Short Communication: Expression of Host Restriction Factors by Memory CD4+ T Cells Differs Between Healthy Donors and HIV-1-Infected Individuals with Effective Antiretroviral Therapy.
ABSTRACT: Much has been learnt from the functions of host restriction factors during acute and chronic HIV-1 infection, but far less is known about their role in HIV-1-infected individuals in which viral load is stably suppressed with antiretroviral therapy (ART). In this study transcriptional expression of 42 host restriction factors was determined for memory CD4+ T cells sorted from 10 uninfected and 21 HIV-1-infected individuals, treated with suppressive ART and for which the viral reservoir was quantified. No significant associations were observed between restriction factor expression and HIV-1 reservoir size, quantified by measurement of HIV-1 Gag DNA using droplet digital polymerase chain reaction, and by measurement of replication-competent inducible virus using quantitative viral outgrowth assays. Expression of eight of the restriction factors differed significantly, and with a false discovery rate of <10%, between ART-suppressed and uninfected individuals. APOBEC3G, ISG15, LGALS3BP, RNASEL, and MX2 were upregulated in the ART-suppressed individuals, likely because of increased levels of immune activation observed in virally suppressed compared with uninfected individuals. In contrast CDKN1A, TRIM11, and BRD4 were expressed at lower levels in ART-suppressed than uninfected individuals. This suggests perturbation of the CD4+ memory T cell compartment, in which a viral reservoir persists in HIV-1-infected individuals with effective ART. Modulation of restriction factor expression, or overrepresentation of cell subsets that intrinsically express these restriction factors at lower levels could result in the distinct expression of restriction factors observed in treated infected individuals.
Project description:The eradication of HIV necessitates elimination of the HIV latent reservoir. Identifying host determinants governing latency and reservoir size in the setting of antiretroviral therapy (ART) is an important step in developing strategies to cure HIV infection. We sought to determine the impact of cell-intrinsic immunity on the HIV latent reservoir.We investigated the relevance of a comprehensive panel of established anti-HIV-1 host restriction factors to multiple established virologic and immunologic measures of viral persistence in HIV-1-infected, ART-suppressed individuals.We measured the mRNA expression of 42 anti-HIV-1 host restriction factors, levels of cell-associated HIV-1 RNA, levels of total pol and 2-long terminal repeat (2-LTR) circle HIV-1 DNA and immunophenotypes of CD4 T cells in 72 HIV-1-infected individuals on suppressive ART (23 individuals initiated ART less than 1 year post-infection, and 49 individuals initiated ART greater than 1 year post-infection). Correlations were analysed using nonparametric tests.The enhanced expression of a few select host restriction factors, p21, schlafen 11 and PAF1, was strongly associated with reduced CD4 T-cell associated HIV RNA during ART (P?<?0.001). In addition, our data suggested that ART perturbs the regulatory relationship between CD4 T-cell activation and restriction factor expression. Lastly, cell-intrinsic immune responses were significantly enhanced in individuals who initiated ART during early versus chronic infection and may contribute to the reduced reservoir size observed in these individuals.Intrinsic immune responses modulate HIV persistence during suppressive ART and may be manipulated to enhance the efficacy of ART and promote viral eradication through reversal of latency in vivo.
Project description:Several host-encoded antiviral factors suppress HIV-1 replication in a cell-autonomous fashion in vitro. The relevance of these defenses to the control of HIV-1 in vivo remains to be elucidated. We hypothesized that cellular restriction of HIV-1 replication plays a significant role in the observed suppression of HIV-1 in "elite controllers", individuals who maintain undetectable levels of viremia in the absence of antiretroviral therapy (ART). We comprehensively compared the expression levels of 34 host restriction factors and cellular activation levels in CD4+ T cells and sorted T cell subsets between elite controllers, HIV-1-infected (untreated) non-controllers, ART-suppressed, and uninfected individuals.Expression of schlafen 11, a codon usage-based inhibitor of HIV-1 protein synthesis, was significantly elevated in CD4+ T cells from elite controllers as compared to both non-controllers (p = 0.048) and ART-suppressed individuals (p = 0.024), with this effect most apparent in central memory CD4+ T cells. Schlafen 11 expression levels were comparable between controllers and uninfected individuals. Cumulative restriction factor expression was positively correlated with CD4+ T cell activation (r² = 0.597, p < 0.0001), viral load (r² = 0.34, p = 0.015), and expression of ISG15 (r² = 0.73, p < 0.0001), a marker of interferon exposure. APOBEC3C, APOBEC3D, CTR9, TRIM26, and TRIM32 were elevated in elite controllers with respect to ART-suppressed individuals, while levels were comparable to uninfected individuals and non-controllers.Host restriction factor expression typically scales with cellular activation levels. However, the elevated mRNA and protein expression of schlafen 11, despite low activation and viral load, violates the global pattern and may be a signature characteristic of HIV-1 elite control.
