A novel small molecule approach for the treatment of propionic and methylmalonic acidemias.

Propionic Acidemia (PA) and Methylmalonic Acidemia (MMA) are inborn errors of metabolism affecting the catabolism of valine, isoleucine, methionine, threonine and odd-chain fatty acids. These are multi-organ disorders caused by the enzymatic deficiency of propionyl-CoA carboxylase (PCC) or methylmalonyl-CoA mutase (MUT), resulting in the accumulation of propionyl-coenzyme A (P-CoA) and methylmalonyl-CoA (M-CoA in MMA only). Primary metabolites of these CoA esters include 2-methylcitric acid (MCA), propionyl-carnitine (C3), and 3-hydroxypropionic acid, which are detectable in both PA and MMA, and methylmalonic acid, which is detectable in MMA patients only (Chapman et al., 2012). We deployed liver cell-based models that utilized PA and MMA patient-derived primary hepatocytes to validate a small molecule therapy for PA and MMA patients. The small molecule, HST5040, resulted in a dose-dependent reduction in the levels of P-CoA, M-CoA (in MMA) and the disease-relevant biomarkers C3, MCA, and methylmalonic acid (in MMA). A putative working model of how HST5040 reduces the P-CoA and its derived metabolites involves the conversion of HST5040 to HST5040-CoA driving the redistribution of free and conjugated CoA pools, resulting in the differential reduction of the aberrantly high P-CoA and M-CoA. The reduction of P-CoA and M-CoA, either by slowing production (due to increased demands on the free CoA (CoASH) pool) or enhancing clearance (to replenish the CoASH pool), results in a net decrease in the CoA-derived metabolites (C3, MCA and MMA (MMA only)). A Phase 2 study in PA and MMA patients will be initiated in the United States.

A Phase IIa Study Evaluating Safety, Pharmacokinetics, and Antiviral Activity of GSK2838232, a Novel, Second-generation Maturation Inhibitor, in Participants With Human Immunodeficiency Virus Type 1 Infection.

BACKGROUND: GSK2838232 is a second-generation, potent, small-molecule, oral human immunodeficiency virus type 1 (HIV-1) maturation inhibitor for once-daily administration boosted with a pharmacoenhancer. METHODS: The phase 2a, proof-of-concept study was an open-label, adaptive dose-ranging design. Safety, pharmacokinetics, and efficacy of GSK2838232 boosted by cobicistat were evaluated in individuals with HIV-1 infection. The study participants (N = 33) received GSK2838232 once daily across a range of doses (20-200 mg) with cobicistat 150 mg for 10 days. RESULTS: GSK2838232 was safe and well tolerated with no clinically meaningful changes in safety parameters or adverse events. Exposure (maximum concentration and area under the concentration-time curve from time zero to the concentration at 24 hours postdose) increased 2- to 3-fold with repeated dosing in an approximately dose-proportional manner, reaching steady-state by day 8 with a half-life (t½) from 16.3 to 19.2 hours. Clearance and t½ values were not dependent on dose. Viral load declined from baseline with all GSK2838232 doses. Mean maximum declines from baseline to day 11 in HIV-1 RNA log10 copies/mL with the 20-mg, 50-mg, 100-mg, and 200-mg cohorts were -0.67, -1.56, -1.32, and -1.70, respectively. CD4+ cell counts increased at doses ≥50 mg. CONCLUSIONS: GSK2838232 with cobicistat was well tolerated and exhibited efficacy as a short-term monotherapy in participants with HIV-1. This positive proof-of-concept study supports the continued development of GSK2838232 for the treatment of HIV as part of combination antiretroviral therapy. CLINICAL TRIALS REGISTRATION: NCT03045861.

Discovery of a novel and potent class of anti-HIV-1 maturation inhibitors with improved virology profile against gag polymorphisms.

A new class of betulin-derived α-keto amides was identified as HIV-1 maturation inhibitors. Through lead optimization, GSK8999 was identified with IC50 values of 17nM, 23nM, 25nM, and 8nM for wild type, Q369H, V370A, and T371A respectively. When tested in a panel of 62 HIV-1 isolates covering a diversity of CA-SP1 genotypes including A, AE, B, C, and G using a PBMC based assay, GSK8999 was potent against 57 of 62 isolates demonstrating an improvement over the first generation maturation inhibitor BVM. The data disclosed here also demonstrated that the new α-keto amide GSK8999 has a mechanism of action consistent with inhibition of the proteolytic cleavage of CA-SP1.

