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Agonists of thyroid hormone receptor β (THR-β) decreased LDL cholesterol (LDL-C) and triglyceride (TG) levels in human clinical trials for patients with dyslipidemia. The authors present the highly potent and selective compound ALG-055009 ( 14 ) as a potential best in class THR-β agonist. The high metabolic stability and good permeability translated well in vivo to afford a long in vivo half-life pharmacokinetic profile with limited liability for DDI, and it overcomes certain drawbacks seen in recent clinical candidates.

Nucleic acid polymers (NAPs) are an attractive treatment modality for chronic hepatitis B (CHB), with REP2139 and REP2165 having shown efficacy in CHB patients. A subset of patients achieve functional cure, whereas the others exhibit a moderate response or are non-responders. NAP efficacy has been difficult to recapitulate in animal models, with the duck hepatitis B virus (DHBV) model showing some promise but remaining underexplored for NAP efficacy testing. Here we report on an optimized in vivo DHBV duck model and explore several characteristics of NAP treatment. REP2139 was efficacious in reducing DHBV DNA and DHBsAg levels in approximately half of the treated ducks, whether administered intraperitoneally or subcutaneously. Intrahepatic or serum NAP concentrations did not correlate with efficacy, nor did the appearance of anti-DHBsAg antibodies. Furthermore, NAP efficacy was only observed in experimentally infected ducks, not in endogenously infected ducks (vertical transmission). REP2139 add-on to entecavir treatment induced a deeper and more sustained virological response compared to entecavir monotherapy. Destabilized REP2165 showed a different activity profile with a more homogenous antiviral response followed by a faster rebound. In conclusion, subcutaneous administration of NAPs in the DHBV duck model provides a useful tool for in vivo evaluation of NAPs. It recapitulates many aspects of this class of compound's efficacy in CHB patients, most notably the clear division between responders and non-responders., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yannick Debing, Hannah Vanrusselt, Santhosh Kumar Thatikonda, Vivek Rajwanshi, Vikrant Gohil, Jin Hong, Dieudonné Buh Kum, Abel Acosta Sanchez, Sushmita Chanda, Lawrence M. Blatt, Andreas Jekle, Julian A. Symons, David B. Smith, Pierre Raboisson, Tse-I Lin, and Leonid Beigelman are or were directly or indirectly employed by Aligos Therapeutics and may own stock. Lars Degrauwe, Daniel Apolônio Silva de Oliveira, Christopher K. Kariuki, Ebanja Joseph Ebwanga, Shahbaz Bashir, Wouter Merckx, and Jan Paeshuyse declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.

Importance: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B., Competing Interests: H.V., D.K.B., C.L., A.A.S., L.B., L.M.B., J.A.S., J.D., P.R., A.J., S.V., and Y.D. are or were directly or indirectly employed by Aligos Therapeutics and may own stock. V.T., N.C., S.M., T.F.B., and E.R.V. declare no conflicts of interest.

Background and Aims: Effective therapies leading to a functional cure for chronic hepatitis B are still lacking. Class A capsid assembly modulators (CAM-As) are an attractive modality to address this unmet medical need. CAM-As induce aggregation of the HBV core protein (HBc) and lead to sustained HBsAg reductions in a chronic hepatitis B mouse model. Here, we investigate the underlying mechanism of action for CAM-A compound RG7907., Approach and Results: RG7907 induced extensive HBc aggregation in vitro , in hepatoma cells, and in primary hepatocytes. In the adeno-associated virus (AAV)-HBV mouse model, the RG7907 treatment led to a pronounced reduction in serum HBsAg and HBeAg, concomitant with clearance of HBsAg, HBc, and AAV-HBV episome from the liver. Transient increases in alanine transaminase, hepatocyte apoptosis, and proliferation markers were observed. These processes were confirmed by RNA sequencing, which also uncovered a role for interferon alpha and gamma signaling, including the interferon-stimulated gene 15 (ISG15) pathway. Finally, the in vitro observation of CAM-A-induced HBc-dependent cell death through apoptosis established the link of HBc aggregation to in vivo loss of infected hepatocytes., Conclusions: Our study unravels a previously unknown mechanism of action for CAM-As such as RG7907 in which HBc aggregation induces cell death, resulting in hepatocyte proliferation and loss of covalently closed circular DNA or its equivalent, possibly assisted by an induced innate immune response. This represents a promising approach to attain a functional cure for chronic hepatitis B., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Respiratory syncytial virus (RSV) infection is the leading cause of infant hospitalizations and mortality. Lumicitabine, an oral nucleoside analog was studied for the treatment of RSV. The phase 1b and phase 2b studies reported here assessed the safety, pharmacokinetics, and pharmacodynamics of lumicitabine in infants/neonates hospitalized with RSV. In the phase 1b study, infants (≥1 to ≤12 months) and neonates (<28 days) received a single-ascending or multiple-ascending doses (single loading dose [LD] then 9 maintenance doses [MD] of lumicitabine, or placebo [3:1]). In the phase 2b study, infants/children (28 days to ≤36 months old) received lumicitabine 40/20 mg/kg, 60/40 mg/kg LD/MD twice-daily or placebo (1:1:1) for 5 days. Safety, pharmacokinetics, and efficacy parameters were assessed over 28 days. Lumicitabine was associated with a dose-related increase in the incidence and severity of reversible neutropenia. Plasma levels of ALS-008112, the active nucleoside analog, were dose-proportional with comparable mean exposure levels at the highest doses in both studies. There were no significant differences between the lumicitabine groups and placebo in reducing viral load, time to viral non-detectability, and symptom resolution. No emergent resistance-associated substitutions were observed at the RSV L-gene positions of interest. In summary, lumicitabine was associated with a dose-related increase in the incidence and severity of reversible neutropenia and failed to demonstrate antiviral activity in RSV-infected hospitalized infants. This contrasts with the findings of the previous RSV-A adult challenge study where significant antiviral activity was noted, without incidence of neutropenia. Trial registration ClinicalTrials.gov Identifier: NCT02202356 (phase 1b); NCT03333317 (phase 2b)., Competing Interests: Abbie Oey, Matthew McClure, Julian A. Symons, Sushmita Chanda, John Fry, Dymphy Huntjens, and James Witek are current or former employees of Janssen Research & Development and may own stock/stock options in Johnson & Johnson. Patrick Smith works for Certara, a consulting firm in integrated drug development and has directly consulted with a variety of not‐for‐profit global health organizations, biotechnology, and pharmaceutical companies and governments with an interest in medical countermeasures against respiratory virus infections. Kathia Luciani, Michael Fayon, Kulkanya Chokephaibulkit, Rattapon Uppala, Jolanta Bernatoniene, Kenji Furuno, and Thorsten Stanley have no potential conflicts of interest to disclose. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Oey et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC 50 ) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. IMPORTANCE Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro . The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors.

