Immune efficacy of an adenoviral vector-based swine influenza vaccine against antigenically distinct H1N1 strains in mice Antivir. Res. (IF 4.271) Pub Date : 2017-09-20 Yunpu Wu, Dawei Yang, Bangfeng Xu, Wenhua Liang, Jinyu Sui, Yan Chen, Huanliang Yang, Hualan Chen, Ping Wei, Chuanling Qiao
Avian-like H1N1 swine influenza viruses are prevalent in pigs and have occasionally crossed the species barrier and infected humans, which highlights the importance of preventing swine influenza. Human adenovirus serotype 5 (Ad5) has been tested in human influenza vaccine clinical trials and has exhibited a reliable safety profile. Here, we generated a replication-defective, recombinant adenovirus (designated as rAd5-avH1HA) expressing the hemagglutinin gene of an avian-like H1N1 virus (A/swine/Zhejiang/199/2013, ZJ/199/13). Using a BALB/c mouse model, we showed that a two-dose intramuscular administration of recombinant rAd5-avH1HA induced high levels of hemagglutination inhibition antibodies and prevented homologous H1N1 and heterologous H1N1 virus-induced weight loss, as well as viral replication in the nasal turbinates and lungs of mice. Furthermore, a prime–boost immunization strategy trial with a recombinant plasmid (designated as pCAGGS-HA) followed by rAd5-avH1HA vaccine provided effective protection against homologous and heterologous H1N1 virus infection in mice. These results indicate that rAd5-avH1HA is an efficacious genetically engineered vaccine candidate against H1N1 swine influenza. Future studies should examine its immune efficacy in pigs.
A novel candidate MS2 phage VLP vaccine displaying a tandem HPV L2 peptide offers similar protection in mice to Gardasil-9 Antivir. Res. (IF 4.271) Pub Date : 2017-09-20 Lukai Zhai, Julianne Peabody, Yuk-Ying Susana Pang, John Schiller, Bryce Chackerian, Ebenezer Tumban
Human papillomaviruses (HPVs) cause approximately 5% of cancer cases worldwide. Fortunately, three prophylactic vaccines have been approved to protect against HPV infections. Gardasil-9, the most recent HPV vaccine, is predicted to offer protection against the HPV types that cause ∼90% of cervical cancer, 86% of HPV-associated penile cancers, and ∼93% of HPV-associated head & neck cancers. As an alternative to Gardasil-9, we developed and tested a novel candidate vaccine targeting conserved epitopes in the HPV minor capsid protein, L2. We displayed a tandem HPV31/16L2 peptide (amino acid 17–31) or consensus peptides from HPV L2 (amino acid 69–86 or 108–122) on the surface of bacteriophage MS2 virus-like particles (VLPs). Mice immunized with the MS2 VLPs displaying the tandem peptide or immunized with a mixture of VLPs (displaying the tandem peptide and consensus peptide 69–86) elicited high titer antibodies against individual L2 epitopes. Moreover, vaccinated mice were protected from cervicovaginal infection with HPV pseudoviruses 16, 18, 31, 33, 45, and 58 at levels similar to mice immunized with Gardasil-9. These results suggest that immunization with a tandem, L2 peptide or a low valency mixture of L2 peptide-displaying VLPs can provide broad protection against multiple HPV types.
Recurrent herpetic keratitis despite antiviral prophylaxis: A virological and pharmacological study Antivir. Res. (IF 4.271) Pub Date : 2017-09-20
Recurrent herpes simplex keratitis (HSK) is a leading infectious cause of blindness in industrialized countries. Antiviral prophylaxis (AVP) may fail to prevent recurrence of HSK due to viral resistance, inadequate dosing, or poor patient compliance. In this prospective multicenter study, we enrolled immunocompetent patients with recurrent HSK despite AVP. Ocular samples were tested by PCR for herpes simplex virus 1 (HSV-1). HSV-1 drug resistance was assessed with a genotypic assay based on UL23 and UL30 gene sequencing. After curative full dose valacyclovir (VACV) treatment was started, peak and trough acyclovir (ACV) plasma concentrations were measured, and patient compliance to AVP was assessed with a questionnaire.The study sample was comprised of 43 patients. Six (14%) patients were positive for HSV-1 using PCR, of whom 5 (83%) harbored genotypically ACV-resistant (ACVR) virus, due to mutations in UL23 (n = 4) or UL30 (n = 1). Disease duration was statistically significantly longer in patients with viral resistance compared to other HSK patients [35.5 ± 23.4 years (range, 6.8–68.4 years) versus 11.1 ± 12.3 years (range, 0.8–56.3 year) respectively; Mann-Whitney p = 0.01)].While patients were treated with full dose VACV, trough ACV plasma concentrations were below the threshold for ACV sensitivity in 9.5% of cases, and compliance was poor in 5.3% of cases.To summarize, HSV-1 resistance to ACV seems to be a significant cause of failure of prophylaxis in patients with HSK and is associated with longer disease duration. Most PCR-positive samples contained genotypically ACVR virus and identification may aid in adapting treatment. Incomplete 24-h drug coverage may also explain some cases of failure of prophylaxis.
