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Journal of Virology, November 2004, p. 12344-12354, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12344-12354.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo

Erika Schlaepfer,1 Annette Audigé,1 Barbara von Beust,2 Vania Manolova,3 Markus Weber,4 Helene Joller,5 Martin F. Bachmann,3 Thomas M. Kundig,2 and Roberto F. Speck1*

Division of Infectious Diseases and Hospital Epidemiology, Department of Internal Medicine,1 Clinics of Dermatology,2 Department of Visceral Surgery,4 Institute of Clinical Immunology, University Hospital of Zurich, Zurich,5 Cytos Biotechnology, Schlieren-Zurich, Switzerland3

Received 26 March 2004/ Accepted 25 June 2004


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ABSTRACT
 
Oligodeoxynucleotides (ODNs) with immunomodulatory motifs control a number of microbial infections in animal models, presumably by acting through toll-like receptor 9 (TLR9) to induce a number of cytokines (e.g., alpha interferon and tumor necrosis factor alpha). The immunomodulatory motif consists of unmethylated sequences of cytosine and guanosine (CpG motif). ODNs without CpG motifs do not trigger TLR9. We hypothesized that triggering of TLR9 generates a cellular environment unfavorable for human immunodeficiency virus (HIV) replication. We tested this hypothesis in human lymphocyte cultures and found that phosphorothioate-modified ODN CpG2006 (type B ODNs) inhibited HIV replication nearly completely and prevented the loss of CD4+ T cells. ODNs CpG2216 and CpG10 (type A ODNs) were less effective. CpG2006 blocked HIV replication in purified CD4+ T cells and T-cell lines; CpG10 was ineffective in this setting, indicating that type A ODNs may inhibit HIV replication in CD4+ T-cell lines indirectly through a separate cell subset. However, control ODNs without CpG motifs also showed anti-HIV effects, indicating that these effects are nonspecific and not due to TLR9 triggering. The mechanism of action is not clear. CpG2006 and its control ODN blocked syncytium formation in a cell fusion-based assay, but CpG10, CpG2216, and their control ODNs did not. The latter types interfered with the HIV replication cycle during disassembly or reverse transcription. In contrast, CpG2006 and CpG2216 specifically induced cytokines critical to initiation of the innate immune response. In summary, the nonspecific anti-HIV activity of CpG ODNs, their ability to stimulate HIV replication in latently infected cells, potentially resulting in their elimination, and their documented ability to link the innate and adaptive immune responses make them attractive candidates for further study as anti-HIV drugs.


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INTRODUCTION
 
Antiretroviral therapy has significantly reduced the morbidity and mortality associated with infection with human immunodeficiency virus type 1 (HIV-1) in the United States and Europe (13, 38). However, antiretroviral therapy has crucial limitations, including serious side effects, the emergence of resistant HIV strains (reviewed in reference 40), and high costs (28). Furthermore, interruption of antiretroviral therapy treatment is followed by a quick rebound of HIV replication (12). Thus, there has been considerable interest in immune response modifiers to treat HIV by enhancing the endogenous HIV-specific immune response. Cytokine-based therapies, such as interleukin-2, gamma interferon (IFN-{gamma}), or granulocyte-macrophage colony-stimulating factor, benefit patients but cannot control HIV without antiretroviral therapy (reviewed in reference 44). These cytokines act on selective targets (e.g., interleukin-2 on T lymphocytes) but do not orchestrate the immune response, including cytokine production and cellular activation, in a manner that produces physiological changes.

Identification of the toll-like receptors (TLRs) has provided a novel way to stimulate the immune system. TLRs are a large family of pattern recognition receptors that recognize conserved molecular targets on diverse microorganisms, including viral RNAs, bacterial DNA, and microbial cell wall components, and induce complex changes in the microenvironment (reviewed in references 25, 49, and 53). TLRs are expressed in a cell-specific manner on a variety of immunological cell types. In humans, B lymphocytes express TLR1 and TLRs 6 to 10 (10, 22), plasmacytoid dendritic cells (PDC) express TLRs 1, 6, 7, and 9, and myeloid dendritic cells (MDC) express TLRs 1, 2, and 3 (24, 26). Thus, TLR triggering results in cell-specific changes (17, 31, 45, 50, 52, 54).

Synthetic oligodeoxynucleotides (ODNs) are generated in response to structural properties of the physiological ligands of TLR9, bacterial DNA rich in unmethylated cytosine-guanosine dinucleotide (CpG) motifs (21, 32). These motifs are abundant in bacterial DNA and underrepresented and mostly methylated in human DNA. Thus, the prototype CpG ODN (denoted CpG) is 20 to 30 nucleotides long, displays two to three CpG motifs, and is unmethylated (31). Furthermore, modifications in the backbone of ODNs, such as with phosphorothioate, stabilize the DNA and contribute to the immunomodulatory activity (51). Recognition of the biological activities of CpGs has led to the generation of a whole panel of synthetic ODNs with optimized CpG motifs (19, 30). CpGs are classified by their ability to induce large amounts of IFN-{alpha} in PDC (CpG type A, prototype 2216) or to promote survival, activation, and maturation of B cells and PDC (CpG type B, prototype 2006) (19, 33).