A functional cure of chronic hepatitis B requires eliminating the hepatitis B virus (HBV)-encoded surface antigen (HBsAg), which can suppress immune responses. STOPS are phosphorothioated single-stranded oligonucleotides containing novel chemistries that significantly reduce HBsAgs produced by HBV-infected liver cells. The STOPS molecule ALG-10000 functions inside cells to reduce the levels of multiple HBV-encoded molecules. However, it does not bind HBV molecules. An affinity resin coupled with ALG-10000 was found to bind several proteins from liver cells harboring replicating HBV. Silencing RNAs targeting host factors SRSF1, HNRNPA2B1, GRP78 (HspA5), RPLP1, and RPLP2 reduced HBsAg levels and other HBV molecules that are concomitantly reduced by STOPS. Host proteins RPLP1/RPLP2 and GRP78 function in the translation of membrane proteins, protein folding, and degradation. ALG-10000 and the knockdowns of RPLP1/2 and GRP78 decreased the levels of HBsAg and increased their ubiquitination and proteasome degradation. GRP78, RPLP1, and RPLP2 affected HBsAg production only when HBsAg was expressed with HBV regulatory sequences, suggesting that HBV has evolved to engage with these STOPS-interacting molecules. The STOPS inhibition of HBsAg levels in HBV-infected cells occurs by sequestering cellular proteins needed for proper expression and folding of HBsAg., Competing Interests: All authors declare no competing interests., (© 2021 The Author(s).)

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC 50  = 7 nM) without affecting the activity of human cathepsin L (IC 50  > 10 μM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log 10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log 10 (TCID 50 /mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of direct antiviral agents. We previously reported the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay (Gurard-Levin et al., 2020). The assay was further improved by adding the rhinovirus HRV3C protease to the same well as the SARS-CoV-2 3CLpro enzyme. High substrate specificity for each enzyme allowed the proteases to be combined in a single assay reaction without interfering with their individual activities. This novel duplex assay was used to profile a diverse set of reference protease inhibitors. The protease inhibitors were grouped into three categories based on their relative potency against 3CLpro and HRV3C including those that are: equipotent against 3CLpro and HRV3C (GC376 and calpain inhibitor II), selective for 3CLpro (PF-00835231, calpain inhibitor XII, boceprevir), and selective for HRV3C (rupintrivir). Structural analysis showed that the combination of minimal interactions, conformational flexibility, and limited bulk allows GC376 and calpain inhibitor II to potently inhibit both enzymes. In contrast, bulkier compounds interacting more tightly with pockets P2, P3, and P4 due to optimization for a specific target display a more selective inhibition profile. Consistently, the most selective viral protease inhibitors were relatively weak inhibitors of human cathepsin L. Taken together, these results can guide the design of cysteine protease inhibitors that are either virus-specific or retain a broad antiviral spectrum against coronaviruses and rhinoviruses., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THRβ agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THRβ-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THRβ and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolism in vitro using human hepatic cells and in vivo using a rat model. Treatment with GC-1 upregulated the transcription of CPT1A in the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely, ANGPTL4 and DIO1. In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liver Dio1 and Me1 RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THRβ agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: XGL, SKS, AJ, KG, D. Misner, SC, SM, CW, AS, LMB, LNB, JAS, and JD are current employees of Aligos Therapeutics, Inc. TL, PR, D. McGowan, and KV are current employees of Aligos Belgium BV. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC 50  = 0.060 μM), calpain inhibitors II and XII (IC 50 ~20-25 μM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human β-coronavirus OC-43 or SARS-CoV-2 in vitro, but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Respiratory syncytial virus (RSV) causes high morbidity, with mortality rates approaching or exceeding that of influenza in adult and infant patient populations, respectively. Lumicitabine (ALS-008176 or JNJ-64041575) is an oral nucleoside analogue prodrug in clinical development to treat RSV infections. This prodrug converts to plasma-circulating ALS-8112, and then to the 5'-active nucleoside triphosphate (NTP) form within host cells. We conducted an RSV-A challenge study in healthy adults to evaluate lumicitabine's activity during an active RSV infection., Objectives: To develop a semi-mechanistic mathematical model describing RSV kinetics, and the pharmacokinetics (PK) and pharmacodynamics (PD) of lumicitabine during treatment., Methods: Nasopharyngeal viral load and concentrations of ALS-8112 and ALS-8144 (uridine metabolite) were measured frequently over the study duration. Population viral kinetic and PK/PD models were developed using NONMEM. The RSV life-cycle was described using a target-cell-limited model that included a physiological delay., Results: The estimated clearances of ALS-8112 and ALS-8144 were 54.2 and 115 L/h/70 kg, respectively. A semi-physiological model was linked to predict ALS-8112 conversion to active intracellular NTP. Extensive and rapid RSV reduction occurred after lumicitabine treatment (EC50 = 1.79 μM), with >99% viral inhibition at 2 h after loading dose. Simulated NTP exposures and time to EC50 attainment suggested that rapid therapeutic effects and reduced dosing frequency are achievable in adult and paediatric patients., Conclusions: The semi-mechanistic model characterizes RSV kinetics and the antiviral effectiveness of lumicitabine in an adult challenge population. This model is applicable to guide dose selection in adult and paediatric patients.

