IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease.

Interleukin (IL)-1R3 is the co-receptor in three signaling pathways that involve six cytokines of the IL-1 family (IL-1α, IL-1ß, IL-33, IL-36α, IL-36ß and IL-36γ). In many diseases, multiple cytokines contribute to disease pathogenesis. For example, in asthma, both IL-33 and IL-1 are of major importance, as are IL-36 and IL-1 in psoriasis. We developed a blocking monoclonal antibody (mAb) to human IL-1R3 (MAB-hR3) and demonstrate here that this antibody specifically inhibits signaling via IL-1, IL-33 and IL-36 in vitro. Also, in three distinct in vivo models of disease (crystal-induced peritonitis, allergic airway inflammation and psoriasis), we found that targeting IL-1R3 with a single mAb to mouse IL-1R3 (MAB-mR3) significantly attenuated heterogeneous cytokine-driven inflammation and disease severity. We conclude that in diseases driven by multiple cytokines, a single antagonistic agent such as a mAb to IL-1R3 is a therapeutic option with considerable translational benefit.

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection.

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model, we further demonstrate that pDCs, while not supporting SARS-CoV-2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL-6, IL-8, CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways, we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL-6 response, suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection.

Longitudinal evaluation of SARS-CoV-2 T cell immunity over 2 years following vaccination and infection.

BACKGROUND: Within a year of the SARS-CoV-2 pandemic, vaccines inducing a robust humoral and cellular immune response were implemented worldwide. However, emergence of novel variants and waning vaccine induced immunity led to implementation of additional vaccine boosters. METHODS: This prospective study evaluated the temporal profile of cellular and serological responses in a cohort of 639 SARS-CoV-2 vaccinated participants, of whom a large proportion experienced a SARS-CoV-2 infection. All participants were infection naïve at the time of their first vaccine dose. Proportions of SARS-CoV-2 Spike-specific T cells were determined after each vaccine dose using the Activation Induced Markers (AIM) assay, while levels of circulating SARS-CoV-2 antibodies were determined by the Meso Scale serology assay. RESULTS: We found a significant increase in SARS-CoV-2 Spike-specific CD4+ and CD8+ T cell responses following the third dose of a SARS-CoV-2 mRNA vaccine as well as enhanced CD8+ T cell responses after the fourth dose. Further, increased age was associated with a poorer response. Finally, we observed that SARS-CoV-2 infection boosts both the cellular and humoral immune response, relative to vaccine-induced immunity alone. CONCLUSION: Our findings highlight the boosting effect on T cell immunity of repeated vaccine administration. The combination of multiple vaccine doses and SARS-CoV-2 infections maintains population T cell immunity although with reduced levels in the elderly.

Revaccination of patients with systemic lupus erythematosus or rheumatoid arthritis without an initial COVID-19 vaccine response elicits seroconversion in half of the patients.

OBJECTIVES: To investigate the effect of COVID-19 mRNA revaccination (two doses) on the antibody response in patients with rheumatic diseases (RD) who were initial vaccine non-responders. Further, to examine if B-cell levels or T-cell responses before revaccination predicted seroconversion. METHODS: From a RD cohort vaccinated with the standard two-dose COVID-19 vaccinations, we enrolled cases without detectable antibody responses (n=17) and controls with detectable antibody response (n=29). Blood donors (n=32) were included as additional controls. Samples were collected before and six weeks after completed revaccination. Total antibodies and specific IgG, IgA, and IgM against SARS-CoV-2 spike protein, SARS-CoV-2 neutralising antibodies, and SARS-CoV-2 reacting CD4+ and CD8+ T-cells were measured before and after revaccination. B-cells (CD19+CD45+) were quantified before revaccination. RESULTS: Forty-seven percent of cases had detectable neutralising antibodies after revaccination. However, antibody levels were significantly lower than in controls and blood donors. Revaccination induced an antibody class switch in cases with a decrease in IgM and increase in IgG. No significant difference was observed in T-cell responses before and after revaccination between the three groups. Only 29% of cases had measurable B-cells compared to 100% of controls and blood donors. Fifty percent of revaccinated cases who seroconverted had measurable B-cells before revaccination. CONCLUSIONS: Forty-seven percent of initial non-responders seroconverted after two-dose revaccination but still had lower levels of SARS-CoV-2 antibodies compared with controls and blood donors. RD patients without a detectable serological response after the initial COVID-19 mRNA vaccine had a T-cell response similar to immunocompetent controls and blood donors.