The use of synthetic ODNs to trigger TLR9 in animals has shown promising results in constraining viruses (herpesviruses [18] and Friend helper retrovirus [37]), bacteria (Listeria monocytogenes and Francisella tularensis) (reviewed in reference 31) and Mycobacterium avium (20), and parasites (Leishmania major and malaria) (reviewed in reference 31). Importantly, ODNs initiated complete protective immunity against reinfection by herpesvirus (18), L. monocytogenes, and F. tularensis in mice (14), suggesting the generation of an adaptive immune response in addition to potent stimulation of innate immunity.

The use of synthetic oligonucleotides as antiviral agents is not a new concept interest (reviewed in reference 16). Oligonucleotides have been designed mainly to act as antisense to HIV RNA sequences. Additional antiviral activities of oligonucleotides include inhibition of HIV adsorption to cells by virtue of their polyanionic nature, inhibition of HIV-encoded enzymes, and inhibition of HIV transcription. The recent discovery that specific structures trigger TLR9 may expand their applicability for treating diseases. In two studies examining oligonucleotides without CpG motifs in SCID mice transplanted with human peripheral blood leukocytes or human fetal thymus and liver in vivo, oligonucleotides revealed distinct anti-HIV activity from potent inhibition to inactivity, depending on the viral isolate (2, 48). In a murine model infected with Friend virus, immunostimulatory ODNs containing CpG motifs significantly enhanced virus-specific cellular immune responses and prevention of retrovirus-induced disease when given 4 days after infection (37).

In the present study, we hypothesized that the immunostimulatory ODNs trigger changes that make the cellular microenvironment unfavorable for HIV replication. We examined the effects of ODNs that trigger TLR9 on HIV replication in lymphoid tissue infected ex vivo. Specifically, we examined three prototype ODN CpGs for their anti-HIV effects: CpG2006 (CpG type B) and CpG2216 and CpG10 (both CpG type A) (23, 33).


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MATERIALS AND METHODS
 
Cells, cell lines, and reagents. Peripheral blood mononuclear cells (PBMC) from random donors were isolated by Ficoll-Hypaque density gradient centrifugation (Nycomed) and cultured at 10 x 106 cells/ml in RPMI (BioWhittaker, Verviers, Belgium), with 10% fetal calf serum (PAA Laboratories, Vienna, Austria), 1% penicillin-streptomycin (Gibco), 1% glutamine (Gibco), and 10 U of interleukin-2 (NIH AIDS Research and Reference Reagent Program) per ml. For generating monocyte-derived macrophages, we first enriched monocytes from whole blood with the RosettSep human monocyte enrichment cocktail (15028, StemCell Technologies, Vancouver, British Columbia, Canada), according to the manufacturer's instructions. Subsequently, 5 x 104 monocytes per well of a 96-well plate were kept in RPMI with 5% fetal calf serum, 5% human AB serum (H1513, Sigma-Aldrich), 1% glutamine, and 1% penicillin-streptomycin for 1 week until differentiation into monocyte-derived macrophages. MT-2, Sup-T1, and A2.01 cell lines expressing CD4 were grown in RPMI with 10% fetal calf serum, glutamine (Life Technologies, Paisley, Scotland), and penicillin-streptomycin.

The stimulatory ODNs were CpG2006 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'), CpG2216 (5'-GGgggacgatcgtcGGGGGg-3') (33), and CpG10 (5'-gggggggggggacgatcgtcgggggggggg-3') (23). The controls were CpG2006/89 (5'-tcgtcgttttgtcgttttgtcgtt-3'), ODN2006/90 (5'-TCGTGCTTTTGTGCTTTTGTGCTT-3'), ODN2006/91 (5'-tcgtgcttttgtgcttttgtgctt-3'), ODN2243 (5'-GGgggagcatgctcGGGGGg-3'; gc control ODN of ODN CpG2216) (33), CpG12 (5'-ggggggggggggaacgttgggggggggggg-3') (23), ODN30 (5'-gggggggggggggggggggggggggggggg-3') (23), and CpG2117 (5'-TC*GTC*GTTTTGTC*GTTTTGTC*GTT-3') (C* signifies a methylated cytidine) (Microsynth GmbH, Balgach, Switzerland). Lowercase and capital letters indicate sites modified by phosphodiester ODNs and phosphorothioate ODNs, respectively. Italics indicate the immunomodulatory CpG motif. CpG2006, CpG2006/89, ODN2006/90, ODN2006/91, and CpG2117 were used at a concentration of 5 µg/ml, and CpG10, CpG12, and ODN30 were used at a concentration of 10 µg/ml. Zidovudine (NIH AIDS Research and Reference Reagent Program) was used at 5 µM.

Lymphoid tissue. Acquisition and processing of lymphoid tissue were approved by the local ethical committee. Human tonsils from otherwise healthy adult patients (18 to 70 years of age, median age of 28 years) were obtained from the Department of Ear, Nose, and Throat Surgery at the University Hospital Zurich within 1 to 5 h after tonsillectomy. Lymph nodes of intestinal origin were obtained from organ donors from the Department of Surgery, University Hospital Zurich. Human lymphocyte aggregate cultures (HLAC) were prepared by transferring minced tissue and tissue fragments into a cell strainer (70 µM; Falcon) and grinding the tissue through the sieve with a syringe plunger. Erythrocytes were lysed with ACK cell-lysing buffer (BioWhittaker). Lymphoid cells were washed and transferred to a 96-well plate at a concentration of 107 cells/ml. HLAC were cultured in RPMI 1640 containing 15% fetal calf serum, 100 U of penicillin per ml, 100 µg of streptomycin per ml, 2.5 µg of fungizone per ml, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% nonessential amino acids. Viability was assessed by trypan blue exclusion.

