal epithelial barrier likely within hours3 to access target cells in the submucosa including CD4 T cells, dendritic cells (DCs), and macrophages.4 Small local founder populations of infected cells are generated5,6 that then undergo local expansion to produce enough virus and infected cells that can migrate to proximal lymphoid tissue to establish a systemic infection.5,7-9 More recently, sequencing viruses in heterosexual transmission pairs and in acute HIV infection provided evidence that a single virus (or infected cell) initiated productive infection in close to 80% of the individuals tested, with the remaining individuals infected by between 2 and 5 viruses.10,11 This underscores the challenges faced in preventing HIV infection and spread. Productive infection of a cell is initiated through attachment of HIV envelope to CD4. This interaction triggers conformational changes in the viral envelope that enable HIV to bind to its coreceptor CCR5 or CXCR4.12-14 Further conformational changes in the envelope lead to the formation of a fusion pore mediating infection of the target cell.15,16
The availability of CD4 and CCR5 molecules on immature DCs render them vulnerable to infection via fusion. Although immature DCs produce much less virus compared with CD4 T cells,17-19 limited amounts of de novo-produced virions are efficiently transmitted (in cis) to resting CD4 T cells,19 especially effector memory CD4 T cells abundant in mucosal tissues.20 Additionally, immature and mature DCs express C-type lectin receptors and may express undefined molecules that facilitate binding and internalization.21-25 Such captured virus can be rapidly (within hours) and efficiently transferred (in trans) to CD4 T cells upon cell-cell contact, referred to as infectious or virological synapses,26-29 a process that is independent of productive DC infection.25,27 Typically the efficiency of HIV transmission can be increased by maturation of DCs,18,27,30,31 indicating that mature DCs in the local and draining lymphoid tissues might further facilitate HIV spread to CD4 T cells. Not surprisingly, numerous studies have highlighted an important role for DC-mediated HIV transmission during mucosal HIV infection, which likely involves the multiple modalities of DC-driven infection.3,6,17,32-38 Thus, topical microbicides that contain agents targeting specific attachment and fusion events need to be extremely efficient in preventing these critical virus-cell interactions.
During each stage of the sequential entry process, HIV is vulnerable to blockade by compounds that bind to viral envelope or the cellular receptors CD4 and CCR5 or CXCR4. Recent in vitro studies showed that DC-mediated infection in trans or cis are sensitive to fusion inhibitors.7,28,39 The virological synapse is also sensitive to a CCR5 antagonist (TAK779) and several viral envelope-binding neutralizing antibodies.28,40 Other small molecule CCR5 ligands being tested in the macaque model show promise.41-44 An envelope-binding small molecule inhibitor (BMS-C) showed in vitro activity against HIV isolates from multiple genetic subtypes.45 Because of the central role of DCs in initiating HIV infection, we were interested to more extensively compare the efficiency of 3 inhibitors that antagonize distinct steps of virus entry, to prevent DC infection and DC-driven viral spread. T-1249, a gp41 peptide fusion inhibitor, is a 39-amino acid synthetic peptide and blocks viral fusion with the cell membrane by inhibiting late-stage conformational changes within gp41.46 BMS-C is a small molecule attachment inhibitor that binds to gp120 to inhibit CD4 binding and subsequent conformational changes associated with coreceptor binding,47 whereas CMPD167 is a CCR5-specific receptor antagonist.48,49 Applying these 3 viral entry inhibitors, we specifically compared distinct viral transfer mechanisms (trans vs cis) and a DC-T-cell coculture setting that involves cell-free virus, allowing both infection mechanisms to occur simultaneously along with direct infection of CD4 T cells. We demonstrate the greater potency of BMS-C in preventing DC and DC-driven infection without perturbing innate DC function.
MATERIALS AND METHODS
The HIV entry inhibitors T-1249,46 BMS-C,47 and CMPD16748,49 were kindly provided by John P. Moore. For all inhibitors, working stocks at a maximum stock concentration of 0.1 mM (CMPD167, in DMSO), and 503.6 μM (T-1249, in PBS) were prepared and kept at −80°C until use. After thawing, aliquots were kept at 4°C for a maximum of 4 weeks.
