Systemic inflammation in pregnant women with HIV: relationship with HIV treatment regimen and preterm delivery.

OBJECTIVE: : HIV treatment regimen during pregnancy was associated with preterm delivery (PTD) in the PROMISE 1077 BF trial. Systemic inflammation among pregnant women with HIV could help explain differences in PTD by treatment regimen. We assessed associations between inflammation, treatment regimen, and PTD. DESIGN/METHODS: : A nested 1:1 case-control study (N = 362) was conducted within a multi-country randomized trial comparing three HIV regimens in pregnant women: zidovudine alone, or combination antiretroviral therapy (ART) with lopinavir/ritonavir and either zidovudine or tenofovir. Cases were women with PTD (<37 weeks of gestational age). The following inflammatory biomarkers were measured in plasma samples using immunoassays: soluble CD14 (sCD14) and sCD163, intestinal fatty acid-binding protein, interleukin (IL)-6, interferon γ, and tumor necrosis factor α. We fit regression models to assess associations between second trimester biomarkers (measured before ART initiation at 13-23 weeks of gestational age and 4 weeks later), treatment regimen, and PTD. We also assessed whether inflammation was a mediator in the relationship between ART regimen and PTD. RESULTS: : Persistently high interleukin-6 was associated with increased PTD. Compared to zidovudine alone, the difference in biomarker concentration between week 0 and week 4 was significantly higher (p < 0.05) for both PI-based regimens. However, the estimated proportion of the ART effect on increased PTD mediated by persistently high biomarker levels was ≤5% for all biomarkers. CONCLUSIONS: : Persistently high IL-6 during pregnancy was associated with PTD. While PI-based ART was associated with increases in inflammation, factors other than inflammation likely explain the increased PTD in ART-based regimens compared to zidovudine alone.

Association of Soluble Markers of Inflammation With Peri-coronary Artery Inflammation in People With and Without HIV Infection and Without Cardiovascular Disease.

Background: Inflammation is linked to elevated cardiovascular disease (CVD) risk in people with HIV (PWH) on antiretroviral therapy (ART). Fat attenuation index (FAI) is a measure of peri-coronary inflammation that independently predicts CVD risk in HIV-uninfected persons. Whether FAI is associated with soluble inflammatory markers is unknown. Methods: Plasma levels of inflammatory markers were measured in 58 PWH and 16 controls without current symptoms or prior known CVD who underwent coronary computed tomography angiography and had FAI measurements. A cross-sectional analysis was performed, and associations of markers with FAI values of the right coronary artery (RCA) and left anterior descending artery (LAD) were assessed using multivariable regression models adjusted for the potential confounders age, sex, race, low-density lipoprotein cholesterol, body mass index, and use of lipid-lowering medication. Results: Several inflammatory markers had significant associations with RCA or LAD FAI in adjusted models, including sCD14, sCD163, TNFR-I, and TNFR-II, CCL5, CX3CL1, IP-10. Conclusions: The associations between indices of systemic and peri-coronary inflammation are novel and suggest that these systemic markers and FAI together are promising noninvasive biomarkers that can be applied to assess asymptomatic CVD in people with and without HIV; they also may be useful tools to evaluate effects of anti-inflammatory interventions.

Interleukin 6 Blockade With Tocilizumab Diminishes Indices of Inflammation That Are Linked to Mortality in Treated Human Immunodeficiency Virus Infection.

BACKGROUND: People with human immunodeficiency virus (PWH) are at increased risk for comorbidities, and plasma interleukin 6 (IL-6) levels are among the most robust predictors of these outcomes. Tocilizumab (TCZ) blocks the receptor for IL-6, inhibiting functions of this cytokine. METHODS: This was a 40-week, placebo-controlled, crossover trial (NCT02049437) where PWH on stable antiretroviral therapy (ART) were randomized to receive 3 monthly doses of TCZ or matching placebo intravenously. Following a 10-week treatment period and a 12-week washout, participants were switched to the opposite treatment. The primary endpoints were safety and posttreatment levels of C-reactive protein (CRP) and CD4+ T-cell cycling. Secondary endpoints included changes in inflammatory indices and lipid levels. RESULTS: There were 9 treatment-related toxicities of grade 2 or greater during TCZ administration (mostly neutropenia) and 2 during placebo administration. Thirty-one of 34 participants completed the study and were included in a modified intent-to-treat analysis. TCZ reduced levels of CRP (median decrease, 1819.9 ng/mL, P < .0001; effect size, 0.87) and reduced inflammatory markers in PWH, including D-dimer, soluble CD14, and tumor necrosis factor receptors. T-cell cycling tended to decrease in all maturation subsets after TCZ administration, but was only significant among naive CD4 T cells. Lipid levels, including lipid classes that have been related to cardiovascular disease risk, increased during TCZ treatment. CONCLUSIONS: TCZ is safe and decreases inflammation in PWH; IL-6 is a key driver of the inflammatory environment that predicts morbidity and mortality in ART-treated PWH. The clinical significance of lipid elevations during TCZ treatment requires further study. Clinical Trials Registration. NCT02049437.

