Enhancing Interferon Regulatory Factor 7 Mediated Antiviral Responses and Decreasing Nuclear Factor Kappa B Expression Limit HIV-1 Replication in Cervical Tissues

Establishment of a productive HIV-1 infection in the female reproductive tract likely depends on the balance between anti-viral and pro-inflammatory responses leading to activation and proliferation of HIV target cells. Immune modulators that boost anti-viral and depress pro-inflammatory immune responses may decrease HIV-1 infection or replication. Polyinosinic: polycytidylic [Poly (I:C)] has been reported to down-regulate HIV-1 replication in immune cell subsets and lymphoid tissues, yet the scope and mechanisms of poly (I:C) regulation of HIV-1 replication in the cervicovaginal mucosa, the main portal of viral entry in women remain unknown. Using a relevant, underexplored ex vivo cervical tissue model, we demonstrated that poly (I:C) enhanced Interferon Regulatory Factor (IRF)7 mediated antiviral responses and decreased tissue Nuclear Factor Kappa B (NF κ B) RNA expression. This pattern of cellular transcription factor expression correlated with decreased HIV-1 transcription and viral release. Reducing IRF7 expression up-regulated HIV-1 and NFκB transcription, providing proof of concept for the critical involvement of IRF7 in cervical tissues. By combining poly (I:C) with a suboptimal concentration of tenofovir, the leading anti-HIV pro-phylactic microbicide candidate, we demonstrated an earlier and greater decrease in HIV replication in poly (I:C)/tenofovir treated tissues compared with tissues treated with tenofovir alone, indicating overall improved efficacy. Poly (I:C) decreases HIV-1 replication by stimulating IRF7 mediated antiviral responses while reducing NFκB expression. Early during the infection, poly (I:C) improved the anti-HIV-1 activity of suboptimal concentrations of tenofovir likely to be present during periods of poor adherence i.e. inconsistent or inadequate drug use. Understanding interactions between anti-viral and pro-inflammatory immune responses in the genital mucosa will provide crucial insights for the identification of targets that can be harnessed to develop preventative combination strategies to improve the efficacy of topical or systemic antiviral prophylactic agents and protect women from HIV-1 and other sexually transmitted infections

Human Immunodeficiency Virus Reactivation by Phorbol Esters or T-Cell Receptor Ligation Requires both PKCα and PKCθ

Latently human immunodeficiency virus (HIV)-infected memory CD4(+) T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKCθ, PKCθA148E, and PKCα, PKCαA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4(+) T cells and that both PKCθA148E and PKCαA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKCα and PKCθ with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKCα or PKCθ. Since suppression of PKCα and PKCθ together has no greater inhibitory effect on HIV reactivation than inhibition of PKCα alone, our data confirm that PKCα and PKCθ act in sequence. The requirement for PKCα and PKCθ for prostratin-induced HIV reactivation and the ability of selective PKCα or PKCθ agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design

Enhancing Interferon Regulatory Factor 7 Mediated Antiviral Responses and Decreasing Nuclear Factor Kappa B Expression Limit HIV-1 Replication in Cervical Tissues.

Establishment of a productive HIV-1 infection in the female reproductive tract likely depends on the balance between anti-viral and pro-inflammatory responses leading to activation and proliferation of HIV target cells. Immune modulators that boost anti-viral and depress pro-inflammatory immune responses may decrease HIV-1 infection or replication. Polyinosinic:polycytidylic [Poly (I:C)] has been reported to down-regulate HIV-1 replication in immune cell subsets and lymphoid tissues, yet the scope and mechanisms of poly (I:C) regulation of HIV-1 replication in the cervicovaginal mucosa, the main portal of viral entry in women remain unknown. Using a relevant, underexplored ex vivo cervical tissue model, we demonstrated that poly (I:C) enhanced Interferon Regulatory Factor (IRF)7 mediated antiviral responses and decreased tissue Nuclear Factor Kappa B (NFκB) RNA expression. This pattern of cellular transcription factor expression correlated with decreased HIV-1 transcription and viral release. Reducing IRF7 expression up-regulated HIV-1 and NFκB transcription, providing proof of concept for the critical involvement of IRF7 in cervical tissues. By combining poly (I:C) with a suboptimal concentration of tenofovir, the leading anti-HIV prophylactic microbicide candidate, we demonstrated an earlier and greater decrease in HIV replication in poly (I:C)/tenofovir treated tissues compared with tissues treated with tenofovir alone, indicating overall improved efficacy. Poly (I:C) decreases HIV-1 replication by stimulating IRF7 mediated antiviral responses while reducing NFκB expression. Early during the infection, poly (I:C) improved the anti-HIV-1 activity of suboptimal concentrations of tenofovir likely to be present during periods of poor adherence i.e. inconsistent or inadequate drug use. Understanding interactions between anti-viral and pro-inflammatory immune responses in the genital mucosa will provide crucial insights for the identification of targets that can be harnessed to develop preventative combination strategies to improve the efficacy of topical or systemic antiviral prophylactic agents and protect women from HIV-1 and other sexually transmitted infections