Isolation and Culture of Primary Leukocytes
Human monocyte-derived DCs were generated from highly enriched populations of CD14 monocytes and autologous CD4 T cells isolated from CD14-negative cell fractions, as previously described, with the minor modification that PBS supplemented with 1% human serum (Sigma, St. Louis, MO) and 2 mM EDTA (Sigma) was used as the buffer.28 As needed, cells were activated on day 5 for 48 hours with the cocktail of prostaglandin E2 (PGE2), IL-1, IL-6, and tumor necrosis factor α (TNF-α) as described.50 For immature DC infection, cells were used on day 5. When comparing immature and mature DCs in the cocultures, the cells were cultured in parallel and day 7 DCs used. Comparable levels of HIV infection are seen for day 5 or day 7 immature DCs. CD4 lymphocytes were sorted from CD14-negative cells by negative selection using the CD4 lymphocyte isolation kit (Miltenyi Biotech, Auburn, CA). Cells were cultured in complete RPMI-1640 (R1) as described.28 R10 contained 10% fetal bovine serum (GIBCO-BRL Life Technologies, Grand Island, NY).
Flow Cytometric Analysis
Phenotypic characterization of immature and mature DCs including purity controls of generated DCs and CD4 T-cell populations (purity ≥97% for DCs and T cells) were routinely monitored by 2-color flow cytometry as previously reported.51 DCs were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-HLA-DR against a panel of phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) characterizing an immature or mature DC phenotype: anti-CD25, anti-CD80, anti-CD86 (all from BD/Pharmingen, San Jose, CA), and anti-CD83 (Immunotech-Beckman-Coulter, Marseille, France). A PE-conjugated anti-CD3 mAb (BD/Pharmingen) was used to detect contaminating T cells in DC preparations. The purity of CD4 T-cell populations was evaluated by stained cells with a FITC-conjugated anti-CD3 mAb against PE-conjugated mAbs anti-CD4 and anti-CD8 (all from BD/Pharmingen). Isotype controls IgG2a-FITC, IgG1-PE, and IgG2b-PE (all from BD/Pharmingen) were included in each experiment. Cell samples were acquired using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and data analysis was carried out using FlowJo software (Tree Star, Ashland, OR).
Virus Inhibition Assays
Sucrose gradient purified infectious HIV BaL was kindly provided by the AIDS and Cancer Virus Program (SAIC Frederick, National Cancer Institute at Frederick, Frederick, MD). HIV BaL (lot # P3953, P4022, P4126, P4143, P4237) was propagated (in SUPT1-CCR5 Cl.30 or Jurkat-TAT-CCR5 cells) and purified as described.52 Aliquots of virus stocks were thawed, and single use aliquots were stored at −80°C before the titer of virus was verified by titration on TZM-bl cells.53 Virus stocks were treated with 10 mg/mL DNase (Roche Applied Science, Indianapolis, IN) for 30 minutes at 37°C before use. For the trans phase of viral transfer, immature and mature DCs were pulsed with HIV (8 × 103 50% tissue culture infective dose (TCID50) per 1 × 105 DCs) for 2 hours at 37°C in a 15 ml conical tube (pretreated with R10 for 2 minutes on ice) at a concentration of 106 DCs/100 μL (with a maximum of 1 × 107 cells/tube). During the last 30 minutes of incubation staphylococcal enterotoxin B (SEB) peptide (Sigma; S4881) at a final concentration of 0.5 mg/mL was added before cells were washed 4 times with ice-cold R1, the viable cells recounted by trypan blue exclusion and cell numbers adjusted to 2.5 × 106 cells/mL. For the cis phase of viral transfer virus-pulsed immature DCs were recultured at a concentration of 1 × 106 cells/mL in a total volume of 3 mL per well in a 6-well plate (in R1 with IL-4/GM-CSF) for additional 48 hours before virus-exposed DCs were collected, incubated with SEB peptide, washed, and cell numbers adjusted as described for the trans phase. For viral replication in DC-T-cell mixtures (mix), immature and mature DCs were pretreated with SEB peptide, washed, and cell numbers adjusted (as above). T cells (3 × 105 per well) were seeded in a 96-well flat bottom plate and the inhibitors (T-1249, BMS-C, or CMPD167; 0.06 to 250 nM) added just before addition of the virus/SEB-pulsed or SEB-pulsed DCs (1 × 105 cells/well). Virus (8 × 103 TCID50) was added directly to the DC-T-cell cocultures that contained SEB-treated DCs. For immature DC infections, cells (3 × 105 per well) were seeded in a 96-well flat bottom plate and inhibitors (T-1249, BMS-C, or CMPD167; 0.06 to 250 nM) added before addition of 2.4 × 104 TCID50 virus per well. Samples were set up in duplicate. After 7 days of culture cells were harvested, washed, and lysed. Samples were stored at −80°C until quantitative PCR (qPCR) analyses.