Omicron Variant-Specific Serological Imprinting Following BA.1 or BA.4/5 Bivalent Vaccination and Previous SARS-CoV-2 Infection: A Cohort Study.

BACKGROUND: Continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outpaces monovalent vaccine cross-protection to new viral variants. Consequently, bivalent coronavirus disease 2019 (COVID-19) vaccines including Omicron antigens were developed. The contrasting immunogenicity of the bivalent vaccines and the impact of prior antigenic exposure on new immune imprinting remains to be clarified. METHODS: In the large prospective ENFORCE cohort, we quantified spike-specific antibodies to 5 Omicron variants (BA.1 to BA.5) before and after BA.1 or BA.4/5 bivalent booster vaccination to compare Omicron variant-specific antibody inductions. We evaluated the impact of previous infection and characterized the dominant antibody responses. RESULTS: Prior to the bivalent fourth vaccine, all participants (N = 1697) had high levels of Omicron-specific antibodies. Antibody levels were significantly higher in individuals with a previous polymerase chain reaction positive (PCR+) infection, particularly for BA.2-specific antibodies (geometric mean ratio [GMR] 6.79, 95% confidence interval [CI] 6.05-7.62). Antibody levels were further significantly boosted in all individuals by receiving either of the bivalent vaccines, but greater fold inductions to all Omicron variants were observed in individuals with no prior infection. The BA.1 bivalent vaccine generated a dominant response toward BA.1 (adjusted GMR 1.31, 95% CI 1.09-1.57) and BA.3 (1.32, 1.09-1.59) antigens in individuals with no prior infection, whereas the BA.4/5 bivalent vaccine generated a dominant response toward BA.2 (0.87, 0.76-0.98), BA.4 (0.85, 0.75-0.97), and BA.5 (0.87, 0.76-0.99) antigens in individuals with a prior infection. CONCLUSIONS: Vaccination and previous infection leave a clear serological imprint that is focused on the variant-specific antigen. Importantly, both bivalent vaccines induce high levels of Omicron variant-specific antibodies, suggesting broad cross-protection of Omicron variants.

Long-term humoral and cellular immunity after primary SARS-CoV-2 infection: a 20-month longitudinal study.

BACKGROUND: SARS-CoV-2 remains a world-wide health issue. SARS-CoV-2-specific immunity is induced upon both infection and vaccination. However, defining the long-term immune trajectory, especially after infection, is limited. In this study, we aimed to further the understanding of long-term SARS-CoV-2-specific immune response after infection. RESULTS: We conducted a longitudinal cohort study among 93 SARS-CoV-2 recovered individuals. Immune responses were continuously monitored for up to 20 months after infection. The humoral responses were quantified by Spike- and Nucleocapsid-specific IgG levels. T cell responses to Spike- and non-Spike epitopes were examined using both intercellular cytokine staining (ICS) assay and Activation-Induced marker (AIM) assay with quantification of antigen-specific IFNγ production. During the 20 months follow-up period, Nucleocapsid-specific antibody levels and non-Spike-specific CD4 + and CD8 + T cell frequencies decreased in the blood. However, a majority of participants maintained a durable immune responses 20 months after infection: 59% of the participants were seropositive for Nucleocapsid-specific IgG, and more than 70% had persisting non-Spike-specific T cells. The Spike-specific response initially decreased but as participants were vaccinated against COVID-19, Spike-specific IgG levels and T cell frequencies were boosted reaching similar or higher levels compared to 1 month post-infection. The trajectory of infection-induced SARS-CoV-2-specific immunity decreases, but for the majority of participants it persists beyond 20 months. The T cell response displays a greater durability. Vaccination boosts Spike-specific immune responses to similar or higher levels as seen after primary infection. CONCLUSIONS: For most participants, the response persists 20 months after infection, and the cellular response appears to be more long-lived compared to the circulating antibody levels. Vaccination boosts the S-specific response but does not affect the non-S-specific response. Together, these findings support the understanding of immune contraction, and with studies showing the immune levels required for protection, adds to the knowledge of durability of protection against future SARS-CoV-2.

