TLR9 agonism differentially impacts human NK cell-mediated direct killing and antibody-dependent cell-mediated cytotoxicity.

There are two known mechanisms by which natural killer (NK) cells recognize and kill diseased targets: (i) direct killing and (ii) antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated an indirect NK cell activation strategy for the enhancement of human NK cell killing function. We did this by leveraging the fact that toll-like receptor 9 (TLR9) agonism within pools of human peripheral blood mononuclear cells (PBMCs) results in a robust interferon signaling cascade that leads to NK cell activation. After TLR9 agonist stimulation, NK cells were enriched and incorporated into assays to assess their ability to kill tumor cell line targets. Notably, differential impacts of TLR9 agonism were observed-direct killing was enhanced while ADCC was not increased. To ensure that the observed differential effects were not attributable to differences between human donors, we recapitulated the observation using our Natural Killer-Simultaneous ADCC and Direct Killing Assay (NK-SADKA) that controls for human-to-human differences. Next, we observed a treatment-induced decrease in NK cell surface CD16-known to be shed by NK cells post-activation. Given the essential role of CD16 in ADCC, such shedding could account for the observed differential impact of TLR9 agonism on NK cell-mediated killing capacity.

BET inhibition reforms the immune microenvironment and alleviates T cell dysfunction in chronic lymphocytic leukemia.

Redundant tumor microenvironment (TME) immunosuppressive mechanisms and epigenetic maintenance of terminal T cell exhaustion greatly hinder functional antitumor immune responses in chronic lymphocytic leukemia (CLL). Bromodomain and extraterminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T cell function and differentiation. Herein, we report that blocking BET protein function alleviates immunosuppressive networks in the CLL TME and repairs inherent CLL T cell defects. The pan-BET inhibitor OPN-51107 reduced exhaustion-associated cell signatures resulting in improved T cell proliferation and effector function in the Eµ-TCL1 splenic TME. Following BET inhibition (BET-i), TME T cells coexpressed significantly fewer inhibitory receptors (IRs) (e.g., PD-1, CD160, CD244, LAG3, VISTA). Complementary results were witnessed in primary CLL cultures, wherein OPN-51107 exerted proinflammatory effects on T cells, regardless of leukemic cell burden. BET-i additionally promotes a progenitor T cell phenotype through reduced expression of transcription factors that maintain terminal differentiation and increased expression of TCF-1, at least in part through altered chromatin accessibility. Moreover, direct T cell effects of BET-i were unmatched by common targeted therapies in CLL. This study demonstrates the immunomodulatory action of BET-i on CLL T cells and supports the inclusion of BET inhibitors in the management of CLL to alleviate terminal T cell dysfunction and potentially enhance tumoricidal T cell activity.

Development and implementation of natural killer cell simultaneous ADCC and direct killing assay.

Assays to quantify natural killer (NK) cell killing efficacy have traditionally focused on assessing either direct killing or antibody dependent cell-mediated cytotoxicity (ADCC) independently. Due to the probability that immunotherapeutic interventions affect NK cell-mediated direct killing and NK cell-mediated ADCC differently, we developed an assay with the capacity to measure NK cell-mediated direct killing and ADCC simultaneously with cells from the same human donor. Specifically, this design allows for a single NK cell population to be split into several experimental conditions (e.g., direct killing, ADCC), thus controlling for potential confounders associated with human-to-human variation when assessing immunotherapy impacts. Our Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA) allows researchers to reproducibly quantify both direct killing and ADCC by human NK cells. Furthermore, this optimized experimental design allows for concurrent analysis of the NK cells via flow cytometric immunophenotyping of NK cell populations which will facilitate the identification of relationships between NK cell phenotype and the subsequent killing potential. This assay will be valuable for assessing the broader impact(s) of immunotherapy strategies on both modes of NK cell killing.

Impact of a TLR9 agonist and broadly neutralizing antibodies on HIV-1 persistence: the randomized phase 2a TITAN trial.

