Early antiretroviral therapy favors post-treatment SIV control associated with the expansion of enhanced memory CD8+ T-cells.

HIV remission can be achieved in some people, called post-treatment HIV controllers, after antiretroviral treatment discontinuation. Treatment initiation close to the time of infection was suggested to favor post-treatment control, but the circumstances and mechanisms leading to this outcome remain unclear. Here we evaluate the impact of early (week 4) vs. late (week 24 post-infection) treatment initiation in SIVmac251-infected male cynomolgus macaques receiving 2 years of therapy before analytical treatment interruption. We show that early treatment strongly promotes post-treatment control, which is not related to a lower frequency of infected cells at treatment interruption. Rather, early treatment favors the development of long-term memory CD8+ T cells with enhanced proliferative and SIV suppressive capacity that are able to mediate a robust secondary-like response upon viral rebound. Our model allows us to formally demonstrate a link between treatment initiation during primary infection and the promotion of post-treatment control and provides results that may guide the development of new immunotherapies for HIV remission.

Anti-V1/V3-glycan broadly HIV-1 neutralizing antibodies in a post-treatment controller.

HIV-1 broadly neutralizing antibodies (bNAbs) can decrease viremia but are usually unable to counteract autologous viruses escaping the antibody pressure. Nonetheless, bNAbs may contribute to natural HIV-1 control in individuals off antiretroviral therapy (ART). Here, we describe a bNAb B cell lineage elicited in a post-treatment controller (PTC) that exhibits broad seroneutralization and show that a representative antibody from this lineage, EPTC112, targets a quaternary epitope in the glycan-V3 loop supersite of the HIV-1 envelope glycoprotein. The cryo-EM structure of EPTC112 complexed with soluble BG505 SOSIP.664 envelope trimers revealed interactions with N301- and N156-branched N-glycans and the 324GDIR327 V3 loop motif. Although the sole contemporaneous virus circulating in this PTC was resistant to EPTC112, it was potently neutralized by autologous plasma IgG antibodies. Our findings illuminate how cross-neutralizing antibodies can alter the HIV-1 infection course in PTCs and may control viremia off-ART, supporting their role in functional HIV-1 cure strategies.

Primary role of type I interferons for the induction of functionally optimal antigen-specific CD8+ T cells in HIV infection.

BACKGROUND: CD8+ T cells equipped with a full arsenal of antiviral effector functions are critical for effective immune control of HIV-1. It has nonetheless remained unclear how best to elicit such potent cellular immune responses in the context of immunotherapy or vaccination. HIV-2 has been associated with milder disease manifestations and more commonly elicits functionally replete virus-specific CD8+ T cell responses compared with HIV-1. We aimed to learn from this immunological dichotomy and to develop informed strategies that could enhance the induction of robust CD8+ T cell responses against HIV-1. METHODS: We developed an unbiased in vitro system to compare the de novo induction of antigen-specific CD8+ T cell responses after exposure to HIV-1 or HIV-2. The functional properties of primed CD8+ T cells were assessed using flow cytometry and molecular analyses of gene transcription. FINDINGS: HIV-2 primed functionally optimal antigen-specific CD8+ T cells with enhanced survival properties more effectively than HIV-1. This superior induction process was dependent on type I interferons (IFNs) and could be mimicked via the adjuvant delivery of cyclic GMP-AMP (cGAMP), a known agonist of the stimulator of interferon genes (STING). CD8+ T cells elicited in the presence of cGAMP were polyfunctional and highly sensitive to antigen stimulation, even after priming from people living with HIV-1. INTERPRETATION: HIV-2 primes CD8+ T cells with potent antiviral functionality by activating the cyclic GMP-AMP synthase (cGAS)/STING pathway, which results in the production of type I IFNs. This process may be amenable to therapeutic development via the use of cGAMP or other STING agonists to bolster CD8+ T cell-mediated immunity against HIV-1. FUNDING: This work was funded by INSERM, the Institut Curie, and the University of Bordeaux (Senior IdEx Chair) and by grants from Sidaction (17-1-AAE-11097, 17-1-FJC-11199, VIH2016126002, 20-2-AEQ-12822-2, and 22-2-AEQ-13411), the Agence Nationale de la Recherche sur le SIDA (ECTZ36691, ECTZ25472, ECTZ71745, and ECTZ118797), and the Fondation pour la Recherche Médicale (EQ U202103012774). D.A.P. was supported by a Wellcome Trust Senior Investigator Award (100326/Z/12/Z).