Purification of CD4+ T cells from HLAC and PBMC. We recovered CD4+ T cells by capturing them with antibodies coupled to MACS beads, according to the manufacturer's instructions (CD4 Microbeads, 130-045-101; Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the recovered CD4+ T cells was >95% in all experiments, as determined by staining with anti-CD4 antibody and flow cytometry.

Viruses. Viral stocks were obtained by transfection with calcium phosphate (Promega) of 293T cells with pNL4-3, pYU-2, pJRFL, and p89.6 (NIH AIDS Research and Reference Reagent Program) or p49.5. Virus was harvested 48 h after transfection, filtered (0.22 µm), and frozen at –80°C. In assays quantifying amounts of HIV DNA generated within a "one-round" replication, we infected HLAC with NL4-3 which was propagated in PBMC activated with phytohemagglutinin. Viral stocks from PBMC lack plasmid DNA, which could lead to falsely high results in this kind of assay.

HIV p24 antigen enzyme-linked immunosorbent assay. A twin-site sandwich enzyme-linked immunosorbent assay was performed essentially as described (35). Briefly, a polyclonal antibody was adsorbed to a solid phase to capture p24 viral capsid antigen (p24) from a detergent lysate of virions. Bound p24 was visualized with an alkaline phosphatase-conjugated anti-p24 monoclonal antibody and luminescent detection system.

Infectivity assays. All infectivity assays with HLAC or PBMC were performed in triplicate in 96-well round-bottomed plates. ODNs were added 2 days before HIV infection, immediately upon infection, or 4 days after infection of PBMC or HLAC. In a subset of experiments, CpG2006 was added 4 days after establishing HLAC; thus, in these experiments, HIV infection was performed at day 7 of culturing. Cultures were exposed to HIV with p24 concentrations of 1 to 3 ng/sample for 4 to 6 h. Subsequently, the cultures were washed three times with phosphate-buffered saline and resuspended in 200 µl of fresh medium containing the corresponding ODN. Twice a week, fresh medium containing the ODN was added. Supernatants were tested for p24 with an in-house enzyme-linked immunosorbent assay. For interdonor comparisons, we expressed the p24 values over time as the area under the curve (AUC) representing HIV replication and calculated the inhibition by any given drug and concentration by first expressing the AUC of treated cultures as a percentage of that of an untreated, infected control culture and second by subtracting the AUC for treated samples from 100% (42).

For infectivity assays, the MT-2, Sup-T1, and A2.01 T-cell lines stably expressing CD4 (5 x 104 cells/well of a 96-well plate) were treated with CpG2006 or CpG10 for 2 days before infection with pNL4-3 for 6 h. Subsequently, the cells were washed three times, and HIV replication was determined by measuring p24 levels in the supernatant at day 5.

Cell-based fusion assay. To assess the impact of ODNs on fusion of the HIV envelope with the cell membrane, we used a cell-based fusion assay with HeLa cells expressing gp140 from LAI (HeLa-Env/LAI) (43) and HeLa cells expressing CD4 cell surface molecule and the long terminal repeat-driven lacZ gene (HeLa SX CCR5) (29). HeLa-Env/LAI cells also expressed the HIV-1 transactivator Tat, and thus fusion of HeLa-Env/LAI cells and HeLa SX CCR5 will result in transcription of the lacZ gene. The extent of fusion was quantified either by assaying ß-galactosidase activity in cell lysates (E2000, Promega, Madison, Wis.) or by histochemical staining of cells for ß-galactosidase activity (1828673, Roche Moecular Biochemicals, Mannheim, Germany), according to the manufacturer's instructions. HeLa-Env/LAI cells were treated with CpG2006 and ODN 2006/90 at 10 µg/ml, CpG2216, ODN2243, or CpG10 at 20 µg/ml, a monoclonal antibody against CD4 (SIM.2) (34) at 300 ng/ml, or Dp178 (56) at 50 µg/ml for 1 h. Subsequently, a similar number of HeLa SX CCR5 cells were added to allow fusion to take place. ß-Galactosidase activity was assessed 12 h later.

Cytokine measurements. Cytokine concentrations were quantified by enzyme immunoassays (IFN-{alpha}, Bender MedSystems Diagnostics, GMBH, Vienna, Austria; IFN-{gamma}, HyCult Biotechnology, Uden, The Netherlands; tumor necrosis factor alpha, RANTES, MIP-1{alpha}, MIP-1ß, interleukin-6, interleukin-8, and interleukin-12, R&D Systems, Minneapolis, Minn.) according to the manufacturer's instructions.