Celastrol inhibits hepatitis C virus replication by upregulating heme oxygenase-1 via the JNK MAPK/Nrf2 pathway in human hepatoma cells Antivir. Res. (IF 4.271) Pub Date : 2017-09-19 Chin-Kai Tseng, Sung-Po Hsu, Chun-Kuang Lin, Yu-Hsuan Wu, Jin-Ching Lee, Kung-Chia Young
Use of whole genome deep sequencing to define emerging minority variants in virus envelope genes in herpesvirus treated with novel antimicrobial K21 Antivir. Res. (IF 4.271) Pub Date : 2017-09-19 Joshua G. Tweedy, Bhupesh K. Prusty, Ursula A. Gompels
New antivirals are required to prevent rising antimicrobial resistance from replication inhibitors. The aim of this study was to analyse the range of emerging mutations in herpesvirus by whole genome deep sequencing. We tested human herpesvirus 6 treatment with novel antiviral K21, where evidence indicated distinct effects on virus envelope proteins. We treated BACmid cloned virus in order to analyse mechanisms and candidate targets for resistance. Illumina based next generation sequencing technology enabled analyses of mutations in 85 genes to depths of 10,000 per base detecting low prevalent minority variants (<1%). After four passages in tissue culture the untreated virus accumulated mutations in infected cells giving an emerging mixed population (45–73%) of non-synonymous SNPs in six genes including two envelope glycoproteins. Strikingly, treatment with K21 did not accumulate the passage mutations; instead a high frequency mutation was selected in envelope protein gQ2, part of the gH/gL complex essential for herpesvirus infection. This introduced a stop codon encoding a truncation mutation previously observed in increased virion production. There was reduced detection of the glycoprotein complex in infected cells. This supports a novel pathway for K21 targeting virion envelopes distinct from replication inhibition.
A pilot study to expand treatment of chronic hepatitis C in resource-limited settings Antivir. Res. (IF 4.271) Pub Date : 2017-09-18 Poonam Mathur, Emily Comstock, Edward McSweegan, Natalia Mercer, Nongthombam Suraj Kumar, Shyamasundaran Kottilil
The past five years have seen a revolution in the treatment of chronic hepatitis C, as short duration oral regimens of direct-acting antiviral drugs (DAAs), with nearly 100% cure rates for all genotypes, have replaced longer courses of ribavirin and injected interferon. Although initially very expensive, these DAAs are now becoming available in generic equivalents in countries with large numbers of chronically infected people, such as India. However, a number of obstacles may hinder the delivery of these drugs in resource-limited settings, including lack of access to diagnostic testing and the restriction of treatment to a small number of medical specialists. New approaches are therefore needed to make DAAs available to the estimated 71 million infected people, many of whom disproportionately live in low- or middle-income countries. A recent pilot study (ASCEND) of hepatitis C management in a low-income population in Washington, D.C., demonstrated that trained nurse practitioners, primary care physicians and hepatologists were equally successful in diagnosing and treating patients, indicating that such an approach might be successful in resource-limited regions of the world. Members of the Global Virus Network have received funding to carry out a similar training project in a region of India with a high prevalence of hepatitis C. This paper reviews the challenges of delivering DAA therapy in low- and middle-income countries, describes plans for performing and evaluating the effectiveness of a training program in India, and discusses future needs for the eventual elimination of hepatitis C.