Project description:HIV persists in cellular and anatomical reservoirs during antiretroviral therapy (ART). Viral persistence is ensured by a variety of mechanisms including ongoing viral replication and proliferation of latently infected cells. In this review, we summarize recent findings establishing a link between the unresolved levels of inflammation observed in virally suppressed individuals on ART and the mechanisms responsible for HIV persistence.Residual levels of viral replication during ART are associated with persistent low levels of immune activation, suggesting that unresolved inflammation can promote the replenishment of the HIV reservoir in tissues. In addition, the recent findings that the latent HIV reservoir is maintained by continuous proliferation of latently infected cells provide another mechanism by which residual inflammation could contribute to HIV persistence.Residual inflammation during ART is likely to be a critical parameter contributing to HIV persistence. Therefore, reducing inflammation may be an efficient way to interfere with the maintenance of the HIV reservoir in virally suppressed individuals on ART.
Project description:Despite viral control, basal chronic inflammation and its related comorbidities remain unsolved problems among HIV-infected individuals. Soluble factors derived from myeloid cells have emerged as potent markers associated with HIV-related comorbidities and mortality. In the present report, we explored the relationship between soluble programmed death-ligand 1 (sPD-L1) and HIV-1 infection, antiretroviral therapy (ART), CD4/CD8 ratio, viral load (VL), and sexually transmitted coinfections.A prospective observational study on 49 HIV-1 infected adults.We found sPD-L1 levels were significantly higher in 49 HIV infected subjects than in 30 uninfected adults (1.05?ng/ml vs 0.52?ng/ml; P?<?.001). In this line, sPD-L1 levels were found to be elevated in 16 HIV infected subjects with undetectable VL compared with the uninfected subjects (0.75?ng/ml vs 0.52?ng/ml; P = .02). Thirteen ART-treated individuals with virological failure exhibited the highest sPDL1 levels, which were significantly higher than both 20 ART naïve infected individuals (1.68?ng/ml vs 0.87?ng/ml; P?=?.003) and the 16 ART-treated individuals with suppressed viremia (1.68?ng/ml vs 0.79?ng/ml; P?=?002). Entire cohort data showed a statistically significant positive correlation between VL and sPD-L1 levels in plasma (r?=?0.3; P?=?036).Our findings reveal sPDL-1 as a potential biomarker for HIV infection especially interesting in those individuals with virological failure.
Project description:A human immunodeficiency virus (HIV) infection cure requires an understanding of the cellular and anatomical sites harboring virus that contribute to viral rebound upon treatment interruption. Despite antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are reported in HIV-infected individuals on ART. Biomarkers for macrophage activation and neuronal damage in cerebrospinal fluid (CSF) of HIV-infected individuals demonstrate continued effects of HIV in brain and suggest that the central nervous system (CNS) may serve as a viral reservoir. Using a simian immunodeficiency virus (SIV)/macaque model for HIV encephalitis and AIDS, we evaluated whether infected cells persist in brain despite ART. Eight SIV-infected pig-tailed macaques were virally suppressed with ART, and plasma and CSF viremia levels were analyzed longitudinally. To assess whether virus persisted in brain macrophages (BrMΦ) in these macaques, we used a macrophage quantitative viral outgrowth assay (MΦ-QVOA), PCR, and in situ hybridization (ISH) to measure the frequency of infected cells and the levels of viral RNA and DNA in brain. Viral RNA in brain tissue of suppressed macaques was undetectable, although viral DNA was detected in all animals. The MΦ-QVOA demonstrated that the majority of suppressed animals contained latently infected BrMΦ. We also showed that virus produced in the MΦ-QVOAs was replication competent, suggesting that latently infected BrMΦ are capable of reestablishing productive infection upon treatment interruption. This report provides the first confirmation of the presence of replication-competent SIV in BrMΦ of ART-suppressed macaques and suggests that the highly debated issue of viral latency in macrophages, at least in brain, has been addressed in SIV-infected macaques treated with ART.IMPORTANCE Resting CD4+ T cells are currently the only cells that fit the definition of a latent reservoir. However, recent evidence suggests that HIV/SIV-infected macrophages persist despite ART. Markers of macrophage activation and neuronal damage are observed in the CSF of HIV-infected individuals and of SIV-infected macaques on suppressive ART regimens, suggesting that the CNS has continued virus infection and latent infection. A controversy exists as to whether brain macrophages represent a latent source of replication-competent virus capable of reestablishing infection upon treatment interruption. In this study, we demonstrated the presence of the latent macrophage reservoir in brains of SIV-infected ART-treated macaques and analyzed the reservoir using our established outgrowth assay to quantitate macrophages harboring replication-competent SIV genomes. Our results support the idea of the existence of other latent reservoirs in addition to resting CD4+ T cells and underscore the importance of macrophages in developing strategies to eradicate HIV.