Inducing antiretroviral therapy (ART)-free virological control is a critical step toward a human immunodeficiency virus type 1 (HIV-1) cure. In this phase 2a, placebo-controlled, double-blinded trial, 43 people (85% males) with HIV-1 on ART were randomized to (1) placebo/placebo, (2) lefitolimod (TLR9 agonist)/placebo, (3) placebo/broadly neutralizing anti-HIV-1 antibodies (bNAbs) or (4) lefitolimod/bNAb. ART interruption (ATI) started at week 3. Lefitolimod was administered once weekly for the first 8 weeks, and bNAbs were administered twice, 1 d before and 3 weeks after ATI. The primary endpoint was time to loss of virologic control after ATI. The median delay in time to loss of virologic control compared to the placebo/placebo group was 0.5 weeks (P = 0.49), 12.5 weeks (P = 0.003) and 9.5 weeks (P = 0.004) in the lefitolimod/placebo, placebo/bNAb and lefitolimod/bNAb groups, respectively. Among secondary endpoints, viral doubling time was slower for bNAb groups compared to non-bNAb groups, and the interventions were overall safe. We observed no added benefit of lefitolimod. Despite subtherapeutic plasma bNAb levels, 36% (4/11) in the placebo/bNAb group compared to 0% (0/10) in the placebo/placebo group maintained virologic control after the 25-week ATI. Although immunotherapy with lefitolimod did not lead to ART-free HIV-1 control, bNAbs may be important components in future HIV-1 curative strategies. ClinicalTrials.gov identifier: NCT03837756 .

Designing peptide amphiphiles as novel antibacterials and antibiotic adjuvants against gram-negative bacteria.

Gram-negative strains are intrinsically resistant to most antibiotics due to the robust and impermeable characteristic of their outer membrane. Self-assembling cationic peptide amphiphiles (PAs) have the ability to disrupt bacteria membranes, constituting an excellent antibacterial alternative to small molecule drugs that can be used alone or as antibiotic adjuvants to overcome bacteria resistance. PA1 (C16KHKHK), self-assembled into micelles, which exhibited low antibacterial activity against all strains tested, and showed strong synergistic antibacterial activity in combination with Vancomycin with a Fractional Inhibitory Concentration index (FICi) of 0.15 against E. coli. The molecules, PA2 (C16KRKR) and PA3 (C16AAAKRKR), also self-assembled into micelles, displayed a broad-spectrum antibacterial activity against all strains tested, and low susceptibility to resistance development over 21 days. Finally, PA1, PA 2 and PA3 displayed low cytotoxicity against mammalian cells, and PA2 showed a potent antibacterial activity and low toxicity in preliminary in vivo models using G. mellonella. The results show that PAs are a great platform for the future development of effective antibiotics to slow down the antibiotic resistance and can act as antibiotic adjuvants with synergistic mechanism of action, which can be repurposed for use with existing antibiotics commonly used to treat gram-positive bacteria to treat infections caused by gram-negative bacteria.

Early intervention with 3BNC117 and romidepsin at antiretroviral treatment initiation in people with HIV-1: a phase 1b/2a, randomized trial.

Attempts to reduce the human immunodeficiency virus type 1 (HIV-1) reservoir and induce antiretroviral therapy (ART)-free virologic control have largely been unsuccessful. In this phase 1b/2a, open-label, randomized controlled trial using a four-group factorial design, we investigated whether early intervention in newly diagnosed people with HIV-1 with a monoclonal anti-HIV-1 antibody with a CD4-binding site, 3BNC117, followed by a histone deacetylase inhibitor, romidepsin, shortly after ART initiation altered the course of HIV-1 infection ( NCT03041012 ). The trial was undertaken in five hospitals in Denmark and two hospitals in the United Kingdom. The coprimary endpoints were analysis of initial virus decay kinetics and changes in the frequency of CD4+ T cells containing intact HIV-1 provirus from baseline to day 365. Secondary endpoints included changes in the frequency of infected CD4+ T cells and virus-specific CD8+ T cell immunity from baseline to day 365, pre-ART plasma HIV-1 3BNC117 sensitivity, safety and tolerability, and time to loss of virologic control during a 12-week analytical ART interruption that started at day 400. In 55 newly diagnosed people (5 females and 50 males) with HIV-1 who received random allocation treatment, we found that early 3BNC117 treatment with or without romidepsin enhanced plasma HIV-1 RNA decay rates compared to ART only. Furthermore, 3BNC117 treatment accelerated clearance of infected cells compared to ART only. All groups had significant reductions in the frequency of CD4+ T cells containing intact HIV-1 provirus. At day 365, early 3BNC117 + romidepsin was associated with enhanced HIV-1 Gag-specific CD8+ T cell immunity compared to ART only. The observed virological and immunological effects of 3BNC117 were most pronounced in individuals whose pre-ART plasma HIV-1 envelope sequences were antibody sensitive. The results were not disaggregated by sex. Adverse events were mild to moderate and similar between the groups. During a 12-week analytical ART interruption among 20 participants, 3BNC117-treated individuals harboring sensitive viruses were significantly more likely to maintain ART-free virologic control than other participants. We conclude that 3BNC117 at ART initiation enhanced elimination of plasma viruses and infected cells, enhanced HIV-1-specific CD8+ immunity and was associated with sustained ART-free virologic control among persons with 3BNC117-sensitive virus. These findings strongly support interventions administered at the time of ART initiation as a strategy to limit long-term HIV-1 persistence.