Pentagalloylglucose, a highly bioavailable polyphenolic compound present in Cortex moutan, efficiently blocks hepatitis C virus entry Antivir. Res. (IF 4.271) Pub Date : 2017-09-18 Patrick Behrendt, Paula Perin, Nicolas Menzel, Dominic Banda, Stephanie Pfaender, Marco P. Alves, Volker Thiel, Phillip Meulemann, Che C. Colpitts, Luis M. Schang, Florian W.R. Vondran, Anggakusuma, Michael P. Manns, Eike Steinmann, Thomas Pietschmann
Approximately 142 million people worldwide are infected with hepatitis C virus (HCV). Although potent direct acting antivirals are available, high costs limit access to treatment. Chronic hepatitis C virus infection remains a major cause of orthotopic liver transplantation. Moreover, re-infection of the graft occurs regularly. Antivirals derived from natural sources might be an alternative and cost-effective option to complement therapy regimens for global control of hepatitis C virus infection.We tested the antiviral properties of a mixture of different Chinese herbs/roots named Zhi Bai Di Huang Wan (ZBDHW) and its individual components on HCV. One of the ZBDHW components, Penta-O-Galloyl-Glucose (PGG), was further analyzed for its mode of action in vitro, its antiviral activity in primary human hepatocytes as well as for its bioavailability and hepatotoxicity in mice.ZBDHW, its component Cortex Moutan and the compound PGG efficiently block entry of HCV of all major genotypes and also of the related flavivirus Zika virus. PGG does not disrupt HCV virion integrity and acts primarily during virus attachment. PGG shows an additive effect when combined with the well characterized HCV inhibitor Daclatasvir. Analysis of bioavailability in mice revealed plasma levels above tissue culture IC50 after a single intraperitoneal injection.In conclusion, PGG is a pangenotypic HCV entry inhibitor with high bioavailability. The low cost and wide availability of this compound make it a promising candidate for HCV combination therapies, and also emerging human pathogenic flaviviruses like ZIKV.
STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease Antivir. Res. (IF 4.271) Pub Date : 2017-09-18 Tobias Schöne, Lena Lisbeth Grimm, Naoki Sakai, Linlin Zhang, Rolf Hilgenfeld, Thomas Peters
West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3pro, we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3pro. Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC50 ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3pro. To identify the matching protein binding site, chemical shift perturbation studies employing 1H,15N-TROSY-HSQC experiments with uniformly 2H,15N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3pro inhibitors.
Broad-spectrum non-nucleoside inhibitors for caliciviruses Antivir. Res. (IF 4.271) Pub Date : 2017-07-27 Natalie E. Netzler, Daniel Enosi Tuipulotu, Auda A. Eltahla, Jennifer H. Lun, Salvatore Ferla, Andrea Brancale, Nadya Urakova, Michael Frese, Tanja Strive, Jason M. Mackenzie, Peter A. White
Viruses of the Caliciviridae cause significant and sometimes lethal diseases, however despite substantial research efforts, specific antivirals are lacking. Broad-spectrum antivirals could combat multiple viral pathogens, offering a rapid solution when no therapies exist. The RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target as it is essential for viral replication and lacks mammalian homologs. To focus the search for pan-Caliciviridae antivirals, the RdRp was probed with non-nucleoside inhibitors (NNIs) developed against hepatitis C virus (HCV) to reveal both allosteric ligands for structure-activity relationship enhancement, and highly-conserved RdRp pockets for antiviral targeting. The ability of HCV NNIs to inhibit calicivirus RdRp activities was assessed using in vitro enzyme and murine norovirus cell culture assays. Results revealed that three NNIs which bound the HCV RdRp Thumb I (TI) site also inhibited transcriptional activities of six RdRps spanning the Norovirus, Sapovirus and Lagovirus genera of the Caliciviridae. These NNIs included JTK-109 (RdRp inhibition range: IC50 4.3–16.6 μM), TMC-647055 (IC50 range: 18.8–45.4 μM) and Beclabuvir (IC50 range: 23.8–>100 μM). In silico studies and site-directed mutagenesis indicated the JTK-109 binding site was within the calicivirus RdRp thumb domain, in a pocket termed Site-B, which is highly-conserved within all calicivirus RdRps. Additionally, RdRp inhibition assays revealed that JTK-109 was antagonistic with the previously reported RdRp inhibitor pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) tetrasodium salt (PPNDS), that also binds to Site-B. Moreover, like JTK-109, PPNDS was also a potent inhibitor of polymerases from six viruses spanning the three Caliciviridae genera tested (IC50 range: 0.1–2.3 μM). Together, this study demonstrates the potential for de novo development of broad-spectrum antivirals that target the highly-conserved RdRp thumb pocket, Site-B. We also revealed three broad-spectrum HCV NNIs that could be used as antiviral scaffolds for further development against caliciviruses and other viruses.
