iconstar paper   HIV Articles  
Back grey arrow rt.gif
Short-Course Toll-Like Receptor 9 Agonist [MGN1703 ] Treatment Impacts Innate Immunity and Plasma Viremia in Individuals With Human Immunodeficiency Virus Infection
  Download the PDF here
CID June 15 2017 - Line Vibholm,1,2,a Mariane H. Schleimann,1,2,a Jesper F. HOjen,1,2 Thomas Benfield,3 Rasmus Offersen,1,2 Katrine Rasmussen,1 Rikke Olesen,1 Anders Dige,2,4 JOrgen Agnholt,2,4 Judith Grau,5 Maria Buzon,5 Burghardt Wittig,6 Mathias Lichterfeld,7 Andreas Munk Petersen,8,9 Xutao Deng,10,11 Mohamed Abdel-Mohsen,10,11,12 Satish K. Pillai,10,11 Sofie Rutsaert,13 Wim Trypsteen,13 Ward De Spiegelaere,13,14 Linos Vandekerchove,13 Lars Ostergaard,1,2 Thomas A. Rasmussen,1 Paul W. Denton,1,2 Martin Tolstrup,1,2 and Ole S. SOgaard1,2

Treatment with latency reversing agents (LRAs) enhances human immunodeficiency virus type 1 (HIV-1) transcription in vivo but leads to only modest reductions in the size of the reservoir, possibly due to insufficient immune-mediated elimination of infected cells. We hypothesized that a single drug molecule-a novel Toll-like receptor 9 (TLR9) agonist, MGN1703-could function as an enhancer of innate immunity and an LRA in vivo.
We conducted a single-arm, open-label study in which 15 virologically suppressed HIV-1-infected individuals on antiretroviral therapy received 60 mg MGN1703 subcutaneously twice weekly for 4 weeks. We characterized plasmacytoid dendritic cell, natural killer (NK), and T-cell activation using flow cytometry on baseline and after 4 weeks of treatment. HIV-1 transcription was quantified by measuring plasma HIV-1 RNA during MGN1703 administration.
In accordance with the cell type-specific expression of TLR9, MGN1703 treatment led to pronounced activation of plasmacytoid dendritic cells and substantial increases in plasma interferon-α2 levels (P < .0001). Consistently, transcription of interferon-stimulated genes (eg, OAS1, ISG15, Mx1; each P < .0001) were upregulated in CD4+ T cells as demonstrated by RNA sequencing. Further, proportions of activated cytotoxic NK cells and CD8+ T cells increased significantly during MGN1703 dosing, suggesting an enhancement of cellular immune responses. In 6 of 15 participants, plasma HIV-1 RNA increased from <20 copies/mL to >1500 copies/mL (range, 21-1571 copies/mL) during treatment.
TLR9 agonist treatment in HIV infection has a dual potential by increasing HIV-1 transcription and enhancing cytotoxic NK cell activation, both of which are key outcomes in HIV-1 eradication therapy.