Background: AL-794 is an orally active prodrug of ALS-033719, which selectively inhibits the endonuclease domain of influenza virus A and B polymerase., Methods: In a phase 1, double-blinded, randomized, placebo-controlled study, healthy subjects were inoculated intranasally with influenza virus (A/Perth/16/2009 H3N2) after confirmation of infection or on day 4. Subjects received 50 mg of AL-794, 150 mg of AL-794, or placebo twice daily for 5 days. Viral load, influenza symptoms, pharmacokinetics, and safety were evaluated., Results: A total of 61 subjects were inoculated. In 42 infected subjects, the mean peak viral load for 50-mg AL-794 recipients, 150-mg AL-794 recipients, and placebo recipients was 3.54, 2.77, and 3.72 log10 50% tissue culture infectious doses (TCID50)/mL, respectively. The mean influenza viral load area under the curve in the corresponding treatment groups was 137, 87.5, and 142 log10 TCID50/mL·h, respectively, and the median time to virus nondetection was 117, 75.3, and 108 hours, respectively. AL-794 was well tolerated, and no viral resistance to ALS-033719 was identified., Conclusion: Following oral administration of AL-794, significant dose-dependent antiviral activity was noted, with a greater decrease in viral load, symptoms, and mucus weight at the 150-mg dose, compared with the 50-mg dose, and no safety concerns for either dose or placebo., Clinical Trials Registration: NCT02588521.

2017 marked the 30th anniversary of the approval of zidovudine (AZT) as the first HIV/AIDS therapy. Since then, more than eighty antiviral drugs have received FDA approval, half of which treat HIV infection. Here, we provide a retrospective analysis of approved antiviral drugs, including therapeutics against other major chronic infections such as hepatitis B and C, and herpes viruses, over the last thirty years. During this time, only a few drugs were approved to treat acute viral infections, mainly influenza. Analysis of these approved antiviral drugs based on molecular class and mode of action shows that a large majority are small molecules and direct-acting agents as opposed to proteins, peptides, or oligonucleotides and host-targeting therapies. In addition, approvals of combination therapies accelerated over the last five years. We also provide a prospective study of future potential antiviral therapies, based on current clinical research pipelines across the pharmaceutical industry. Comparing past drug approvals with current clinical candidates hints at the future evolution in antiviral therapies and reveals how antiviral medicines are often discovered. Overall, this work helps forecast future trends and innovation in the field of antiviral research and development., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addition to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using negative-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3'-terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clinically relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resolution structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented negative-strand RNA viruses.

Background: This double-blind, first-in-human Phase I study evaluated pharmacokinetics, safety and tolerability of AL-794 (prodrug of ALS-033719), a potent endonuclease inhibitor of influenza A and B in healthy volunteers., Methods: Healthy adult volunteers were randomized to AL-794 (50-2,000 mg single ascending doses, fasting) or placebo (5 cohorts, n=6:2 AL-794: placebo/cohort) in part 1, and AL-794 (50-600 mg multiple ascending doses, twice-daily, fed or fasted) or placebo (3 cohorts, n=8:2 AL-794: placebo/cohort) for 7 days in part 2. In part 3, 8 healthy volunteers from part 1 received 450 mg AL-794 (n=6) or placebo (n=2) following a high-fat meal. All dosing was done with an oral suspension. Blood and urine samples for pharmacokinetics were collected at scheduled times and analysed for ALS-033719 and ALS-033927 (inactive glucuronide) plasma concentrations using LC-MS/MS., Results: ALS-033719 plasma concentrations increased dose proportionately up to 150 mg but less than proportionately above 150 mg. Steady-state was generally achieved by the third dose. ALS-033719 exposure increased following administration with a standard meal (19%-33%) or high-fat meal (3-3.6-fold). ALS-033927 was the major metabolite observed. Renal elimination was negligible (0.2%). Seventeen AL-794-treated healthy volunteers reported ≥1 treatment-emergent adverse event (TEAE; part 1: n=6, 24%; part 2: n=11, 69%). The most common TEAEs were headache (part 1: n=3; part 2: n=5) and dizziness (part 1: n=2; part 2: n=6)., Conclusions: AL-794 up to 200 mg twice daily achieved ALS-033719 exposures which are expected to be efficacious and were generally tolerated. Further studies are planned to characterize safety and antiviral activity.

Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3'-deoxy or 2'-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (K i ) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2' or 4' mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5'-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, prompting us to investigate dCK recognition of other 2'- and 4'-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure-activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery.

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.

ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.

BACKGROUND Respiratory syncytial virus (RSV) infection is a cause of substantial morbidity and mortality. There is no known effective therapy. METHODS We conducted a randomized, double-blind, clinical trial in healthy adults inoculated with RSV. Participants received the oral nucleoside analogue ALS-008176 or placebo 12 hours after confirmation of RSV infection or 6 days after inoculation. Treatment was administered every 12 hours for 5 days. Viral load, disease severity, resistance, and safety were measured throughout the 28-day study period, with measurement beginning before inoculation. The primary end point was the area under the curve (AUC) for viral load, which was assessed immediately before administration of the first dose through the 12th day after inoculation in participants infected with RSV. RESULTS A total of 62 participants received placebo or one of three ALS-008176 dosing regimens: 1 loading dose of 750 mg followed by 9 maintenance doses of 500 mg (group 1), 1 loading dose of 750 mg followed by 9 maintenance doses of 150 mg (group 2), or 10 doses of 375 mg (group 3). In the 35 infected participants (23 of whom were treated with ALS-008176), the AUCs for viral load for groups 1, 2, and 3 and the placebo group were 59.9, 73.7, 133.4, and 500.9 log10 plaque-forming-unit equivalents × hours per milliliter, respectively (P≤0.001). The time to nondetectability on polymerase-chain-reaction assay (P<0.001), the peak viral load (P≤0.001), the AUC for symptom score (P<0.05), and the AUC for mucus weight were lower in all groups receiving ALS-008176 than in the placebo group. Antiviral activity was greatest in the two groups that received a loading dose--viral clearance was accelerated (P≤0.05), and the AUC for viral load decreased by 85 to 88% as compared with the placebo group. Within this small trial, no viral rebound or resistance was identified. There were no serious adverse events, and there was no need for premature discontinuation of the study drug. CONCLUSIONS In this RSV challenge study, more rapid RSV clearance and a greater reduction of viral load, with accompanying improvements in the severity of clinical disease, were observed in the groups treated with ALS-008176 than in the placebo group. (Funded by Alios BioPharma; ClinicalTrials.gov number, NCT02094365.).

Objectives: To determine whether preoperative endothelial dysfunction provides risk stratification for perioperative renal injury in patients undergoing noncardiac surgery. The relationship between perioperative renal injury and myocardial injury after noncardiac surgery (MINS) was explored secondarily., Design: An observational study., Setting: Two academic medical centers., Participants: A total of 218 patients scheduled to undergo intermediate or high-risk noncardiac surgery., Interventions: None., Measurements and Main Results: Endothelial dysfunction was identified preoperatively by a Reactive Hyperemia-Peripheral Arterial Tonometry (RH-PAT) index. Renal injury was defined by peak delta serum creatinine (ΔSCr) or creatinine-based kidney disease: Improving global outcomes acute kidney injury (AKI) criteria within 7 days postoperatively. MINS was defined by peak troponin ≥0.04 µg/L within 3 days postoperatively. AKI occurred in 22 patients (10.1%). Median RH-PAT index within the study cohort was 1.64 (range 1.03-4.96) and did not differ between patients with or without AKI. When adjusted for covariates, there was no association between RH-PAT index and either AKI or peak ΔSCr. MINS occurred in 32 patients (14.7%) and was associated independently with the outcome of AKI (odds ratio [OR], 3.7; 95% confidence interval [CI], 1.2-10.8; p = 0.02) and peak ΔSCr (β-regression coefficient 23; 95% CI, 9-37; p = 0.002). Co-occurrence of AKI and MINS portended a marked increase in 30-day mortality (OR, 43; 95% CI, 6-305; p = 0.001) and delayed time to discharge (hazard ratio, 0.27; 95% CI, 0.13-0.54; p = 0.001)., Conclusions: For patients undergoing noncardiac surgery, preoperative endothelial function assessed by noninvasive peripheral arterial tonometry was not associated with perioperative AKI. Perioperative renal injury was associated strongly with MINS, and this may represent a mechanism by which AKI increases adverse outcomes., (Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.)