In-Depth Characterization of Full-Length Archived Viral Genomes after Nine Years of Posttreatment HIV Control.

In the search for control of human immunodeficiency virus type 1 (HIV-1) infection without antiretroviral therapy, posttreatment controllers (PTCs) are models of HIV remission. To better understand their mechanisms of control, we characterized the HIV blood reservoirs of 8 PTCs (median of 9.4 years after treatment interruption) in comparison with those of 13 natural HIV infection controllers (HICs) (median of 18 years of infection) and with those of individuals receiving efficient antiretroviral therapy initiated during either primary HIV infection (PHIs; n = 8) or chronic HIV infection (CHIs; n = 6). This characterization was performed with single-genome amplification and deep sequencing. The proviral diversity, which reflects the history of past viral replication, was lower in the PTCs, PHIs, and aviremic HICs than in the blipper HICs and CHIs. The proportions of intact and defective proviruses among the proviral pool in PTCs were not significantly different from those of other groups. When looking at the quantities of proviruses per million peripheral blood mononuclear cells (PBMCs), they had similar amounts of intact proviruses as other groups but smaller amounts of defective proviruses than CHIs, suggesting a role of these forms in HIV pathogenesis. Two HICs but none of the PTCs harbored only proviruses with deletion in nef; these attenuated strains could contribute to viral control in these participants. We show, for the first time, the presence of intact proviruses and low viral diversity in PTCs long after treatment interruption, as well as the absence of evolution of the proviral quasispecies in subsequent samples. This reflects low residual replication over time. Further data are necessary to confirm these results. IMPORTANCE Most people living with HIV need antiretroviral therapy to control their infection and experience viral relapse in case of treatment interruption, because of viral reservoir (proviruses) persistence. Knowing that proviruses are very diverse and most of them are defective in treated individuals, we aimed to characterize the HIV blood reservoirs of posttreatment controllers (PTCs), rare models of drug-free remission, in comparison with spontaneous controllers and treated individuals. At a median time of 9 years after treatment interruption, which is unprecedented in the literature, we showed that the proportions and quantities of intact proviruses were similar between PTCs and other individuals. Unlike 2/7 spontaneous controllers who harbored only nef-deleted proviruses, which are attenuated strains, which could contribute to their control, no such case was observed in PTCs. Furthermore, PTCs displayed low viral genetic diversity and no evolution of their reservoirs, indicating very low residual replication, despite the presence of intact proviruses.

Transient viral exposure drives functionally-coordinated humoral immune responses in HIV-1 post-treatment controllers.

HIV-1 post-treatment controllers are rare individuals controlling HIV-1 infection for years after antiretroviral therapy interruption. Identification of immune correlates of control in post-treatment controllers could aid in designing effective HIV-1 vaccine and remission strategies. Here, we perform comprehensive immunoprofiling of the humoral response to HIV-1 in long-term post-treatment controllers. Global multivariate analyses combining clinico-virological and humoral immune data reveal distinct profiles in post-treatment controllers experiencing transient viremic episodes off therapy compared to those stably aviremic. Virally-exposed post-treatment controllers display stronger HIV-1 humoral responses, and develop more frequently Env-specific memory B cells and cross-neutralizing antibodies. Both are linked to short viremic exposures, which are also accompanied by an increase in blood atypical memory B cells and activated subsets of circulating follicular helper T cells. Still, most humoral immune variables only correlate with Th2-like circulating follicular helper T cells. Thus, post-treatment controllers form a heterogeneous group with two distinct viral behaviours and associated immune signatures. Post-treatment controllers stably aviremic present "silent" humoral profiles, while those virally-exposed develop functionally robust HIV-specific B-cell and antibody responses, which may participate in controlling infection.

Reprogramming dysfunctional CD8+ T cells to promote properties associated with natural HIV control.