Decreasing IRF7 expression enhances HIV-1 and RelA transcription.

<p>(A) IRF7, (C and F) HIV-1, (B, D and G) IFNα and (E and H) RelA transcription in HIV-1-infected cervical tissues treated with random and IRF7 targeting siRNA were quantified by RT-PCR before (A) and (B) and on days 1 (C, D and E) and 3 (F, G and H) after HIV-1 infection. All data was normalized to GAPDH. Results were consistent among four donors and are shown as the mean ± STDEV from one representative experiment with each condition tested in triplicate. * p<0.05 for tissues treated with random and IRF7 targeting siRNA.</p

Poly (I:C) decreases HIV-1 replication in cervical tissues.

<p>(A) HIV-1 p24 levels (ng/ml) in HIV-1 infected cervical tissues left untreated or treated with poly (I:C) at 20 μg/ml were measured after washing the residual input virus (day 0), and again on days 11 and 21 after infection. Results are shown as the geometric mean ± STDEV from triplicate values of 12 individual donors. *p = 0.00005 and p = 0.07 between untreated and poly (I:C) treated tissues at days 11 and 21 after infection, respectively. (B) Levels of HIV-1 reverse transcription and (C) integration in donor matched HIV-1 infected cervical tissues left untreated or treated with poly (I:C) at 20 μg/ml were quantified by RT-PCR on days 11 and 21 after infection. All data was normalized to human β actin. For HIV-1 reverse transcription and integration, day 11 values in untreated control tissues were set to 1. Day 11 values in poly (I:C) treated tissues or days 21 values in untreated or poly (I:C) treated tissues were normalized to 1. Results are shown as the relative geometric mean ± STDEV from triplicate values of 8 and 12 individual donors on days 11 and day 21 respectively. *p = 0.004 and p = 0.0009 between untreated and poly (I:C) treated tissues at days 11 and 21 after infection, respectively.</p

Poly (I:C) improves the efficacy of TFV in cervical tissues.

<p>(A) HIV-1 p24 levels (ng/ml) in HIV-1 infected cervical tissues left untreated or treated with TFV at 10 μg/ml alone or in combination with poly (I:C) at 20 μg/ml were evaluated after washing the residual input virus (day 0), and again on days 11 and 21 after infection. (B) Levels of HIV-1 reverse transcription and (C) viral integration in donor matched HIV-1 infected cervical tissues left untreated or treated with TFV at 10 μg/ml alone or in combination poly (I:C) at 20 μg/ml were quantified by RT-PCR on days 11 and 21 after infection. All data was normalized to human β-actin. For HIV-1 reverse transcription and integration, day 11 values in untreated control tissues were set to 1. Day 11 values in TFV or TFV/Poly (I:C) treated tissues or days 21 values in untreated; TFV or TFV/Poly (I:C) treated tissues were normalized to 1. Results were consistent among 4 donors and are shown as the mean ± STDEV from one representative experiment with each condition tested in triplicate. * p<0.05 for untreated, poly (I:C) and TFV treated tissues. (D) FACS analysis of single cell suspensions from HIV-1 infected ectocervical tissues left untreated or treated with TFV at 10 μg/ml and stained for CD3 and CD8 on day 11 after infection. CD4⁺ T cells were defined as CD3⁺ and CD8^-. For each panel the percentage of CD4⁺ T cells from the total cell number is depicted in the upper left corner. Results were consistent among 4 donors.</p

Poly (I:C) decreases HIV-1 transcription in cervical tissues by enhancing IRF7 mediated antiviral responses and reducing RelA expression.