Administration of broadly neutralizing anti-HIV-1 antibodies at ART initiation maintains long-term CD8+ T cell immunity.

In simian-human immunodeficiency virus (SHIV)-infected non-human primates, broadly neutralizing antibodies (bNAbs) against the virus appear to stimulate T cell immunity. To determine whether this phenomenon also occurs in humans we measured HIV-1-specific cellular immunity longitudinally in individuals with HIV-1 starting antiviral therapy (ART) with or without adjunctive bNAb 3BNC117 treatment. Using the activation-induced marker (AIM) assay and interferon-γ release, we observe that frequencies of Pol- and Gag-specific CD8+ T cells, as well as Gag-induced interferon-γ responses, are significantly higher among individuals that received adjunctive 3BNC117 compared to ART-alone at 3 and 12 months after starting ART. The observed changes in cellular immunity were directly correlated to pre-treatment 3BNC117-sensitivity. Notably, increased HIV-1-specific immunity is associated with partial or complete ART-free virologic control during treatment interruption for up to 4 years. Our findings suggest that bNAb treatment at the time of ART initiation maintains HIV-1-specific CD8+ T cell responses that are associated with ART-free virologic control.

CD169 (Siglec-1) as a Robust Human Cell Biomarker of Toll-Like Receptor 9 Agonist Immunotherapy.

Immunotherapy is a promising therapeutic area in cancer and chronic viral infections. An important component of immunotherapy in these contexts is the activation of innate immunity. Here we investigate the potential for CD169 (Siglec 1) expression on monocytes to serve as a robust biomarker for activation of innate immunity and, particular, as a proxy for IFN-α production. Specifically, we investigated the effects of Toll-like receptor 9 agonism with MGN1703 (lefitolimod) across experimental conditions ex vivo, in humanized mice, and in clinical trial participants. Ex vivo we observed that the percentage of classical monocytes expressing CD169 increased dramatically from 10% pre-stimulation to 97% 24 hrs after MGN1703 stimulation (p<0.0001). In humanized NOG mice, we observed prominent upregulation of the proportions of monocytes expressing CD169 after two doses of MGN1703 where 73% of classical monocytes were CD169 positive in bone marrow following MGN1703 treatment vs 19% in vehicle treated mice (p=0.0159). Finally, in a clinical trial in HIV-infected individuals receiving immunotherapy treatment with MGN1703, we observed a uniform upregulation of CD169 on monocytes after dosing with 97% of classical monocytes positive for CD169 (p=0.002). Hence, in this comprehensive evaluation ex vivo, in an animal model, and in a clinical trial, we find increases in the percentage of CD169 positive monocytes to be a reliable and robust biomarker of immune activation following TLR9 agonist treatment.

Helminth products modulate innate immune recognition of nucleic acids in systemic lupus erythematosus.

AIM: Current treatment of Systemic Lupus Erythematosus (SLE) is suboptimal and causes broad immunosuppression. Therapeutic use of helminths or helminth products has been suggested for autoimmune diseases such as SLE. In the present study, we evaluated possible immunomodulating effects of adult body fluid (ABF) from Ascaris suum on peripheral blood mononuclear cells (PBMCs) from SLE patients in an ex vivo setup. METHODS: PBMCs from SLE patients and healthy controls (HC) were isolated and stimulated ex vivo with ABF and Toll-like receptor agonists or activators of the stimulator of interferon genes (STING) or mitochondrial antiviral signaling protein (MAVS) pathways. After 24 h of incubation, the cytokine profile was analyzed using ELISA and Meso Scale Discovery techniques. RESULTS: ABF suppressed production of IL-6, TNF-α, CXCL10, and IL-10 by PBMCs from SLE patients and HCs following stimulation with specific agonists. ABF also reduced IFN-у production by stimulated PBMCs from HCs. CONCLUSIONS: Our data show that ABF has an immunomodulatory effect on the production of key cytokines in the pathogenesis of SLE. These results suggest that ABF or ABF components hold potential as a novel treatment option for SLE.