Virus-specific CD8+ T cells play a central role in HIV-1 natural controllers to maintain suppressed viremia in the absence of antiretroviral therapy. These cells display a memory program that confers them stemness properties, high survival, polyfunctionality, proliferative capacity, metabolic plasticity, and antiviral potential. The development and maintenance of such qualities by memory CD8+ T cells appear crucial to achieving natural HIV-1 control. Here, we show that targeting the signaling pathways Wnt/transcription factor T cell factor 1 (Wnt/TCF-1) and mTORC through GSK3 inhibition to reprogram HIV-specific CD8+ T cells from noncontrollers promoted functional capacities associated with natural control of infection. Features of such reprogrammed cells included enrichment in TCF-1+ less-differentiated subsets, a superior response to antigen, enhanced survival, polyfunctionality, metabolic plasticity, less mTORC1 dependency, an improved response to γ-chain cytokines, and a stronger HIV-suppressive capacity. Thus, such CD8+ T cell reprogramming, combined with other available immunomodulators, might represent a promising strategy for adoptive cell therapy in the search for an HIV-1 cure.

Ultrasensitive Detection of p24 in Plasma Samples from People with Primary and Chronic HIV-1 Infection.

HIV-1 Gag p24 has long been identified as an informative biomarker of HIV replication, disease progression, and therapeutic efficacy, but the lower sensitivity of immunoassays in comparison to molecular tests and the interference with antibodies in chronic HIV infection limit its application for clinical monitoring. The development of ultrasensitive protein detection technologies may help in overcoming these limitations. Here, we evaluated whether immune complex dissociation combined with ultrasensitive digital enzyme-linked immunosorbent assay (ELISA) single-molecule array (Simoa) technology could be used to quantify p24 in plasma samples from people with HIV-1 infection. We found that, among different immune complex dissociation methods, only acid-mediated dissociation was compatible with ultrasensitive p24 quantification by digital ELISA, strongly enhancing p24 detection at different stages of HIV-1 infection. We show that ultrasensitive p24 levels correlated positively with plasma HIV RNA and HIV DNA and negatively with CD4-positive (CD4+) T cells in the samples from people with primary and chronic HIV-1 infection. In addition, p24 levels also correlated with plasma D-dimers and interferon alpha (IFN-α) levels. p24 levels sharply decreased to undetectable levels after initiation of combined antiretroviral treatment (cART). However, we identified a group of people who, 48 weeks after cART initiation, had detectable p24 levels despite most having undetectable viral loads. These people had different virological and immunological baseline characteristics compared with people who had undetectable p24 after cART. These results demonstrate that ultrasensitive p24 analysis provides an efficient and robust means to monitor p24 antigen in plasma samples from people with HIV-1 infection, including during antiretroviral treatment, and may provide complementary information to other commonly used biomarkers. IMPORTANCE The introduction of combined antiretroviral treatment has transformed HIV-1 infection into a manageable condition. In this context, there is a need for additional biomarkers to monitor HIV-1 residual disease or the outcome of new interventions, such as in the case of HIV cure strategies. The p24 antigen has a long half-life outside viral particles, and it is, therefore, a very promising marker to monitor episodes of viral replication or transient activation of the viral reservoir. However, the formation of immune complexes with anti-p24 antibodies makes its quantification difficult beyond acute HIV-1 infection. We show here that, upon immune complex dissociation, new technologies allow the ultrasensitive p24 quantification in plasma samples throughout HIV-1 infection at levels close to those of viral RNA and DNA determinations. Our results further indicate that ultrasensitive p24 quantification may have added value when used in combination with other classic clinical biomarkers.

Optimal Maturation of the SIV-Specific CD8+ T Cell Response after Primary Infection Is Associated with Natural Control of SIV: ANRS SIC Study.

Highly efficient CD8+ T cells are associated with natural HIV control, but it has remained unclear how these cells are generated and maintained. We have used a macaque model of spontaneous SIVmac251 control to monitor the development of efficient CD8+ T cell responses. Our results show that SIV-specific CD8+ T cells emerge during primary infection in all animals. The ability of CD8+ T cells to suppress SIV is suboptimal in the acute phase but increases progressively in controller macaques before the establishment of sustained low-level viremia. Controller macaques develop optimal memory-like SIV-specific CD8+ T cells early after infection. In contrast, a persistently skewed differentiation phenotype characterizes memory SIV-specific CD8+ T cells in non-controller macaques. Accordingly, the phenotype of SIV-specific CD8+ T cells defined early after infection appears to favor the development of protective immunity in controllers, whereas SIV-specific CD8+ T cells in non-controllers fail to gain antiviral potency, feasibly as a consequence of early defects imprinted in the memory pool.