Antiretroviral therapy (ART) effectively suppresses human immunodeficiency virus type 1 (HIV-1) [1]. However, replication-competent proviruses persist in long-lived resting memory CD4+ T cells [2-5] and neither ART nor the immune system is capable of eliminating this latent viral reservoir [6]. Proof-of-concept trials testing the "shock and kill" approach [7] to eradicate the reservoir have demonstrated that treatment with latency reversing agents (LRAs) can enhance transcription of HIV-1 RNA from latently infected cells in vivo [8-12]. Yet clinical trials have found no, or only modest, reductions in the size of the HIV-1 reservoir, possibly due to insufficient immune-mediated killing of infected cells [13]. In vivo and ex vivo studies demonstrate that priming of specific effector cells, such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, can enhance their capacity to recognize and kill infected cells [13-17]. This indicates that therapeutic interventions boosting NK and CTL activity could lead to elimination of cells expressing HIV-1 antigens. MGN1703 (lefitolimod), a novel Toll-like receptor 9 (TLR9) agonist, belongs to a class of drugs referred to as immune surveillance reactivators and is currently in phase 3 testing for treatment of metastatic colorectal cancer [18]. Preclinical testing and data from cancer trials demonstrates that MGN1703 activates immune functions through TLR9 stimulation. MGN1703-induced TLR9 signaling leads to increased secretion of a range of cytokines (eg, interferon [IFN] α) and activation of plasmacytoid dendritic cells (pDCs) and B cells [18]. Furthermore, MGN1703 increases activation and cytotoxicity of NK cells in vitro [19]. We recently demonstrated ex vivo that MGN1703 stimulation of peripheral blood mononuclear cells from HIV-1-infected donors enhanced the cellular immune response and increased HIV-1 transcription in CD4+ T cells [20]. Based on our ex vivo findings, we hypothesized that adjunctive MGN1703 treatment in individuals with HIV infection would (1) augment NK and CD8+ T-cell activation and (2) induce plasma viremia. To test our hypotheses, we enrolled 15 virologically suppressed HIV-1-infected individuals on ART to receive MGN1703 for 4 consecutive weeks.
To date, all clinical HIV cure interventions in the context of shock and kill have focused on the ability of a single intervention to either reverse latency or enhance cell-mediated immunity [9-14]. However, in the HIV eradication context, a single agent that is capable of affecting both outcomes has great potential benefits. Here we report the results of the first clinical trial using a single drug, a TLR9 agonist, as adjunctive immune therapeutic treatment in HIV-1-infected individuals on ART with the aim of both enhancing innate immunity and activating the HIV-1 reservoir. Our results demonstrate that MGN1703 treatment increased the activation of pDCs, upregulated levels of cytokines, and enhanced activation of cytotoxic NK cells and effector CD8+ T cells. Importantly, MGN1703 treatment induced plasma HIV-1 RNA blips up to >1500 copies/mL in a subset of participants. Thus, we report the first in vivo evidence that a single drug may both enhance immunity and increase HIV-1 transcription.
IFN-α is essential for immunological control of HIV infection (eg, by induction of antiretroviral restriction factors) [27, 28], and studies have demonstrated that long-term treatment with exogenous IFN-α can lead to viral suppression and a decline in integrated HIV-1 DNA [29], total HIV-1 DNA [30], and CA-US HIV-1 RNA [31]. We observed increased levels of IFN-α2 and a significant upregulation of numerous ISGs. Interestingly, levels of CA-US HIV-1 RNA declined at follow-up. Therefore, we conclude that the repetitive TLR9 stimulation induced an advantageous IFN-α secretion, without causing the severe side effects commonly observed in exogenous IFN-α treatment. However, the biological effects of IFN-α on HIV infection are very complex. Recent data from humanized mice indicate a potential beneficial effect of blocking the IFN-α receptor as mediator of enhanced viral reservoir control [32, 33], whereas studies in nonhuman primates suggest that short courses of INF-2α therapy enhance antiviral gene expression and prevent systemic infection [34]. This strategy needs to be further explored in HIV-infected patients to fully evaluate this modality.
During the acute phase of HIV-1 infection, the NK cell population expands rapidly, especially the cytotoxic subsets [16], and NK cells are possibly responsible for the initial containment of HIV-1 before potent CD8+ T-cell responses develop [35]. In an ex vivo study from our group, NK cells pretreated with MGN1703 demonstrated an HIV-1 specific response, evidenced by inhibition of virus production from autologous HIV-1-infected CD4+ T cells in a p24 viral inhibition assay [20]. In this present clinical study, TLR9 treatment activated cytotoxic CD56dimCD16+ NK cells [24], and downregulated the inhibitory receptor NKG2A, suggesting that MGN1703 might improve immunological control of HIV-1 infection in vivo.