Examination of autoantibodies to type I interferon in patients suffering from long COVID.

Long COVID (LC) is an emerging global health concern. The underlying mechanism and pathophysiology remain unclear. Presence of neutralizing autoantibodies against type 1 interferons (IFN) has been established as a predictor of critical COVID-19. We hypothesized that persistent autoimmune activity with autoantibodies against type 1 IFN may contribute to symptoms in patients with LC. Plasma samples and clinical information were obtained from a Danish LC cohort consisting of adult patients with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Information on symptoms and quality of life was derived from an LC-specific questionnaire and the EQ-5D-5L questionnaire. Detection of type 1 IFN autoantibodies in plasma were performed by ELISA. Samples collected between June, 2020, and September, 2021, from 279 patients were analyzed and compared to a control group of 94 individuals with prior mild SARS-CoV-2 infection who did not develop LC symptoms. In total, five LC patients (1.8%) and 3 (3.2%) of the controls had detectable circulating type 1 IFN autoantibodies. Collectively, prevalence of autoantibodies against type 1 IFN subtypes in our LC cohort were primarily driven by men and did not exceed the prevalence in controls. Thus, in our cohort, anti-type I IFN autoantibodies are unlikely to drive LC symptoms.

Severe Acute Respiratory Syndrome Coronavirus 2 Seroprevalence Survey Among 17 971 Healthcare and Administrative Personnel at Hospitals, Prehospital Services, and Specialist Practitioners in the Central Denmark Region.

BACKGROUND: The objective of this study was to perform a seroprevalence survey on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among Danish healthcare workers to identify high-risk groups. METHODS: All healthcare workers and administrative personnel at the 7 hospitals, prehospital services, and specialist practitioner clinics in the Central Denmark Region were invited to be tested by a commercial SARS-CoV-2 total antibody enzyme-linked immunosorbent assay (Wantai Biological Pharmacy Enterprise Co, Ltd, Beijing, China). RESULTS: A total of 25 950 participants were invited. Of these, 17 971 had samples available for SARS-CoV-2 antibody testing. After adjustment for assay sensitivity and specificity, the overall seroprevalence was 3.4% (95% confidence interval [CI], 2.5%-3.8%). The seroprevalence was higher in the western part of the region than in the eastern part (11.9% vs 1.2%; difference: 10.7 percentage points [95% CI, 9.5-12.2]). In the high-prevalence area, the emergency departments had the highest seroprevalence (29.7%), whereas departments without patients or with limited patient contact had the lowest seroprevalence (2.2%). Among the total 668 seropositive participants, 433 (64.8%) had previously been tested for SARS-CoV-2 RNA, and 50.0% had a positive reverse-transcription polymerase chain reaction (PCR) result. CONCLUSIONS: We found large differences in the prevalence of SARS-CoV-2 antibodies in staff working in the healthcare sector within a small geographical area of Denmark. Half of all seropositive staff had been tested positive by PCR prior to this survey. This study raises awareness of precautions that should be taken to avoid in-hospital transmission. Regular testing of healthcare workers for SARS-CoV-2 should be considered to identify areas with increased transmission.

Characterization of Intact Proviruses in Blood and Lymph Node from HIV-Infected Individuals Undergoing Analytical Treatment Interruption.

