Elucidating the mechanisms underlying the persistence and location of the HIV reservoir is critical for developing cure interventions. While it has been shown that levels of T-cell activation and the ...size of the HIV reservoir are greater in rectal tissue and lymph nodes (LN) than in blood, the relative contributions of T-cell subsets to this anatomic difference are unknown. We measured and compared HIV-1 DNA content, expression of the T-cell activation markers CD38 and HLA-DR, and expression of the exhaustion markers programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) in naive, central memory (CM), transitional memory (TM), and effector memory (EM) CD4
and CD8
T-cells in paired blood and LN samples among 14 people with HIV who were receiving antiretroviral therapy. HIV-1 DNA levels, T-cell immune activation, and TIGIT expression were higher in LN than in blood, especially in CM and TM CD4
T-cell subsets. Immune activation was significantly higher in all CD8
T-cell subsets, and memory CD8
T-cell subsets from LN had higher levels of PD-1 expression, compared with blood, while TIGIT expression levels were significantly lower in TM CD8
T-cells. The differences seen in CM and TM CD4
T-cell subsets were more pronounced among participants with CD4
T-cell counts of <500 cells/μL within 2 years after antiretroviral therapy initiation, thus highlighting increased residual dysregulation in LN as a distinguishing feature of and a potential mechanism for individuals with suboptimal CD4
T-cell recovery during antiretroviral therapy.
This study provides new insights into the contributions of different CD4
and CD8
T-cell subsets to the anatomic differences between LN and blood in individuals with HIV who have optimal versus suboptimal CD4
T-cell recovery. To our knowledge, this is the first study comparing paired LN and blood CD4
and CD8
T-cell differentiation subsets, as well as those subsets in immunological responders versus immunological suboptimal responders.
Before initiation of antiretroviral therapy (ART), HIV-specific CD8+ T cells are dysfunctional and short lived. To better understand the relationship between the HIV reservoir in CD4+ T cells and the ...magnitude and differentiation status of HIV-specific CD8+ T cells, we investigated these cells from acute and chronic HIV-infected individuals after 2 years of ART. Although both the HIV reservoir and the CD8+ T cell responses declined significantly after 2 years of ART, sustained HIV-specific CD8+ T cell responses correlated with a greater reduction of integrated HIV provirus. However, the magnitude of CD8+ T cells specific for HIV Gag, Pol, Nef, and Vif proteins positively associated with the active reservoir size during ART, measured as cell-associated RNA. Importantly, high HIV DNA levels strongly associate with maintenance of short-lived HIV-specific CD8+ T cells, regardless of ART initiation time. Our data suggest that the active reservoir maintains HIV-specific CD8+ T cell magnitude but prevents their differentiation into functional cells.
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•Magnitude of CD8+ T cell response associated with active HIV reservoir size on therapy•Sustained CD8+ T cell responses correlated with a greater HIV provirus decay•Residual HIV reservoir levels associated with maintenance of short-lived CD8+ T cells•Low HIV reservoir associated with generation of functional HIV-specific CD8+ T cells
Takata et al. found that the residual antigen expression by the active HIV reservoir during suppressive antiretroviral therapy correlated with increased HIV-specific CD8+ T cell magnitude but prevented differentiation into functional cells. Thus, targeting the residual HIV reservoir may improve HIV-specific CD8+ T cell functionality needed for an HIV remission.
Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained ...during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA
/p24
cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound.
Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.
We administered Ad26, modified vaccinia Ankara vectors containing mosaic HIV-1 antigens or placebo in 26 individuals who initiated antiretroviral therapy during acute human immunodeficiency virus ...infection as an exploratory study to determine the safety and duration of viremic control after treatment interruption. The vaccine was safe and generated robust immune responses, but delayed time to viral rebound compared to that in placebo recipients by only several days and did not lead to viremic control after treatment interruption (clinical trial NCT02919306).
