Inhibition of HIV type 1 replication in CD4+ and CD14+ cells purified from HIV type 1-infected individuals by the 2-5A agonist immunomodulator, 2-5A(N6B)

Abstract

Two major interferon (IFN)-mediated antiviral defense enzymes are double-stranded (ds)RNA-dependent 2',5'-oligoadenylate (2-5A) synthetase (2-5OAS) and p68 kinase (PKR). When activated by dsRNA, 2-5OAS synthesizes 2-5A, which binds to and activates RNase L. Activated RNase L hydrolyzes single-stranded viral RNA, thereby inhibiting viral protein synthesis. HIV-1 inhibits the IFN-mediated intracellular antiviral pathways. We have reported the synthesis and characterization of a nuclease-resistant 2-5A agonist (2-5A(N6B)) that overcomes the HIV-1 induced blockades by restoring the 2-5OAS/RNase L antiviral pathway (Homan JW, et al., J Acquir Immune Defic Syndr 2002;30:9-20). The objective of this study was to test the effect of 2-5A(N6B) on chronically infected CD4(+) T lymphocytes and CD14(+) monocytes derived from HIV-1-seropositive individuals. Wild-type HIV-1 replication was effectively inhibited by 2-5A(N6B) in CD4(+) T lymphocytes and CD14(+) monocytes purified from HIV-1 seropositive individuals (n = 18) compared to untreated cells. We also assessed the cytotoxicity of 2-5A(N6B) and report that 2-5A(N6B) exerts its anti-HIV-1 activity with no evidence of cytotoxicity (IC(90) > 100,000 nM). Furthermore, 2-5A(N6B) did not alter the cellular RNA profile, affect CCR5 or CXCR4 coreceptor expression, or activate caspase-dependent apoptosis. Evidence is also provided to show that 2-5A(N6B), and naturally occurring 2-5A(4), act as ligands to activate human Toll-like receptor 4. These results indicate that the 2-5A agonist 2-5A(N6B) has the potential to enhance host cell innate and acquired immune defense mechanisms against HIV-1 infection.

Figure 1

Antiproliferative effect of 2-5A^N6B against human CCRF-CEM cells. CCRF-CEM cells were treated for 72 hr with 2-5A^N6B (▪) or naturally occurring 2-5A₄ (•) at the concentrations indicated. Data are plotted as concentration of 2-5A^N6B or 2-5A₄ (X axis) vs. % inhibition (Y axis). Results represent data from two independent experiments.

Figure 2

Inhibition of X4-tropic HIV-1 replication in purified CD4⁺ T lymphocytes by the 2-5A agonist immunomodulator, 2-5A^N6B. CD4+ T cells were purified from peripheral blood (98% purity by FACS analysis). CD4⁺ T cells were incubated in the absence or presence of 2-5A^N6B at the concentrations shown for 3 hr prior to infection with HIV-1 (IIIB, m.o.i=1). The excess virus was washed away 24 hr post-infection HIV-1 replication was assessed in culture supernatants by p24 antigen ELISA. p24 antigen concentration at 48 hr (slashed bars) and 96 hr (solid bars) post-infection are shown. Results represent the mean (± standard deviation) of triplicate determinations. * = p < 0.005.

Figure 3

Inhibition of wild type HIV-1 replication in CD4⁺ T lymphocytes (panel A) and CD14⁺ monocytes (panel B) derived from HIV-1 seropositive patients by treatment with 2-5A^N6B. CD4+ lymphocytes and CD14+ monocytes were isolated from HIV-1 seropositive individuals by MACS^R (as described in Materials and Methods). Cells were incubated in the absence or presence of 1000 nM 2-5A^N6B for 5 days. Culture supernatants were collected and HIV-1 replication was quantified by p24 antigen ELISA. Results represent the average of triplicate determinations. Paired patient data are shown. Closed circles represent p24 antigen in individual study subjects; open circles represent the group means.

Figure 4

Flow cytometric analysis of CXCR4 (panels A,B) and CCR5 (panels C,D) co-receptor expression following in vitro treatment of PBMC with 2-5A^N6B. Healthy donor PBMC were incubated in the absence or presence of 2-5A^N6B (100 nM) prior to infection with HIV-1. PBMC were harvested 24 hr post-infection and labeled with anti-CXCR4 and anti-CCR5 antibodies as described in Materials and Methods. Results are shown as dotplots with cell populations in the live lymphocyte gate. The expression of cell surface markers was initially gated (forward vs. side-scatter) to examine viable cells.

Figure 4

Flow cytometric analysis of CXCR4 (panels A,B) and CCR5 (panels C,D) co-receptor expression following in vitro treatment of PBMC with 2-5A^N6B. Healthy donor PBMC were incubated in the absence or presence of 2-5A^N6B (100 nM) prior to infection with HIV-1. PBMC were harvested 24 hr post-infection and labeled with anti-CXCR4 and anti-CCR5 antibodies as described in Materials and Methods. Results are shown as dotplots with cell populations in the live lymphocyte gate. The expression of cell surface markers was initially gated (forward vs. side-scatter) to examine viable cells.

Figure 5

Activation of hTLRs by 2-5A^N6B and 2-5A₄. hTLR activation was tested by assessing NF-κB activation in HEK293 cells expressing a given hTLR. HEK293 cells were transfected with empty vector or human TLR2, 3, 4, 5, 7, 8, or 9 and a NF-κB inducible promoter plasmid expressing the secreted alkaline phosphatase reporter gene. Twenty-four hours after transfection, cells were left unstimulated or were stimulated with positive control ligand, 2-5A^N6B or 2-5A₄ (500 μM). The cells were incubated at 37°C for 18 hr. The results are provided as OD₆₂₀ values. Positive control ligands for hTLRs 2, 3, 4, 5, 7, 8 and 9 were: heat killed Listeria monocytogenes (HKLM) at 10⁸ cells/ml, poly(I:C) at 10 μg/ml, E. coli K12 lipopolysaccharide (LPS) at 10 μg/ml, S. typhimurium flagellin at 1 μg/ml, loxoribine at 1 mM, sspolyU/LyoVec at 50 μg/ml, and CpG at 10 μg/ml, respectively. Results represent the mean of duplicate determinations in two independent experiments.

Figure 6

Activation of hTLR4 by 2-5A^N6B and 2-5A₄. Activation of hTLR4 was tested by assessing NF-κB activation in HEK293-hTLR4/MD2-CD14 cells. Cells were transfected with empty vector or hTLR4/CD2-CD14 and a NF-κB inducible promoter plasmid expressing the secreted alkaline phosphatase reporter gene. Twenty-four hours after transfection, cells were left unstimulated or were stimulated with 2-5A^N6B or 2-5A₄ (at 0.5, 5 or 500 μM) or the positive control ligand, E. coli K12 lipopolysaccharide (LPS) (at 10 μg/ml). Cells were incubated at 37°C for 18 hr. The results are provided as OD₆₂₀ values. Results represent the mean of triplicate determinations. * = p < 0.05.