N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus

AbstractThe native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120–gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B

A Phase 2b randomised, controlled, partially blinded trial of the HIV Nucleoside Reverse Transcriptase Inhibitor BMS-986001 (AI467003): Weeks 24 and 48 Efficacy, Safety, Bone and Metabolic Results

Background BMS-986001 is a thymidine analogue nucleoside reverse transcriptase inhibitor (NRTI) designed to maintain in-vitro antiviral activity while minimising off-target effects. We assessed the efficacy and safety of BMS-986001 versus tenofovir disoproxil fumarate in treatment-naive patients with HIV-1. Methods In this phase 2b, randomised, active-controlled trial (AI467003), we recruited treatment-naive (no current or previous exposure to an antiretroviral drug for >1 week) adults (aged at least 18 years) with HIV-1 from 47 sites across Asia, Australia, Europe, North America, South Africa, and South America. Patients with plasma HIV-1 RNA greater than 5000 copies per mL and CD4 counts greater than 200 cells per μL were randomly assigned (2:2:2:3) to receive BMS-986001 100 mg, 200 mg, or 400 mg once a day or to receive tenofovir disoproxil fumarate 300 mg once a day; each allocation was given with efavirenz 600 mg once a day and lamivudine 300 mg once a day. Both patients and investigators were masked to BMS-986001 dose (achieved with similar looking placebo tablets), but not allocation up to and including week 48. The primary endpoints were the proportion of patients with plasma HIV-1 RNA less than 50 copies per mL and safety events (serious adverse events and adverse events leading to discontinuation) through week 24; the main analysis was with a modified intention-to-treat population. Resistance analysis was a secondary endpoint, and additional safety parameters were exploratory endpoints. This trial is registered with ClinicalTrials.gov, number NCT01489046, and the European Clinical Trials Database, number EudraCT 2011-003329-89. Findings Patients were recruited between Jan 25, 2012, and Oct 3, 2012; 757 patients were assessed for eligibility and 301 were randomly assigned to receive either BMS-986001 once a day (67 patients to 100 mg, 67 to 200 mg, and 66 to 400 mg) or tenofovir disoproxil fumarate (n=101). 297 patients received at least one dose of study drug. At week 24, 57 (88%) of 65 patients for whom there were data in the 100 mg group, 54 (81%) of 67 in the 200 mg group, 62 (94%) of 66 in the 400 mg group achieved HIV-1 RNA less than 50 copies per mL, compared with 88 (89%) of 99 in the tenofovir disoproxil fumarate group (modified intention-to-treat population). BMS-986001 was generally well tolerated through week 48. Two patients had BMS-986001-related serious adverse events (atypical drug eruption and thrombocytopenia) and two in the tenofovir disoproxil fumarate group had study drug-related serious adverse events (potential drug-induced liver injury and depression or lipodystrophy) that led to discontinuation. NRTI resistance-associated mutations were reported in four (2%) of 198 patients, and non-NRTI mutations in 17 (9%) of 198 patients receiving BMS-986001 versus none of 99 and one (1%) of 99 patients receiving tenofovir disoproxil fumarate, respectively. Compared with tenofovir disoproxil fumarate, individuals in the BMS-986001 groups showed a smaller decrease in lumbar spine and hip bone mineral density but greater accumulation of limb and trunk fat, subcutaneous and visceral adipose tissue, and increased total cholesterol. Interpretation BMS-986001 had similar efficacy to that of tenofovir disoproxil fumarate and was associated with a smaller decrease in bone mineral density; however, greater resistance and gains in both peripheral and central fat accumulation were recorded for the investigational drug. Bristol-Myers Squibb has discontinued its involvement in the development of BMS-986001, and future decisions on development will be made by Oncolys BioPharma

Efficacy and safety of abatacept in active primary Sjogren's syndrome:results of a phase III, randomised, placebo-controlled trial