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Interleukin 32 (IL-32) is a potent multi-isoform proinflammatory cytokine, which is upregulated in people with HIV (PWH) and is associated with cardiovascular disease (CVD) risk. However, the impact ...of IL-32 isoforms on CD4 T-cell cardiotropism, a mechanism potentially contributing to heart inflammation, remains unknown. Here we show that IL-32 isoforms β and γ induce the generation of CCR4+CXCR3+ double positive (DP) memory CD4 T-cell subpopulation expressing the tyrosine kinase receptor c-Met, a phenotype associated with heart-homing of T cells. Our ex vivo studies on PWH show that the frequency of DP CD4 T cells is significantly higher in individuals with, compared to individuals without, subclinical atherosclerosis and that DP cells from antiretroviral-naive and treated individuals are highly enriched with HIV DNA. Together, these data demonstrate that IL-32 isoforms have the potential to induce heart-homing of HIV-infected CD4 T cells, which may further aggravate heart inflammation and CVD in PWH.
Throughout the SARS-CoV-2 pandemic, several variants of concern (VOCs) have been identified, many of which share recurrent mutations in the spike glycoprotein's receptor-binding domain (RBD). This ...region coincides with known epitopes and can therefore have an impact on immune escape. Protracted infections in immunosuppressed patients have been hypothesized to lead to an enrichment of such mutations and therefore drive evolution towards VOCs. Here, we present the case of an immunosuppressed patient that developed distinct populations with immune escape mutations throughout the course of their infection. Notably, by investigating the co-occurrence of substitutions on individual sequencing reads in the RBD, we found quasispecies harboring mutations that confer resistance to known monoclonal antibodies (mAbs) such as S:E484K and S:E484A. These mutations were acquired without the patient being treated with mAbs nor convalescent sera and without them developing a detectable immune response to the virus. We also provide additional evidence for a viral reservoir based on intra-host phylogenetics, which led to a viral substrain that evolved elsewhere in the patient's body, colonizing their upper respiratory tract (URT). The presence of SARS-CoV-2 viral reservoirs can shed light on protracted infections interspersed with periods where the virus is undetectable, and potential explanations for long-COVID cases.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
Background
Evaluations of human immunodeficiency virus (HIV) curative interventions require reliable and efficient quantification of replication-competent latent reservoirs. The “classic” ...quantitative viral outgrowth assay (QVOA) has been regarded as the reference standard, although prohibitively resource and labor intensive. We compared 6 “next-generation” viral outgrowth assays, using polymerase chain reaction or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA.
Methods
Next-generation QVOAs were compared with classic QVOA using single leukapheresis-derived samples from 5 antiretroviral therapy–suppressed HIV-infected participants and 1 HIV-uninfected control; each laboratory tested blinded batches of 3 frozen and 1 fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and laboratory levels. Models also estimated the effect of testing frozen versus fresh samples.
Results
Next-generation QVOAs had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher infectious units per million values than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95% credible interval CI, 2.1–3.5-fold) for next-generation assays. Between-laboratory variation increased extra-Poisson variation to 3.4-fold (95% CI, 2.6–5.4-fold). Frozen storage did not substantially alter infectious units per million values (−18%; 95% CI, −52% to 39%).
Conclusions
The data offer cautious support for use of next-generation QVOAs as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of latent reservoirs in eradication strategies would benefit from high throughput and scalable assays.
We demonstrated similar estimates of variation for classic and next-generation quantitative viral outgrowth assays (QVOAs), with enhanced sensitivity supporting the use of next-generation QVOAs as a proxy for classic QVOAs in measuring the latent human immunodeficiency virus reservoir.
The lungs are relatively unexplored anatomical human immunodeficiency virus (HIV) reservoirs in the antiretroviral therapy (ART) era. Double negative (DN) T cells are a subset of T cells that lack ...expression of CD4 and CD8 (CD4
CD8
) and may have both regulatory and effector functions during HIV infection. Notably, circulating DN T cells were previously described as cellular HIV reservoirs. Here, we undertook a thorough analysis of pulmonary versus blood DN T cells of people living with HIV (PLWH) under ART. Bronchoalveolar lavage (BAL) fluid and matched peripheral blood were collected from 35 PLWH on ART and 16 uninfected volunteers without respiratory symptoms. Both PLWH and HIV-negative (HIV
) adults displayed higher frequencies of DN T cells in BAL versus blood, and these cells mostly exhibited an effector memory phenotype. In PLWH, pulmonary mucosal DN T cells expressed higher levels of HLA-DR and several cellular markers associated with HIV persistence (CCR6, CXCR3, and PD-1) than blood. We also observed that DN T cells were less senescent (CD28
CD57
) and expressed less immunosuppressive ectonucleotidase (CD73/CD39), granzyme B, and perforin in the BAL fluid than in the blood of PLWH. Importantly, fluorescence-activated cell sorter (FACS)-sorted DN T cells from the BAL fluid of PLWH under suppressive ART harbored HIV DNA. Using the humanized bone marrow-liver-thymus (hu-BLT) mouse model of HIV infection, we observed higher infection frequencies of lung DN T cells than those of the blood and spleen in both early and late HIV infection. Overall, our findings show that HIV is seeded in pulmonary mucosal DN T cells early following infection and persists in these potential cellular HIV reservoirs even during long-term ART.