Affinity for the Interface Underpins Potency of Antibodies Operating In Membrane Environments.

The contribution of membrane interfacial interactions to recognition of membrane-embedded antigens by antibodies is currently unclear. This report demonstrates the optimization of this type of antibodies via chemical modification of regions near the membrane but not directly involved in the recognition of the epitope. Using the HIV-1 antibody 10E8 as a model, linear and polycyclic synthetic aromatic compounds are introduced at selected sites. Molecular dynamics simulations predict the favorable interactions of these synthetic compounds with the viral lipid membrane, where the epitope of the HIV-1 glycoprotein Env is located. Chemical modification of 10E8 with aromatic acetamides facilitates the productive and specific recognition of the native antigen, partially buried in the crowded environment of the viral membrane, resulting in a dramatic increase of its capacity to block viral infection. These observations support the harnessing of interfacial affinity through site-selective chemical modification to optimize the function of antibodies that target membrane-proximal epitopes.

Extremely low viral reservoir in treated chronically HIV-1-infected individuals.

BACKGROUND: Small viral reservoirs are found predominantly in HIV-1 controllers and individuals treated during acute/early HIV-1 infection. However, other HIV+ individuals could naturally also harbour low viral reservoirs. METHODS: We screened 451 HIV-1-infected treated-individuals with suppressed plasma viremia for at least 3 years and stored cryopreserved peripheral blood mononuclear cells (PBMCs). Total HIV-DNA was analysed in PBMCs with ddPCR. Individuals with <50 HIV-DNA copies/106 PBMCs constitute the 'Low Viral Reservoir Treated' cohort (LoViReT). Longitudinal samples were obtained from 12 chronically treated LoViReT and compared to 13 controls (>50 HIV-DNA copies/106 PBMCs) to analyse total HIV-DNA, T-cell and NK-cell populations, HIV-1 specific antibodies, and plasma inflammation markers. FINDINGS: We found that 9.3% of the individuals screened had <50 HIV-DNA copies/106 PBMCs. At least 66% initiated cART during the chronic phase of HIV-1 infection (cp-LoViReT). Cp-LoViReT harboured lower levels of HIV-DNA before cART and after treatment introduction the decays were greater compared to controls. They displayed a marked decline in quantity and avidity in HIV-specific antibodies after initiation of cART. Cp-LoViReT had fewer CD8+ TTM and TEMRA in the absence of cART, and higher CD8+ TN after 18 months on therapy. INTERPRETATION: Treated chronically HIV-1-infected LoViReT represent a new phenotype of individuals characterized by an intrinsically reduced viral reservoir, less impaired CD8+ T-cell compartment before cART, and low circulating HIV-1 antigens despite being treated in the chronic phase of infection. The identification of this unique group of individuals is of great interest for the design of future eradication studies. FUNDING: MSD Spain.

Vulnerability to reservoir reseeding due to high immune activation after allogeneic hematopoietic stem cell transplantation in individuals with HIV-1.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only medical intervention that has led to an HIV cure. Whereas the HIV reservoir sharply decreases after allo-HSCT, the dynamics of the T cell reconstitution has not been comprehensively described. We analyzed the activation and differentiation of CD4+ and CD8+ T cells, and the breadth and quality of HIV- and CMV-specific CD8+ T cell responses in 16 patients with HIV who underwent allo-HSCT (including five individuals who received cells from CCR5Δ32/Δ32 donors) to treat their underlying hematological malignancy and who remained on antiretroviral therapy (ART). We found that reconstitution of the T cell compartment after allo-HSCT was slow and heterogeneous with an initial expansion of activated CD4+ T cells that preceded the expansion of CD8+ T cells. Although HIV-specific CD8+ T cells disappeared immediately after allo-HSCT, weak HIV-specific CD8+ T cell responses were detectable several weeks after transplant and could still be detected at the time of full T cell chimerism, indicating that de novo priming, and hence antigen exposure, occurred during the time of T cell expansion. These HIV-specific T cells had limited functionality compared with CMV-specific CD8+ T cells and persisted years after allo-HSCT. In conclusion, immune reconstitution was slow, heterogeneous, and incomplete and coincided with de novo detection of weak HIV-specific T cell responses. The initial short phase of high T cell activation, in which HIV antigens were present, may constitute a window of vulnerability for the reseeding of viral reservoirs, emphasizing the importance of maintaining ART directly after allo-HSCT.