Screening of an FDA-approved compound library identifies levosimendan as a novel anti-HIV-1 agent that inhibits viral transcription Antivir. Res. (IF 4.271) Pub Date : 2017-08-24 Tsuyoshi Hayashi, Maxime Jean, Huachao Huang, Sydney Simpson, Netty G. Santoso, Jian Zhu
Combination antiretroviral therapy (cART) has been proven to efficiently inhibit ongoing replication of human immunodeficiency virus type 1 (HIV-1), and significantly improve the health outcome in patients of acquired immune deficiency syndrome (AIDS). However, cART is unable to cure HIV-1/AIDS. Even in presence of cART there exists a residual viremia, contributed from the viral reservoirs of latently infected HIV-1 proviruses; this constitutes a major hurdle. Currently, there are multiple strategies aimed at eliminating or permanently silence these HIV-1 latent reservoirs being intensely explored. One such strategy, a recently emerged “block and lock” approach is appealing. For this approach, so-called HIV-1 latency-promoting agents (LPAs) are used to reinforce viral latency and to prevent the low-level or sporadic transcription of integrated HIV-1 proviruses. Although several LPAs have been reported, there is still a question of their suitability to be further developed as a safe and valid therapeutic agent for the clinical use. In this study, we aimed to identify new potential LPAs through the screening an FDA-approved compound library. A new and promising anti-HIV-1 inhibitor, levosimendan, was identified from these screens. Levosimendan is currently used to treat heart failure in clinics, but it demonstrates strong inhibition of TNFα-induced HIV-1 reactivation in multiple cell lines of HIV-1 latency through affecting the HIV-1 Tat-LTR transcriptional axis. Furthermore, we confirmed that in primary CD4+ T cells levosimendan inhibits both the acute HIV-1 replication and the reactivation of latent HIV-1 proviruses. As a summary, our studies successfully identify levosimendan as a novel and promising anti-HIV-1 inhibitor, which should be immediately investigated in vivo given that it is already an FDA-approved drug.
Rapamycin-induced autophagy restricts porcine epidemic diarrhea virus infectivity in porcine intestinal epithelial cells Antivir. Res. (IF 4.271) Pub Date : 2017-08-24 Seongyeol Ko, Min Jeong Gu, Cheol Gyun Kim, Yoon Chul Kye, Younggap Lim, Ji Eun Lee, Byung-Chul Park, Hyuk Chu, Seung Hyun Han, Cheol-Heui Yun
Porcine epidemic diarrhea virus (PEDV) invades porcine intestinal epithelial cells (IECs) and causes diarrhea and dehydration in pigs. In the present study, we showed a suppression of PEDV infection in porcine jejunum intestinal epithelial cells (IPEC-J2) by an increase in autophagy. Autophagy was activated by rapamycin at a dose that does not affect cell viability and tight junction permeability. The induction of autophagy was examined by LC3I/LC3II conversion. To confirm the autophagic-flux (entire autophagy pathway), autophagolysosomes were examined by an immunofluorescence assay. Pre-treatment with rapamycin significantly restricted not only a 1 h infection but also a longer infection (24 h) with PEDV, while this effect disappeared when autophagy was blocked. Co-localization of PEDV and autophagosomes suggests that PEDV could be a target of autophagy. Moreover, alleviation of PEDV-induced cell death in IPEC-J2 cells pretreated with rapamycin demonstrates a protective effect of rapamycin against PEDV-induced epithelial cell death. Collectively, the present study suggests an early prevention against PEDV infection in IPEC-J2 cells via autophagy that might be an effective strategy for the restriction of PEDV, and opens up the possibility of the use of rapamycin in vivo as an effective prophylactic and prevention treatment.