The role of lymphoid tissue as a potential source of HIV-1 rebound following interruption of antiretroviral therapy (ART) is uncertain. To address this issue, we compared the latent viruses obtained from CD4+ T cells in peripheral blood and lymph nodes to viruses emerging during treatment interruption. Latent viruses were characterized by sequencing near-full-length (NFL) proviral DNA and env from viral outgrowth assays (VOAs). Five HIV-1-infected individuals on ART were studied, four of whom participated in a clinical trial of a TLR9 agonist that included an analytical treatment interruption. We found that 98% of intact or replication-competent clonal sequences overlapped between blood and lymph node. In contrast, there was no overlap between 205 latent reservoir and 125 rebound sequences in the four individuals who underwent treatment interruption. However, rebound viruses could be accounted for by recombination. The data suggest that CD4+ T cells carrying latent viruses circulate between blood and lymphoid tissues in individuals on ART and support the idea that recombination may play a role in the emergence of rebound viremia.IMPORTANCE HIV-1 persists as a latent infection in CD4+ T cells that can be found in lymphoid tissues in infected individuals during ART. However, the importance of this tissue reservoir and its contribution to viral rebound upon ART interruption are not clear. In this study, we sought to compare latent HIV-1 from blood and lymph node CD4+ T cells from five HIV-1-infected individuals. Further, we analyzed the contribution of lymph node viruses to viral rebound. We observed that the frequencies of intact proviruses were the same in blood and lymph node. Moreover, expanded clones of T cells bearing identical proviruses were found in blood and lymph node. These latent reservoir sequences did not appear to be the direct origin of rebound virus. Instead, latent proviruses were found to contribute to the rebound compartment by recombination.

Multiple Homozygous Variants in the STING-Encoding TMEM173 Gene in HIV Long-Term Nonprogressors.

Among HIV-infected individuals, long-term nonprogressor (LTNP) patients experience slow CD4 T cell decline and almost undetectable viral load for several years after primary acquisition of HIV. Type I IFN has been suggested to play a pathogenic role in HIV pathogenesis, and therefore diminished IFN responses may underlie the LTNP phenotype. In this study, we examined the presence and possible immunological role of multiple homozygous single-nucleotide polymorphisms in the stimulator of IFN genes (STING) encoding gene TMEM173 involved in IFN induction and T cell proliferation in HIV LTNP patients. We identified LTNPs through the Danish HIV Cohort and performed genetic analysis by Sanger sequencing, covering the R71H-G230A-R293Q (HAQ) single-nucleotide polymorphisms in TMEM173 This was followed by investigation of STING mRNA and protein accumulation as well as innate immune responses and proliferation following STING stimulation and infection with replication-competent HIV in human blood-derived cells. We identified G230A-R293Q/G230A-R293Q and HAQ/HAQ homozygous TMEM173 variants in 2 out of 11 LTNP patients. None of the 11 noncontrollers on antiretroviral treatment were homozygous for these variants. We found decreased innate immune responses to DNA and HIV as well as reduced STING-dependent inhibition of CD4 T cell proliferation, particularly in the HAQ/HAQ HIV LTNP patients, compared with the age- and gender-matched noncontrollers on antiretroviral treatment. These findings suggest that homozygous HAQ STING variants contribute to reduced inhibition of CD4 T cell proliferation and a reduced immune response toward DNA and HIV, which might result in reduced levels of constitutive IFN production. Consequently, the HAQ/HAQ TMEM173 genotype may contribute to the slower disease progression characteristic of LTNPs.

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.

Estimating Initial Viral Levels during Simian Immunodeficiency Virus/Human Immunodeficiency Virus Reactivation from Latency.

