ABSTRACT
Upon infection, HIV disseminates throughout the human body within 1-2 weeks. However, its early cellular targets remain poorly characterized. We used a single-cell approach to retrieve the phenotype and TCR sequence of infected cells in blood and lymphoid tissue from individuals at the earliest stages of HIV infection. HIV initially targeted a few proliferating memory CD4+ T cells displaying high surface expression of CCR5. The phenotype of productively infected cells differed by Fiebig stage and between blood and lymph nodes. The TCR repertoire of productively infected cells was heavily biased, with preferential infection of previously expanded and disseminated clones, but composed almost exclusively of unique clonotypes, indicating that they were the product of independent infection events. Latent genetically intact proviruses were already archived early in infection. Hence, productive infection is initially established in a pool of phenotypically and clonotypically distinct T cells, and latently infected cells are generated simultaneously.
Subject(s)
HIV Infections , HIV-1 , Latent Infection , Humans , CD4-Positive T-Lymphocytes/metabolism , HIV-1/genetics , Latent Infection/metabolism , Latent Infection/pathology , Receptors, Antigen, T-Cell/metabolism , Virus LatencyABSTRACT
Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulate the immune response against many RNA viruses. We previously identified that a small molecule compound, KIN1148, led to the activation of IFN regulatory factor 3 (IRF3) and served to enhance protection against influenza A virus (IAV) A/California/04/2009 infection. We have now determined direct binding of KIN1148 to RIG-I to drive expression of IFN regulatory factor 3 and NF-κB target genes, including specific immunomodulatory cytokines and chemokines. Intriguingly, KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I IFN treatment. When administered in combination with a vaccine against IAV, KIN1148 induces both neutralizing Ab and IAV-specific T cell responses compared with vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal A/California/04/2009 and H5N1 IAV challenge. Importantly, KIN1148 also augments human CD8+ T cell activation. Thus, we have identified a small molecule RIG-I agonist that serves as an effective adjuvant in inducing noncanonical RIG-I activation for induction of innate immune programs that enhance adaptive immune protection of antiviral vaccination.
Subject(s)
Influenza A Virus, H5N1 Subtype , Influenza A virus , Influenza Vaccines , Influenza, Human , Humans , Animals , Mice , DEAD Box Protein 58/metabolism , Influenza A Virus, H5N1 Subtype/metabolism , Interferon Regulatory Factor-3/metabolism , Adjuvants, Immunologic , Antiviral Agents/pharmacology , Immunity, InnateABSTRACT
Activation of the NF-κB signaling pathway by Protein Kinase C (PKC) agonists is a potent mechanism for human immunodeficiency virus (HIV) latency disruption in vitro. However, significant toxicity risks and the lack of evidence supporting their activity in vivo have limited further evaluation of PKC agonists as HIV latency-reversing agents (LRA) in cure strategies. Here we evaluated whether GSK445A, a stabilized ingenol-B derivative, can induce HIV/simian immunodeficiency virus (SIV) transcription and virus production in vitro and demonstrate pharmacological activity in nonhuman primates (NHP). CD4+ T cells from people living with HIV and from SIV+ rhesus macaques (RM) on antiretroviral therapy (ART) exposed in vitro to 25 nM of GSK445A produced cell-associated viral transcripts as well as viral particles at levels similar to those induced by PMA/Ionomycin, indicating that GSK445A can potently reverse HIV/SIV latency. Importantly, these concentrations of GSK445A did not impair the proliferation or survival of HIV-specific CD8+ T cells, but instead, increased their numbers and enhanced IFN-γ production in response to HIV peptides. In vivo, GSK445A tolerability was established in SIV-naïve RM at 15 µg/kg although tolerability was reduced in SIV-infected RM on ART. Increases in plasma viremia following GSK445A administration were suggestive of increased SIV transcription in vivo. Collectively, these results indicate that GSK445A is a potent HIV/SIV LRA in vitro and has a tolerable safety profile amenable for further evaluation in vivo in NHP models of HIV cure/remission.