HIV post-treatment controllers have distinct immunological and virological features.

HIV post-treatment controllers (PTCs) are rare individuals who maintain low levels of viremia after stopping antiretroviral therapy (ART). Understanding the mechanisms of HIV post-treatment control will inform development of strategies aiming at achieving HIV functional cure. In this study, we evaluated 22 PTCs from 8 AIDS Clinical Trials Group (ACTG) analytical treatment interruption (ATI) studies who maintained viral loads ≤400 copies/mL for ≥24 wk. There were no significant differences in demographics or frequency of protective and susceptible human leukocyte antigen (HLA) alleles between PTCs and post-treatment noncontrollers (NCs, n = 37). Unlike NCs, PTCs demonstrated a stable HIV reservoir measured by cell-associated RNA (CA-RNA) and intact proviral DNA assay (IPDA) during analytical treatment interruption (ATI). Immunologically, PTCs demonstrated significantly lower CD4+ and CD8+ T cell activation, lower CD4+ T cell exhaustion, and more robust Gag-specific CD4+ T cell responses and natural killer (NK) cell responses. Sparse partial least squares discriminant analysis (sPLS-DA) identified a set of features enriched in PTCs, including a higher CD4+ T cell% and CD4+/CD8+ ratio, more functional NK cells, and a lower CD4+ T cell exhaustion level. These results provide insights into the key viral reservoir features and immunological profiles for HIV PTCs and have implications for future studies evaluating interventions to achieve an HIV functional cure.

Association of Cytomegalovirus Serostatus With Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine Responsiveness in Nursing Home Residents and Healthcare Workers.

Background: Latent cytomegalovirus (CMV) infection is immunomodulatory and could affect mRNA vaccine responsiveness. We sought to determine the association of CMV serostatus and prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with antibody (Ab) titers after primary and booster BNT162b2 mRNA vaccinations in healthcare workers (HCWs) and nursing home (NH) residents. Methods: Nursing home residents (N = 143) and HCWs (N = 107) were vaccinated and serological responses monitored by serum neutralization activity against Wuhan and Omicron (BA.1) strain spike proteins, and by bead-multiplex immunoglobulin G immunoassay to Wuhan spike protein and its receptor-binding domain (RBD). Cytomegalovirus serology and levels of inflammatory biomarkers were also measured. Results: Severe acute respiratory syndrome coronavirus 2-naive CMV seropositive (CMV+) HCWs had significantly reduced Wuhan-neutralizing Ab (P = .013), anti-spike (P = .017), and anti-RBD (P = .011) responses 2 weeks after primary vaccination series compared with responses among CMV seronegative (CMV-) HCWs, adjusting for age, sex, and race. Among NH residents without prior SARS-CoV-2 infection, Wuhan-neutralizing Ab titers were similar 2 weeks after primary series but were reduced 6 months later (P = .012) between CMV+ and CMV- subjects. Wuhan-neutralizing Ab titers from CMV+ NH residents who had prior SARS-CoV-2 infection consistently trended lower than titers from SARS-CoV-2 experienced CMV- donors. These impaired Ab responses in CMV+ versus CMV- individuals were not observed after booster vaccination or with prior SARS-CoV-2 infection. Conclusions: Latent CMV infection adversely affects vaccine-induced responsiveness to SARS-CoV-2 spike protein, a neoantigen not previously encountered, in both HCWs and NH residents. Multiple antigenic challenges may be required for optimal mRNA vaccine immunogenicity in CMV+ adults.