Bis(benzofuran–thiazolidinone)s and bis(benzofuran–thiazinanone)s as inhibiting agents for chikungunya virus Antivir. Res. (IF 4.271) Pub Date : 2017-08-19 Jih Ru Hwu, Nitesh K. Gupta, Shwu-Chen Tsay, Wen-Chieh Huang, Irina C. Albulescu, Kristina Kovacikova, Martijn J. van Hemert
Evaluation of antiviral activity of piperazine against Chikungunya virus targeting hydrophobic pocket of alphavirus capsid protein Antivir. Res. (IF 4.271) Pub Date : 2017-08-24 Megha Aggarwal, Ramanjit Kaur, Amrita Saha, Rajat Mudgal, Ravi Yadav, Paban Kumar Dash, Manmohan Parida, Pravindra Kumar, Shailly Tomar
Small heterocyclic molecules such as piperazine are potential pharmacotherapeutic agents and binding of these molecules to the hydrophobic pocket of capsid protein (CP) offers a new perspective for therapeutic intervention. Here, we report the crystal structure of CP from Aura virus (AVCP) in complex with piperazine at 2.2 Å resolution. Piperazine binds to the conserved hydrophobic pocket of CP where dioxane based antivirals bind. Comparative structural studies of the piperazine-bound AVCP structure with the apo, active and dioxane-bound AVCP structures provide insights into the conformational variations in the pocket. Additionally, the molecular docking studies showed that piperazine binds into the hydrophobic pocket of Chikungunya virus CP (CVCP) with more affinity than with AVCP. Furthermore, the antiviral activity of piperazine against Chikungunya virus (CHIKV) was investigated by plaque reduction and immunofluorescence assays. The AVCP-piperazine complex may serve as a lead scaffold for structure-based design of piperazine derivatives as alphaviral inhibitors. The antiviral properties of piperazine provide its usefulness for further investigations towards the development of piperazine based anti-alphaviral drugs.
Identification of broadly neutralizing monoclonal antibodies against Crimean-Congo hemorrhagic fever virus Antivir. Res. (IF 4.271) Pub Date : 2017-08-24 Marko Zivcec, Lisa I.W. Guerrero, César G. Albariño, Éric Bergeron, Stuart T. Nichol, Christina F. Spiropoulou
Despite the serious public health impact of Crimean-Congo hemorrhagic fever (CCHF), the efficacy of antivirals targeting the causative agent, CCHF virus (CCHFV), remains debatable. Neutralizing monoclonal antibodies (MAbs) targeting the CCHFV glycoprotein Gc have been reported to protect mice against challenge with the prototype CCHFV strain, IbAr10200. However, due to extensive sequence diversity of CCHFV glycoproteins, it is unknown whether these MAbs neutralize other CCHFV strains. We initially used a CCHF virus-like particle (VLP) system to generate 11 VLP moieties, each possessing a glycoprotein from a genetically diverse CCHFV strain isolated in either Africa, Asia, the Middle East, or southeastern Europe. We used these VLPs in biosafety level 2 conditions to efficiently screen MAb cross-neutralization potency. Of the 16 MAbs tested, 3 (8A1, 11E7, and 30F7) demonstrated cross-neutralization activity with most CCHF VLPs, with 8A1 neutralizing all VLPs tested. Although binding studies suggest that none of the MAbs compete for the same epitope, combining 11E7, 30F7, or both 11E7 and 30F7 with 8A1 had no additive effect on increasing neutralization in this system. To confirm our findings from the VLP system, the 3 MAbs capable of strain cross-neutralization were confirmed to effectively neutralize 5 diverse CCHFV strains in vitro. Passaging CCHFV strains in the presence of sub-neutralizing concentrations of MAbs did not generate escape mutants resistant to subsequent neutralization. This study demonstrates the utility of the VLP system for screening neutralizing MAbs against multiple CCHFV strains, and provides the first evidence that a single MAb can effectively neutralize a number of diverse CCHFV strains in vitro, which may lead to development of future CCHF therapeutics.