Immunostaining and flow cytometry. Cells were stained simultaneously with monoclonal antibodies (Becton Dickinson, Oxford, United Kingdom) for the cell-surface markers CD4 (phycoerythrin, 555347), CD8 (fluorescein isothiocyanate, 555634), CD25 (Cy-Chrome, 555433), and CD45RA (allophycocyanin, 550855) or CD4, CD8, and CXCR4 (Cy, 555975). For staining PDC or MDC, we used lineage cocktail 1 (40565). All monoclonal antibodies were used at a dilution of 1:10. Flow cytometry was performed on a FACSCalibur (Becton Dickinson), and data were analyzed with Cellquest or FlowJo software (Tree Star).

Quantitative PCR for measuring proviral HIV DNA. Cells (106) were lysed in 0.1 ml of lysis buffer. HIV DNA concentrations were measured in duplicate by real-time PCR (i-cycler; Bio-Rad) as described (8.). Forward and reverse primer sequences specific for HIV-1 were located within the gag region and consisted of 5'-GCAGCCATGCAAATGTTAAAAGAG-3' and 5'-TCCCCTTGGTTCTCTCATCTGG-3'. Serial dilutions of linearized full-length HIV plasmid ppNL4-3 were used as external standards. DNA (5 µl) was added to 40 µl of HotStartTaq master mix (Qiagen) supplemented with 1 µM each primer, 0.3 µM fluorescent probe, and 3 mM MgCl2. Cycling conditions were 95°C for 15 min, followed by 50 cycles at 95°C for 10 s each, and 60°C for 1 min. Copy numbers were calculated by interpolation from a standard curve with software provided by the manufacturer.

Quantitative PCR for measuring TLR9 mRNA. Quantitative PCR was performed as described (4). In brief, for measuring TLR9 mRNA, we used commercially available primers and probes (Assays-on-demand; Applied Biosystems). Hydroxymethyl bilane synthase (GenBank X04217) was used as a housekeeping gene and designed as an MGB (3' minus groove binder) probe (9). Data generated by real-time quantitative PCR were analyzed by determining the mean normalized gene expression for every sample with the software application Q-Gene (calculation procedure 2 for mean normalized gene expression) (36).

Statistical analysis. For multiple comparisons of repeated measures, we used the nonparametric Friedman test. For comparisons of groups, we used the Wilcoxon signed rank test.


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RESULTS
 
ODNs block HIV replication in tonsillar HLAC. Vigorous HIV replication occurred in control HLAC, but treatment of HLAC with CpG type B (CpG2006) for 2 days suppressed HIV replication nearly completely. The CpG type A (CpG2216 and CpG10) were less effective (Fig. 1). Suppression by CpG2006 was independent of the coreceptor preference of the HIV strains. Control ODNs to CpG2006 had different anti-HIV activities, with ODN2006/90 and CpG2117 blocking HIV to a similar degree as CpG2006 and with CpG2006/89 and ODN2006/91 showing only moderate anti-HIV activity. ODN2243 was as anti-HIV active as CpG2116. The guanosine-rich ODNs CpG12 and ODN30 also blocked HIV in the same range.



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  		FIG. 1. CpG2006, CpG2216, and CpG10 block HIV replication in HLAC of tonsillar tissue. (A) pNL4-3, a CXCR4-tropic HIV strain, (B) 49.5 and (C) JRFL, both CCR5-tropic HIV strains, and (D) 89.6, a dual-tropic HIV strain. However, ODN controls for CpG2006, CpG2216, and CpG10 without CpG motifs (i.e., ODN2006/90, ODN2243, and ODN30) also block HIV replication, pointing to a nonspecific interference of ODNs with HIV. The rather modest HIV blocking after treatment with the phosphodiester version of CpG2006, CpG2006/89, indicates the importance of the phosphorothioate backbone, especially for CpG2006. The results of treatments are represented as percent HIV inhibition as related to an untreated, infected control culture (for strains pNL4-3 and 49.5: CpG2006, n = 15 and 11, respectively; CpG10, n = 11 and 7, respectively; CpG2006/89, n = 2 and 2, respectively; ODN2006/90, n = 5 and 2, respectively; ODN2006/91, n = 3 and 2, respectively; CpG12, n =3 and 3, respectively; ODN30, n = 3 and 3, respectively; CpG2117, n = 2; CpG2216, n = 3; ODN2243, n = 3; for strains JRFL and 89.6, n = 3 for all ODNs tested).

CpG2006 and CpG10 preserve CD4+ T-cell levels. Infection with pNL4-3 resulted in a profound depletion of CD4+ T cells in HLAC of tonsillar tissue and mesenteric lymph nodes (Fig. 2). However, in cultures treated with CpG2006, CD4+ T-cell levels were consistently preserved, and in those treated with CpG10, the cells were preserved to a lesser extent. Staining for CD4+ and CD8+ T cells was not sufficiently sensitive to detect CD4+ T-cell loss after infection with CCR5-tropic HIV strains. In contrast, when we stained for CD4+ CCR5+ cells, we observed that infection of control cultures with CCR5-tropic viruses resulted in a loss of this cell subset and that cultures pretreated with TLR ligands maintained cell counts (data not shown).