The size and culturability of patient-generated SARS-CoV-2 aerosol.

BACKGROUND: Aerosol transmission of COVID-19 is the subject of ongoing policy debate. Characterizing aerosol produced by people with COVID-19 is critical to understanding the role of aerosols in transmission. OBJECTIVE: We investigated the presence of virus in size-fractioned aerosols from six COVID-19 patients admitted into mixed acuity wards in April of 2020. METHODS: Size-fractionated aerosol samples and aerosol size distributions were collected from COVID-19 positive patients. Aerosol samples were analyzed for viral RNA, positive samples were cultured in Vero E6 cells. Serial RT-PCR of cells indicated samples where viral replication was likely occurring. Viral presence was also investigated by western blot and transmission electron microscopy (TEM). RESULTS: SARS-CoV-2 RNA was detected by rRT-PCR in all samples. Three samples confidently indicated the presence of viral replication, all of which were from collected sub-micron aerosol. Western blot indicated the presence of viral proteins in all but one of these samples, and intact virions were observed by TEM in one sample. SIGNIFICANCE: Observations of viral replication in the culture of submicron aerosol samples provides additional evidence that airborne transmission of COVID-19 is possible. These results support the use of efficient respiratory protection in both healthcare and by the public to limit transmission.

Poor compliance with an antibiotic directive-A call for intensified monitoring.

BACKGROUND: In April 2017, the Central Denmark Region Antibiotic Stewardship Committee issued a directive to reduce the general use of piperacillin-tazobactam and prescribe narrow-spectrum antibiotics for mild and moderate pneumonia. The directive was distributed to all regional hospital clinicians. METHODS: Electronic medical records were used to obtain de-identified details of all antibiotics administered (together with diagnosis codes) to all in-hospital patients (pre-directive and post-directive) in the nine regional hospitals. Average moving range statistical process control charts were used to analyze pre-directive and post-directive variation in antibiotic usage patterns. RESULTS: Upon the distribution of the directive, a period of decline of the overall usage of piperacillin-tazobactam ensued. Rather than benzylpenicillin, as recommended for pneumonia, the initial decline in piperacillin/tazobactam usage was accompanied by increased use of cefuroxime. CONCLUSIONS: A steward-directed reduction in piperacillin-tazobactam usage was accompanied by less desirable usage of a broad-spectrum alternative. Future antibiotic stewardship initiatives will hopefully benefit from close monitoring and timely feedback to clinicians. A dialogue with clinicians based on near real-time data is predicted to improve antibiotic stewardship actions.

Comparable human reconstitution following Cesium-137 versus X-ray irradiation preconditioning in immunodeficient NOG mice.

Humanized mouse models are used extensively in research involving human pathogens and diseases. However, most of these models require preconditioning. Radio-active sources have been used routinely for this purpose but safety issues have motivated researchers to transition to chemical or X-ray based preconditioning. In this study, we directly compare 350 kV X-ray and Cs-137 low-dose precondition of NOG mice before human stem cell transplantation. Based on flow cytometry data, we found that engraftment of human cells into the mouse bone marrow was similar between radiation sources. Likewise, human engraftment in the peripheral blood was comparable between Cs-137 and three different X-ray doses with equal chimerization kinetics. In primary lymphoid organs such as the thymus and lymph nodes, and spleen, liver and lung, human-to-mouse chimerization was also comparable between irradiation sources. Development of different CD4 and CD8 T cells as well as these cells' maturation stages, i.e. from naïve to effector and memory subsets were generally analogous. Based on our results, we conclude that there are no discernable differences between the two sources in the low-dose spectrum investigated. However, while we encourage the transition to X-ray-based sources, we recommend all research groups to consider technical specifications and dose-finding studies.

Sialylation and fucosylation modulate inflammasome-activating eIF2 Signaling and microbial translocation during HIV infection.