Pro-inflammatory and pro-resolving lipid mediators of inflammation in HIV: effect of aspirin intervention.

BACKGROUND: Persons with HIV (PWH) have an increased risk of cardiovascular disease (CVD) compared to HIV-seronegative individuals (SN). Inflammation contributes to this risk but the role of lipid mediators, with central roles in inflammation, in HIV infection remain to be established; further aspirin reduces CVD risk in the general population through production of some of these anti-inflammatory lipid mediators, but they have not been studied in PWH. METHODS: We evaluated the relationship between plasma lipid mediators (i.e. 50 lipid mediators including classic eicosanoids and specialized pro-resolving mediators (SPMs)) and HIV status; and the impact of aspirin in PWH on regulating these autacoids. Plasma samples were obtained from 110 PWH receiving antiretroviral therapy (ART) from a randomized trial of aspirin (ACTG-A5331) and 107 matched SN samples (MACS-WIHS Combined Cohort). FINDINGS: PWH had lower levels of arachidonic acid-derived pro-inflammatory prostaglandins (PGs: PGE2 and PGD2) and thromboxanes (Tx: TxB2), and higher levels of select pro-resolving lipid mediators (e.g. RvD4 and MaR2n-3 DPA) compared to SN. At the interval tested, aspirin intervention was observed to reduced PGs and Tx, and while we did not observe an increase in aspirin triggered mediators, we observed the upregulation of other SPM in aspirin treated PWH, namely MaR2n-3 DPA. INTERPRETATION: Together these observations demonstrate that plasma lipid mediators profiles, some with links to systemic inflammation and CVD risk, become altered in PWH. Furthermore, aspirin intervention did not increase levels of aspirin-triggered pro-resolving lipid mediators, consistent with other reports of an impaired aspirin response in PWH. FUNDING: NIH.

Methotrexate Inhibits T Cell Proliferation but Not Inflammatory Cytokine Expression to Modulate Immunity in People Living With HIV.

Inflammation associated with increased risk of comorbidities persists in people living with HIV (PWH) on combination antiretroviral therapy (ART). A recent placebo-controlled trial of low-dose methotrexate (MTX) in PWH found that numbers of total CD4 and CD8 T cells decreased in the low-dose MTX arm. In this report we analyzed T cell phenotypes and additional plasma inflammatory indices in samples from the trial. We found that cycling (Ki67+) T cells lacking Bcl-2 were reduced by MTX but plasma inflammatory cytokines were largely unaffected. In a series of in vitro experiments to further investigate the mechanisms of MTX activity, we found that MTX did not inhibit effector cytokine production but inhibited T cell proliferation downstream of mTOR activation, mitochondrial function, and cell cycle entry. This inhibitory effect was reversible with folinic acid, suggesting low-dose MTX exerts anti-inflammatory effects in vivo in PWH largely by blocking T cell proliferation via dihydrofolate reductase inhibition, yet daily administration of folic acid did not rescue this effect in trial participants. Our findings identify the main mechanism of action of this widely used anti-inflammatory medicine in PWH and may provide insight into how MTX works in the setting of other inflammatory conditions.

Randomized Trial of Ruxolitinib in Antiretroviral-Treated Adults With Human Immunodeficiency Virus.