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  		FIG. 2. Immunostimulatory ODNs prevent CD4+ T-cell loss in HLAC infected ex vivo with HIV. To assess CD4+ T-cell preservation, 7 or 12 to 14 days after infection, HLAC cells were stained with antibodies against CD4 and CD8. CD4+ T-cell depletion was indicated by changes in the ratio of CD4+ to CD8+ T cells for each sample; this was expressed as a percentage of that of a noninfected control culture. Data represent the results of six independent experiments with tonsillar tissue. *, result of one experiment with a mesenteric lymph node; #, results of CD4+ T-cell depletion at day 7; {square}, untreated HLAC infected with pNL4-3, {diamondsuit}, pretreated with CpG2006; •, pretreated with CpG10). Inset: Inhibition of replication is tightly correlated with the preservation of CD4+ T cells. Thus, in these experiments with these compounds, quantification of viral replication also gives information about the cytopathic effects of HIV infection.

We also examined whether the different ODNs affected the CD4/CD8 ratio in uninfected HLAC by staining for CD4 and CD8 cell surface markers after culturing the HLAC for 12 to 14 days. CD4/CD8 ratios were similar regardless of the treatment, rendering it highly unlikely that ODNs change the absolute numbers of CD4 and CD8 lymphocytes in this ex vivo system [median (25%, 75% interquartiles) of CD4/CD8 ratio after CpG2006, 5.6 (4.5, 7.5); ODN2006/90, 6.5 (5.1, 7.8); CpG10, 6.3 (5.7, 8.2); CpG2216, 5.8 (4.9, 6.7); ODN2243 6 (5.3, 7.2); and untreated 7.1 (4.5, 10.4); n = 4, Friedman test, P = 0.25)].

Dose-dependent suppression of HIV replication. The suppression of HIV replication by CpG2006 and CpG10 depended on the dose (Fig. 3A, B). When they were added before or immediately after HIV infection, both compounds suppressed HIV replication (Fig. 3C, D). In contrast, when they were added 4 days after HIV infection, they were clearly less effective. Culturing HLAC in normal medium for 4 days before adding CpG2006 did not affect CpG's ability to block HIV (mean ± SE of inhibition, pNL4-3, 85.4% ± 4.9%, n = 5; 49.5 70% ± 11%, n = 3). In this setup, CpG10 again showed less pronounced anti-HIV activity (inhibitory effect in the two experiments performed, 47% and 72%).



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  		FIG. 3. CpG2006 and CpG10 show dose- and time-dependent suppression of HIV replication after infection with strain 49.5. (A) The usual working concentration of CpG2006 was 5 µg/ml ({diamondsuit}). At 0.5 µg/ml ({blacktriangledown}), CpG2006 was only partially effective; at 0.05 µg/ml ({blacktriangleup}), its effect was lost. (B) A similar pattern was found with CpG10. A working concentration of 10 µg/ml ({diamondsuit}) effectively inhibited HIV replication. The effect was partially lost at 1 µg/ml ({blacktriangledown}) and entirely lost at 0.1 µg/ml ({blacktriangleup}) (n = 2). (C, D) To examine the time constraint of these compounds on HIV replication, we compared their activities when added at different times: over the entire culture period (•), only before ({triangleup}), immediately after ({triangledown}), or 4 days after HIV infection ({circ}). CpG2006 (C) and CpG10 (D) displayed anti-HIV activity when added immediately after but lost their effects when added 4 days after HIV infection. {square}, infected untreated control culture. Infections with strain pNL4-3 showed similar results (data not shown).

ODNs display anti-HIV activities in PBMC and HLAC of mesenteric lymph nodes. In PBMC, CpG2006 and CpG10 nearly completely suppressed pNL4-3 replication. They were less effective in blocking strain 49.5 (Table 1).


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  		TABLE 1. Inhibition of HIV replicationa

In HLAC from mesenteric lymph nodes pretreated with CpG2006 and CpG10, the median inhibition of pNL4-3 replication was 98% (min, 90%; max, 98%; n = 3) and 57% (min, 25%; max, 88%; n = 2), respectively. Surprisingly, HLAC from mesenteric lymph nodes were permissive to infection with CCR5-tropic HIV strains in only one of four experiments. In this particular experiment, we observed complete blockage (100%) of the CCR5-tropic strain, YU-2, after treatment with CpG2006.

Activity of immunostimulatory ODN against HIV in purified primary CD4+ T cells, T-cell lines, and monocyte-derived macrophages. Purified CD4+ T cells were completely resistant to HIV replication when pretreated with CpG2006 (Fig. 4A). Similar results were obtained with the T-cell lines MT-2 (Fig. 4B) and with SupT1 and A2.01 (data not shown). In contrast to CpG2006, CpG10 displayed no anti-HIV activity in T-cell lines (n = 1). In monocyte-derived macrophages, pretreatment with ODN, particularly with CpG2006, showed only modest anti-HIV activity (median, 46.8%; interquartiles, 8.6% to 76.2%), whereas pretreatment with CpG10 had no activity (Fig. 4C).



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  		FIG. 4. ODN anti-HIV activity in primary CD4+ T cells, T-cell lines, and monocyte-derived macrophages (MDM). (A) CpG2006 completely blocked HIV replication in primary CD4+ T cells purified from PBMC (>99% pure) (n = 1); similar results were obtained with purified CD4+ T cells from an experiment with tonsillar tissue (data not shown) {square}, infection with pNL4-3 of untreated PBMC, {diamondsuit}, infection of CpG2006-treated PBMC. (B) Impact of ODNs on HIV replication in MT2 cell lines. CpG2006 displayed substantial anti-HIV activity in MT2 cell lines (n = 2), and CpG10 was inactive. The control indicates untreated MT2 cells infected with pNL4-3. (C) In monocyte-derived macrophages, CpG2006 and CpG10 revealed modest and no anti-HIV activity, respectively. (D) Primary CD4+ T cells from PBMC and T-cell lines express TLR9 mRNA. The amounts of TLR9 mRNA were determined by quantitative PCR.