Antiapoptotic Clone 11-Derived Peptides Induce In Vitro Death of CD4+ T Cells Susceptible to HIV-1 Infection.

HIV-1 successfully establishes long-term infection in its target cells despite viral cytotoxic effects. We have recently shown that cell metabolism is an important factor driving CD4+ T cell susceptibility to HIV-1 and the survival of infected cells. We show here that expression of antiapoptotic clone 11 (AAC-11), an antiapoptotic factor upregulated in many cancers, increased with progressive CD4+ T cell memory differentiation in association with the expression of cell cycle, activation, and metabolism genes and was correlated with susceptibility to HIV-1 infection. Synthetic peptides based on the LZ domain sequence of AAC-11, responsible for its interaction with molecular partners, were previously shown to be cytotoxic to cancer cells. Here, we observed that these peptides also blocked HIV-1 infection by inducing the death of HIV-1-susceptible primary CD4+ T cells across all T cell subsets. The peptides targeted metabolically active cells and had the greatest effect on effector and transitional CD4+ T cell memory subsets. Our results suggest that the AAC-11 survival pathway is potentially involved in the survival of HIV-1-infectible cells and provide proof of principle that some cellular characteristics can be targeted to eliminate the cells offering the best conditions to sustain HIV-1 replication.IMPORTANCE Although antiretroviral treatment efficiently blocks HIV multiplication, it cannot eliminate cells already carrying integrated proviruses. In the search for an HIV cure, the identification of new potential targets to selectively eliminate infected cells is of the outmost importance. We show here that peptides derived from antiapoptotic clone 11 (AAC-11), whose expression levels correlated with susceptibility to HIV-1 infection of CD4+ T cells, induced cytotoxicity in CD4+ T cells showing the highest levels of activation and metabolic activity, conditions known to favor HIV-1 infection. Accordingly, CD4+ T cells that survived the cytotoxic action of the AAC-11 peptides were resistant to HIV-1 replication. Our results identify a new potential molecular pathway to target HIV-1 infection.

The proportion of CD57+ cells among effector CD8+ T cells is lower in HIV controllers compared with antiretroviral therapy-treated patients.

BACKGROUND: HIV infection has often been linked to faster immune ageing. We sought to determine whether or not treatment-naive spontaneous HIV-1 controllers (HICs) and ART-exposed patients differ with regard to the expression of cell senescence markers. METHODS: Eighty-eight chronically infected HICs and ART-exposed patients (median time since infection: 15 years) with an undetectable plasma HIV RNA load (at least for the previous 2 years) were included. We used flow cytometry to measure immunosenescence markers (KLRG-1 and CD57) expression in fresh blood samples collected from patients and healthy donors. RESULTS: For the CD8 T-cell population as a whole, the ART-exposed but not the HIC patients exhibited a much higher proportion of KLRG-1 and CD57 CD8 T cells than healthy blood donors. For the CD8 T-cell subsets, HICs had a lower proportion of CD57 effector CD8 T cells than ART patients or healthy blood donors, whereas the proportions of KLRG-1 effector were similar. A similar trend was observed for terminal effectors. No impact of age, sex or standard parameters of infection (CD4 percentage, protective HLA allele, viral blips) was observed. The difference in the proportion of CD57 cells between HICs and ART was observed more specifically in long-term infected patients (>20 years). However, whenever considering the CD57 effector memory and effector subsets, the cytotoxic granule content was greater in HICs than in ART. CONCLUSION: The proportion of CD57 effector CD8 T cells is lower in HICs than in ART-exposed patients. This profile may be beneficial by ensuring limited senescence associated with consistent cytotoxic potential.