Objectives To evaluate efficacy and safety of abatacept in adults with active primary Sjogren's syndrome (pSS) in a phase III, randomised, double-blind, placebo-controlled trial. Methods Eligible patients (moderate-to-severe pSS [2016 ACR/European League Against Rheumatism (EULAR) criteria], EULAR Sjogren's Syndrome Disease Activity Index [ESSDAI] >= 5, anti-SS-related antigen A/anti-Ro antibody positive) received weekly subcutaneous abatacept 125 mg or placebo for 169 days followed by an open-label extension to day 365. Primary endpoint was mean change from baseline in ESSDAI at day 169. Key secondary endpoints were mean change from baseline in EULAR Sjogren's Syndrome Patient Reported Index (ESSPRI) and stimulated whole salivary flow (SWSF) at day 169. Other secondary clinical endpoints included glandular functions and patient-reported outcomes. Selected biomarkers and immune cell phenotypes were examined. Safety was monitored. Results Of 187 patients randomised, 168 completed double-blind period and 165 continued into open-label period. Mean (SD) baseline ESSDAI and ESSPRI total scores were 9.4 (4.3) and 6.5 (2.0), respectively. Statistical significance was not reached for primary (ESSDAI - 3.2 abatacept vs -3.7 placebo, p=0.442) or key secondary endpoints (ESSPRI, p=0.337; SWSF, p=0.584). No clinical benefit of abatacept over placebo at day 169 was seen with other clinical and PRO endpoints. Relative to baseline, abatacept was associated with significant differences vs placebo in some disease-relevant biomarkers (including IgG, IgA, IgM-rheumatoid factor) and pathogenic cell subpopulations (post hoc analyses). No new safety signals were identified. Conclusions Abatacept treatment did not result in significant clinical efficacy compared with placebo in patients with moderate-to-severe pSS, despite evidence of biological activity

Composite of Relevant Endpoints for Sjogren's Syndrome (CRESS):development and validation of a novel outcome measure

Background Recent randomised controlled trials (RCTs) in primary Sjogren's syndrome used the European League Against Rheumatism (EULAR) Sjogren's Syndrome Disease Activity Index (ESSDAI) as their primary endpoint. Given the heterogeneous and complex nature of primary Sjogren's syndrome, it might be more appropriate to also assess other clinically relevant disease features. We aimed to develop a novel composite endpoint for assessing treatment efficacy in patients with primary Sjogren's syndrome: the Composite of Relevant Endpoints for Sjogren's Syndrome (CRESS). Methods A multidisciplinary expert team selected clinically relevant items and candidate measurements for inclusion in the composite score. For each measurement, cutoff points for response to treatment were chosen based on expert opinion, previously published data on minimal clinically important improvements, and trial data, primarily the week-24 data of the single-centre ASAP-III trial of abatacept versus placebo. CRESS was validated using data from three independent RCTs: one trial of rituximab (TRACTISS), one of abatacept (multinational trial), and one of tocilizumab (ETAP). We calculated the number and percentage of patients who were responders in the separate CRESS items, and the percentage of responders based on the total CRESS at the primary endpoint visits (week 48 for TRACTISS, week 24 for the other two trials). Patients with fewer than three items available for evaluating CRESS response were imputed as non-responders. Findings Based on expert opinion, five complementary items were selected to assess response: (1) systemic disease activity by Clinical ESSDAI (less than 5 points); (2) patient-reported symptoms by EULAR Sjogren's Syndrome Patient Reported Index, assessed by a decrease of at least 1 point or at least 15% from baseline; (3) tear gland item by Schirmer's test and ocular staining score, assessed by an increase of at least 5 mm or decrease of at least 2 points, respectively, in patients with abnormal Schirmer's test or ocular staining score findings at baseline, or, in patients with normal baseline values, assessed by no change to abnormal for both; (4) salivary gland item, assessed by unstimulated whole saliva secretion (increase of at least 25%) and salivary gland ultrasonography (decrease of at least 25%); and (5) serology, assessed by rheumatoid factor (decrease of at least 25%) and IgG (decrease of at least 10%). Total CRESS response is defined as response on at least three of five items. Post-hoc assessment of phase 3 trial data showed that CRESS response rates at the primary endpoint visits were 60% (24 of 40) for abatacept versus 18% (seven of 39) for placebo (p Interpretation The CRESS is a feasible, well-balanced, composite endpoint for use in trials of primary Sjogren's syndrome. As a next step, the CRESS will require validation in a prospective RCT. Copyright (C) 2021 Elsevier Ltd. All rights reserved

Influence of polymorphisms in TNF-α and IL1β on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1β mediated multi-cellular signaling in liver microenvironment

Background and aimsAlcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p.Materials and methodsBio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required.ResultsThe combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1β/IL8/MCP1/IL6/TGFβ) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1β were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1β was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1β-31CT+TT than TNF-α-238GG/IL1β-31CC. The TNF-α/IL1β promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1β over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFβ and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1β were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFβR2/MCP1, and ICAM1 respectively.ConclusionThus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1β gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1β and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients

Cyclooxygenase-1 and -2 Are Required for Production of Infectious Pseudorabies Virus