CpG2006 and CpG10 do not change expression levels of CD4, CXCR4, or CD25. Percentages of CD4+ T lymphocytes expressing CXCR4 two days after treatment with CpG2006, CpG10, and controls were 91.9 ± 2.3, 91.7 ± 2.4, and 93.6 ± 2.4, respectively (n = 7 for each). Mean intensities of CXCR4 expression of the CD4+ T lymphocytes were 609 ± 12,605 ± 17.5, and 625 ± 13.1 after treatment with CpG2006, CpG10 or controls, respectively (not significant, Kruskal-Wallis test). We also found no difference between activation levels of treated and control cultures as measured by the expression levels of CD25 (CD4+ T cells: CpG2006, 13.4 ± 2.8%, n = 3; CpG10, 15.6 ± 4.1%, n = 3; untreated, 16.0 ± 4.9%, n = 2).

Intracellular HIV DNA decreased substantially after CpG2006 or CpG10. To examine the effects of CpG 2006 or CpG10 on "one-round" HIV replication, we harvested pretreated tonsillar cells 6 h after a challenge with pNL4-3 and determined HIV DNA levels. CpG2006 and CpG10 dramatically reduced the number of copies of the HIV genome (Fig. 5).



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  		FIG. 5. Effects of CpG2006 and CpG10 on HIV DNA synthesis in a one-round replication assay. Immunostimulatory ODN reduced the amount of HIV DNA produced in HLAC infected by the replication-competent HIV strain pNL4-3. Immediately after exposure of HLAC to pNL4-3, the infection was stopped by adding zidovudine (5 µmol). One representative experiment is shown; all samples were done in triplicate (n = 4).

Phosphorothioate-modified ODNs block syncytium formation; phosphodiester ODN do not. We examined these reactions histochemically and by measuring ß-galactosidase activity in cell lysates. We found convincing evidence that CpG2006 and CpG2006/90 block fusion between HeLa-Env/LAI cells and HeLa cells expressing CD4 at 5 µg/ml. The blocking effect is similar to that caused by monoclonal antibodies against CD4, SIM.2, or Dp178 and is lost at 1 µg/ml or less of CpG2006 (Fig. 6). In contrast, fusion between HeLa-Env/LAI and HeLa-CD4 cells was not prevented by the pure phosphodiester ODN CpG10 or the phosphodiester ODNs CpG2216 and ODN2243, in which a few oxygen molecules are replaced by sulfurs.




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  		FIG. 6. Phosphorothioate-modified ODNs block syncytium formation, and phosphodiester ODNs do not. HeLa-Env/LAI cells expressing HIV envelope from HIV strain LAI were treated with the various compounds for 1 h and subsequently mixed with HeLa cells expressing CD4 and the lacZ gene. Fusion between these two cell lines were assessed by (Top) histochemical staining and (Bottom) lysing the cell cultures and quantifying ß-galactosidase activity. SIM.2 is a monoclonal antibody against CD4 and blocks syncytium formation. Dp178 is a fusion inhibitor, also known as T20. One representative experiment is shown (n = 2).

ODNs induce major changes in the cytokine profiles. We examined cytokine profiles 1 and 2 days after exposing HLAC to the ODNs CpG2006, ODN2006/90, CpG10, CpG2216, and ODN2243. CpG2006 and CpG2216 markedly upregulated levels of IFN-{alpha}, tumor necrosis factor alpha, MIP-1{alpha}, MIP-1ß, RANTES, and interleukin-6 compared to tissue treated with the control ODNs ODN2006/90 and ODN2243. After treatment with CpG10, cytokine levels were the same as those in supernatants of untreated HLAC (Fig. 7).



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  		FIG. 7. Cytokine levels in the supernatants of HLAC 1 and 2 days after treatment with ODNs. Treatment with CpG2006 and CpG2216 resulted in characteristic induction of a number of cytokines, whereas the control ODNs CpG2006/90 and CpG2243 did not. One representative experiment of three is shown.

Effects of IFN-{alpha} on HIV replication. CpG2216 is a strong inducer of IFN-{alpha}. We wondered if IFN-{alpha} reproduces the anti-HIV effect observed in this experimental setting. Median IFN-{alpha} induction after CpG2216 at day 1 was 974 pg/ml (min, 510 pg/ml; max, 2,470 pg/ml) and at day 2 was 1,056 pg/ml (min, 804 pg/ml; max, 2,528 pg/ml; n = 3). To ensure that we were above the expected induction of IFN-{alpha}, recombinant IFN-{alpha} was added at 10 ng/ml over the entire culture period. IFN-{alpha} resulted in only minor HIV inhibition, with a median of 25.05% (min, 23.4%; max, 36%; n = 3), which does not explain the potent inhibition observed with CpG2006 or CpG2216.