Immature DC Assays for Cytokine/Chemokine Analysis
Candida albicans (strain SC5314, obtained from the American Type Culture Collection) was cultured and maintained as previously described.54 After overnight amplification in Sabouraud dextrose broth (Sigma) at 30°C, Candida was washed 4 times in PBS before viable Candida yeasts were counted by trypan blue exclusion and resuspended in R1. Immature DCs (3 × 105/well of a 96-well flat bottom plate) were cultured in the presence and absence of 3 × 105 Candida albicans yeast. Amphotericin B (5 μg/mL, Sigma) was added to all conditions to limit Candida overgrowth. Viral entry inhibitors were added at a final concentration of 250 nM/well. Cells were cultured at 37°C, and supernatants harvested 24 hrs or 7 days later. Harvested supernatants were spun and transferred to fresh plates to remove any cellular debris and immediately frozen at −80°C until further analysis. Cytokines and chemokines were detected using a Beadlyte 24-Plex Detection System as previously described.54
HIV gag qPCR
qPCR was performed as previously described28 with the minor modification that HIV copies were normalized on cell numbers by using qPCR for albumin gene copy number. Albumin (Alb) forward (F) and reverse (R) primer/probe sequences were AlbF: TGC ATG AGA AAA CGC CAG TAA, AlbR: ATG GTC GCC TGT TCA CCA A, and AlbP: 5′ FAM-TGA CAG AGT CAC CAA ATG CTG CAC AGA A-TAMRA 3′. Standards for quantification of viral and albumin copy numbers were set up by adding titrated quantities of the plasmid HIV AD8 NL43 DNA into a constant genomic background of SUPT1/CCR5 CL.30 cells. For albumin copies, known numbers of lysed uninfected SUPT1/CCR5 CL.30 cells were serially diluted in lysis buffer.
Data were analyzed using GraphPad Prism software version 5.03 (GraphPad InStat Inc, San Diego, CA). Results of experiments have been summarized as the arithmetic mean and standard error of the mean (SEM). When 2 groups were compared, the null hypothesis of no group difference was evaluated with the nonparametric Mann-Whitney, the Wilcoxon Signed-Ranked, or the Student t test. The 50% or 90% inhibitory concentration (IC50 and IC90) values of different compounds were modeled with nonlinear regression employing a constant slope algorithm. The conventional measure of P < 0.05 was used to determine whether experimental differences were statistically significant.
Establishing a Sensitive Infection Assay to Test Inhibitors
To evaluate the efficacy of viral entry inhibitors to prevent HIV infection of immature DCs and DC-driven infection of CD4 T cells, it was necessary to establish a system with high reproducibility and sensitivity to detect low levels of viral replication. We used sensitive qPCR to monitor viral HIV late gag proviral DNA as a quantitative measure of productive infection.28,55 Although qPCR was sensitive down to detecting one copy of proviral DNA, immature DCs and cocultures of immature DCs with resting CD4 T cells required a minimum of about 4000 (for DCs) or 2000 (for cocultures) TCID50 per 1 × 105 DCs for reliable detection of low copy numbers of viral DNA (data not shown). Therefore, we increased the viral inoculum to 8000 TCID50 per 1 × 105 DCs for more consistent viral amplification across different donors. To increase levels of viral replication in cocultures, DCs were pre-exposed to an SEB peptide to uniformly activate the T cells. SEB-activation of DC-T-cell cocultures has been used previously.56-58 SEB did not impact the DC phenotype in the DC or DC-T-cell cocultures (data not shown).