Inhibition of the ribonuclease H activity of HIV-1 reverse transcriptase by GSK5750 correlates with slow enzyme-inhibitor dissociation.

Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor ß-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.

Antiviral characteristics of GSK1265744, an HIV integrase inhibitor dosed orally or by long-acting injection.

GSK1265744 is a new HIV integrase strand transfer inhibitor (INSTI) engineered to deliver efficient antiviral activity with a once-daily, low-milligram dose that does not require a pharmacokinetic booster. The in vitro antiviral profile and mechanism of action of GSK1265744 were established through integrase enzyme assays, resistance passage experiments, and cellular assays with site-directed molecular (SDM) HIV clones resistant to other classes of anti-HIV-1 agents and earlier INSTIs. GSK1265744 inhibited HIV replication with low or subnanomolar efficacy and with a selectivity index of at least 22,000 under the same culture conditions. The protein-adjusted half-maximal inhibitory concentration (PA-EC50) extrapolated to 100% human serum was 102 nM. When the virus was passaged in the presence of GSK1265744, highly resistant mutants with more than a 10-fold change (FC) in EC50 relative to that of the wild-type were not observed for up to 112 days of culture. GSK1265744 demonstrated activity against SDM clones containing the raltegravir (RAL)-resistant Y143R, Q148K, N155H, and G140S/Q148H signature variants (FC less than 6.1), while these mutants had a high FC in the EC50 for RAL (11 to >130). Either additive or synergistic effects were observed when GSK1265744 was tested in combination with representative anti-HIV agents, and no antagonistic effects were seen. These findings demonstrate that, similar to dolutegravir, GSK1265744 is differentiated as a new INSTI, having a markedly distinct resistance profile compared with earlier INSTIs, RAL, and elvitegravir (EVG). The collective data set supports further clinical development of GSK1265744.

The Effect of Repeated Ketamine Infusion Over Facial Emotion Recognition in Treatment-Resistant Depression: A Preliminary Report.

In contrast to improvement in emotion recognition bias by traditional antidepressants, the authors report preliminary findings that changes in facial emotion recognition are not associated with response of depressive symptoms after repeated ketamine infusions or relapse during follow-up in treatment-resistant depression.

Neurocognitive performance and serial intravenous subanesthetic ketamine in treatment-resistant depression.

The N-methyl-D-aspartate glutamate receptor antagonist ketamine has demonstrated rapid antidepressant effects in treatment-resistant depression (TRD). However, evaluation of ketamine's neurocognitive aspects in TRD has started to be explored. This study aims to (1) examine baseline neurocognitive performance and change in severity of depressive symptoms through six ketamine infusions, (2) examine the neurocognitive effects after completion of serial infusions and whether changes were associated to relapse to depression. Six IV infusions of 0.5 mg/Kg ketamine over 40 min were conducted on a Monday-Wednesday-Friday schedule during a 12-d period on 15 patients with TRD followed by a 4-wk observational period. Neurocognitive functioning was assessed using the CogState battery at baseline and at each follow-up visit. Tasks were designed to test attention, memory (working, visual, and verbal), speed of processing, and set shifting. The likelihood of response through six infusions was greater among depressed subjects with lower attention at baseline (F(1,13)=5.59, p=0.034). Significant improvement was found in scores of visual memory (F(4,33.82)=5.12, p=0.002), simple working memory (F(4, 24.85)=3.29, p=0.027) and complex working memory (F(4, 32.76)=4.18, p=0.008) after the last ketamine infusion. However, neurocognitive changes were accounted for by improvement in the severity of depressive symptom. The acute neurocognitive effect after completion of repeated infusions was not associated with the likelihood of subsequent relapse during follow-up. Our findings suggest a potential baseline neurocognitive predictor of ketamine response and the apparently lack of short-term neurocognitive impairment after completion of six ketamine infusions in TRD.

Naphthyridinone (NTD) integrase inhibitors 4. Investigating N1 acetamide substituent effects with C3 amide groups.