BACKGROUND: Inflammation is associated with end-organ disease and mortality for people with human immunodeficiency virus (PWH). Ruxolitinib, a Jak 1/2 inhibitor, reduces systemic inflammation for individuals without human immunodeficiency virus (HIV) and HIV reservoir markers ex vivo. The goal of this trial was to determine safety and efficacy of ruxolitinib for PWH on antiretroviral therapy (ART). METHODS: AIDS Clinical Trials Group (ACTG) A5336 was an open-label, multisite, randomized controlled trial (RCT). Participants were randomly assigned (2:1) using centralized software to ruxolitinib (10 mg twice daily) plus stable ART for 5 weeks vs ART alone, stratified by efavirenz use. Eligible participants were suppressed on ART for ≥2 years, without comorbidities, and had >350 CD4+ T cells/µL. Primary endpoints were premature discontinuation, safety events, and change in plasma interleukin 6 (IL-6). Secondary endpoints included other measures of inflammation/immune activation and HIV reservoir. RESULTS: Sixty participants were enrolled from 16 May 2016 to 10 January 2018. Primary safety events occurred in 2.5% (1 participant) for ruxolitinib and 0% for controls (P = .67). Three participants (7.5%) prematurely discontinued ruxolitinib. By week 5, differences in IL-6 (mean fold change [FC], 0.93 vs 1.10; P = .18) and soluble CD14 (mean FC, 0.96 vs 1.08; relative FC, 0.96 [90% confidence interval {CI}, .90-1.02]) levels for ruxolitinib vs controls was observed. Ruxolitinib reduced CD4+ T cells expressing HLA-DR/CD38 (mean difference, -0.34% [90% CI, -.66% to -.12%]) and Bcl-2 (mean difference, -3.30% [90% CI, -4.72% to -1.87%]). CONCLUSIONS: In this RCT of healthy, virologically suppressed PWH on ART, ruxolitinib was well-tolerated. Baseline IL-6 levels were normal and showed no significant reduction. Ruxolitinib significantly decreased markers of immune activation and cell survival. Future studies of Jak inhibitors should target PWH with residual inflammation despite suppressive ART. CLINICAL TRIALS REGISTRATION: NCT02475655.

Changes in Immune Activation During Pregnancy and the Postpartum Period in Treated HIV Infection.

BACKGROUND: Pregnant women with HIV (PWWH) have high postpartum morbidity and mortality from infections like tuberculosis. Immunologic changes during pregnancy and postpartum periods may contribute to these risks, particularly the immunoregulatory kynurenine pathway of tryptophan catabolism, which contributes to both HIV and tuberculosis pathogenesis and increases in the early postpartum period. METHODS: Women with HIV initiating antiretroviral therapy (ART) in the Uganda AIDS Rural Treatment Outcomes (UARTO) cohort who were pregnant at enrollment or became pregnant during observation were studied (n = 54). Plasma kynurenine/tryptophan (KT) ratio, soluble CD14 (sCD14), sCD163, sCD27, interferon-inducible protein 10 (IP-10), D-dimer, interleukin-6, and intestinal fatty-acid binding protein levels were assessed through the first year of ART and at 3-month intervals throughout pregnancy and 1 year postpartum. Biomarker changes were assessed with linear mixed models adjusted for ART duration. Hemoglobin concentration changes were used to estimate pregnancy-related changes in plasma volume. RESULTS: The median pre-ART CD4 count was 134. D-dimer increased through the third trimester before returning to baseline postpartum, while most other biomarkers declined significantly during pregnancy, beyond what would be expected from pregnancy-associated plasma volume expansion. IP-10 and sCD14 remained suppressed for at least 12 months postpartum. KT ratio was the only biomarker that increased above prepregnancy baseline postpartum (mean + 30%; P < .001) and remained higher than baseline for ≥9 months (P ≤ .045 for all time points). CONCLUSIONS: Several immune activation markers decline during pregnancy and remain suppressed postpartum, but the kynurenine pathway of tryptophan catabolism increases above baseline for ≥9 months postpartum. The mechanisms underlying postpartum kynurenine pathway activity are incompletely understood but may contribute to increased tuberculosis risk in this setting.

Markers of inflammation and immune activation are associated with lung function in a multi-center cohort of persons with HIV.

OBJECTIVES: Studies have shown that people with HIV (PWH) may be at increased risk for chronic lung diseases and lung function abnormalities, which may be associated with immune activation. We tested the association of a panel of 12 immune activation and inflammation biomarkers with spirometry and single-breath diffusing capacity for carbon monoxide (DLco). DESIGN: Cross-sectional, observational study. METHODS: Participants were enrolled from the Inflammation, Aging, Microbes and Obstructive Lung Disease cohort of PWH at two US sites. Biomarkers were examined and standardized spirometry and DLco testing were performed. We tested associations between each biomarker and lung function, examined individually and in combination, using multi-variable linear and logistic regression. RESULTS: Among 199 participants, median forced expiratory volume in 1 s (FEV1) was normal (90% predicted) and median DLco was abnormal (69% predicted). The most common lung function abnormality (57%) was a normal FEV1 to forced vital capacity ratio with an abnormal DLco of 80% or less predicted (iso↓DLco). Two markers (IL-6, high-sensitivity C-reactive protein) were associated with FEV1% predicted, whereas eight markers (soluble CD14, soluble CD163, inducible protein-10, soluble CD27, IL-6, soluble tumor necrosis factor receptors 1 and 2, D-dimer) were associated with DLco% predicted. Compared with those participants with normal spirometry and DLco, five markers (soluble CD14, soluble CD163, interferon gamma inducible protein-10, soluble tumor necrosis factor receptors 1 and 2) were associated with iso↓DLco. CONCLUSION: Among PWH, different markers of immune activation and inflammation are associated with FEV1% predicted than with DLco% predicted and with an iso↓DLco, representing possible unique pathways of chronic lung disease. Identifying plausible drivers of these inflammatory pathways may clarify mechanisms underlying impaired lung function in HIV infection and may identify therapeutic avenues.