Human immunodeficiency virus (HIV) viremia rebounds rapidly after treatment interruption, and a variety of strategies are being explored to reduce or control viral reactivation posttreatment. This viral rebound arises from reactivation of individual latently infected cells, which spread during ongoing rounds of productive infection. The level of virus produced by the initial individual reactivating cells is not known, although it may have major implications for the ability of different immune interventions to control viral rebound. Here we use data from both HIV and simian immunodeficiency virus (SIV) treatment interruption studies to estimate the initial viral load postinterruption and thereby the initial individual reactivation event. Using a barcoded virus (SIVmac239M) to track reactivation from individual latent cells, we use the observed viral growth rates and frequency of reactivation to model the dynamics of reactivation to estimate that a single reactivated latent cell can produce an average viral load equivalent to ∼0.1 to 0.5 viral RNA (vRNA) copies/ml. Modeling of treatment interruption in HIV suggests an initial viral load equivalent of ∼0.6 to 1 vRNA copies/ml. These low viral loads immediately following latent cell reactivation provide a window of opportunity for viral control by host immunity, before further replication allows viral spread. This work shows the initial levels of viral production that must be controlled in order to successfully suppress HIV reactivation following treatment interruption.IMPORTANCE Current treatment for HIV is able to suppress viral replication and prevent disease progression. However, treatment cannot eradicate infection, because the virus lies silent within latently infected cells. If treatment is stopped, the virus usually rebounds above the level of detection within a few weeks. There are a number of approaches being tested aimed at either eradicating latently infected cells or controlling the virus if it returns. Studying both the small pool of latently infected cells and the early events during viral reactivation is difficult, because these involve very small levels of virus that are difficult to measure directly. Here, we combine experimental data and mathematical modeling to understand the very early events during viral reactivation from latency in both HIV infection of humans and SIV infection of monkeys. We find that the initial levels of virus are low, which may help in designing therapies to control early viral reactivation.

Anti-HIV-1 ADCC Antibodies following Latency Reversal and Treatment Interruption.

There is growing interest in utilizing antibody-dependent cellular cytotoxicity (ADCC) to eliminate infected cells following reactivation from HIV-1 latency. A potential barrier is that HIV-1-specific ADCC antibodies decline in patients on long-term antiretroviral therapy (ART) and may not be sufficient to eliminate reactivated latently infected cells. It is not known whether reactivation from latency with latency-reversing agents (LRAs) could provide sufficient antigenic stimulus to boost HIV-1-specific ADCC. We found that treatment with the LRA panobinostat or a short analytical treatment interruption (ATI), 21 to 59 days, was not sufficient to stimulate an increase in ADCC-competent antibodies, despite viral rebound in all subjects who underwent the short ATI. In contrast, a longer ATI, 2 to 12 months, among subjects enrolled in the Strategies for Management of Antiretroviral Therapy (SMART) trial robustly boosted HIV-1 gp120-specific Fc receptor-binding antibodies and ADCC against HIV-1-infected cells in vitro These results show that there is a lag between viral recrudescence and the boosting of ADCC antibodies, which has implications for strategies toward eliminating latently infected cells.IMPORTANCE The "shock and kill" HIV-1 cure strategy aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. Several latency reversing agents (LRAs) have been examined in vivo, but LRAs alone have not been able to achieve HIV-1 remission and prevent viral rebound following analytical treatment interruption (ATI). In this study, we examined whether LRA treatment or ATI can provide sufficient antigenic stimulus to boost HIV-1-specific functional antibodies that can eliminate HIV-1-infected cells. Our study has implications for the antigenic stimulus required for antilatency strategies and/or therapeutic vaccines to boost functional antibodies and assist in eliminating the latent reservoir.

Correction: HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days-Implications for HIV Remission.

[This corrects the article DOI: 10.1371/journal.ppat.1005000.][This corrects the article DOI: 10.1371/journal.ppat.1005740.][This corrects the article DOI: 10.1371/journal.ppat.1005679.].

Short-Course Toll-Like Receptor 9 Agonist Treatment Impacts Innate Immunity and Plasma Viremia in Individuals With Human Immunodeficiency Virus Infection.

BACKGROUND.: 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. METHODS.: 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. RESULTS.: 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. CONCLUSIONS.: 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. CLINICAL TRIALS REGISTRATION.: NCT02443935.

A Novel Toll-Like Receptor 9 Agonist, MGN1703, Enhances HIV-1 Transcription and NK Cell-Mediated Inhibition of HIV-1-Infected Autologous CD4+ T Cells.