Type 1 interferons (IFNs) have been shown to be efficacious against hepatitis C virus (HCV), hepatitis B virus (HBV), and some cancers with a significant drawback of short drug exposure. We have significantly improved the pharmacokinetic (PK) of consensus interferon (CIFN) by glycoengineering. We generated AL-624 by introducing 4 glycosylation sites. AL-624 was expressed, purified, and fractionated to yield 2-Gly, 3-Gly, and 4-Gly. In a rat PK study, AL-624 4-Gly exhibited a 6-fold increase of area under curve (AUC) and more than an 11-fold increase in time to half life (T1/2) over CIFN, suggesting the potential for weekly dosing (QW). In Yellow fever virus hamster model, QW of 4-Gly achieved similar efficacy to daily dosing (QD) CIFN and QW Peg-IFN-α-2a in overall survival rate and reduction of alanine aminotransferase (ALT) level. Further refinement resulted in development of AL-683 by addition of external glycosylation sites and its mouse homologue. AL-683 maintains undiminished biological potency in HCV replicon. In mouse PK/pharmacodynamic (PD) studies, AL-683 homologue has a ∼37-fold improvement in T1/2 and a ∼33-fold improvement in AUC when compared with the unglycosylated mouse IFN-α-1. Significantly improved PD responses were also observed. The significant improvement of AL-683 PK over AL-624 suggests a bimonthly dosing regimen for AL-683. The possibility for once-a-month dosing could be realized by further optimization of manufacturing conditions.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

Respiratory syncytial virus (RSV) is a leading pathogen of childhood and is associated with significant morbidity and mortality. To date, ribavirin is the only approved small molecule drug, which has limited use. The only other RSV drug is palivizumab, a monoclonal antibody, which is used for RSV prophylaxis. Clearly, there is an urgent need for small molecule RSV drugs. This article reports the design, synthesis, anti-RSV activity, metabolism, and pharmacokinetics of a series of 4'-substituted cytidine nucleosides. Among tested compounds 4'-chloromethyl-2'-deoxy-2'-fluorocytidine (2c) exhibited the most promising activity in the RSV replicon assay with an EC50 of 0.15 μM. The 5'-triphosphate of 2c (2c-TP) inhibited RSV polymerase with an IC50 of 0.02 μM without appreciable inhibition of human DNA and RNA polymerases at 100 μM. ALS-8176 (71), the 3',5'-di-O-isobutyryl prodrug of 2c, demonstrated good oral bioavailability and a high level of 2c-TP in vivo. Compound 71 is a first-in-class nucleoside RSV polymerase inhibitor that demonstrated excellent anti-RSV efficacy and safety in a phase 2 clinical RSV challenge study.

A number of important human infections are caused by positive-strand RNA viruses, yet almost none can be treated with small molecule antiviral therapeutics. One exception is the chronic infection caused by hepatitis C virus (HCV), against which new generations of potent inhibitors are being developed. One of the main molecular targets for anti-HCV drugs is the viral RNA-dependent RNA polymerase, NS5B. This review summarizes the search for nucleoside and nucleotide analogs that inhibit HCV NS5B, which led to the FDA approval of sofosbuvir in 2013. Advances in anti-HCV therapeutics have also stimulated efforts to develop nucleoside analogs against other positive-strand RNA viruses. Although it remains to be validated in the clinic, the prospect of using nucleoside analogs to treat acute infections caused by RNA viruses represents an important paradigm shift and a new frontier for future antiviral therapies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Myocardial injury after non-cardiac surgery (MINS) is a common complication with associated serious morbidity and mortality. Endothelial dysfunction might play an important role in MINS, and its rapid assessment could provide a novel method of risk stratification before surgery., Methods: We studied 238 subjects scheduled to undergo intermediate or high-risk surgery in a two-centre prospective study to determine whether preoperative endothelial dysfunction identified by a reactive hyperaemia-peripheral arterial tonometry (RH-PAT) index could provide effective risk stratification for MINS, defined as serum troponin ≥0.04 μg litre(-1), within 3 postoperative days., Results: The primary outcome occurred in 35 subjects (14.7%). Endothelial dysfunction was defined as an RH-PAT index of ≤1.22. Adjusted for age, Lee index and a composite measure of the extent of surgery, endothelial dysfunction was associated with MINS [odds ratio 10.1, 95% confidence interval (CI) 3.3-30.9, P=0.001] and increased time to discharge from hospital after surgery (hazard ratio 0.39, 95% CI 0.23-0.65, P=0.001). Endothelial dysfunction identified MINS with a sensitivity of 31%, a specificity of 96%, and a positive diagnostic likelihood ratio of 8.0. Risk classification for MINS was improved by the addition of RH-PAT-defined endothelial dysfunction to the Lee index (c-statistic increased from 0.69 to 0.77; integrated discrimination improvement 0.11, P=0.003). However, prognostic utility varied widely between sites., Conclusions: For patients undergoing non-cardiac surgery, non-invasive assessment of endothelial function might enhance preoperative risk stratification for perioperative myocardial injury. However, unexplained large inter-site variation in prognostic utility could limit widespread application and needs to be further understood.