An emerging paradigm suggests that gut glycosylation is a key force in maintaining the homeostatic relationship between the gut and its microbiota. Nevertheless, it is unclear how gut glycosylation contributes to the HIV-associated microbial translocation and inflammation that persist despite viral suppression and contribute to the development of several comorbidities. We examined terminal ileum, right colon, and sigmoid colon biopsies from HIV-infected virally-suppressed individuals and found that gut glycomic patterns are associated with distinct microbial compositions and differential levels of chronic inflammation and HIV persistence. In particular, high levels of the pro-inflammatory hypo-sialylated T-antigen glycans and low levels of the anti-inflammatory fucosylated glycans were associated with higher abundance of glycan-degrading microbial species (in particular, Bacteroides vulgatus), a less diverse microbiome, higher levels of inflammation, and higher levels of ileum-associated HIV DNA. These findings are linked to the activation of the inflammasome-mediating eIF2 signaling pathway. Our study thus provides the first proof-of-concept evidence that a previously unappreciated factor, gut glycosylation, is a force that may impact the vicious cycle between HIV infection, microbial translocation, and chronic inflammation.

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%-50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

Fimepinostat, a novel dual inhibitor of HDAC and PI3K, effectively reverses HIV-1 latency ex vivo without T cell activation.

OBJECTIVES: To test the potential of fimepinostat (CUDC-907), a dual inhibitor of histone deacetylases (HDAC) and phosphatidylinositol-3-kinases (PI3K), to reverse human immunodeficiency virus type 1 (HIV-1) latency in infected cell lines and in CD4+ T cells from HIV-1-infected donors on long-term combination antiretroviral therapy (cART). METHODS: Latently HIV-1-infected J-lat Tat-GFP and ACH-2 cell lines were stimulated with clinically relevant concentrations of fimepinostat using the HDAC inhibitors (HDACi) panobinostat and romidepsin for comparison. Next, CD4+ T cells from donors living with HIV-1 on long-term cART were stimulated ex vivo and cell-associated unspliced HIV-1 RNA was measured to quantify changes in HIV-1 transcription. Finally, the impact of fimepinostat on T cell activation (CD69 expression) and proliferation (Ki67 expression) was determined using peripheral blood mononuclear cells from uninfected donors. RESULTS: We found fimepinostat to be a potent latency-reversing agent. This was true in two latently infected cell lines as well as ex vivo in CD4+ T cells isolated from donors living with HIV-1. Relative to therapeutic dosing levels, fimepinostat showed latency-reversing potential comparable to romidepsin, which is the most potent HDACi tested in HIV-1 cure-related trials. Interestingly, in contrast to romidepsin, fimepinostat stimulation resulted in decreased T cell activation and had no negative impact on T cell proliferation. CONCLUSIONS: At therapeutic concentration, the dual HDAC and PI3K inhibitor fimepinostat was a potent HIV-1 latency-reversing agent and it did not induce T cell activation and proliferation. The potential of fimepinostat as a latency-reversing agent warrants further investigation.

Impacts of HIV Cure Interventions on Viral Reservoirs in Tissues.

HIV reservoirs persist in infected individuals despite combination antiretroviral therapy and can be identified in secondary lymphoid tissues, in intestinal tissues, in the central nervous system as well as in blood. Clinical trials have begun to explore effects of small molecule interventions to perturb the latent viral infection, but only limited information is available regarding the impacts of HIV cure-related clinical interventions on viral reservoirs found in tissues. Of the 14 HIV cure-related clinical trials since 2012 that have evaluated the effects of small molecule interventions in vivo, four trials have examined the impacts of the interventions in peripheral blood as well as other tissues that harbor persistent HIV. The additional tissues examined include cerebral spinal fluid, intestines and lymph nodes. We provide a comparison contrast analyses of the data across anatomical compartments tested in these studies to reveal where peripheral blood analyses reflect outcomes in other tissues as well as where the data reveal differences between tissue outcomes. We also summarize the current knowledge on these topics and highlight key open questions that need to be addressed experimentally to move the HIV cure research field closer to the development of an intervention strategy capable of eliciting long-term antiretroviral free remission of HIV disease.

TLR9 agonist MGN1703 enhances B cell differentiation and function in lymph nodes.