Pneumoproteins are associated with pulmonary function in HIV-infected persons.

BACKGROUND: COPD is a common HIV comorbidity, and HIV-infected individuals have a higher incidence and earlier onset of COPD compared to HIV-uninfected individuals. While the pathogenesis of HIV-associated COPD is largely unknown, chronic inflammation may contribute. Four pneumoproteins known to be markers of lung injury and inflammation have been associated with COPD in HIV-uninfected individuals: PARC/CCL-18, SP-D, CC-16, and sRAGE. OBJECTIVE: To determine whether these pneumoproteins are also associated with pulmonary function and COPD Assessment Test (CAT) scores in HIV-infected individuals. METHODS: Associations between plasma pneumoprotein levels and pulmonary function were determined in a cross-sectional study of otherwise healthy HIV-infected individuals enrolled between September 2016 and June 2017. Covariates included HIV-associated (antiretroviral therapy, CD4 count, and viral load) and COPD-associated (smoking and BMI) covariates. RESULTS: Among 65 participants, 78.5% were male, 50.8% had undetectable viral load, and 76.9% were ever-smokers. Mean post-bronchodilator FEV1/FVC was 0.71, and mean DLco%predicted was 61%. Higher PARC/CCL-18 was associated with lower DLco%predicted and higher CAT score. Higher CC-16 was associated with lower DLco%predicted and lower FVC%predicted. CONCLUSIONS: This exploratory analysis is the first to characterize associations between these four pneumoproteins and pulmonary function in an HIV-infected cohort. Our findings suggest the pathogenesis of HIV-associated COPD may differ from that of non-HIV-associated COPD due to HIV-specific inflammatory changes affecting DLco. PARC/CCL-18 is associated with structural and functional pulmonary abnormalities and may be an important COPD biomarker candidate in HIV infection. Our study is a preliminary step toward finding clinically relevant COPD biomarkers in high-risk populations.

HIV infection is associated with elevated biomarkers of immune activation in Ugandan adults with pneumonia.

INTRODUCTION: Pneumonia is an important cause of morbidity and mortality in persons living with human immunodeficiency virus (HIV) infection. How immune activation differs among HIV-infected and HIV-uninfected adults with pneumonia is unknown. METHODS: The Inflammation, Aging, Microbes, and Obstructive Lung Disease (I AM OLD) Cohort is a prospective cohort of adults with pneumonia in Uganda. In this cross-sectional analysis, plasma was collected at pneumonia presentation to measure the following 12 biomarkers: interleukin 6 (IL-6), soluble tumor necrosis factor receptors 1 and 2 (sTNFR-1 and sTNFR-2), high sensitivity C-reactive protein (hsCRP), fibrinogen, D-dimer, soluble CD27 (sCD27), interferon gamma-inducible protein 10 (IP-10), soluble CD14 (sCD14), soluble CD163 (sCD163), hyaluronan, and intestinal fatty acid binding protein. We asked whether biomarker levels differed between HIV-infected and HIV-uninfected participants, and whether higher levels of these biomarkers were associated with mortality. RESULTS: One hundred seventy-three participants were enrolled. Fifty-three percent were HIV-infected. Eight plasma biomarkers-sTNFR-1, sTNFR-2, hsCRP, D-dimer, sCD27, IP-10, sCD14, and hyaluronan-were higher among participants with HIV infection, after adjustment for pneumonia severity. Higher levels of 8 biomarkers-IL-6, sTNFR-1, sTNFR-2, hsCRP, IP-10, sCD14, sCD163, and hyaluronan-were associated with increased 2-month mortality. CONCLUSIONS: As in other clinical contexts, HIV infection is associated with a greater degree of immune activation among Ugandan adults with pneumonia. Some of these are also associated with short-term mortality. Further study is needed to explore whether these biomarkers might predict poor long-term outcomes-such as the development of obstructive lung disease-in patients with HIV who have recovered from pneumonia.