Project description:Several highly potent and broadly neutralizing monoclonal antibodies against HIV have recently been isolated from B cells of infected individuals. However, the effects of these antibodies on the persistent viral reservoirs in HIV-infected individuals receiving antiretroviral therapy (ART) are unknown. We show that several HIV-specific monoclonal antibodies--in particular, PGT121, VRC01, and VRC03--potently inhibited entry into CD4(+) T cells of HIV isolated from the latent viral reservoir of infected individuals whose plasma viremia was well controlled by ART. In addition, we demonstrate that HIV replication in autologous CD4(+) T cells derived from infected individuals receiving ART was profoundly suppressed by three aforementioned and other HIV-specific monoclonal antibodies. These findings have implications for passive immunotherapy as an approach toward controlling plasma viral rebound in patients whose ART is withdrawn.
Project description:A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4+ T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4+ T cells, and individually SDF-1β, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1β, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4+ T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies.IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4+ T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells.
Project description:BACKGROUND:Future strategies aiming to achieve HIV-1 remission are likely to target individuals with small reservoir size. SETTING:We retrospectively investigated factors associated with HIV-1 DNA levels in European, perinatally HIV-infected children starting antiretroviral therapy (ART) <6 months of age. METHODS:Total HIV-1 DNA was measured from 51 long-term suppressed children aged 6.3 years (median) after initial viral suppression. Factors associated with log10 total HIV-1 DNA were analyzed using linear regression. RESULTS:At ART initiation, children were aged median [IQR] 2.3 [1.2-4.1] months, CD4% 37 [24-45] %, CD8% 28 [18-36] %, log10 plasma viral load (VL) 5.4 [4.4-5.9] copies per milliliter. Time to viral suppression was 7.98 [4.6-19.3] months. After suppression, 13 (25%) children had suboptimal response [?2 consecutive VL 50-400 followed by VL <50] and/or experienced periods of virological failure [?2 consecutive VL ?400 followed by VL <50]. Median total HIV-1 DNA was 43  copies/10 PBMC. Younger age at therapy initiation was associated with lower total HIV-1 DNA (adjusted coefficient [AC] 0.12 per month older, P = 0.0091), with a month increase in age at ART start being associated with a 13% increase in HIV DNA. Similarly, a higher proportion of time spent virally suppressed (AC 0.10 per 10% higher, P = 0.0022) and the absence of viral failure/suboptimal response (AC 0.34 for those with fail/suboptimal response, P = 0.0483) were associated with lower total HIV-1 DNA. CONCLUSIONS:Early ART initiation and a higher proportion of time suppressed are linked with lower total HIV-1 DNA. Early ART start and improving adherence in perinatally HIV-1-infected children minimize the size of viral reservoir.
Project description:The phenotypic characterization of the cells in which HIV persists during antiretroviral therapy (ART) remains technically challenging. We developed a simple flow cytometry-based assay to quantify and characterize infected cells producing HIV proteins during untreated and treated HIV infection. By combining two antibodies targeting the HIV capsid in a standard intracellular staining protocol, we demonstrate that p24-producing cells can be detected with high specificity and sensitivity in the blood from people living with HIV. In untreated individuals, the frequency of productively infected cells strongly correlated with plasma viral load. Infected cells preferentially displayed a transitional memory phenotype and were enriched in Th17, peripheral Tfh and regulatory T cells subsets. These cells also preferentially expressed activation markers (CD25, HLA-DR, Ki67), immune checkpoint molecules (PD-1, LAG-3, TIGIT, Tim-3) as well as the integrins ?4?7 and ?4?1. In virally suppressed individuals on ART, p24-producing cells were only detected upon stimulation (median frequency of 4.3 p24+ cells/106 cells). These measures correlated with other assays assessing the size of the persistent reservoir including total and integrated HIV DNA, Tat/rev Induced Limiting Dilution Assay (TILDA) and quantitative viral outgrowth assay (QVOA). In ART-suppressed individuals, p24-producing cells preferentially displayed a transitional and effector memory phenotype, and expressed immune checkpoint molecules (PD-1, TIGIT) as well as the integrin ?4?1. Remarkably, ?4?1 was expressed by more than 70% of infected cells both in untreated and ART-suppressed individuals. Altogether, these results highlight a broad diversity in the phenotypes of HIV-infected cells in treated and untreated infection and suggest that strategies targeting multiple and phenotypically distinct cellular reservoirs will be needed to exert a significant impact on the size of the reservoir.
Project description:Clonal expansions occur in the persistent HIV reservoir as shown by the duplication of proviral integration sites. However, the source of the proliferation of HIV-infected cells remains unclear. Here, we analyze the TCR repertoire of single HIV-infected cells harboring translation-competent proviruses in longitudinal samples from eight individuals on antiretroviral therapy (ART). When compared to uninfected cells, the TCR repertoire of reservoir cells is heavily biased: expanded clonotypes are present in all individuals, account for the majority of reservoir cells and are often maintained over time on ART. Infected T cell clones are detected at low frequencies in the long-lived central memory compartment and overrepresented in the most differentiated memory subsets. Our results indicate that clonal expansions highly contribute to the persistence of the HIV reservoir and suggest that reservoir cells displaying a differentiated phenotype are the progeny of infected central memory cells undergoing antigen-driven clonal expansion during ART.