Combination therapy with brincidofovir and valganciclovir against species C adenovirus infection in the immunosuppressed Syrian hamster model allows for substantial reduction of dose for both compounds Antivir. Res. (IF 4.271) Pub Date : 2017-08-04 Karoly Toth, Ann E. Tollefson, Jacqueline F. Spencer, Baoling Ying, William S.M. Wold
Adenovirus infections of immunocompetent adults are usually mild and resolve without serious sequelae. However, adenovirus infections of immunocompromised patients often develop into life-threatening multi-organ disease. Pediatric hematopoietic transplant patients are especially threatened, with high incidence of infection and high mortality rates. Presently, there is no drug specifically approved by the FDA to treat adenovirus infections; thus there is an urgent need to develop effective antivirals against the virus. Previously, we demonstrated that brincidofovir and valganciclovir were efficacious against lethal intravenous challenge with human type 5 adenovirus in the Syrian hamster model. Here, we tested the in vivo efficacy of the combination of these two drugs and showed that the combination of brincidofovir and valganciclovir is more efficacious than either drug alone, thus potentially allowing decreased patient exposure to the drugs while maintaining antiviral efficacy. As antiviral compounds often have toxic side effects, a decrease in dose or duration of therapy allowed by the combination could also improve tolerability.
Evaluation of antiviral effect of type I, II, and III interferons on direct-acting antiviral-resistant hepatitis C virus Antivir. Res. (IF 4.271) Pub Date : 2017-08-31 Atsushi Hamana, Yuki Takahashi, Takuro Uchida, Makiya Nishikawa, Michio Imamura, Kazuaki Chayama, Yoshinobu Takakura
Treatment of hepatitis C virus (HCV) infection has greatly improved in the last 5 years because of the identification of direct-acting antivirals (DAAs). However, concerns exist regarding the emergence of drug resistance-associated substitutions (RASs). In this study, we evaluated the in vivo antiviral effect of three classes of interferons (IFNs), namely, types I, II, and III IFNs, on DAA-resistant HCVs. IFN-α2, IFN-γ, and IFN-λ1 were selected as typical types I, II, and III IFNs, respectively. Human hepatocyte-transplanted chimeric mice were infected with NS3-D168, NS5A-L31-, and NS5A-Y93-mutated HCVs, and the antiviral effect of IFN-α2, IFN-γ, and IFN-λ1 on these HCV RASs was examined. Chimeric mice infected with NS3- and NS5A-mutated HCVs were hydrodynamically injected with IFN-expressing plasmids to evaluate the antiviral effect of IFNs. Serum concentrations of IFNs were maintained for at least 42 days. We found that serum HCV level significantly decreased and serum and hepatic HCV levels reached below detection limit in 5/5 and 3/5 chimeric mice injected with IFN-γ- and IFN-λ1-expressing plasmids, respectively. The antiviral effect of IFN-α2 on DAA-resistant HCVs was weaker than that of IFN-γ and IFN-λ1. Serum ALT levels showed a small and transient increase in mice injected with the IFN-γ-expressing plasmid but not in mice injected with the IFN-λ1-expressing plasmid. However, no apparent histological damage was observed in the liver sections of mice injected with the IFN-γ-expressing plasmid. These results indicate that IFN-γ and IFN-λ1 are an attractive therapeutic option for treating infection caused by NS3- and NS5A-mutated HCV.
Searching for synergy: Identifying optimal antiviral combination therapy using Hepatitis C virus (HCV) agents in a replicon system Antivir. Res. (IF 4.271) Pub Date : 2017-09-04 Justin J. Pomeroy, George L. Drusano, Jaime L. Rodriquez, Ashley N. Brown
Direct acting antiviral agents (DAAs) are potent inhibitors of Hepatitis C virus (HCV) that have revolutionized the treatment landscape for this important viral disease. There are currently four classes of DAAs that inhibit HCV replication via distinct mechanisms of action: nonstructural protein (NS) 3/4a protease inhibitors, NS5A inhibitors, NS5B nucleoside polymerase inhibitors, and NS5B non-nucleoside polymerase inhibitors. Combination therapy with two or more DAAs has great potential to further enhance antiviral potency. The purpose of this study was to identify optimal combinations of DAAs against genotype 1 HCV replicons that maximized the inhibition of replicon replication. All possible two-drug combinations were evaluated against genotype 1a and 1b HCV replicons using a 96-well plate luciferase-based assay in triplicate. The Greco Universal Response Surface Area mathematical model was fit to the luciferase data to identify drug-drug interactions (i.e.: synergy, additivity, and antagonism) for antiviral effect against both genotypes. This information was used to rank-order combinations of DAAs based on their ability to inhibit replicon replication against genotype 1a and 1b HCV. These preclinical findings can provide information as to which antiviral regimens should move on in the development process.