Soluble Urokinase Plasminogen Activator Receptor Is Predictive of Non-AIDS Events During Antiretroviral Therapy-mediated Viral Suppression.

BACKGROUND: Despite effective antiretroviral therapy (ART), human immunodeficiency virus (HIV) infection remains associated with higher morbidity and mortality, driven, in part, by increased inflammation. Our objective was to identify associations between levels of plasma biomarkers of chronic inflammation, microbial translocation, and monocyte activation, with occurrence of non-AIDS events. METHODS: Participants (141 cases, 310 matched controls) were selected from a longitudinal observational trial; all were virally suppressed on ART at year 1 and thereafter. Soluble urokinase plasminogen activator receptor (suPAR), lipopolysaccharide binding protein (LBP), beta-D-glucan (BDG), intestinal fatty-acid binding protein, oxidized low-density lipoproteins, and soluble CD163 were measured pre-ART, after 1-year of ART, and pre-event. At each time point, conditional logistic regression analysis assessed associations of the biomarkers with events and adjusted for relevant covariates to calculate odds ratios (ORs) according to 1 interquartile range (IQR) difference. RESULTS: At all time points, higher levels of suPAR were associated with increased risk of non-AIDS events (OR per 1 IQR was 1.7 before ART-initiation, OR per 1 IQR was 2.0 after 1 year of suppressive ART, and OR 2.1 pre-event). Higher levels of BDG and LBP at year 1 and pre-event (but not at baseline) were associated with increased risk of non-AIDS events. No associations were observed for other biomarkers. CONCLUSIONS: Elevated levels of suPAR were strongly, consistently, and independently predictive of non-AIDS events at every measured time point. Interventions that target the suPAR pathway should be investigated to explore its role in the pathogenesis of non-AIDS-related outcomes in HIV infection.

A Randomized Placebo Controlled Trial of Aspirin Effects on Immune Activation in Chronically Human Immunodeficiency Virus-Infected Adults on Virologically Suppressive Antiretroviral Therapy.

BACKGROUND: Immune activation persists despite suppressive antiretroviral therapy (ART) in human immunodeficiency virus (HIV) infection and predicts non-Acquired Immune Deficiency Syndrome (AIDS) comorbidities including cardiovascular disease. Activated platelets play a key role in atherothrombosis and inflammation, and platelets are hyperactivated in chronic HIV infection. Aspirin is a potent inhibitor of platelet activation through the cyclooxygenase-1 (COX-1) pathway. We hypothesized that platelet activation contributes to immune activation and that aspirin would reduce immune activation and improve endothelial function in ART-suppressed HIV-infected individuals. METHODS: In this prospective, double-blind, randomized, placebo-controlled 3-arm trial of 121 HIV-infected participants on suppressive ART for >48 weeks, we evaluated the effects of 12 weeks of daily aspirin 100 mg, aspirin 300 mg, or placebo on soluble and cellular immune activation markers, flow-mediated dilation (FMD) of the brachial artery, and serum thromboxane B2, a direct readout of platelet COX-1 inhibition. RESULTS: The 300-mg and 100-mg aspirin arms did not differ from placebo in effects on soluble CD14, interleukin (IL)-6, soluble CD163, D-dimer, T-cell or monocyte activation, or the other immunologic endpoints measured. Endothelial function, as measured by FMD, also was not significantly changed when comparing the 300-mg and 100-mg aspirin arms to placebo. CONCLUSIONS: Aspirin treatment for 12 weeks does not have a major impact on soluble CD14, IL-6, soluble CD163, D-dimer, T-cell or monocyte activation, or FMD, suggesting that inhibition of COX-1-mediated platelet activation does not significantly improve HIV-related immune activation and endothelial dysfunction. Although future studies are needed to further identify the causes and consequences of platelet activation in ART-treated HIV infection, interventions other than COX-1 inhibition will need to be explored to directly reduce immune activation in treated HIV infection.

Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in Simian Immunodeficiency Virus SIVmac239.

UNLABELLED: We previously showed that the simian immunodeficiency virus SIVmac239 is susceptible to human immunodeficiency virus (HIV) integrase (IN) strand transfer inhibitors (INSTIs) and that the same IN drug resistance mutations result in similar phenotypes in both viruses. Now we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 wild-type (WT) and E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q T97A, E92Q Y143R, R263K H51Y, and G140S Q148R recombinant substitution-containing IN enzymes were produced, and each of the characteristics strand transfer, 3'-processing activity, and INSTI inhibitory constants was assessed in cell-free assays. The results show that the G118R and G140S Q148R substitutions decreased Km' and Vmax'/Km' for strand transfer compared to those of the WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q Y143R, and G140S Q148R. Both viruses and enzymes containing Q148R and G140S Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV. IMPORTANCE: Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV integrase resistance-associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.

The dolutegravir R263K resistance mutation in HIV-1 integrase is incompatible with the emergence of resistance against raltegravir.

OBJECTIVE: Although the integrase inhibitor dolutegravir (DTG) has demonstrated greater resilience than other antiretroviral drugs at withstanding the emergence of HIV-1 resistance mutations, such substitutions can develop, albeit rarely, in treatment-experienced integrase inhibitor-naïve individuals. The most common substitution in integrase under those circumstances is R263K whereas another substitution that was selected against DTG in tissue culture was G118R. The objective of this study was to determine the effects of these DTG-specific resistance substitutions on the ability of HIV-1 to become resistant against either of two other integrase inhibitors, raltegravir (RAL) and elvitegravir (EVG). DESIGN AND METHODS: We performed tissue culture selection experiments using DTG-resistant viruses containing integrase substitutions at positions R263K, H51Y/R263K, E138K/R263K, G118R and H51Y/G118R in the presence of increasing concentrations of either RAL or EVG. Changes in integrase sequences were monitored by genotyping. RESULTS: The presence of the R263K substitution delayed the emergence of resistance against RAL whereas the simultaneous presence of either the H51Y or E138K secondary substitutions in combination with R263K somewhat mitigated this inhibitory effect. In contrast, resistance against EVG appeared earlier than in wild-type virus in viruses containing the R263K and E138K/R263K DTG-associated resistance substitutions. CONCLUSION: The DTG-resistant R263K substitution antagonized the development of HIV-1 resistance against RAL while partially facilitating the occurrence of resistance against EVG.

HIV-1 Group O Resistance Against Integrase Inhibitors.

BACKGROUND: HIV-1 group O (HIV-O) is a rare variant that is characterized by a high number of natural polymorphisms in the integrase coding region that may impact on susceptibility to integrase strand transfer inhibitors (INSTIs) and on the emergence of resistance substitutions. We previously reported that HIV-O is more susceptible to RAL than HIV-1 group M (HIV-M). METHODS: The aim of this study was to assess pathways of resistance to INSTIs in group 0 variants. Accordingly, we selected for resistance to each of raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) in cord blood mononuclear cells using HIV group O subtypes A and B, an HIV-O divergent isolate, and HIV-1 group M (subtype B, which served as a reference). Site-directed mutagenesis was performed on the pCOM2.5 HIV group 0 infectious clone to ascertain the impact of INSTI resistance substitutions at positions Q148R, N155H, and R263K within integrase on susceptibility to INSTIs and infectiousness. RESULTS: Cell culture selections of group O variants yielded similar patterns of resistance to RAL, EVG, and DTG as observed for subtype B. In the DTG selections, subtype B yielded S153Y, whereas a natural S153A polymorphism sometimes led to A153V in group O. The pCMO2.5/Q148R and pCMO2.5/N155H variants displayed far higher levels of resistance to DTG (>1000 FC) than was seen for group M viruses. CONCLUSIONS: HIV-O harboring Q148R and N155H shows higher resistance to DTG compared with HIV-M subtype B.

Dolutegravir inhibits HIV-1 Env evolution in primary human cells.