Type I IFNs are unusually pleiotropic cytokines that bind to a single heterodimeric receptor and have potent antiviral, antiproliferative, and immune modulatory activities. The diverse effects of the type I IFNs are of differential therapeutic importance; in cancer therapy, an enhanced antiproliferative effect may be beneficial, whereas in the therapy of viral infections (such as hepatitis B and hepatitis C), the antiproliferative effects lead to dose limiting bone marrow suppression. Studies have shown that various members of the natural IFN-alpha family and engineered variants, such as IFN-con1, vary in the ratios between various IFN-mediated cellular activities. We used DNA shuffling to explore and confirm the hypothesis that one could simultaneously increase the antiviral and Th1-inducing activity and decrease the antiproliferative activity. We report IFN-alpha hybrids wherein the ratio of antiviral:antiproliferative and Th1-inducing: antiproliferative potencies are markedly increased with respsect to IFN-con1 (75- and 80-fold, respectively). A four-residue motif that overlaps with the IFNAR1 binding site and is derived by cross breeding with a pseudogene contributes significantly to this phenotype. These IFN-alphas have an activity profile that may result in an improved therapeutic index and, consequently, better clinical efficacy for the treatment of chronic viral diseases such as hepatitis B virus, human papilloma virus, HIV, or chronic hepatitis C.

Previous studies have investigated the role of volatile anaesthetic agents in myocardial protection during coronary artery bypass graft (CABG) surgery, and some have identified beneficial effects. However, these studies have been too small to identify a significant effect on myocardial infarction (MI) or mortality. We undertook a systematic overview and meta-analysis of all randomized trials comparing volatile with non-volatile anaesthesia in CABG surgery. We identified 27 trials that included 2979 patients. There was no significant difference in myocardial ischaemia, MI, intensive care unit length of stay or hospital mortality between the groups (all P>0.05). Post-bypass, patients randomized to receive volatile anaesthetics had 20% higher cardiac indices (P=0.006), significantly lower troponin I serum concentrations (P=0.002) and lesser requirement for inotropic support (P=0.004) compared with those randomized to receive i.v. anaesthetics. Duration of mechanical ventilation was reduced by 2.7 h (P=0.04), and there was a 1 day decrease in hospital length of stay (P<0.001). Some of these outcomes were based on a smaller number of trials because of incomplete data, largely because the individual trials focused on one or more surrogate endpoints. We found some evidence that volatile anaesthetic agents provide myocardial protection in CABG surgery, but larger adequately powered trials with agreed, defined outcomes need to be done to fully assess a possible beneficial effect of volatile anaesthetic agents on the risk of MI and mortality.

The regulation of the human IL-1alpha gene was studied using a series of 5' deletion promoter chloramphenicol acetyltransferase (CAT) reporter constructs. The IL-1alpha promoter from -967 to +64 produced no significant expression of CAT. Progressive 5' deletion indicated the presence of a repressor binding site between -477 and -305 bp as deletion in this region resulted in CAT expression. Electrophoretic mobility shift assay (EMSA) analysis confirmed that protein(s) bound to this region and DNaseI footprinting localized the binding site to between -448 and -435. Deletion of the IL-1alpha promoter to -42 resulted in reduced CAT expression suggesting the presence of a positive regulatory element in this region. EMSA experiments using IL-1alpha promoter DNA from -163 to +64 demonstrated protein binding to this region and DNaseI footprinting demonstrated protection between -59 and -40. Transcriptional activity of the IL-1alpha promoter was also tested using an in vitro transcription assay. Reactions using -163, -100 and -52 promoter templates all produced a correctly sized transcript but deletion to -42 resulted in no transcript production. Analysis of the promoter indicated that a potential Sp1 binding site existed in the region from -52 to -45. An EMSA using an anti-Sp1 antibody indicated that Sp1 specifically bound to the -52 to +64 region.

Many poxviruses express a secreted protein that binds CC chemokines with high affinity and has been called viral CC chemokine inhibitor (vCCI). This protein is unrelated to any known cellular protein, yet can compete with host cellular CC chemokine receptors to modulate host inflammatory and immune responses. Although several strains of vaccinia virus (VV) express a vCCI, the best characterized VV strains Western Reserve and Copenhagen do not. In this study, we have expressed the vCCI from VV strain Lister in a recombinant Western Reserve virus (v Delta B8R-35K) and characterized its binding properties in vitro and its effect on virulence in vivo relative to wild-type virus (v Delta B8R) or a revertant virus (v Delta B8R-R) where Lister 35-kDa had been removed. Cells infected with v Delta B8R-35K secreted a 35-kDa protein that bound the CC chemokine macrophage-inflammatory protein 1 alpha. Expression of vCCI attenuated the virus in a murine intranasal model, characterized by reduced mortality and weight loss, decreased virus replication and spread, and a reduced recruitment of inflammatory cells into the lungs of VV-infected mice. The CC chemokines macrophage-inflammatory protein 1 alpha, eotaxin, and macrophage chemotactic protein 1 were detected in bronchoalveolar lavage fluids from v Delta B8R-infected mice; however, bronchoalveolar lavage fluids from v Delta B8R-35K-infected mice had lower levels of chemokines and a reduced chemotactic activity for murine leukocytes in vitro. These observations suggest that vCCI plays an important role in regulating leukocyte trafficking to the lungs during VV infection by binding to CC chemokines and blocking their chemotactic activities.