Dynamics in HIV-DNA levels over time in HIV controllers.

INTRODUCTION: HIV controllers (HIC) maintain viraemia at low levels without antiretroviral treatment and have small HIV reservoirs. Nevertheless, they are heterogeneous regarding their risk of infection progression. The study of reservoirs can help elucidate this control. This study aimed to explore the factors implicated in the pathogenesis of HIV infection that are potentially associated with HIV reservoirs and their dynamics in HIC. METHODS: Individuals living with HIV included in the ANRS-CODEX cohort with at least two HIV-DNA measurements between 2009 and 2016 were selected. The total HIV-DNA levels had been quantified prospectively from blood samples. Mixed-effect linear models estimated the HIV-DNA dynamics over time. RESULTS: The median (interquartile range (IQR)) HIV-DNA level was 1.5 (1.3 to 1.9) log copies/million peripheral blood mononuclear cells at inclusion (n = 202 individuals). These low levels showed heterogeneity among HIC. Lower levels were then associated with the protective HLA-B*27/B*57 alleles and/or lower HIV-RNA level at inclusion, negative hepatitis C virus serology, lower HIV-suppressive capacity of specific CD8 T cells and lower levels of immune activation and inflammation. Interestingly, mathematical modelling of the dynamics of HIV-DNA over time (840 measurements) showed that the number of infected cells decreased in 46% of HIC (follow-up: 47.6 months) and increased in 54% of HIC. A multivariate analysis indicated that HLA-B*27/B*57 alleles, a low level of HIV-RNA and a low level of HIV-DNA at inclusion were markers independently associated with this decrease. CONCLUSIONS: These results offer new insights into the mechanisms of long-term control in HIC. In half of HIC, the decrease in HIV-DNA level could be linked to tighter viral control and progressive loss of infected cells. These findings allow the identification of HIC with a low risk of progression who may not need treatment.

Metabolic plasticity of HIV-specific CD8+ T cells is associated with enhanced antiviral potential and natural control of HIV-1 infection.

Spontaneous control of human immunodeficiency virus (HIV) is generally associated with an enhanced capacity of CD8+ T cells to eliminate infected CD4+ T cells, but the molecular characteristics of these highly functional CD8+ T cells are largely unknown. In the present study, using single-cell analysis, it was shown that HIV-specific, central memory CD8+ T cells from spontaneous HIV controllers (HICs) and antiretrovirally treated non-controllers have opposing transcriptomic profiles. Genes linked to effector functions and survival are upregulated in cells from HICs. In contrast, genes associated with activation, exhaustion and glycolysis are upregulated in cells from non-controllers. It was shown that HIV-specific CD8+ T cells from non-controllers are largely glucose dependent, whereas those from HICs have more diverse metabolic resources that enhance both their survival potential and their capacity to develop anti-HIV effector functions. The functional efficiency of the HIV-specific CD8+ T cell response in HICs is thus engraved in their memory population and related to their metabolic programme. Metabolic reprogramming in vitro through interleukin-15 treatment abrogated the glucose dependency and enhanced the antiviral potency of HIV-specific CD8+ T cells from non-controllers.

Cellular Metabolism Is a Major Determinant of HIV-1 Reservoir Seeding in CD4+ T Cells and Offers an Opportunity to Tackle Infection.

HIV persists in long-lived infected cells that are not affected by antiretroviral treatment. These HIV reservoirs are mainly located in CD4+ T cells, but their distribution is variable in the different subsets. Susceptibility to HIV-1 increases with CD4+ T cell differentiation. We evaluated whether the metabolic programming that supports the differentiation and function of CD4+ T cells affected their susceptibility to HIV-1. We found that differences in HIV-1 susceptibility between naive and more differentiated subsets were associated with the metabolic activity of the cells. Indeed, HIV-1 selectively infected CD4+ T cells with high oxidative phosphorylation and glycolysis, independent of their activation phenotype. Moreover, partial inhibition of glycolysis (1) impaired HIV-1 infection in vitro in all CD4+ T cell subsets, (2) decreased the viability of preinfected cells, and (3) precluded HIV-1 amplification in cells from HIV-infected individuals. Our results elucidate the link between cell metabolism and HIV-1 infection and identify a vulnerability in tackling HIV reservoirs.