A series of N1 acetamide substituted naphthyridinone HIV-1 integrase inhibitors have been explored to understand structure-activity relationships (SAR) with various C3 amide groups. Investigations were evaluated using integrase enzyme inhibition, antiviral activity and protein binding effects to optimize the sub-structures. Lipophilicity was also incorporated to understand ligand lipophilic efficiency as a function of the structural modifications. Three representative analogs were further examined in a peripheral blood mononuclear cell (PBMC) antiviral assay as well as in vitro and in vivo drug metabolism and pharmacokinetic studies.

Emotion recognition processing as early predictor of response to 8-week citalopram treatment in late-life depression.

BACKGROUND: In subjects with depression, exposure to antidepressants improves recognition of positive emotions. This phenomenon, which occurs early in the course of treatment, has been proposed as the initial step in the mechanism of action to subsequent therapeutic effects of antidepressants. To this date, it has not been well examined among older depressed patients. METHOD: Older subjects with non-psychotic major depressive disorder were treated with citalopram in an 8-week open-label study. The main predictor of response and remission was the change in emotion recognition between baseline and day 7. Covariates included executive functions, baseline anxiety level, medical comorbidity, level of subjective stress, serum citalopram level, and level of social support. RESULTS: Twenty-seven patients were considered for final analysis. Overall, accuracy of emotion recognition significantly improved between baseline (75%) and day 7 (83%) (X(2) = 34.50, df = 1, p < 0.001). Improvement to identify happy expressions occurred at 25% and 50% intensity with ceiling effect at 0%, 75%, and 100%. Change in emotion processing was marginally significant in predicting antidepressant response at day 56. Multivariate analysis showed that emotion processing is a significant predictor of response and remission when considered along with perceived level of social support. CONCLUSIONS: Recognition of mildly intense happy expression, which improved early in the course of citalopram treatment, predicts subsequent antidepressant response and remission when considered along with perception of social support. Further studies would be necessary to examine specific neural substrates in the affective network involved in the acute therapeutic action of antidepressant in late-life depression.

Parenteral platforms for tunable, long-acting administration of a highly hydrophobic antiretroviral drug.

GSK2838232 (GSK8232) is a second-generation maturation inhibitor (MI) developed for the treatment of HIV with excellent broad-spectrum virological profiles. The compound has demonstrated promising clinical results as an orally administered agent. Additionally, the compound's physical and pharmacological properties present opportunities for exploitation as long-acting parenteral formulations. Despite unique design constraints including solubility and dose of GSK8232, we report on three effective tunable drug delivery strategies: active pharmaceutical ingredient (API) suspensions, ionic liquids, and subdermal implants. Promising sustained drug release profiles were achieved in rats with each approach. Additionally, we were able to tune drug release rates through a combination of passive and active strategies, broadening applicability of these formulation approaches beyond GSK8232. Taken together, this report is an important first step to advance long-acting formulation development for critical HIV medicines that do not fit the traditional profile of suitable long-acting candidates.

Choosing the right antibody for resistin-like molecule (RELM/FIZZ) family members.

The family of resistin-like molecules (RELM), also known as found in inflammatory zone (FIZZ), consists of four members in mouse (RELMα/FIZZ1/HIMF, RELMß/FIZZ2, Resistin/FIZZ3, and RELMγ/FIZZ4) and two members in human (resistin and RELMß). The importance of these proteins in many aspects of physiology and pathophysiology, especially inflammatory processes, is rapidly evolving in the literature, and many investigators are beginning to work in this field. Most published studies focus on only one isoform, do not evaluate other isoforms that might be present, and have not tested for the specificity of the antibody used. Because RELM isoforms have high sequence and structural similarity and both distinct and overlapping functions, it is important to use a specific antibody to distinguish each isoform in the study. We constructed and established HEK 293 cell lines that constitutively express each isoform. Using these cell lines, we determined the specificity of antibodies (both commercially available and laboratory-made) to each isoform by Western blot and immunofluorescence. Some of the antibodies showed specificity in Western blotting but were not applicable in immunofluorescence. Others showed cross reactivity in Western blot assays. Our results indicate that RELM antibody specificity should be taken into account when using them in research and interpreting data obtained with them.

Naphthyridinone (NTD) integrase inhibitors: N1 protio and methyl combination substituent effects with C3 amide groups.

Substituent effects of a series of N1 protio and methyl naphthyridinone HIV-1 integrase strand-transfer inhibitors has been explored. The effects of combinations of the N1 substituent and C3 amide groups was extensively studied to compare enzyme inhibition, antiviral activity and protein binding effects on potency. The impact of substitution on ligand efficiency was considered and several compounds were advanced into in vivo pharmacokinetic studies ultimately leading to the clinical candidate GSK364735.