OBJECTIVE: In treatment-naive HIV-positive individuals, the integrase strand-transfer inhibitor dolutegravir (DTG) has not been associated with emergent drug-resistance mutations, neither against this drug nor against other antiretroviral drugs that were used in combination with it. This is in contrast to all other antiretroviral drugs tested so far, including the integrase strand-transfer inhibitors raltegravir (RAL) and elvitegravir that can lead to treatment failure with the emergence of drug-resistance mutations. DESIGN: These observations suggest that DTG may provide an additional protection against resistance compared to other drugs by decreasing HIV-1 genetic evolution. METHODS: Here, we tested this hypothesis by measuring the genetic and amino-acid diversity of Env/gp160 from two HIV-1 primary isolates that were grown in the presence of increasing concentrations of DTG or RAL over the course of 38-55 weeks. RESULTS: The results show that treatment with DTG led to less HIV-1 genetic and amino-acid diversification over time, as compared to treatment with RAL or the absence of drug. CONCLUSION: These results may help to explain the absence of emergent resistance mutations in treatment-naive individuals treated with DTG.

Evolution of a novel pathway leading to dolutegravir resistance in a patient harbouring N155H and multiclass drug resistance.

OBJECTIVES: Dolutegravir has been recently approved for treatment-naive and -experienced HIV-infected subjects, including integrase inhibitor (INI)-experienced patients. Dolutegravir is a second-generation INI that can overcome many prior raltegravir and elvitegravir failures. Here, we report the evolution of resistance to dolutegravir in a highly treatment-experienced patient harbouring the major N155H mutation consequent to raltegravir treatment failure. METHODS: Genotypic and phenotypic analyses were done on longitudinal samples to determine viral resistance to INIs. Integrase amino acid sequence interactions with raltegravir and dolutegravir were assessed by molecular modelling and docking simulations. RESULTS: Five mutations (A49P, L68FL, T97A, E138K and L234V) were implicated in emergent dolutegravir resistance, with a concomitant severe compromise in viral replicative capacity. Molecular modelling and docking simulations revealed that dolutegravir binding to integrase was affected by these acquired dolutegravir mutations. CONCLUSIONS: Our findings identify a novel mutational pathway involving integrase mutations A49P and L234V, leading to dolutegravir resistance in a patient with the N155H raltegravir mutation.

HIV-1 group O integrase displays lower susceptibility to raltegravir and has a different mutational pathway for resistance than HIV-1 group M.

INTRODUCTION: HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase gene, e.g. positions L74I, S153A, G163Q and T206S. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on susceptibility to integrase inhibitors and emergence of resistance associated mutations. Viruses and Methods: We cloned and purified integrase proteins from each of HIV-1 Group O clades A (HIV-O/A) and B (HIV-O/B), a HIV-O divergent strain (HIV-O/Div), and HIV-1 group M (subtype B, HIV-M/B) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of varying concentrations of several INSTIs. The inhibition constant (Ki) and IC50 were calculated and compared for HIV-M and HIV-O integrases. Selections for resistance-related mutations were performed using cord blood mononuclear cells and increasing concentration of INSTIs. RESULTS: HIV-O integrase and viruses were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. During selection, we observed different pathways of resistance depending on the drug and clade. Mutations selected in HIV-O can be classified as follows: (1) mutations described for HIV-M such as T97A, Q148R, V151A/I (RAL), T66I, E92Q, E157Q (EVG) and M50I, R263K (DTG) and (2) signature mutations for HIV-O (i.e. not described in HIV-M) F121C (HIV-O/B for RAL), V75I (HIV-O/A for RAL) and S153V (HIV-O/A for DTG). Only the HIV-O/Div selected the Q148R mutation for RAL and R263K+M50I for DTG, as previously described for HIV-M. None of the HIV-O viruses selected either N155H or Y143C. The selection of the specific S153V mutation could be explained at the nucleotide level: HIV-O at this position contains an alanine and substitution of alanine to valine (153AGGC→153VGTC) is easier than substitution of alanine to tyrosine (153AGGC→153YTAC), with only a transversion needed instead of one transition plus one transversion. CONCLUSIONS: This is the first report of susceptibility and resistance in vitro to INSTIs for HIV-O. Our study confirmed the impact of HIV-O polymorphism, on susceptibility to INSTIs and the emergence of resistance mutations.