The anti-caspase inhibitor Q-VD-OPH prevents AIDS disease progression in SIV-infected rhesus macaques.

Apoptosis has been proposed as a key mechanism responsible for CD4+ T cell depletion and immune dysfunction during HIV infection. We demonstrated that Q-VD-OPH, a caspase inhibitor, inhibits spontaneous and activation-induced death of T cells from SIV-infected rhesus macaques (RMs). When administered during the acute phase of infection, Q-VD-OPH was associated with (a) reduced levels of T cell death, (b) preservation of CD4+/CD8+ T cell ratio in lymphoid organs and in the gut, (c) maintenance of memory CD4+ T cells, and (d) increased specific CD4+ T cell response associated with the expression of cytotoxic molecules. Although therapy was limited to the acute phase of infection, Q-VD-OPH-treated RMs showed lower levels of both viral load and cell-associated SIV DNA as compared with control SIV-infected RMs throughout the chronic phase of infection, and prevented the development of AIDS. Overall, our data demonstrate that Q-VD-OPH injection in SIV-infected RMs may represent an adjunctive therapeutic agent to control HIV infection and delaying disease progression to AIDS.

Ultrasensitive HIV-1 p24 Assay Detects Single Infected Cells and Differences in Reservoir Induction by Latency Reversal Agents.

The existence of HIV reservoirs in infected individuals under combined antiretroviral therapy (cART) represents a major obstacle toward cure. Viral reservoirs are assessed by quantification of HIV nucleic acids, a method which does not discriminate between infectious and defective viruses, or by viral outgrowth assays, which require large numbers of cells and long-term cultures. Here, we used an ultrasensitive p24 digital assay, which we report to be 1,000-fold more sensitive than classical enzyme-linked immunosorbent assays (ELISAs) in the quantification of HIV-1 Gag p24 production in samples from HIV-infected individuals. Results from ultrasensitive p24 assays were compared to those from conventional viral RNA reverse transcription-quantitative PCR (RT-qPCR)-based assays and from outgrowth assay readout by flow cytometry. Using serial dilutions and flow-based single-cell sorting, we show that viral proteins produced by a single infected cell can be detected by the ultrasensitive p24 assay. This unique sensitivity allowed the early (as soon as day 1 in 43% of cases) and more efficient detection and quantification of p24 in phytohemagglutinin-L (PHA)-stimulated CD4+ T cells from individuals under effective cART. When seven different classes of latency reversal agents (LRA) in resting CD4+ T cells from HIV-infected individuals were tested, the ultrasensitive p24 assay revealed differences in the extent of HIV reactivation. Of note, HIV RNA production was infrequently accompanied by p24 protein production (19%). Among the drugs tested, prostratin showed a superior capacity in inducing viral protein production. In summary, the ultrasensitive p24 assay allows the detection and quantification of p24 produced by single infected CD4+ T cells and provides a unique tool to assess early reactivation of infectious virus from reservoirs in HIV-infected individuals.IMPORTANCE The persistence of HIV reservoirs in infected individuals under effective antiretroviral treatment represents a major obstacle toward cure. Different methods to estimate HIV reservoirs exist, but there is currently no optimal assay to measure HIV reservoirs in HIV eradication interventions. In the present study, we report an ultrasensitive digital ELISA platform for quantification of the HIV-1 protein p24. This method was employed to assess the early reactivation of infectious virus from reservoirs in HIV-1-infected individuals. We found that viral proteins produced by a single infected cell can be detected by an ultrasensitive p24 assay. This unprecedented resolution showed major advantages in comparison to other techniques currently used to assess viral replication in reactivation studies. In addition, such a highly sensitive assay allows discrimination of drug-induced reactivation of productive HIV based on protein expression. The present study heralds new opportunities to evaluate the HIV reservoir and the efficacy of drugs used to target it.

Anoxia and glucose supplementation preserve neutrophil viability and function.

Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours in culture media at 37°C under anoxic conditions with 3 mM glucose and 32 µg/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use.