We have recently shown that cyclooxygenase-2 (COX-2) transcription is markedly induced after herpes simplex virus type 1 and pseudorabies virus (PRV) infections of rat embryonic fibroblast (REF) cells (N. Ray and L. W. Enquist, J. Virol. 78:3489-3501, 2004). For this study, we investigated the role of cyclooxygenase induction in the replication and growth of PRV. We demonstrate here a concordant increase in COX-2 mRNA and protein levels after the infection of REF cells. Inhibitors blocking the activity of cyclooxygenases caused a dramatic reduction in PRV growth. Viral growth could be restored if prostaglandin E(2), the final product of COX-2 activity, was added simultaneously with the COX inhibitors. Immediate-early protein IE180, major capsid protein VP5, and glycoprotein expression were slightly reduced in the presence of COX-2 inhibitors, but expression of the early protein EP0 was not affected by COX inhibition. Viral DNA replication was marginally reduced in the presence of a COX-1/2 inhibitor, but there was no defect in viral DNA cleavage. Electron microscopy analysis revealed an increased number of unusual empty capsid structures in the nuclei of cells infected with PRV in the presence of a COX-1/2 inhibitor. These capsid structures shared some characteristics with procapsids but had a novel appearance by negative staining. Our data establish a role for COX-1 and COX-2 in facilitating the efficient growth and replication of PRV in primary cells

HR-2 Mutations in Human Immunodeficiency Virus Type 1 gp41 Restore Fusion Kinetics Delayed by HR-1 Mutations That Cause Clinical Resistance to Enfuvirtide ▿

Enfuvirtide (ENF) prevents the entry of human immunodeficiency virus type 1 (HIV-1) into cells by binding to the HR-1 region of the viral envelope (Env) protein gp41 subunit. Resistance to ENF arises via mutations in the drug binding site in HR-1. In addition, HR-2 mutations are commonly observed in ENF-resistant Env proteins, though their role remains unclear. We explored the mechanistic basis for clinical resistance to ENF and the role of HR-2 mutations. Using panels of ENF resistance-associated mutants for two patients, we found that mutations in HR-1 slowed the fusion kinetics and that mutations in HR-2 restored fusion rates. We assessed the differences in the rates of fusion of these mutants from a temperature-arrested state and observed similar trends, suggesting that the step of delay occurs after coreceptor engagement. Sensitivity to neutralizing antibodies was unchanged by the HR-1 and HR-2 mutants in each panel. Since this result was in contrast to those of a previous in vitro analysis where enhanced sensitivity to neutralization was demonstrated for heterologous Envs with ENF resistance-associated HR-1 changes, we examined the context dependence of HR-1 and HR-2 mutations by transferring the mutations seen in one patient into the Env context of another. These studies revealed that some, but not all, HR-1 mutations, when placed out of context (i.e., in a patient Env where they did not originally arise), enhance sensitivity to neutralizing antibodies. However, in most cases, HR-1 mutations in ENF-treated patients evolve in a manner that preserves pretreatment neutralization sensitivity so as to evade the pressures of the immune system

Clinical Resistance to Enfuvirtide Does Not Affect Susceptibility of Human Immunodeficiency Virus Type 1 to Other Classes of Entry Inhibitors

The clinical use of the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide (ENF) can select for drug-resistant HIV-1 strains bearing mutations in the HR1 region of the viral envelope (Env) protein. We analyzed the properties of multiple Env proteins isolated from five patients who experienced an initial decline in viral load after ENF therapy followed by subsequent rebound due to emergence of ENF-resistant HIV-1. Prior to ENF therapy, each patient harbored genetically and phenotypically diverse Env proteins that used CCR5 and/or CXCR4 to elicit membrane fusion. Coreceptor usage patterns of the Envs isolated from two patients underwent homogenization following ENF therapy, whereas in the other three patients, recombination appeared to allow the introduction of a single HR1 sequence with ENF resistance mutations into phenotypically distinct Env proteins. Analysis of individual Env clones also revealed that prior to ENF therapy, there was sometimes marked heterogeneity in the susceptibility of individual Env proteins to coreceptor inhibitors. After virologic failure, all Envs acquired resistance to ENF but exhibited no consistent change in their sensitivity to the fusion inhibitor T-1249 or to coreceptor inhibitors. In summary, using patient-derived Env proteins, we found that ENF failure was associated with emergence of high-level resistance to ENF due largely to mutations in HR1 but that susceptibility to other entry inhibitors was unaffected, that in these late-stage patients there was greater clonal variability to coreceptor than to fusion inhibitors, and that recombination events in vivo could sometimes restore Env genotypic and phenotypic heterogeneity by introducing drug-resistant gp41 sequences into heterologous gp120 backgrounds