Subject(s)
Diterpenes/pharmacology , HIV , Protein Kinase C/drug effects , Simian Immunodeficiency Virus , Virus Activation/drug effects , Virus Latency/drug effects , Animals , Humans , Macaca mulatta , Protein Kinase C/metabolism , RNA, Viral/drug effects , Transcription, GeneticABSTRACT
INTRODUCTION: Immune dysregulation persists in people with HIV (PWH) on antiretroviral therapy (ART) and may lead to accelerated vascular ageing and cardiovascular disease (CVD). While delayed time to initiation of ART has been linked to worse cardiovascular outcomes, the effect of ART initiation during acute infection on these outcomes is not well understood. METHODS: Participants were enrolled from the SEARCH010/RV254 acute HIV (AHI) and HIV-NAT chronic HIV (CHI) cohorts in Thailand. Participants with 6-year follow-up and viral suppression (viral load < 50 copies/µL) at follow-up were included. Both unmatched cohorts and age and gender-matched cohorts were analysed. Demographics, HIV laboratories, and cardiovascular risk factors from enrolment and 6-year follow-up were obtained from electronic records. Framingham Risk Score (FRS), vascular age (VA), vascular age deviation (VAD), and 10-year atherosclerotic cardiovascular disease (ASCVD) risk were calculated from previously published equations. Vascular outcomes in AHI and CHI cohorts were compared, and univariable and multivariable linear regression analyses were used to investigate risk factors associated with worse vascular scores. RESULTS: In all, 373 AHI participants and 608 CHI participants were identified. AHI participants were of younger age, had a higher prevalence of syphilis and a lower prevalence of prior hepatitis B, tuberculosis, diabetes, and hypertension. Higher CD4 T-cell and lower CD8 T-cell counts were seen in the AHI cohort at enrolment and 6-year follow-up. In all participants, the AHI cohort had a lower median FRS (p < 0.001) and VA (p < 0.001), but higher VAD (p < 0.001). However, in matched cohorts, no differences were found in FRS-based outcomes. In all participants, higher VAD after 6 years of ART was associated with higher body mass index (p < 0.001) and higher CD4 count (p < 0.001), which persisted in multivariable analysis. When FRS components were analysed individually, CD4 count was associated only with male sex and cholesterol. CONCLUSIONS: We did not identify differences in FRS-based vascular outcomes at 6 years in matched cohorts of participants who started ART during AHI versus CHI. We identified a correlation between higher CD4 count and worse FRS-based vascular outcomes, which may be driven by underlying metabolic risk factors. Further study is needed to confirm these findings and evaluate underlying mechanisms.
Subject(s)
HIV Infections , Humans , Male , HIV Infections/drug therapy , HIV Infections/complications , Female , Adult , Middle Aged , Thailand/epidemiology , Risk Factors , Cardiovascular Diseases/epidemiology , Viral Load , CD4 Lymphocyte Count , Risk Assessment , Cohort Studies , Anti-Retroviral Agents/therapeutic use , Anti-HIV Agents/therapeutic useABSTRACT
OBJECTIVE: We examined individual differences in CD4/CD8 T-cell ratio trajectories and associated risk profiles from acute HIV infection (AHI) through 144 weeks of antiretroviral therapy (ART) using a data-driven approach. METHODS: A total of 483 AHI participants began ART during Fiebig I-V and completed follow-up evaluations for 144 weeks. CD4+, CD8+, and CD4/CD8 T-cell ratio trajectories were defined followed by analyses to identify associated risk variables. RESULTS: Participants had a median viral load (VL) of 5.88 copies/ml and CD4/CD8 T-cell ratio of 0.71 at enrollment. After 144 weeks of ART, the median CD4/CD8 T-cell ratio was 1.3. Longitudinal models revealed five CD4/CD8 T-cell ratio subgroups: group 1 (3%) exhibited a ratio >1.0 at all visits; groups 2 (18%) and 3 (29%) exhibited inversion at enrollment, with normalization 4 and 12 weeks after ART, respectively; and groups 4 (31%) and 5 (18%) experienced CD4/CD8 T-cell ratio inversion due to slow CD4+ T-cell recovery (group 4) or high CD8+ T-cell count (group 5). Persistent inversion corresponded to ART onset after Fiebig II, higher VL, soluble CD27 and TIM-3, and lower eosinophil count. Individuals with slow CD4+ T-cell recovery exhibited higher VL, lower white blood cell count, lower basophil percent, and treatment with standard ART, as well as worse mental health and cognition, compared with individuals with high CD8+ T-cell count. CONCLUSIONS: Early HIV disease dynamics predict unfavorable CD4/CD8 T-cell ratio outcomes after ART. CD4+ and CD8+ T-cell trajectories contribute to inversion risk and correspond to specific viral, immune, and psychological profiles during AHI. Adjunctive strategies to achieve immune normalization merit consideration.