Human cardiovascular disease model predicts xanthine oxidase inhibitor cardiovascular risk.

Some health concerns are often not identified until late into clinical development of drugs, which can place participants and patients at significant risk. For example, the United States Food and Drug Administration (FDA) labeled the xanthine oxidase inhibitor febuxostat with a"boxed" warning regarding an increased risk of cardiovascular death, and this safety risk was only identified during Phase 3b clinical trials after its approval. Thus, better preclinical assessment of drug efficacy and safety are needed to accurately evaluate candidate drug risk earlier in discovery and development. This study explored whether an in vitro vascular model incorporating human vascular cells and hemodynamics could be used to differentiate the potential cardiovascular risk associated with molecules that have similar on-target mechanisms of action. We compared the transcriptomic responses induced by febuxostat and other xanthine oxidase inhibitors to a database of 111 different compounds profiled in the human vascular model. Of the 111 compounds in the database, 107 are clinical-stage and 33 are FDA-labelled for increased cardiovascular risk. Febuxostat induces pathway-level regulation that has high similarity to the set of drugs FDA-labelled for increased cardiovascular risk. These results were replicated with a febuxostat analog, but not another structurally distinct xanthine oxidase inhibitor that does not confer cardiovascular risk. Together, these data suggest that the FDA warning for febuxostat stems from the chemical structure of the medication itself, rather than the target, xanthine oxidase. Importantly, these data indicate that cardiovascular risk can be evaluated in this in vitro human vascular model, which may facilitate understanding the drug candidate safety profile earlier in discovery and development.

Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

In Vitro antiretroviral properties of S/GSK1349572, a next-generation HIV integrase inhibitor.

S/GSK1349572 is a next-generation HIV integrase (IN) inhibitor designed to deliver potent antiviral activity with a low-milligram once-daily dose requiring no pharmacokinetic (PK) booster. In addition, S/GSK1349572 demonstrates activity against clinically relevant IN mutant viruses and has potential for a high genetic barrier to resistance. S/GSK1349572 is a two-metal-binding HIV integrase strand transfer inhibitor whose mechanism of action was established through in vitro integrase enzyme assays, resistance passage experiments, activity against viral strains resistant to other classes of anti-HIV agents, and mechanistic cellular assays. In a variety of cellular antiviral assays, S/GSK1349572 inhibited HIV replication with low-nanomolar or subnanomolar potency and with a selectivity index of 9,400. The protein-adjusted half-maximal effective concentration (PA-EC(50)) extrapolated to 100% human serum was 38 nM. When virus was passaged in the presence of S/GSK1349572, highly resistant mutants were not selected, but mutations that effected a low fold change (FC) in the EC(50) (up to 4.1 fold) were identified in the vicinity of the integrase active site. S/GSK1349572 demonstrated activity against site-directed molecular clones containing the raltegravir-resistant signature mutations Y143R, Q148K, N155H, and G140S/Q148H (FCs, 1.4, 1.1, 1.2, and 2.6, respectively), while these mutants led to a high FC in the EC(50) of raltegravir (11- to >130-fold). Either additive or synergistic effects were observed when S/GSK1349572 was tested in combination with representative approved antiretroviral agents; no antagonistic effects were seen. These findings demonstrate that S/GSK1349572 would be classified as a next-generation drug in the integrase inhibitor class, with a resistance profile markedly different from that of first-generation integrase inhibitors.

Combining symmetry elements results in potent naphthyridinone (NTD) HIV-1 integrase inhibitors.

A series of naphthyridinone HIV-1 integrase strand-transfer inhibitors have been designed based on a psdeudo-C2 symmetry element present in the two-metal chelation pharmacophore. A combination of two distinct inhibitor binding modes resulted in potent inhibition of the integrase strand-transfer reaction in the low nM range. Effects of aryl and N1 substitutions are disclosed including the impact on protein binding adjusted antiviral activity.

Identification of 2,2-Dimethylbutanoic Acid (HST5040), a Clinical Development Candidate for the Treatment of Propionic Acidemia and Methylmalonic Acidemia.