We have previously demonstrated that MGN1703 increases HIV-1 transcription ex vivo [20]. In the present study, we found that 6 of 15 participants had increased plasma HIV-1 RNA during the MGN1703 treatment period. Contrary to findings from trials aiming to reverse HIV-1 latency by histone deacetylase (HDAC) inhibitor administration [10-14], we did not observe increases in CA-US HIV-1 RNA in MGN1703-treated individuals. However, this was not surprising given that HDAC inhibitors interrupt epigenetic gene regulation in all cells (including HIV-1-infected CD4+ T cells) while MGN1703 only directly activates TLR9-expressing cells (eg pDCs). Because peripheral blood does not have the same well-defined architecture as lymphoid tissues, the cell-to-cell interaction between TLR9-activated pDCs and latently infected CD4+ memory T cells is limited. MGN1703 treatment induced activation of all memory CD4+ and CD8+ T-cell subsets in peripheral blood, including both CD4+ TEM and TCM cells. These cells frequently harbor latent HIV-1 [4] and are homing to immunological tissues [36]. Thus, we speculate that the TLR9-mediated activation of latently infected CD4+ T cells residing in lymphoid tissues resulted in the production of HIV virions observed in plasma.
While participants were closely monitored and multiple virological and immunological parameters were investigated, a few limitations of this study should be mentioned. The lack of a control group reduced our ability to demonstrate that the observed plasma viremia was not caused by natural variation. However, a previous study from our group revealed that levels of plasma HIV-1 RNA is generally very low in ART-treated individuals, as only 2 of 26 individuals had blips of quantifiable viremia over a 6-month period during which a total of 225 plasma HIV-1 RNA quantifications were performed (Ole SOgaard, written personal communication, January 2017). Both cohorts are similar with respect to time on ART and level of viral suppression prior to enrollment. Thus, the virological signal detected in this current MGN1703-treated cohort is beyond that expected by natural variation. The discrepancy between the observed increases in pVL and the stability in CA-US HIV-1 RNA from peripheral blood CD4+ T cells during MGN1703 highlights a second limitation of our study: the general reliance upon peripheral blood-based measures of intervention outcomes, as lymphoid tissues represent key sites for harboring replication-competent and infectious HIV-1 in patients on ART [37-40]. Last, we only treated the participants for a 4-week period and protective CD4+ T-cell, CTL, and B-cell responses take weeks to fully develop. Thus, the limited duration of the TLR9 treatment might offer an explanation to the unaffected reservoir measurements.
In addition to TLR9 agonists, TLR7 agonists are also being investigated in HIV cure-related research [41]. The TLR7 agonist GS-9620 induce a pDC-dependent anti-HIV activity in vitro but also leads to an increase in the proinflammatory cytokines such as IL-6 [42]. Interestingly, a recent nonhuman primate study suggested that the TLR7 agonist GS-986 in combination with Ad26/MVA vectored therapeutic simian immunodeficiency virus vaccination improved immunologic control and delayed viral rebound compared to placebo-treated animals whereas animals receiving GS-986 alone appeared to have no improvement in virological control or change in the size of the viral reservoir. Collectively, these findings suggest that TLR agonists might have several possible roles to play in HIV cure-related research [43].
Whether immunity against HIV-1 infection can be augmented by therapeutic strategies is a focal point in current HIV research. We found that MGN1703 induced a balanced, targeted reactivation of cells and secretion of cytokines that are known to be critical for controlling HIV-1 infection [44] without objective clinical signs of general inflammation (eg, fever). Four weeks of MGN1703 treatment was considered safe and well tolerated in HIV-infected individuals, as only mild AEs occurred and all study subjects completed the treatment. In conclusion, this is the first study to report that a single-drug intervention increases HIV-1 transcription and enhances innate immunity altogether. To further enhance the anti-HIV-1 effect of MGN1703, a combination of immune therapeutic strategies with more potent LRAs and/or additional immune-based interventions could be a path for future HIV eradication studies.

  iconpaperstack View Older Articles   Back to Top   www.natap.org