Cell-line dependent antiviral activity of sofosbuvir against Zika virus Antivir. Res. (IF 4.271) Pub Date : 2017-09-11 Noreen Mumtaz, Leah C. Jimmerson, Lane R. Bushman, Jennifer J. Kiser, Georgina Aron, Chantal B.E.M. Reusken, Marion P.G. Koopmans, Jeroen J.A. van Kampen
The recent epidemic of Zika virus (ZIKV) in the Americas and its association with fetal and neurological complications has shown the need to develop a treatment. Repurposing of drugs that are already FDA approved or in clinical development may shorten drug development timelines in case of emerging viral diseases like ZIKV. Initial studies have shown conflicting results when testing sofosbuvir developed for treatment of infections with another Flaviviridae virus, hepatitis C virus. We hypothesized that the conflicting results could be explained by differences in intracellular processing of the compound. We assessed the antiviral activity of sofosbuvir and mericitabine against ZIKV using Vero, A549, and Huh7 cells and measured the level of the active sofosbuvir metabolite by mass spectrometry. Mericitabine did not show activity, while sofosbuvir inhibited ZIKV with an IC50 of ∼4 μM, but only in Huh7 cells. This correlated with differences in intracellular concentration of the active triphosphate metabolite of sofosbuvir, GS-461203 or 007-TP, which was 11–342 times higher in Huh7 cells compared to Vero and A549 cells. These results show that a careful selection of cell system for repurposing trials of prodrugs is needed for evaluation of antiviral activity. Furthermore, the intracellular levels of 007-TP in tissues and cell types that support ZIKV replication in vivo should be determined to further investigate the potential of sofosbuvir as anti-ZIKV compound.
Human polyclonal antibodies produced in transchromosomal cattle prevent lethal Zika virus infection and testicular atrophy in mice Antivir. Res. (IF 4.271) Pub Date : 2017-09-08 Derek R. Stein, Joseph W. Golden, Bryan D. Griffin, Bryce M. Warner, Charlene Ranadheera, Leanne Scharikow, Angela Sloan, Kathy L. Frost, Darwyn Kobasa, Stephanie A. Booth, Matthew Josleyn, John Ballantyne, Eddie Sullivan, Jin-an Jiao, Hua Wu, Zhongde Wang, Jay W. Hooper, David Safronetz
Raltegravir blocks the infectivity of red-fluorescent-protein (mCherry)-labeled HIV-1JR-FL in the setting of post-exposure prophylaxis in NOD/SCID/Jak3−/− mice transplanted with human PBMCs Antivir. Res. (IF 4.271) Pub Date : 2017-09-08 Hiromi Ogata-Aoki, Nobuyo Higashi-Kuwata, Shin-ichiro Hattori, Hironori Hayashi, Matthew Danish, Manabu Aoki, Chiemi Shiotsu, Yumi Hashiguchi, Akinobu Hamada, Hisataka Kobayashi, Hironobu Ihn, Seiji Okada, Hiroaki Mitsuya
Employing NOD/SCID/Jak3−/− mice transplanted with human PBMCs (hNOJ mice) and replication-competent, red-fluorescent-protein (mCherry; mC)-labeled HIV-1JR-FL (HIVmC), we examined whether early antiretroviral treatment blocked the establishment of HIV-1 infection. The use of hNOJ mice and HIVmC enabled us to visually locate infection foci and to examine the early dynamics of HIVmC infection without using a large amount of antiretroviral unlike in non-human primate models. Although when raltegravir (RAL) administration was begun 1 day after intraperitoneal (ip) inoculation of HIVmC, no plasma p24 or plasma HIV-1-RNA (pRNA) were detected in 10 of 12 hNOJ (hNOJmCRAL+) mice over 14-day observation, all 10 untreated hNOJmC (hNOJmCRAL−) mice became positive for p24 and pRNA and had significantly swollen lymph nodes in peritoneal cavity and abundant p24+/mC+/CD3+/CD4+ T cells and p24+/mC+/CD68+ monocytes/macrophages were identified in their omenta and mesenteric lymphoid tissues/lymph nodes. In 12 hNOJmCRAL+ mice, no significantly swollen lymph nodes were seen compared to hNOJmCRAL− mice; however, in the omentum of the 2 hNOJmCRAL+ mice that were positive for pRNA and in site RNA, mC+/p24+/CD3+/CD83+ cells were identified, suggesting that viral breakthrough occurred later in the observation period. The present data suggest that the use of hNOJ mouse model and HIVmC may shed light in the study of early-phase dynamics of HIV-1 infection and cellular events in post-exposure/pre-exposure prophylaxis.