Reservoirs of HIV during ART are the primary reasons why HIV/AIDS remains an incurable disease. Indeed, HIV remains latent and unreachable by antiretrovirals in cellular and anatomical sanctuaries, preventing its eradication. The lungs have received very little attention compared to other anatomical reservoirs despite being immunological effector sites exhibiting characteristics ideal for HIV persistence. Furthermore, PLWH suffer from a high burden of pulmonary non-opportunistic infections, suggesting impaired pulmonary immunity despite ART. Meanwhile, various immune cell populations have been proposed to be cellular reservoirs in blood, including CD4
CD8
DN T cells, a subset that may originate from CD4 downregulation by HIV proteins. The present study aims to describe DN T cells in human and humanized mice lungs in relation to intrapulmonary HIV burden. The characterization of DN T cells as cellular HIV reservoirs and the lungs as an anatomical HIV reservoir will contribute to the development of targeted HIV eradication strategies.
Infusion of the broadly neutralizing antibody VRC01 has been evaluated in individuals chronically infected with HIV-1. Here, we studied how VRC01 infusions affected viral rebound after cessation of ...antiretroviral therapy (ART) in 18 acutely treated and durably suppressed individuals. Viral rebound occurred in all individuals, yet VRC01 infusions modestly delayed rebound and participants who showed a faster decay of VRC01 in serum rebounded more rapidly. Participants with strains most sensitive to VRC01 or with VRC01 epitope motifs similar to known VRC01-susceptible strains rebounded later. Upon rebound, HIV-1 sequences were indistinguishable from those sampled at diagnosis. Across the cohort, participant-derived Env showed different sensitivity to VRC01 neutralization (including 2 resistant viruses), yet neutralization sensitivity was similar at diagnosis and after rebound, indicating the lack of selection for VRC01 resistance during treatment interruption. Our results showed that viremia rebounded despite the absence of HIV-1 adaptation to VRC01 and an average VRC01 trough of 221 μg/mL. Although VRC01 levels were insufficient to prevent a resurgent infection, knowledge that they did not mediate Env mutations in acute-like viruses is relevant for antibody-based strategies in acute infection.
HIV persists because a reservoir of latently infected CD4 T cells do not express viral proteins and are indistinguishable from uninfected cells. One approach to HIV cure suggests that reactivating ...HIV will activate cytotoxic pathways; yet when tested in vivo, reactivating cells do not die sufficiently to reduce cell-associated HIV DNA levels. We recently showed that following reactivation from latency, HIV infected cells generate the HIV specific cytotoxic protein Casp8p41 which is produced by HIV protease cleaving procaspase 8. However, cell death is prevented, possibly due to low procaspase 8 expression. Here, we tested whether increasing procaspase 8 levels in CD4 T cells will produce more Casp8p41 following HIV reactivation, causing more reactivated cells to die. Screening 1277 FDA approved drugs identified 168 that increased procaspase 8 expression by at least 1.7-fold. Of these 30 were tested for anti-HIV effects in an acute HIVIIIb infection model, and 9 drugs at physiologic relevant levels significantly reduced cell-associated HIV DNA. Primary CD4 T cells from ART suppressed HIV patients were treated with one of these 9 drugs and reactivated with αCD3/αCD28. Four drugs significantly increased Casp8p41 levels following HIV reactivation, and decreased total cell associated HIV DNA levels (flurbiprofen: p = 0.014; doxycycline: p = 0.044; indomethacin: p = 0.025; bezafibrate: P = 0.018) without effecting the viability of uninfected cells. Thus procaspase 8 levels can be increased pharmacologically and, in the context of HIV reactivation, increase Casp8p41 causing death of reactivating cells and decreased HIV DNA levels. Future studies will be required to define the clinical utility of this or similar approaches.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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