As previously reported,18,19,59 immature DCs alone exhibited low-level infection with R5 HIV BaL compared with the significantly greater replication seen in the presence of CD4 T cells (Fig. 1). The latter was true for both modes of DC-mediated transmission of virus to the T cells, and when virus was added directly to DC-T-cell mixtures. All immature DC-T-cell cocultures replicated virus to similar levels, independent of mode of infection (trans, cis, or cell-free). Notably, under coculture conditions, the immature DCs promoted more virus replication than their mature counterparts in trans infection systems and in the cell-free virus infected DC-T-cell mixtures (P < 0.02).
BMS-C Is Most Effective at Blocking HIV Infection in DC and DC-T-cell cocultures
The ability of CMPD167, BMS-C, and T-1249 to block DC infection, and the different modes of DC-driven infection in DC-T cell co-cultures was measured. No toxicity was detected at any of the inhibitor doses tested (data not shown). Although each inhibitor effectively blocked HIV infection across all settings, inhibitor-dependent and cell system-dependent differences were observed. A complete block of infection in immature DCs required 250 nM of each inhibitor, which was 16-fold greater than for the various DC-T-cell cocultures (Fig. 2). Although all 3 compounds show comparable efficiency in the higher dose range (250 nM to 15.6 nM), the dose-response curve of the CCR5 inhibitor CMPD167 markedly deviated from the others at doses below 15.6 nM. In fact, DC infection was significantly enhanced in the presence of low dose of CMPD167 (0.06 nM; P < 0.005) (Fig. 2A). A similar effect was observed for concentrations lower than 0.06 nM (data not shown). Furthermore, area under the curve analyses highlight significant differences between the activity of CMPD167 and T-1249 (P < 0.04) or BMS-C (P < 0.02) (Fig. 2B). On the other hand, the inhibitors exhibited similar levels of potency against HIV infection of the various DC-T-cell environments (Fig. 2A), despite immature DCs driving more virus replication than mature DCs (Fig. 1).
Although all inhibitors completely blocked HIV infection in different cell culture settings, the inhibitory doses varied within and across cultures. The most dramatic differences between inhibitors were observed in blocking the low level infection in immature DCs, whereas in contrast, dose-response curves of all 3 inhibitors were similar for the DC-mediated transfer in cis (Fig. 2A). To examine this more closely, we calculated the IC50 and IC90 values of the inhibitors in each biological system (Fig. 3). Significantly more T-1249 (P values from 0.008 to 0.02 for 50% and P values from 0.008 to 0.04 for 90% inhibition) and CMPD167 (P values from 0.0002 to 0.07 for 50% and P values from 0.001 to 0.03 for 90% inhibition) was needed to block immature DC infection compared with all other DC-T-cell systems. In contrast, comparable amounts of BMS-C were required to block infection of immature DCs and the immature DC-T-cell cocultures. However, ∼10-fold less BMS-C was necessary to prevent trans-mediated infection of mature DC-T cocultures and mature DC-T-cell mixtures (P < 0.03; IC50 = 0.19 nM and 0.14 nM, respectively) compared with that required to inhibit immature DC infection (IC50 = 1.58 nM). Thus, although similar amounts of the inhibitors were needed to block the different modes of infection in the various DC-T-cell systems, BMS-C was more efficient in preventing immature DC infection.