UNLABELLED: Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a "shock-and-kill" approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703 ex vivo exhibited increased secretion of interferon alpha (IFN-α) (P= 0.005) and CXCL10 (P= 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56(dim)CD16(+)NK cells (P= 0.001) as well as higher proportions of CD107a-positive (P= 0.002) and IFN-γ-producing (P= 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4(+)and CD8(+)T cells. Furthermore, CD4(+)T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (P= 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4(+)T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (P= 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4(+)T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials. IMPORTANCE: We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family are due to its novel "dumbbell-shape" structure made of covalently closed, natural DNA. In our study, we found that incubation of peripheral blood mononuclear cells with MGN1703 results in natural killer cell activation and increased natural killer cell function, which significantly inhibited the spread of HIV in a culture of autologous CD4(+)T cells. Furthermore, we discovered that MGN1703-mediated activation can enhance HIV-1 transcription in CD4(+)T cells, suggesting that this molecule may serve a dual purpose in HIV-1 eradication therapy: enhanced immune function and latency reversal. These findings provide a strong preclinical basis for the inclusion of MGN1703 in an HIV eradication clinical trial.

HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days--Implications for HIV Remission.

HIV infection can be effectively controlled by anti-retroviral therapy (ART) in most patients. However therapy must be continued for life, because interruption of ART leads to rapid recrudescence of infection from long-lived latently infected cells. A number of approaches are currently being developed to 'purge' the reservoir of latently infected cells in order to either eliminate infection completely, or significantly delay the time to viral recrudescence after therapy interruption. A fundamental question in HIV research is how frequently the virus reactivates from latency, and thus how much the reservoir might need to be reduced to produce a prolonged antiretroviral-free HIV remission. Here we provide the first direct estimates of the frequency of viral recrudescence after ART interruption, combining data from four independent cohorts of patients undergoing treatment interruption, comprising 100 patients in total. We estimate that viral replication is initiated on average once every ≈6 days (range 5.1- 7.6 days). This rate is around 24 times lower than previous thought, and is very similar across the cohorts. In addition, we analyse data on the ratios of different 'reactivation founder' viruses in a separate cohort of patients undergoing ART-interruption, and estimate the frequency of successful reactivation to be once every 3.6 days. This suggests that a reduction in the reservoir size of around 50-70-fold would be required to increase the average time-to-recrudescence to about one year, and thus achieve at least a short period of anti-retroviral free HIV remission. Our analyses suggests that time-to-recrudescence studies will need to be large in order to detect modest changes in the reservoir, and that macaque models of SIV latency may have much higher frequencies of viral recrudescence after ART interruption than seen in human HIV infection. Understanding the mean frequency of recrudescence from latency is an important first step in approaches to prolong antiretroviral-free viral remission in HIV.

The Depsipeptide Romidepsin Reverses HIV-1 Latency In Vivo.

UNLABELLED: Pharmacologically-induced activation of replication competent proviruses from latency in the presence of antiretroviral treatment (ART) has been proposed as a step towards curing HIV-1 infection. However, until now, approaches to reverse HIV-1 latency in humans have yielded mixed results. Here, we report a proof-of-concept phase Ib/IIa trial where 6 aviremic HIV-1 infected adults received intravenous 5 mg/m2 romidepsin (Celgene) once weekly for 3 weeks while maintaining ART. Lymphocyte histone H3 acetylation, a cellular measure of the pharmacodynamic response to romidepsin, increased rapidly (maximum fold range: 3.7­7.7 relative to baseline) within the first hours following each romidepsin administration. Concurrently, HIV-1 transcription quantified as copies of cell-associated un-spliced HIV-1 RNA increased significantly from baseline during treatment (range of fold-increase: 2.4­5.0; p = 0.03). Plasma HIV-1 RNA increased from <20 copies/mL at baseline to readily quantifiable levels at multiple post-infusion time-points in 5 of 6 patients (range 46­103 copies/mL following the second infusion, p = 0.04). Importantly, romidepsin did not decrease the number of HIV-specific T cells or inhibit T cell cytokine production. Adverse events (all grade 1­2) were consistent with the known side effects of romidepsin. In conclusion, romidepsin safely induced HIV-1 transcription resulting in plasma HIV-1 RNA that was readily detected with standard commercial assays demonstrating that significant reversal of HIV-1 latency in vivo is possible without blunting T cell-mediated immune responses. These finding have major implications for future trials aiming to eradicate the HIV-1 reservoir. TRIAL REGISTRATION: clinicaltrials.gov NTC02092116.