BACKGROUND: TLR9 agonists are being developed as immunotherapy against malignancies and infections. TLR9 is primarily expressed in B cells and plasmacytoid dendritic cells (pDCs). TLR9 signalling may be critically important for B cell activity in lymph nodes but little is known about the in vivo impact of TLR9 agonism on human lymph node B cells. As a pre-defined sub-study within our clinical trial investigating TLR9 agonist MGN1703 (lefitolimod) treatment in the context of developing HIV cure strategies (NCT02443935), we assessed TLR9 agonist-mediated effects in lymph nodes. METHODS: Participants received MGN1703 for 24 weeks concurrent with antiretroviral therapy. Seven participants completed the sub-study including lymph node resection at baseline and after 24 weeks of treatment. A variety of tissue-based immunologic and virologic parameters were assessed. FINDINGS: MGN1703 dosing increased B cell differentiation; activated pDCs, NK cells, and T cells; and induced a robust interferon response in lymph nodes. Expression of Activation-Induced cytidine Deaminase, an essential regulator of B cell diversification and somatic hypermutation, was highly elevated. During MGN1703 treatment IgG production increased and antibody glycosylation patterns were changed. INTERPRETATION: Our data present novel evidence that the TLR9 agonist MGN1703 modulates human lymph node B cells in vivo. These findings warrant further considerations in the development of TLR9 agonists as immunotherapy against cancers and infectious diseases. FUND: This work was supported by Aarhus University Research Foundation, the Danish Council for Independent Research and the NovoNordisk Foundation. Mologen AG provided study drug free of charge.

Effects of 24-week Toll-like receptor 9 agonist treatment in HIV type 1+ individuals.

DESIGN: This was an exploratory, single-arm clinical trial that tested the immune enhancement effects of 24-weeks of Toll-like receptor 9 (TLR9) agonist (MGN1703; Lefitolimod; 60 mg × 2 weekly) therapy. METHODS: We enrolled HIV-1-infected individuals on suppressive combination antiretroviral therapy. Safety was assessed throughout the study. The primary outcome was reduction in total CD4 T-cell viral DNA levels. Secondary outcomes included safety, detailed immunological and virological analyses, and time to viral rebound (viral load > 5000 copies/ml) after randomization into an analytical treatment interruption (ATI). RESULTS: A total of 12 individuals completed the treatment phase and nine completed the ATI. Adverse events were limited and consistent with previous reports for MGN1703. Although the dosing regimen led to potent T-cell activation and increased HIV-1-specific T-cell responses, there were no cohort-wide changes in persistent virus (total CD4 T cells viral DNA; P = 0.34). No difference in time to rebound was observed between the ATI arms (log rank P = 0.25). One of nine ATI participants, despite harboring a large replication-competent reservoir, controlled viremia for 150 days via both HIV-1-specific cellular and antibody-mediated immune responses. CONCLUSION: A period of 24 weeks of MGN1703 treatment was safe and improved innate as well as HIV-1-specific adaptive immunity in HIV-1+ individuals. These findings support the incorporation of TLR9 agonism into combination HIV-1 cure strategies. TRIAL NAME AND REGISTRATION: TLR9 Enhancement of antiviral immunity in chronic HIV-1 infection: a phase 1B/2A trial; ClinicalTrials.gov NCT02443935.

cAIMP administration in humanized mice induces a chimerization-level-dependent STING response.

It is well understood that the STING signalling pathway is critical for generating a robust innate immune response to pathogens. Human and mouse STING signalling pathways are not identical, however. For example, mice lack IFI16, which has been proven important for the human STING pathway. Therefore, we investigated whether humanized mice are an appropriate experimental platform for exploring the human STING signalling cascade in vivo. We found that NOG mice reconstituted with human cord blood haematopoietic stem cells (humanized NOG mice) exhibit human STING signalling responses to an analogue of the cyclic di-nucleotide cGAMP. There was an increase in the proportions of monocytes in the lungs of mice receiving cGAMP analogue. The most robust levels of STING expression and STING-induced responses were observed in mice exhibiting the highest levels of human chimerization. Notably, differential levels of STING in lung versus spleen following cGAMP analogue treatment suggest that there are tissue-specific kinetics of STING activation and/or degradation in effector versus inductive sites. We also examined the mouse innate immune response to cGAMP analogue treatment. We detected that mouse cells in the immunodeficient NOG mice responded to the cGAMP analogue and they do so with distinct kinetics from the human response. In conclusion, humanized NOG mice represent a valuable experimental model for examining in vivo human STING responses.