In Vivo Anti-HIV Activity of the Heparin-Activated Serine Protease Inhibitor Antithrombin III Encapsulated in Lymph-Targeting Immunoliposomes

Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Several serpins have been reported to modulate HIV pathogenesis, or exhibit potent anti-HIV activity in vitro, but the efficacy of serpins as therapeutic agents for HIV in vivo has not yet been demonstrated. In the present study, we show that heparin-activated antithrombin III (hep-ATIII), a member of the serpin family, significantly inhibits lentiviral replication in a non-human primate model. We further demonstrate greater than one log10 reduction in plasma viremia in the nonhuman primate system by loading of hep-ATIII into anti-HLA-DR immunoliposomes, which target tissue reservoirs of viral replication. We also demonstrate the utility of hep-ATIIII as a potential salvage agent for HIV strains resistant to standard anti-retroviral treatment. Finally, we applied gene-expression arrays to analyze hep-ATIII-induced host cell interactomes and found that downstream of hep-ATIII, two independent gene networks were modulated by host factors prostaglandin synthetase-2, ERK1/2 and NFκB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention

Genital Tract Sequestration of SIV following Acute Infection

We characterized the evolution of simian immunodeficiency virus (SIV) in the male genital tract by examining blood- and semen-associated virus from experimentally and sham vaccinated rhesus monkeys during primary infection. At the time of peak virus replication, SIV sequences were intermixed between the blood and semen supporting a scenario of high-level virus "spillover" into the male genital tract. However, at the time of virus set point, compartmentalization was apparent in 4 of 7 evaluated monkeys, likely as a consequence of restricted virus gene flow between anatomic compartments after the resolution of primary viremia. These findings suggest that SIV replication in the male genital tract evolves to compartmentalization after peak viremia resolves

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

<i>In vivo</i> anti-viral activity of non-activated and heparin-activated ATIII in rhesus macaques.

<p>(<b>A</b>) Viral loads as RNA copies/ml of chronically SIV_mac239 infected rhesus macaques treated with 0.8 µmol/kg non-activated ATIII (n = 3) and (<b>B</b>) corresponding log₁₀ reduction of viral load of same treatment group. (<b>C</b>) Viral loads as RNA copies/ml of chronically SIV_mac239 infected rhesus macaques treated with 0.6 µmol/kg heparin-activated ATIII (n = 3) and (<b>D</b>) corresponding log₁₀ reduction of viral load of same treatment group. Administration via i. v. inoculation is shown at indicated time points depicted by arrows. Viral load was measured and compared to animals before treatment (day 0). *, <i>P</i><0.05, paired T-test, compared to pre-treatment (day 0). Data are shown as mean ± S.E.</p

<i>In vivo</i> anti-viral activity of ET-ATIII in rhesus macaques.

<p>Chronically SIV_mac239 infected rhesus macaques (n = 2) were treated with 1.5 ml ET-ATIII (0.3 nmol/kg encapsulated hep-ATIII) at indicated time points depicted by arrows. (<b>A</b>) Viral load of ET-ATIII treated animals as RNA copies/ml. (<b>B</b>) Log₁₀ reduction of viral load. Vehicle liposomes were used as a control. Viral load was measured and compared to animals before treatment (day 0). Data are shown as mean ± S.E.</p

Highest scoring network after interactive network analysis of gene expression changes induced after hep-ATIII treatment of SIV-infected rhesus macaques.

<p>The highest scoring primary transcriptional network activated by hep-ATIII treatment of chronically infected rhesus macaques, at a time point when viral replication is inhibited by hep-ATIII (day 7), is shown. Rx (orange): potential medication treatment options, BM (green): possible biomarkers. Network analysis was performed using Ingenuity 8.0 software. Explanation for symbols is given in <b>Figure S2</b>.</p

IC50 of hep-ATIII in different liposome formulations.

<p>IC50 of hep-ATIII in different liposome formulations.</p

Second-highest scoring network after interactive network analysis of gene expression changes induced after hep-ATIII treatment of SIV-infected rhesus macaques.

<p>The second-highest scored network activated by hep-ATIII treatment of chronically SIV-infected rhesus macaques, at a time point when viral replication is inhibited by hep-ATIII (day 7), is shown. Network analysis was performed using Ingenuity 8.0 software. Explanation for symbols is given in <b>Figure S2</b>.</p

Characterization of heparin-activated ATIII.

<p>(<b>A</b>) Sepharcyl S100 ÄKTA FPLC of purified hep-ATIII. A 9–14 ml fraction was separated, termed as hep-ATIII and used for our experiments. Detection at 260 nm for protein detection and 280 nm for heparin detection is shown. (<b>B</b>) Hep-ATIII purity and molecular weight were also determined by SDS-PAGE and silver staining (Bio-Rad kit) of a 15% slab gel. For molecular weight determination a low-range protein molecular weight marker (Bio-Rad) was used. Lane 1∶2 µg hep-ATIII; lane 2: phosphorylase B (97 kDa), serum albumin (69 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), aprotinin (6.5 kDa).</p