Accumulation of CCL4 in CMPD167-Exposed Immature DC Cultures
As sentinels of the immune system, DCs produce cytokines and chemokines that mediate direct effector and immunoregulatory functions. Although entry antagonists have significant potential to prevent HIV entry into permissive target cells, inhibitors must not trigger the release of inflammatory factors that might exacerbate HIV infection or impede responses to other pathogens. Therefore, we investigated whether these viral entry inhibitors affected DC biology. Exposure of immature DCs to CMPD167, BMS-C, or T-1249 for up to 7 days did not alter their typical pattern of cell surface receptor expression (data not shown) including CD4, which is the main viral entry receptor that mediates HIV infection. However, when supernatants of such cultures were tested for the presence of different cytokines and chemokines, CCL4 levels were significantly increased after 7 days of culture with CMPD167 (P < 0.02) (Fig. 4A, left panel). The increased level of CCL4 after 24 hours of culture with CMPD167 was not significant. Minor increases in the levels of CCL5 after culture with CMPD167 were not significantly different from the control (Fig. 4A, right panel). No other cytokines or chemokines were elevated after culture with any of the inhibitors at these time points (data not shown for IL-1β, IL-1RA, IL-2, IL-2R, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-g, CCL2, CCL3, CXCL8, CXCL9, CXCL10).
To determine if the inhibitors might impair innate DC responses to common pathogens, we measured the impact of each inhibitor on the DC cytokine and chemokine production induced by Candida albicans. Immature DCs produce IL-1β, IL-1RA, IL-2R, IL-6, IL-10, IL-12, TNF-α, CCL2, CCL3, CCL4, CXCL8, and CXCL10 in response to Candida yeast (see Figure, Supplemental Digital Content 1, http://links.lww.com/QAI/A107).54 After 7 days, there was an increase in the production of CCL4 in the presence of CMPD167 (Fig. 4B, left panel), but this was not significant and a marginal increase of CCL5 in the presence of all three compounds (Fig. 4B, right panel). There was no impact on any of the other cytokine and chemokine responses induced by Candida yeast (data not shown). Thus, apart from the higher levels of CCL4 detected in the presence of CMPD167, the inhibitors had minimal impact on innate DC cytokine/chemokine responses.
Dendritic cells are believed to play a central role in the onset of HIV infection at the body surfaces through a variety of mechanisms.2,17,37 Immature and mature DCs can capture and internalize HIV that is then rapidly disseminated to CD4 T cells on DC-T-cell contact.27-29,60 Upon productive infection, immature DCs produce much smaller amounts of virus compared with CD4 T cells, but they are extremely efficient in transmitting progeny virions to CD4 T cells.32,61 Additionally, DCs readily trigger virus growth upon contacting virus-carrying CD4 T cells.62,63 It is proposed that the role of immature DCs is more restricted to local HIV replication at the mucosal surface, while mature DCs might drive trans infection in the lymph nodes.64 Activation of DCs triggered by local pathogens may also facilitate trans infection at body surfaces.
We examined the ability of 3 viral entry inhibitors that are being explored as potential microbicide candidates to prevent cell-free infection of immature DCs and DC-T-cell mixtures, and DC-mediated infection of CD4 T cells, using the blood monocyte-derived DC model. This system provides sufficient cell numbers to carry out side-by-side analyses of the 3 inhibitors. Unlike in an earlier study,61 the levels of virus replication after trans vs cis transfer by DCs were comparable. This might be due to methodology differences, especially the inclusion of the SEB peptide herein to activate the recipient T cells to allow more robust replication of virus in both settings. Similarly, the use of SEB peptide-pulsed immature DCs here likely overcame the greater efficiency in DC-T-cell communication and conjugate formation by mature DCs, facilitating relatively greater replication in the presence of immature DCs than previously observed.28,65 Augmented DC-T-cell communication in the presence of SEB peptide may also provide signals to increase replication in the immature DCs66,67 that would further amplify virus directly and provide additional new virus to spread to T cells.
