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 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

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 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 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

Randomized, placebo controlled phase I trial of safety, pharmacokinetics, pharmacodynamics and acceptability of tenofovir and tenofovir plus levonorgestrel vaginal rings in women - Fig 2

<p><b>(A) TFV in CV aspirate</b>. Blue Bars: TFV IVR, Red Bars: TFV/LNG IVR, Visit 4, pooled across sampling times of 1, 2, 4 and 8 hours post insertion [n = 18 for each IVR group], Visit 5: 24 hours post insertion [n = 20 for TFV and n = 19 for TFVLNG IVRs], Visit 6: LH surge or Menstrual Cycle day 17 [n = 20 for each IVR group], Visit 7: Prior to IVR Removal [n = 19 for each IVR group], Visit 8: 24 hours post removal [n = 15 for TFV and n = 20 for TFVLNG IVRs]. Reference line indicates 1,000 ng/mL, aspirate level associated with 75% reduction in HIV acquisition in CAPRISA 004 study subset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199778#pone.0199778.ref041" target="_blank">41</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199778#pone.0199778.ref042" target="_blank">42</a>] <b>(B) TFV [ng/mg] in vaginal tissue</b>. Blue Bars: TFV IVR, Red Bars: TFV/LNG IVR, Proximal indicates biopsy was obtained close to the IVR, in the posterior vaginal fornix, Distal indicates biopsy was obtained farther from the IVR, in the lower 1/3 of the vagina closer to the introitus. Visit 5: 24 hours post insertion [for proximal biopsies n = 15 for TFV and n = 16 for TFVLNG IVR. For distal biopsies, n = 19 for TFV and n = 20 for TFVLNG IVR]. Visit 7: Prior to IVR Removal [for proximal biopsies n = 15 for TFV and n = 17 for TFVLNG IVR] and [for distal biopsies, n = 19 for TFV and n = 20 for TFVLNG IVR]. Visit 8: 24 hours post removal [n = 9 for TFV and n = 8 for TFVLNG IVR], Visit 9 72 hours post removal [n = 5 for TFV and n = 9 for TFVLNG IVR]. Dashed line indicates TFV level [10 ng/mg] associated with high TFV-DP concentrations of approximately 1,000 fmol/mg [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199778#pone.0199778.ref024" target="_blank">24</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199778#pone.0199778.ref041" target="_blank">41</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199778#pone.0199778.ref042" target="_blank">42</a>]. <b>(C) TFV-DP [fmol/mg] in vaginal tissue</b>. Blue Bars: TFV IVR, Red Bars: TFV/LNG IVR. Proximal indicates biopsy was obtained close to the IVR, in the posterior vaginal fornix. Distal indicates biopsy was obtained farther from the IVR, in the lower 1/3 of the vagina closer to the introitus. Visit 5: 24 hours post insertion [for proximal biopsies, n = 14 for TFV and n = 16 for TFVLNG IVR. For distal biopsies, [n = 19 for TFV and n = 20 for TFVLNG IVR], Visit 7: Prior to IVR Removal [for proximal biopsies, n = 14 for TFV and n = 17 for TFVLNG IVR]. For distal biopsies, n = 19 for both IVRs], Visit 8: 24 hours post removal [n = 8 for TFV and n = 7 for TFVLNG IVR], Visit 9 72 hours post removal [n = 5 for TFV and n = 9 for TFVLNG IVR]. Dashed line indicates levels found to be protective against SHIV transmission in non-human primates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199778#pone.0199778.ref043" target="_blank">43</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199778#pone.0199778.ref044" target="_blank">44</a>] <b>(D) TFV in CV fluid [ng/mg] obtained from lower genital tract swabs</b>. Blue Bars: TFV IVR, Red Bars: TFV/LNG IVR, Visit 4: 1–8 hours post IVR insertion vaginal swab taken near IVR [n = 20 combined observations for TFV IVR and TFVLNG IVR], Visit 5: 24 hours post insertion taken near IVR [n = 20 for both IVRs], ectocervix [n = 20 for both IVRs] and introitus [n = 20 for both IVRs], Visit 7: Prior to IVR removal, taken near IVR [n = 20 for both IVRs], ectocervix [n = 20 for both IVRs] and introitus [n = 20 for TFV and n = 18 for TFV/LNG IVR], Visit 8: 24 hours post removal [n = 19 for TFV and n = 20 for TFVLNG IVR]. Cx = Ectocervix, Int = Introitus, IVR = vaginal near IVR.</p

Comparison of viral replication [P24 antigen production] in ectocervical biopsy tissue obtained from participants at baseline [visit 3] and post-treatment [visit 7] after use of placebo, TFV and TFV/LNG rings.

<p>Comparison of viral replication [P24 antigen production] in ectocervical biopsy tissue obtained from participants at baseline [visit 3] and post-treatment [visit 7] after use of placebo, TFV and TFV/LNG rings.</p

Study overview.

<p>Study overview.</p

CONSORT figure CONRAD A13-128 study population.

<p>CONSORT figure CONRAD A13-128 study population.</p