PDC and MDC are rapidly lost in HLAC, and NK cells are preserved. Immediately after receipt of the fresh tissue at day 1, PDC represented 0.53 ± 0.12% of the total cells (mean ± standard error of the mean), and MDC 0.58 ± 0.27% (n = 4). However, after 7 days of culture, there was a dramatic decrease in PDC (0.025 ± 0.01%) and MDC (0.0075 ± 0.005%) (n = 4). In contrast, NK cells were well preserved over 7 days (day 1, 0.53 ± 0.09%; day 7, 0.22 ± 0.06%; n = 4).


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DISCUSSION
 
In this study, we showed that CpG2006 very effectively inhibited HIV replication in HLAC whether the challenging HIV strain was CCR5, CXCR4, or dual-tropic. CpGs 2216 and 10 were somewhat less effective. CpGs 2216 and 10 appeared to be more potent against CCR5-tropic strains. Not unexpectedly, the antiviral activity correlated with preservation of CD4+ T cells, as demonstrated by the CD4/CD8 ratios in the HLAC.

CpG2006 and CpG10 blocked HIV replication in a dose-dependent manner. They displayed similar anti-HIV activities when added immediately after the HLAC were exposed to HIV compared to exposure of HLAC to ODNs over the entire culture period. However, the effect was lost when the compounds were added 4 days after HIV infection, suggesting that spreading infection had overwhelmed their anti-HIV activities. Alternatively, the cells responding to ODNs may decrease over time in these ex vivo cultures, as was the case for PDC and MDC. Thus, we examined the effects of culturing the HLAC for 4 days before adding the ODNs. CpG2006 retained its anti-HIV activity; CpG10 again showed moderate anti-HIV activity. Thus, a decrease in PDC and MDC in these ex vivo cultures was not critical for the anti-HIV activity of the ODNs.

We found that CpG2006 and CpG10 were effective in all tissues tested, including tonsil, intestinal lymph nodes, and PMBCs (Table 1). Strikingly, CpG2006 was less effective against the CCR5-tropic strain 49.5 in PBMCs. We also examined the activities of ODNs in several cell lines. In purified primary CD4+ T cells and T-cell lines, CpG2006 blocked HIV replication completely. In contrast, CpG10 had no anti-HIV effect in T-cell lines. In macrophages, neither CpG2006 nor CpG10 displayed substantial anti-HIV activity. Thus, the different anti-HIV activities of CpG2006 and CpG10 in different cell culture systems clearly demonstrate the importance of an integrated approach to examine lymphoid tissue with its rich cell context and cell subsets. CpG2006 appeared to block HIV rather widely, and CpG10 was only active within the context of the rich lymphatic cell repertoire.

The universal blocking of HIV replication by CpG2006 raises the possibility that it may interfere directly and nonspecifically with the HIV replication cycle, independent of TLR9 triggering. A BLAST search revealed no alignment between the HIV strain pNL4-3 and CpGs 2006, 2216, and 10, excluding antisense as the molecular mechanism interfering with HIV replication.

Since control ODNs lacking CpG motifs are also active against HIV, nonspecific blocking through CpG2006 appears highly likely. CpG2006 has a phosphorothioate backbone which renders ODNs resistant to DNases (31). Control ODNs ODN2006/90 and CpG2117 with a phosphorothioate backbone but reversed immunomodulatory motif (e.g., CpG to GpC) or methylated cytidine retained anti-HIV activity, whereas control ODNs with a phosphodiester backbones even with the CpG motif (e.g., CpG2006/89 and ODN2006/91) had only marginal anti-HIV effects. Thus, phosphorothioate modification contributes fundamentally to the observed anti-HIV effect of CpG2006, as shown for other phosphorothioate ODNs (47). Furthermore, the ODN examined, rich in guanosines (e.g., ODNs 10, 12, 30, 2216, and 2243) and irrespective of the presence of the CpG motif, displayed substantial anti-HIV activity. This observation is reminiscent of earlier observations that guanosine-rich ODN may interfere with HIV gp120 binding to CD4. No tested compounds adversely affected cell viability.

Oligonucleotides have complex antiviral activities (16), and we wanted to identify their mechanism of action in our system. We first examined if CpG 2006 or 10 affect the HIV viral receptor complex of CD4 and one of the chemokine receptors, CCR5 or CXCR4 (reviewed in reference 7). We quantified CD4 and CXCR4 in HLAC by flow cytometry 2 days after treatment with CpG2006 or CpG10. We found no differences between treated and control cultures, excluding downregulation of the HIV receptor complex, as being critical for the anti-HIV effects of ODNs.

We next looked for an effect of the ODNs on virus-cell fusion. We used a cell-based fusion assay to recapitulate fusion of the HIV envelope with the cell membrane. The assay revealed that CpG2006 as well as its control ODN, ODN2006/90 (reversed immunomodulatory CpG motif), effectively blocked syncytium formation. Previous studies showed that a number of oligonucleotides may interact with the virion envelope to block HIV adsorption (16). Thus, CpG2006's primary molecular mechanism appears to be nonspecific interference with HIV entry and to be independent of triggering of TLR9. The phosphorothioate-modified ODN SdC28, which consists of 28 cytidines, and a number of other ODNs interfere with HIV replication by binding to the positively charged V3 loop of the HIV envelope (3,46). Thus, the phosphorothioate-modified ODNs examined may similarly bind to the HIV envelope.