Subject(s)
Anti-HIV Agents , HIV Infections , Anti-HIV Agents/therapeutic use , CD4 Lymphocyte Count , CD8-Positive T-Lymphocytes , HIV Infections/drug therapy , Hepatitis A Virus Cellular Receptor 2/therapeutic use , Humans , Individuality , Viral LoadABSTRACT
The success of the shock and kill strategy for the HIV cure depends both on the reactivation of the latent reservoir and on the ability of the immune system to eliminate infected cells. As latency reversal alone has not shown any impact in the size of the latent reservoir, ensuring that effector CTLs are able to recognize and kill HIV-infected cells could contribute to reservoir reduction. In this study, we investigated which functional aspects of human CTLs are associated with a better capacity to kill HIV-infected CD4+ T cells. We isolated Gag- and Nef-specific CTL clones with different TCR sequences from the PBMC of donors in acute and chronic infection. High-affinity clonotypes that showed IFN-γ production preserved even when the CD8 coreceptor was blocked, and clones with high Ag sensitivity exhibited higher efficiency at reducing the latent reservoir. Although intrinsic cytotoxic capacity did not differ according to TCR affinity, clonotypes with high TCR affinity showed a better ability to kill HIV-infected CD4+ T cells obtained from in vivo-infected PBMC and subjected to viral reactivation. Strategies aiming to specifically boost and maintain long-living memory CTLs with high TCR affinity in vivo prior to latency-reversing treatment might improve the efficacy of the shock and kill approach to reduce the latent reservoir.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , HIV Infections/immunology , HIV-1/physiology , Receptors, Antigen, T-Cell/immunology , Virus Latency/immunology , CD4-Positive T-Lymphocytes/virology , Humans , Interferon-gamma/immunologyABSTRACT
BACKGROUND: Early antiretroviral therapy (ART) restricts the size of the human immunodeficiency virus (HIV) reservoir in infants. However, whether antiretroviral (ARV) prophylaxis given to exposed vertically infected children exerts similar effects remains unknown. METHODS: We measured total and integrated HIV DNA, as well as the frequency of CD4 T cells producing multiply spliced RNA (msRNA) after stimulation (inducible reservoir) in vertically infected Thai infants. Eighty-five infants were followed longitudinally for up to 3 years. We compared the size of the reservoir in children who received continuous ARV prophylaxis since birth vs those who never received or discontinued prophylaxis before initiating ART. We used samples from a cross-sectional cohort of 37 Thai children who had initiated ART within 6 months of life to validate our findings. RESULTS: Before ART, levels of HIV DNA and the frequencies of cells producing msRNA were significantly lower in infants who received continuous ARV prophylaxis since birth compared to those in whom ARV prophylaxis was discontinued or never initiated (P < .020 and P < .001, respectively). Upon ART initiation, total and integrated HIV DNA levels decayed significantly in both groups (P < .01 in all cases). Interestingly, the initial differences in the frequencies of infected cells persisted during 3 years on ART. The beneficial effect of prophylaxis on the size of the HIV reservoir was confirmed in the cross-sectional study. Importantly, no differences were observed between children who discontinued prophylactic ARVs before starting ART and those who delayed ART initiation without receiving prior prophylaxis. CONCLUSIONS: Neonatal ARV prophylaxis with direct transition to ART durably limits the size of the HIV reservoir.