The compounds used in this study represent the three main classes of HIV entry inhibitors. All three inhibitors prevented R5 HIV replication in the different DC systems, impeding the distinct stages of the sequential cascade of viral fusion. This is in agreement with previous findings by us and others, where fusion peptides28,68-70, CCR5 antagonists,69,70 or other potential entry inhibitors61 readily blocked DC-mediated infection in trans or cis. Recent reports suggest that even neutralizing Abs are more efficient at blocking HIV transfer than originally thought.67,71-74 Similarly, cell-free or cell-to-cell spread between T cells were recently shown to be equally sensitive to viral entry inhibition.40 Inhibition of DC-driven trans and cis infection further suggests that the recently described formation of nanotubes75,76 or cytoplasmic extensions by immature DCs that envelop CD4 T cells77 and are enriched with immature virus particles are freely accessible to viral entry inhibitors. Therefore, even in the presence of virological synapse formation, each step of the viral fusion cascade is vulnerable to the inherent antiviral potency of the different viral entry inhibitors. Immature and mature DCs (1) sequester entrapped virions in unique compartments, (2) exhibit differences in DC-T-cell conjugate formation, and (3) release entrapped particles and/or produce new viruses at CD4 T-cell-DC junctions.28 These features may contribute to differences in the sensitivity of immature or mature DC-driven, and the trans or cis DC-driven virus spread to inhibition.
Another main finding in this study is the enhancement of DC infection at low doses of CMPD167. This possibly relates to increased levels of CCL4 induced by CMPD167 and likely parallels what CCL5 may do in macrophages.69,70,78 An increase in CCL4 levels in DC cultures could also explain previous observations where only partial inhibition of immature DC infection was achieved using CCL5, a natural ligand for CCR5, to block infection. Additionally, CCL4 could encourage the recruitment of additional target cells to augment HIV amplification79 in vivo.
The remarkable low efficiency of CCR5 and fusion inhibitors to block DC infection compared with the various DC-T-cell cocultures is in agreement with previous findings, where immature DC infection was more resistant to complete inhibition by 20-fold or 36-fold for a CCR5 (SCH-C) vs 6 -fold or 10-fold for a fusion peptide inhibitor than PBMCs or trans-mediated infection.69,70 This effect possibly relates to the common sensitivity of CCR5 and fusion inhibitors to the level of cell surface expression of CCR5. Although the cooperation of multiple CCR5 coreceptors is required for infection by HIV,79 high or low levels of CCR5 affect the efficiency of a CCR5 antagonist and can impact the speed of fusion pore formation and, therefore, the efficiency of a fusion peptide inhibitor.80 Unlike in DC infection, HIV receptors CD4 and CCR5 (or CXCR4) on the T cell are recruited to the DC-T-cell contact sites thereby enhancing productive infection events and possibly the inhibition of such events by CCR5 or fusion inhibitors. On the other hand, direct binding to the virus, thereby preventing virus attachment to its target cell, likely explains the high potency of BMS-C to prevent cell-free and DC-driven infection to comparable levels. Although herein inhibitors were only tested against one R5-virus and variations in the potency of the 3 chosen inhibitors across several R5-viruses cannot be excluded, a recent study showed no substantive virus-specific pattern to the actions of the CMPD167, BMS-C, or a fusion peptide against a panel of R5 viruses.45 This is consistent with earlier reports on other entry inhibitors and with the subtype-independent nature of the HIV fusion process.81,82
Several approaches utilizing inhibitors of virus-cell interactions are being explored for topical microbicides,28,40,44,83 particularly CCR5 inhibitors.42,84,85 Although CMPD167 has been shown to inhibit vaginal transmission of an R5 SHIV to macaques,42,85 there might be concern that residual low levels of drug might ultimately enhance infection if animals were exposed to virus at later time points when drug would become limiting. Alternatively, small molecule inhibitors like BMS-C that directly target viral envelope may have considerable advantages compared with CCR5 or fusion peptide inhibitors due to their superior ability to efficiently prevent DC-driven HIV infection and spread coincident with their limited impact on innate immune function. Despite a preferential transmission of R5 HIV during mucosal infection, future studies are needed to evaluate the potency of entry inhibitors against X4-tropic and dual-tropic HIV to advance our understanding for the development of a potent topical prevention strategy. Overall, combination strategies using inhibitors targeting multiple stages of the infection process will likely be most effective against HIV.
The authors would like to thank Julian W. Bess Jr and Jeffrey D. Lifson for providing the HIV used in these studies and for critical review of the article. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL(AD8), HIV AD8 Macrophage-Tropic R586 from Dr Eric O. Freed. We thank Daniel Gawarecki for statistics advice. The assistance and use of the Population Council's Flow Cytometry Facility is gratefully acknowledged.
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