Macromolecular Prodrugs of Ribavirin: Structure-Function Correlation as Inhibitors of Influenza Infectivity.

The requirement for new antiviral therapeutics is an ever present need. Particularly lacking are broad spectrum antivirals that have low toxicity. We develop such agents based on macromolecular prodrugs whereby both the polymer chain and the drug released from the polymer upon cell entry have antiviral effects. Specifically, macromolecular prodrugs were designed herein based on poly(methacrylic acid) and ribavirin. Structure-function parameter space was analyzed via the synthesis of 10 polymer compositions varied by molar mass and drug content. Antiviral activity was tested in cell culture against both low and high pathogenic strains of influenza. Lead compounds were successfully used to counter infectivity of influenza in chicken embryos. The lead composition with the highest activity against influenza was also active against another respiratory pathogen, respiratory syncytial virus, providing opportunity to potentially treat infection by the two pathogens with one antiviral agent. In contrast, structure-function activity against the herpes simplex virus was drastically different, revealing limitations of the broad spectrum antiviral agents based on macromolecular prodrugs.

Innate Immune Activity Correlates with CD4 T Cell-Associated HIV-1 DNA Decline during Latency-Reversing Treatment with Panobinostat.

UNLABELLED: The pharmaceutical reactivation of dormant HIV-1 proviruses by histone deacetylase inhibitors (HDACi) represents a possible strategy to reduce the reservoir of HIV-1-infected cells in individuals treated with suppressive combination antiretroviral therapy (cART). However, the effects of such latency-reversing agents on the viral reservoir size are likely to be influenced by host immune responses. Here, we analyzed the immune factors associated with changes in proviral HIV-1 DNA levels during treatment with the potent HDACi panobinostat in a human clinical trial involving 15 cART-treated HIV-1-infected patients. We observed that the magnitude, breadth, and cytokine secretion profile of HIV-1-specific CD8 T cell responses were unrelated to changes in HIV-1 DNA levels in CD4 T cells during panobinostat treatment. In contrast, the proportions of CD3(-) CD56(+) total NK cells and CD16(+) CD56(dim) NK cells were inversely correlated with HIV-1 DNA levels throughout the study, and changes in HIV-1 DNA levels during panobinostat treatment were negatively associated with the corresponding changes in CD69(+) NK cells. Decreasing levels of HIV-1 DNA during latency-reversing treatment were also related to the proportions of plasmacytoid dendritic cells, to distinct expression patterns of interferon-stimulated genes, and to the expression of the IL28B CC genotype. Together, these data suggest that innate immune activity can critically modulate the effects of latency-reversing agents on the viral reservoir and may represent a target for future immunotherapeutic interventions in HIV-1 eradication studies. IMPORTANCE: Currently available antiretroviral drugs are highly effective in suppressing HIV-1 replication, but the virus persists, despite treatment, in a latent form that does not actively express HIV-1 gene products. One approach to eliminate these cells, colloquially termed the "shock-and-kill" strategy, focuses on the use of latency-reversing agents that induce active viral gene expression in latently infected cells, followed by immune-mediated killing. Panobinostat, a histone deacetylase inhibitor, demonstrated potent activities in reversing HIV-1 latency in a recent pilot clinical trial and reduced HIV-1 DNA levels in a subset of patients. Interestingly, we found that innate immune factors, such as natural killer cells, plasmacytoid dendritic cells, and the expression patterns of interferon-stimulated genes, were most closely linked to a decline in the HIV-1 DNA level during treatment with panobinostat. These data suggest that innate immune activity may play an important role in reducing the residual reservoir of HIV-1-infected cells.