Subject(s)
Anti-Retroviral Agents , HIV Infections , Anti-Retroviral Agents/therapeutic use , CD4-Positive T-Lymphocytes , Child , Cross-Sectional Studies , HIV , HIV Infections/drug therapy , HIV Infections/prevention & control , Humans , Infant , Infant, NewbornABSTRACT
The low frequency of latently HIV-infected cells in vivo limits the testing of potential HIV cure strategies using cells from successfully suppressed individuals. To date, primary cell models of latency use cells infected in vitro Primary CD4+ T cell models carrying an individual's endogenous HIV reservoir that recapitulate in vivo conditions of HIV latency are still outstanding. We developed a primary CD4+ T cell model of HIV latency derived from memory CD4+ T cells isolated from virally suppressed HIV-infected individuals that recapitulates HIV-1 latency and viral reactivation events. This model is based on the expansion of primary CD4+ T cells up to 300-fold in cell number. These cells reestablish a resting state without active virus production after extended culture and maintain a stable number of total HIV proviruses. The ability of these cells to respond to various classes of latency-reversing agents is similar to that of ex vivo CD4+ T cells directly isolated from blood. Importantly, viral outgrowth assays confirmed the ability of these expanded cells to produce replication-competent endogenous virus. In sum, this model recapitulates ex vivo viral reactivation conditions, captures the variability between individuals with different HIV reservoirs, and provides large numbers of cells for testing multiple agents from a single donor. The use of this novel model will allow accurate exploration of novel cure approaches aimed either at promoting viral reactivation or maintaining sustained latency.IMPORTANCE Primary cell models of HIV latency have been very useful to identify mechanisms contributing to HIV latency and to evaluate potential HIV cure strategies. However, the current models utilize in vitro infection with exogenous virus that does not fully recapitulate virus reactivation profiles of endogenous HIV in in vivo-infected CD4+ T cells. In contrast, obtaining sufficient amounts of CD4+ T cells from HIV-infected individuals to interrogate the HIV reservoir in vitro requires leukapheresis. In the model we propose here, in vitro expansion and extended culture of primary CD4+ T cells isolated from virally suppressed HIV-infected individuals enable obtaining large numbers of cells harboring endogenous latent HIV reservoirs without performing leukapheresis. This model captures the variability of HIV reservoirs seeded in different individuals and should be useful to evaluate future HIV cure strategies.
Subject(s)
Virus Latency/physiology , Virus Replication/physiology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , HIV Infections/metabolism , HIV Infections/virology , HIV Seropositivity , HIV-1/metabolism , HIV-1/physiology , Humans , Models, Biological , Primary Cell Culture , Proviruses , Virus ActivationABSTRACT
The recent Zika virus (ZIKV) epidemic has created an urgent need for a safe and effective vaccine. There is still a dearth of knowledge about ZIKV immunity, but years of investigation into the immunobiology of other flaviviruses has helped to accelerate the development of a ZIKV vaccine. Although the humoral immune response generates the primary correlate of protection from disease, robust T cell responses could enhance ZIKV vaccine efficacy. Additionally, pre-existing immunity to related flaviviruses could generate cross-reactive T cells that may affect immune responses upon vaccination. In this review, we summarize the key discoveries in the area of flavivirus T cell immunity and postulate on how these findings can inform ZIKV vaccine strategies for inducing protective immunity.
Subject(s)
T-Lymphocytes/immunology , Viral Vaccines/immunology , Zika Virus Infection/prevention & control , Zika Virus/immunology , Animals , Dengue Virus/immunology , Humans , Immunity, Heterologous/immunology , Zika Virus Infection/immunology , Zika Virus Infection/virologyABSTRACT
PURPOSE OF REVIEW: The purpose of this review is to summarize the current knowledge on the role of CD4+ T lymphocytes leading to HIV assault and persistence in the central nervous system (CNS) and the elimination of HIV-infected CNS resident cells by CD8+ T lymphocytes. RECENT FINDINGS: HIV targets the CNS early in infection, and HIV-infected individuals suffer from mild forms of neurological impairments even under antiretroviral therapy (ART). CD4+ T cells and monocytes mediate HIV entry into the brain and constitute a source for HIV persistence and neuronal damage. HIV-specific CD8+ T cells are also massively recruited in the CNS in acute infection to control viral replication but cannot eliminate HIV-infected cells within the CNS. This review summarizes the involvement of CD4+ T cells in seeding and maintaining HIV infection in the brain and describes the involvement of CD8+ T cells in HIV neuropathogenesis, playing a role still to be deciphered, either beneficial in eliminating HIV-infected cells or deleterious in releasing inflammatory cytokines.
Subject(s)
CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/immunology , Central Nervous System/pathology , HIV Infections/pathology , HIV-1/immunology , CD4-Positive T-Lymphocytes/immunology , Central Nervous System/virology , Cytokines/immunology , HIV Infections/immunology , Humans , MonocytesABSTRACT
The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory.