The reasons behind the lack of anti-HIV activity of CpG2006 in monocyte-derived macrophages are unknown. A distinct monocyte-derived macrophage surface composition or an alternative HIV entry mechanism might be responsible. Indeed, it appears that some control ODNs resulted in stimulation of HIV. Could it be that they activate monocyte-derived macrophages and thereby foster HIV replication? These questions will require additional studies.

In contrast, CpGs 10 and 2216 and ODN2243 did not prevent syncytium formation between HeLa-Env/LAI and HeLa-CD4 cells, suggesting that they work after viral entry. To further define the block in the HIV replication cycle, we quantified HIV DNA copies intracellularly in a one-round HIV replication assay in HLAC, in which the spreading HIV infection was halted by adding zidovudine immediately after infection (42). CpG2006 and CpG10 both decreased HIV DNA levels. From these findings in the fusion assay and the one replication cycle, CpG10 appears to interfere at the level of disassembly or reverse transcription of the HIV replication cycle. Since we have not examined virus-cell membrane fusion, we cannot exclude the possibility that CpG2216 and CpG10 act on virus entry after receptor binding but before virus-cell membrane fusion, as reported for the amphotericin B derivative MS8209 (39). The inhibition of HIV in HLAC, while having no effect in the cell-cell fusion assay, suggests that CpG10 may indirectly mediate the observed anti-HIV effects in HLAC. Since primary CD4+ T cells and all T-cell lines screened by quantitative PCR express a low level of TLR9, the absence of TLR9 will not by itself explain the molecular mechanism of action of CpG10.

We also examined the role of cytokines in the anti-HIV activity of ODNs. ODNs with CpG motifs are potent inducers of cytokines (e.g., IFN-{gamma}, tumor necrosis factor alpha, interleukin-12, and interleukin-6) that are believed to be critical for the antimicrobial activity of the CpGs (18, 31, 55, 58). We found prominent induction of IFN-{alpha}, tumor necrosis factor alpha, MIP-1{alpha}, MIP-1ß, and RANTES in HLAC treated with CpG2006 and CpG2216 at days 1 and 2 compared to ODNs with no CpG motifs or untreated HLAC. Thus, while CpG2006 and CpG2216 appear to nonspecifically block HIV replication, the CpG motif is required to trigger TLR9-mediated cytokine changes. IFN-{alpha} has potent antiviral activities (27). We wondered if IFN-{alpha} contributes to the overall anti-HIV activities in HLAC treated with CpG2006 or CpG2216. Recombinant IFN-{alpha} at concentrations exceeding the levels induced by these ODNs decreased HIV replication only slightly. Therefore, while IFN-{alpha} may be beneficial overall in the context of an immune response, it is certainly not the driving force in this experimental setting.

TLR2/TLR9 signaling results in HIV long terminal repeat trans-activation and HIV replication in HIV transgenic mouse spleen cells (5, 15). Treatment of latently HIV-infected T-cell lines with CpG ODNs such as CpG2006 stimulates HIV replication. No effects were evident when ODNs without CpG motifs were used. CpG-mediated TLR9 triggering resulted in enhanced expression of the transcription factor NF{kappa}B (41; reviewed in reference 49), which increases initiation and elongation of viral transcription (6).

Based on our own and other published data, we propose a model in which TLR9 ligands may block HIV infection nonspecifically but stimulate HIV replication specifically in latently infected cells through TLR9 triggering. The diverse effects of ODN CpG triggering of TLR9 are very interesting; they might be able to prevent new infections and simultaneously to eradicate latently infected cells by stimulating HIV replication. Importantly, they may link the innate and adaptive immune responses. Thus, future in vivo studies will have to show whether ODNs with immunomodulatory CpGs will be as promising as they are in vitro assays and whether the generation of ODNs combining antisense, nonspecific interference with HIV replication and the incorporation of immunostimulatory CpG motifs will indeed improve the antiviral action of ODNs.


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ACKNOWLEDGMENTS
 
We acknowledge M. Hersberger and M. Fischer for technical advice and D. Nadal, M. Opravil, S. Rampini, and R. Weber for critical reviews of the manuscript. The HeLa SX CCR5 cell line was generously provided by T. Klimkait (Institute of Medical Microbiology, Basel, Switzerland), and HeLa-Env/LAI was generously provided by M. Alizon (Department of Cell Biology, Institut Cochin, Paris, France). p49.5 was kindly provided by B. Chesebro (Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Mont.). pYU-2, pNL4-3, and p89.6 were obtained from B. H. Hahn (University of Alabama, Birmingham), M. A. Martin (Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Md.) and R. G. Collman (University of Pennsylvania School of Medicine, Philadelphia, Penn.), respectively, through the NIH AIDS Research and Reference Reagent Program. Zidovudine and interleukin-2 were obtained from the AIDS Research and Reference Reagent Program.

This work was supported by the EMDO Foundation, the OPO Foundation, and the Stiftung für wissenschaftliche Forschung an der Universität Zürich.

The authors declare that they have no competing financial interests.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases and Hospital Epidemiology, Department of Internal Medicine, University Hospital of Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. Phone: 41 44 255 37 75. Fax: 41 44 255 32 91. E-mail: roberto.speck{at}usz.ch. Back


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Journal of Virology, November 2004, p. 12344-12354, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12344-12354.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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