Advances in technology have resulted in the development of several depth-of-anaesthesia monitors. Whether any of these monitors can reduce the incidence of awareness is an important issue for anaesthetists and their patients. We therefore surveyed a random selection of anaesthetists, asking for their opinions of awareness and depth-of-anaesthesia monitoring in current clinical practice. Approximately half (52%) of the anaesthetists surveyed had experienced a patient with awareness. Anaesthetists considered that they had a lower incidence of awareness in their own practice when compared with others, 1:5000 vs. 1:10 000 (p < 0.001). Anaesthetists rated awareness on an 11-point scale as only a moderate problem, median (interquartile range) 5 (2-7). Older anaesthetists were less likely to rate the importance of awareness highly (p = 0.009) and to use awareness monitoring (p = 0.001). Anaesthetists are prepared to use depth-of-anaesthesia monitoring more widely if it can be shown to prevent most cases of awareness in routine practice.

A bioassay that measured the interleukin (IL)-12-induced production of interferon (IFN)-gamma from mouse splenocytes was used to identify a soluble factor in the supernatants of vaccinia virus (VV)-infected cells that inhibited the production of IFN-gamma. This soluble factor was expressed by 14 out of 16 VV strains including the Western Reserve (WR) strain, but strains Copenhagen and Tashkent and a mutant of strain WR called 6/2 lacked this activity. The gene encoding this activity was identified as C12L by transferring DNA present in VV WR but missing in VV WR 6/2 into VV Copenhagen and testing for expression of the soluble factor. The C12L protein shows amino acid similarity to IL-18 binding proteins that are encoded by poxviruses, mice and humans, and C12L protein produced from VV or baculovirus inhibited the biological activity of mouse IL-18 in vitro. Thus the inhibition of IL-12-induced IFN-gamma production was due to indirect effects of C12L on IL-18, illustrating the synergistic action of these pro-inflammatory cytokines. To study the role of the C12L protein in the virus life-cycle, we constructed a deletion mutant lacking the C12L gene and a revertant virus in which the gene was reinserted into the deletion mutant. In vitro the replication and plaque size of these viruses were indistinguishable. However, infection of BALB/c mice by the intranasal route showed that the deletion mutant was attenuated and induced lower weight loss and signs of illness compared to controls.

The vaccinia virus (VV) N1L gene encodes a protein of 14 kDa that was identified previously in the concentrated supernatant of virus-infected cells. Here we show that the protein is present predominantly (>90%) within cells rather than in the culture supernatant and it exists as a non-glycosylated, non-covalent homodimer. The N1L protein present in the culture supernatant was uncleaved at the N terminus and was released from cells more slowly than the VV A41L gene product, a secreted glycoprotein that has a conventional signal peptide. Bioinformatic analyses predict that the N1L protein is largely alpha-helical and show that it is conserved in many VV strains, in other orthopoxviruses and in members of other chordopoxvirus genera. However, database searches found no non-poxvirus proteins with significant amino acid similarity to N1L. A deletion mutant lacking the N1L gene replicated normally in cell culture, but was attenuated in intranasal and intradermal murine models compared to wild-type and revertant controls. The conservation of the N1L protein and the attenuated phenotype of the deletion mutant indicate an important role in the virus life-cycle.

Vaccinia virus (VV) strain Western Reserve gene B8R encodes a 43 kDa glycoprotein that is secreted from infected cells early in infection as a homodimer. This protein has amino acid similarity with the extracellular domain of cellular IFN-gamma receptor (IFN-gammaR) and binds and inhibits IFN-gamma from a wide range of species. Here we demonstrate that the B8R protein also inhibits equine IFN-gamma. The 5' end of the B8R mRNA has been mapped by primer extension analysis and the contribution of IFN-gammaRs to VV virulence was studied by the construction of a deletion mutant lacking the B8R gene (vDeltaB8R) and a revertant virus (vB8R-R) in which the B8R gene was re-inserted into the deletion mutant. A recombinant virus that expressed a soluble form of the mouse IFN-gammaR was also constructed and studied. The virulence of these viruses was tested in rodent models of infection. In mice, the loss of the VV IFN-gammaR did not affect virulence compared with WT and revertant viruses, consistent with the low affinity of the VV IFN-gammaR for mouse IFN-gamma. However, expression of the mouse soluble IFN-gammaR increased virus virulence slightly. In rabbit skin, loss of the VV IFN-gammaR produced lesions with histological differences compared with WT and revertant viruses. Lastly, the affinity constants of the VV IFN-gammaR for human and mouse IFN-gamma were determined by surface plasmon resonance.

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma interferon (IFN-gamma), IFN-alpha/beta, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-alpha/beta. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-alpha/beta with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-alpha/beta. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-alpha, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-alpha/beta receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-alpha/beta, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-alpha/beta activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-alpha/beta against virus infections.

Poxviruses employ many strategies to evade and neutralize the host immune response. In this study, we have identified two vaccinia virus ORFs, termed A46R and A52R, that share amino acid sequence similarity with the Toll/IL-1 receptor (TIR) domain, a motif that defines the IL-1/Toll-like receptor (TLR) superfamily of receptors, which have a key role in innate immunity and inflammation. When expressed in mammalian cells, the protein products of both ORFs were shown to interfere specifically with IL-1 signal transduction. A46R partially inhibited IL-1-mediated activation of the transcription factor NFkappaB, and A52R potently blocked both IL-1- and TLR4-mediated NFkappaB activation. MyD88 is a TIR domain-containing adapter molecule known to have a central role in both IL-1 and TLR4 signaling. A52R mimicked the dominant-negative effect of a truncated version of MyD88 on IL-1, TLR4, and IL-18 signaling but had no effect on MyD88-independent signaling pathways. Therefore, A46R and A52R are likely to represent a mechanism used by vaccinia virus of suppressing TIR domain-dependent intracellular signaling.