Subject(s)
B-Lymphocytes/immunology , HIV Infections/immunology , Immunologic Memory/immunology , T-Lymphocytes, Helper-Inducer/immunology , Adult , Anti-HIV Agents/therapeutic use , Coculture Techniques , Cohort Studies , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , HIV-1/immunology , Humans , Lymphocyte Activation/immunology , Male , Viral LoadABSTRACT
Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.
Subject(s)
B-Lymphocytes/immunology , HIV Infections/immunology , HIV , Interleukin-2/immunology , T-Lymphocytes, Helper-Inducer/immunology , Adult , Antibodies, Viral/immunology , Antibody Formation , B-Lymphocytes/virology , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , Chronic Disease , Humans , Immunologic Memory , Middle Aged , Signal Transduction , T-Lymphocytes, Helper-Inducer/virology , Young AdultABSTRACT
UNLABELLED: Replication of the integrated HIV-1 genome is tightly regulated by a series of cellular factors. In previous work we showed that transactivation of the HIV-1 promoter is regulated by the cellular splicing factor SRSF1. Here we report that SRSF1 can downregulate the replication of B, C, and D subtype viruses by >200-fold in a cell culture system. We show that viral transcription and splicing are inhibited by SRSF1 expression. Furthermore, SRSF1 deletion mutants containing the protein RNA-binding domains but not the arginine serine-rich activator domain can downregulate viral replication by >2,000-fold with minimal impact on cell viability and apoptosis. These data suggest a therapeutic potential for SRSF1 and its RNA-binding domains. IMPORTANCE: Most drugs utilized to treat the HIV-1 infection are based on compounds that directly target proteins encoded by the virus. However, given the high viral mutation rate, the appearance of novel drug-resistant viral strains is common. Thus, there is a need for novel therapeutics with diverse mechanisms of action. In this study, we show that the cellular protein SRSF1 is a strong inhibitor of viral replication. Furthermore, expression of the SRSF1 RNA-binding domains alone can inhibit viral replication by >2,000-fold in multiple viral strains without impacting cell viability. Given the strong antiviral properties of this protein, the RNA-binding domains, and the minimal effects observed on cell metabolism, further studies are warranted to assess the therapeutic potential of peptides derived from these sequences.
Subject(s)
HIV-1/immunology , HIV-1/physiology , Host-Pathogen Interactions , Nuclear Proteins/metabolism , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Virus Replication , Antiviral Agents/metabolism , Cell Line , DNA Mutational Analysis , Humans , RNA Splicing , Serine-Arginine Splicing Factors , Transcription, GeneticABSTRACT
UNLABELLED: The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. IMPORTANCE: The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Antibodies, Viral/biosynthesis , DEAD-box RNA Helicases/immunology , Influenza Vaccines/immunology , Oligoribonucleotides/administration & dosage , Orthomyxoviridae Infections/prevention & control , Vaccines, Virus-Like Particle/immunology , Adjuvants, Immunologic/genetics , Animals , DEAD Box Protein 58 , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/genetics , Dendritic Cells/immunology , Dendritic Cells/virology , Female , HEK293 Cells , Hemagglutinins, Viral/chemistry , Hemagglutinins, Viral/genetics , Hemagglutinins, Viral/immunology , Humans , Immunity, Cellular/drug effects , Immunity, Humoral/drug effects , Immunization , Influenza A Virus, H5N1 Subtype/drug effects , Influenza A Virus, H5N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/pathogenicity , Influenza Vaccines/administration & dosage , Influenza Vaccines/genetics , Mice , Mice, Inbred BALB C , Neuraminidase/chemistry , Neuraminidase/genetics , Neuraminidase/immunology , Oligoribonucleotides/genetics , Oligoribonucleotides/immunology , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/mortality , Orthomyxoviridae Infections/virology , Primary Cell Culture , Receptors, Immunologic , Survival Analysis , Th1-Th2 Balance/drug effects , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/geneticsABSTRACT
UNLABELLED: The cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5'-triphosphate (5'ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5'ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5'pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5'pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replication in vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo prolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5'pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses. IMPORTANCE: The development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral response in vitro and in vivo and was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5'pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.