Propionic acidemia (PA) and methylmalonic acidemia (MMA) are rare autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, caused by a deficiency in the enzymes P-CoA carboxylase and methylmalonyl-CoA (M-CoA) mutase, respectively. PA and MMA are classified as intoxication-type inborn errors of metabolism because the intramitochondrial accumulation of P-CoA, M-CoA, and other metabolites results in secondary inhibition of multiple pathways of intermediary metabolism, leading to organ dysfunction and failure. Herein, we describe the structure-activity relationships of a series of short-chain carboxylic acids which reduce disease-related metabolites in PA and MMA primary hepatocyte disease models. These studies culminated in the identification of 2,2-dimethylbutanoic acid (10, HST5040) as a clinical candidate for the treatment of PA and MMA. Additionally, we describe the in vitro and in vivo absorption, distribution, metabolism, and excretion profile of HST5040, data from preclinical studies, and the synthesis of the sodium salt of HST5040 for clinical trials.

Pharmacovirological impact of an integrase inhibitor on human immunodeficiency virus type 1 cDNA species in vivo.

Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients.

A Phase 1 Study to Assess the Relative Bioavailability, Food Effect, and Safety of a Tablet Formulation of GSK2838232, a Novel HIV Maturation Inhibitor in Healthy Participants After Single and Repeated Doses.

GSK2838232 is a novel, potent HIV-1 maturation inhibitor for use in regimen-based combination antiretroviral therapy from a once-daily oral dose boosted with a pharmacoenhancer (ritonavir or cobicistat). This phase 1 study in healthy participants was conducted in 2 parts. Part 1 (n = 14) assessed the relative bioavailability of single doses of a 200-mg GSK2838232 tablet and capsule formulation boosted with 100 mg ritonavir in fed and fasted (tablet-only) subjects. Part 2 (n = 10) assessed the pharmacokinetics of repeated 500-mg once-daily doses of GSK2838232 without a pharmacoenhancing boosting agent. In part 1, GSK2838232 demonstrated comparable bioavailability following a single dose of 200 mg GSK2838232 as capsule and tablet formulations in combination with ritonavir (RTV) under fed conditions, with lower intrasubject variability observed for the tablet formulation. In part 2, following administration of 500 mg GSK2838232 once daily for 11 days under fed conditions, Cmax , AUC0-τ , and Cτ showed a small degree of accumulation (1.2- to 1.3-fold) of GSK2838232. The median tmax was approximately 4 hours on both day 1 and day 11 when given with food. The mean t½ was approximately 23 hours on day 11. Steady-state concentrations were achieved by day 3 with a geometric mean steady-state Cτ on day 11 of 28 ng/mL. The tablet formulation was generally well tolerated as a single 200-mg dose with RTV under fed and fasted conditions and following administration of multiple daily doses (11 days) of 500 mg unboosted.

CTLA-4 and PD-1 dual blockade induces SIV reactivation without control of rebound after antiretroviral therapy interruption.

The primary human immunodeficiency virus (HIV) reservoir is composed of resting memory CD4+ T cells, which often express the immune checkpoint receptors programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which limit T cell activation via synergistic mechanisms. Using simian immunodeficiency virus (SIV)-infected, long-term antiretroviral therapy (ART)-treated rhesus macaques, we demonstrate that PD-1, CTLA-4 and dual CTLA-4/PD-1 immune checkpoint blockade using monoclonal antibodies is well tolerated, with evidence of bioactivity in blood and lymph nodes. Dual blockade was remarkably more effective than PD-1 blockade alone in enhancing T cell cycling and differentiation, expanding effector-memory T cells and inducing robust viral reactivation in plasma and peripheral blood mononuclear cells. In lymph nodes, dual CTLA-4/PD-1 blockade, but not PD-1 alone, decreased the total and intact SIV-DNA in CD4+ T cells, and SIV-DNA and SIV-RNA in B cell follicles, a major site of viral persistence during ART. None of the tested interventions enhanced SIV-specific CD8+ T cell responses during ART or viral control after ART interruption. Thus, despite CTLA-4/PD-1 blockade inducing robust latency reversal and reducing total levels of integrated virus, the degree of reservoir clearance was still insufficient to achieve viral control. These results suggest that immune checkpoint blockade regimens targeting PD-1 and/or CTLA-4, if performed in people living with HIV with sustained aviremia, are unlikely to induce HIV remission in the absence of additional interventions.