Poly-ICLC, a TLR3 Agonist, Induces Transient Innate Immune Responses in Patients With Treated HIV-Infection: A Randomized Double-Blinded Placebo Controlled Trial

Abstract

Objective: Toll-like receptor-3 agonist Poly-ICLC has been known to activate immune cells and induce HIV replication in pre-clinical experiments. In this study we investigated if Poly-ICLC could be used for disrupting HIV latency while simultaneously enhancing innate immune responses. Design: This was a randomized, placebo-controlled, double-blinded trial in aviremic, cART-treated HIV-infected subjects. Participants (n = 15) were randomized 3:1 to receive two consecutive daily doses of Poly-ICLC (1.4 mg subcutaneously) vs. placebo. Subjects were observed for adverse events, immune activation, and viral replication. Methods: Besides primary outcomes of safety and tolerability, several longitudinal immune parameters were evaluated including immune cell phenotype and function via flowcytometry, ELISA, and transcriptional profiling. PCR assays for plasma HIV-1 RNA, CD4+ T cell-associated HIV-1 RNA, and proviral DNA were performed to measure HIV reservoirs and latency. Results: Poly-ICLC was overall safe and well-tolerated. Poly-ICLC-related adverse events were Grade 1/2, with the exception of one Grade 3 neutropenia which was short-lived. Mild Injection site reactions were observed in nearly all participants in the Poly-ICLC arm. Transcriptional analyses revealed upregulation of innate immune pathways in PBMCs following Poly-ICLC treatment, including strong interferon signaling accompanied by transient increases in circulating IP-10 (CXCL10) levels. These responses generally peaked by 24-48 h after the first injection and returned to baseline by day 8. CD4+ T cell number and phenotype were unchanged, plasma viral control was maintained and no significant effect on HIV reservoirs was observed. Conclusions: These finding suggest that Poly-ICLC could be safely used for inducing transient innate immune responses in treated HIV+ subjects indicating promise as an adjuvant for HIV therapeutic vaccines. Trial Registration: www.ClinicalTrials.gov, identifier: NCT02071095.

Keywords: HIV-1; adjuvant; poly-ICLC; toll-like receptor ligand; vaccine.

Figures

Figure 1

Study Schema. Following screening, eligible subjects are randomized 3:1 to receive Poly-ICLC (1.4 mg SQ) vs. Placebo on days 1 and 2. Participants returned for follow up visits on days 4 and 8, weeks 4, 16, and 48. Unblinding of the study occurred after all subjects completed the week 16 follow up visit as specified by the protocol.

Figure 2

Study flow diagram.

Figure 3

No overall change in CD4+ T cells counts in the treatment arm. CD4+ T cell counts (cells/mm3) for each subject are shown over the course of the study. Values for subjects in Arm A (Poly-ICLC) and Arm B (Placebo) are depicted by solid and dashed lines, respectively.

Figure 4

Transient upregulation of pro-inflammatory genes in subjects who received Poly-ICLC, with strong induction of interferon pathway. Transcriptional responses were evaluated longitudinally in subjects' PBMCs using NanoString Technologies (nCounter® gene expression panel, human inflammation kit). (A) Fold change (FC) from baseline of significantly upregulated genes from subjects in Arm A (Poly-ICLC) are depicted graphically over time through day 8. Transient upregulation of several genes occurred (N = 31), generally peaking at 24 h and returning to baseline shortly thereafter. No significant changes were observed in subjects' PBMCs in Arm B (Placebo) (not shown). (B) Heat map of all significantly induced genes in Arm A (Poly-ICLC) represented as a FC over baseline. Most of the highly upregulated genes were found to be interferon-stimulated genes (ISGs) FDR < 1, FC ≥ ±1.5. (C) In concordance with strong upregulation of ISGs, plasma levels of IP-10 were transiently upregulated in subjects in Arm A Poly-ICLC (N = 12) vs. those in Arm B Placebo (N = 3). *p < 0.001.

Figure 5

Markers of immune activation and exhaustion on CD4+ and CD8+ T cells following Poly-ICLC administration. In order to monitor for the secondary induction of generalized immune activation following Poly-ICLC administration, surface expression of CD38, HLA-DR, PD-1, and CTLA-4 was evaluated at each time point via flowcytometry on subjects'; (A) CD8+ T cells and (B) CD4+ T cells. No longitudinal changes were found to be statistically significant following Poly-ICLC administration with the exception of transient upregulation of CD38 on CD8+ T cells at Day 4. *p < 0.001.

Figure 6

No changes in DC and NK cell numbers and activation following Poly-ICLC treatment. DC and NK cell subsets were enumerated as described following Poly-ICLC vs. placebo. Percentage of mDCs, pDCs, total NK cells, CD56 bright, CD56 dim, and CD56 negative cells in PBMCs for each subject are shown over the course of the study as measured by flowcytometry. Values for subjects in Arm A (Poly-ICLC) and Arm B (Placebo) are depicted by solid and dashed lines, respectively. Though multiple NK cell subsets declined following Poly-ICLC, the only CD56 dim NK cells were found to be statistically significant. (*p < 0.05, FDR <1).

Figure 7

Longitudinal assessment of cell associated HIV-1 RNA and DNA values show no significant changes overall following discrete dosing of Poly-ICLC. (A) Following RNA extraction from purified CD4+ T cells at baseline and multiple time points following Poly-ICLC, rtPCR for HIV-1 RNA was performed in quadruplicate. Cell-associated HIV-1 RNA copy number is expressed as copy number per 1 μg of RNA in purified CD4+ T cells. The left graph depicts pooled values from all participants in Arm A (Poly-ICLC) and Arm B (placebo), with the mean values and standard deviation. The right graph depicts this data as individual values over time for each participant in Arm A and Arm B. (B) Following DNA extraction from purified CD4+ T cells at baseline and at week 4, rtPCR for HIV-1 DNA was performed in quadruplicate. Cell-associated HIV-1 DNA copy number is expressed as copy number per 1 μg of DNA in purified CD4+ T cells. Left graph depicts pooled values from all participants in Arm A and Arm B. The right graph shows individual values for each participant in Arm A and Arm B. Values for subjects in Arm A (Poly-ICLC) and Arm B (Placebo) are depicted by solid and dashed lines, respectively.