Subject(s)
Chikungunya virus/immunology , DEAD-box RNA Helicases/immunology , Dengue Virus/immunology , Influenza A Virus, H1N1 Subtype/immunology , RNA, Viral/agonists , RNA, Viral/immunology , Virus Diseases/immunology , Animals , Cell Line , Chikungunya virus/chemistry , Chikungunya virus/genetics , DEAD Box Protein 58 , DEAD-box RNA Helicases/genetics , Dengue Virus/chemistry , Dengue Virus/genetics , Humans , Influenza A Virus, H1N1 Subtype/chemistry , Influenza A Virus, H1N1 Subtype/genetics , Mice , Mice, Inbred BALB C , Nucleic Acid Conformation , RNA, Viral/genetics , Receptors, Immunologic , Virus Diseases/genetics , Virus Diseases/virologyABSTRACT
Immediate-early host-virus interactions that occur during the first weeks after HIV infection have a major impact on disease progression. The mechanisms underlying the failure of HIV-specific CD8 T-cell response to persist and control viral replication early in infection are yet to be characterized. In this study, we performed a thorough phenotypic, gene expression and functional analysis to compare HIV-specific CD8 T cells in acutely and chronically infected subjects. We showed that HIV-specific CD8 T cells in primary infection can be distinguished by their metabolic state, rate of proliferation, and susceptibility to apoptosis. HIV-specific CD8 T cells in acute/early HIV infection secreted less IFN-γ but were more cytotoxic than their counterparts in chronic infection. Importantly, we showed that the levels of IL-7R expression and the capacity of HIV-specific CD8 T cells to secrete IL-2 on antigenic restimulation during primary infection were inversely correlated with the viral set-point. Altogether, these data suggest an altered metabolic state of HIV-specific CD8 T cells in primary infection resulting from hyperproliferation and stress induced signals, demonstrate the discordant function of HIV-specific CD8 T cells during early/acute infection, and highlight the importance of T-cell maintenance for viral control.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Gene Expression Regulation/immunology , HIV Infections/metabolism , Acute Disease , Antibody-Dependent Cell Cytotoxicity/genetics , Antibody-Dependent Cell Cytotoxicity/immunology , Apoptosis , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Proliferation , Chronic Disease , HIV/physiology , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/pathology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Interleukin-2/biosynthesis , Interleukin-2/immunology , Receptors, Interleukin-7/genetics , Receptors, Interleukin-7/immunology , Time Factors , Viral LoadABSTRACT
Persistent exposure to cognate Ag leads to the functional impairment and exhaustion of HIV-specific CD8 T cells. Ag withdrawal, attributable either to antiretroviral treatment or the emergence of epitope escape mutations, causes HIV-specific CD8 T cell responses to wane over time. However, this process does not continue to extinction, and residual CD8 T cells likely play an important role in the control of HIV replication. In this study, we conducted a longitudinal analysis of clonality, phenotype, and function to define the characteristics of HIV-specific CD8 T cell populations that persist under conditions of limited antigenic stimulation. Ag decay was associated with dynamic changes in the TCR repertoire, increased expression of CD45RA and CD127, decreased expression of programmed death-1, and the emergence of polyfunctional HIV-specific CD8 T cells. High-definition analysis of individual clonotypes revealed that the Ag loss-induced gain of function within HIV-specific CD8 T cell populations could be attributed to two nonexclusive mechanisms: 1) functional improvement of persisting clonotypes; and 2) recruitment of particular clonotypes endowed with superior functional capabilities.
Subject(s)
Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/virology , HIV/immunology , CD8-Positive T-Lymphocytes/immunology , Clone Cells , Gene Expression Regulation/immunology , Humans , Interleukin-7 Receptor alpha Subunit , Leukocyte Common Antigens , Longitudinal Studies , Programmed Cell Death 1 ReceptorABSTRACT
Here, we present a protocol to evaluate the killing capacity and functional profile of human HIV-specific CD8 T cells. We describe steps for culturing peripheral blood mononuclear cells (PBMCs) from patients with HIV on antiretroviral therapy (ART) with HIV peptides ex vivo and quantifying HIV-specific CD8 T cell killing using flow cytometry. We then detail procedures for integrating the established killing assay with intracellular cytokine staining (ICS) and assessing CD8 T cell function. This protocol can provide insights into CD8 T cell-mediated immunity against HIV. For complete details on the use and execution of this protocol, please refer to Mbitikon-Kobo et al.,1 Noto et al.,2 and Gubser et al.3.