<p>(A) Levels of HIV-1, (B) RelA, (C) IRF7, (D) IRF3, (E) RIG-1, (F) TLR3, (G) IFNα and (H) IFNβ transcription in HIV-1-infected cervical tissues left untreated or treated with poly (I:C) at 20 μg/ml were quantified by RT-PCR on days 3 and 5 after infection. All data was normalized to glyceraldehyde 3-phophate (GAPDH). For each gene, day 3 values in untreated control tissues were set to 1. Day 5 values in untreated control tissues or days 3 and 5 values in poly (I:C) treated tissues were normalized to 1. Results were consistent among three donors and are shown as the mean ± STDEV from one representative experiment with each condition tested in triplicate. * p<0.05 for untreated and poly (I:C) treated tissues.</p

Poly (I:C) decreases HIV-1 replication in PBMCs.

<p>HIV-1 p24 levels (ng/ml) in HIV-1 infected PBMCs left untreated or treated with poly (I:C) at 20 μg/ml were measured after washing the residual input virus (day 0), and again on days 3, 5 and 7 after infection. Results are shown as the mean ± STDEV from three experiments with each condition tested in triplicate. * p = 0.008, p = 0.01 and p = 0.009 for days 3, 5 and 7 after infection between untreated and poly (I:C) treated PBMCs.</p

Poly (I:C) decreases HIV-1 transcription by increasing IRF7 expression in PBMCs.

<p>(A) Levels of HIV-1, (B) RelA, (C) IRF7, (D) IRF3, (E) RIG-1, and (F) TLR3 transcription in HIV-1 infected PBMCs left untreated or treated with poly (I:C) at 20 μg/ml were quantified by RT-PCR on days 3 and 5 after infection. All data was normalized to GAPDH. For each gene, day 3 values in untreated control tissues were set to 1. Day 5 values in untreated control tissues or days 3 and 5 values in poly (I:C) treated tissues were normalized to 1. Results were consistent among three donors and are shown as the mean ± STDEV from one representative experiments with each condition tested in triplicate. * p<0.05 for untreated and poly (I:C) treated cells.</p

Randomized, placebo controlled phase I trial of safety, pharmacokinetics, pharmacodynamics and acceptability of tenofovir and tenofovir plus levonorgestrel vaginal rings in women.

To prevent the global health burdens of human immunodeficiency virus [HIV] and unintended/mistimed pregnancies, we developed an intravaginal ring [IVR] that delivers tenofovir [TFV] at ~10mg/day alone or with levonorgestrel [LNG] at ~20μg/day for 90 days. We present safety, pharmacokinetics, pharmacodynamics, acceptability and drug release data in healthy women. CONRAD A13-128 was a randomized, placebo controlled phase I study. We screened 86 women; 51 were randomized to TFV, TFV/LNG or placebo IVR [2:2:1] and 50 completed all visits, using the IVR for approximately 15 days. We assessed safety by adverse events, colposcopy, vaginal microbiota, epithelial integrity, mucosal histology and immune cell numbers and phenotype, cervicovaginal [CV] cytokines and antimicrobial proteins and changes in systemic laboratory measurements, and LNG and TFV pharmacokinetics in multiple compartments. TFV pharmacodynamic activity was measured by evaluating CV fluid [CVF] and tissue for antiviral activity using in vitro models. LNG pharmacodynamic assessments were timed based on peak urinary luteinizing hormone levels. All IVRs were safe with no significant colposcopic, mucosal, immune and microbiota changes and were acceptable. Among TFV containing IVR users, median and mean CV aspirate TFV concentrations remained above 100,000 ng/mL 4 hours post IVR insertion and mean TFV-diphosphate [DP] concentrations in vaginal tissue remained above 1,000 fmol/mg even 3 days post IVR removal. CVF of women using TFV-containing IVRs completely inhibited [94-100%] HIV infection in vitro. TFV/LNG IVR users had mean serum LNG concentrations exceeding 300 pg/mL within 1 hour, remaining high throughout IVR use. All LNG IVR users had a cervical mucus Insler score <10 and the majority [95%] were anovulatory or had abnormal cervical mucus sperm penetration. Estimated in vivo TFV and LNG release rates were within expected ranges. All IVRs were safe with the active ones delivering sustained high concentrations of TFV locally. LNG caused changes in cervical mucus, sperm penetration, and ovulation compatible with contraceptive efficacy. The TFV and TFV/LNG rings are ready for expanded 90 day clinical testing. Trial registration ClinicalTrials.gov #NCT02235662