Vaccinia virus comprises the live vaccine that was used for vaccination against smallpox. Following the eradication of smallpox, vaccinia virus was developed as an expression vector that is now used widely in biological research and vaccine development. In recent years vaccinia virus and other poxviruses have been found to express a collection of proteins that block parts of the host response to infection. Some of these proteins are secreted from the infected cell where they bind and neutralise host cytokines, chemokines and interferons (IFN). In this paper three such proteins that bind interleukin (IL)-1 beta, type I IFNs and CC chemokines are described. The study of these immunomodulatory molecules is enhancing our understanding of virus pathogenesis, yielding fundamental information about the immune system, and providing new molecules that have potential application for the treatment of immunological disorders or infectious diseases.

To counteract the host immune response, poxviruses have evolved secreted factors that bind cytokines and thereby neutralize their biological activities. The vaccinia virus B8R gene encodes a protein that neutralizes interferon-gamma (IFN-gamma) from several mammals including man, cow, rat, and rabbit but not mice. We now report that the activity of the B8R gene product is not restricted to cytokines of mammals: it also efficiently neutralized chicken IFN-gamma. B8R blocked chicken IFN-gamma-mediated induction of guanylate binding protein RNA in the chicken fibroblast cell line CEC-32 and secretion of nitric oxide in HD-11 cells. Radiolabeled baculovirus-expressed B8R efficiently bound to immobilized recombinant chicken IFN-gamma. Scatchard analysis revealed a binding constant of chicken IFN-gamma to B8R of approximately 0.5 nM. A mutant form of chicken IFN-gamma which lacks the 18 C-terminal amino acids and which has lost more than 99% of its biological activity was able to block the IFN-gamma-neutralizing effect of B8R. Binding studies showed that the mutant protein bound radiolabeled B8R only about threefold less well than wild-type chicken IFN-gamma but failed to compete with wild-type chicken IFN-gamma for binding to the cellular receptor. These results suggest that the extreme C terminus of chicken IFN-gamma is crucial for binding to its cellular receptor but less important for recognition by the viral cytokine receptor., (Copyright 1998 Academic Press.)

Chemokines direct migration of immune cells into sites of inflammation and infection. Chemokine receptors are seven-transmembrane domain proteins that, in contrast to other cytokine receptors, cannot be easily engineered as soluble chemokine inhibitors. Poxviruses encode several soluble cytokine receptors to evade immune surveillance, providing new strategies for immune modulation. Here we show that vaccinia virus and other orthopoxviruses (cowpox and camelpox) express a secreted 35-kDa chemokine binding protein (vCKBP) with no sequence similarity to known cellular chemokine receptors. The vCKBP binds CC, but not CXC or C, chemokines with high affinity (Kd = 0.1-15 nM for different CC chemokines), blocks the interaction of chemokines with cellular receptors, and inhibits chemokine-induced elevation of intracellular calcium levels and cell migration in vitro, thus representing a soluble inhibitor that binds and sequesters chemokines. The potential of vCKBP as a therapeutic agent in vivo was illustrated in a guinea pig skin model by the blockade of eotaxin-induced eosinophil infiltration. a feature of allergic inflammatory reactions. Furthermore, vCKBP may enable the rational design of antagonists to neutralize pathogens that use chemokine receptors to initiate infection, such as HIV or the malarial parasite.

Vaccinia virus and other poxviruses express a wide variety of proteins which are non-essential for virus replication in culture but help the virus to evade the host response to infection. Examples include proteins which oppose apoptosis, synthesise steroids, capture chemokines, counteract complement, interfere with interferon and intercept interleukins. This review provides an overview of such proteins, with an emphasis on work from our laboratory, and illustrates how the study of these proteins can increase our understanding of virus pathogenesis, the function of the immune system and how to make safer and more immunogenic poxvirus-based vaccines.

Tumor necrosis factor alpha (TNF alpha) is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious diseases. For example, plasma levels of TNF alpha are positively correlated with severity and mortality in malaria and leishmaniasis. We have previously described a polymorphism at -308 in the TNF alpha promoter and shown that the rare allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3-DQ2, which is associated with autoimmunity and high TNF alpha production. Homozygosity for TNF2 carries a sevenfold increased risk of death from cerebral malaria. Here we demonstrate, with reporter genes under the control of the two allelic TNF promoters, that TNF2 is a much stronger transcriptional activator than the common allele (TNF1) in a human B cell line. Footprint analysis using DNase I and B cell nuclear extract showed the generation of a hypersensitive site at -308 and an adjacent area of protection. There was no difference in affinity of the DNA-binding protein(s) between the two alleles. These results show that this polymorphism has direct effects on TNF alpha gene regulation and may be responsible for the association of TNF2 with high TNF alpha phenotype and more severe disease in infections such as malaria and leishmaniasis.