DHEA prevents ribavirin-induced anemia via inhibition of glucose-6-phosphate dehydrogenase Antivir. Res. (IF 4.271) Pub Date : 2017-09-08 Lynda Handala, Barbara Domange, Hakim Ouled-Haddou, Loïc Garçon, Eric Nguyen-Khac, Francois Helle, Sandra Bodeau, Gilles Duverlie, Etienne Brochot
Ribavirin has been widely used for antiviral therapy. Unfortunately, ribavirin-induced anemia is often a cause of limiting or interrupting treatment. Our team has observed that dehydroepiandrosterone (DHEA) has a protective effect against in vitro and in vivo ribavirin-induced hemolysis. The aim of this study was to better understand this effect as well as the underlying mechanism(s).DHEA was able to reduce in vitro intraerythrocytic ATP depletion induced by ribavirin. Only 1% of ATP remained after incubation with ribavirin (2 mM) at 37 °C for 24 h vs. 37% if DHEA (200 μM) was added (p < 0.01). DHEA also helped erythrocytes conserve their size, with a shrinkage of only 10% vs 40% at 24 h with ribavirin alone (p < 0.01), and reduced phosphatidylserine exposure at the outer membrane, i.e. 27% vs 40% at 48 h, (p < 0.05). DHEA also inhibits ribavirin-induced hemolysis, i.e. 34% vs 46.5% at 72 h (p < 0.01).DHEA is an inhibitor of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme in the hexose monophosphate shunt connected to the glycolytic pathway which is the only energy supplier of the red blood cell in the form of ATP. We have confirmed this inhibitory effect in the presence of ribavirin. All these observations suggest that ribavirin-induced hemolysis was initiated by ATP depletion, and that the inhibitory effect of DHEA on G6PD was able to rescue enough ATP to limit this hemolysis. This mechanism could be important for improving the therapeutic management of patients treated with ribavirin.
Comparison of three dimensional synergistic analyses of percentage versus logarithmic data in antiviral studies Antivir. Res. (IF 4.271) Pub Date : 2017-07-01 Donald F. Smee, Mark N. Prichard
Cell culture antiviral experiments were conducted in order to understand the relationship between percentage data generated by plaque reduction (PR) and logarithmic data derived by virus yield reduction (VYR) assays, using three-dimensional MacSynergy II software. The relationship between percentage and logarithmic data has not been investigated previously. Interpretation of drug-drug interactions is based on a Volume of Synergy (VS) calculation, which can be positive (synergy), negative (antagonistic), or neutral (no or minimal interaction). Interactions of two known inhibitors of vaccinia virus replication, cidofovir and 6-azauridine, used in combination by PR assay yielded a VS value of 265, indicative of strong synergy. By VYR, the VS value was only 37, or weak synergy using the same criterion, even though profound log10 reductions in virus titer occurred at multiple drug combinations. These results confirm that the differences in VS values is dependent of the measurement scale, and not that the degree of synergy differed between the assays. We propose that for logarithmic data, the calculated VS values will be lower for significant synergy and antagonism and that volumes of >10 μM2log10 PFU/ml (or other units such as μM2log10 genomic equivalents/ml or μM2log10 copies/ml) and <-10 μM2log10 PFU/ml are likely to be indicative of strong synergy and strong antagonism, respectively. Data presented here show that the interaction of cidofovir and 6-azauridine was strongly synergistic in vitro.
Antiviral activity of gemcitabine against human rhinovirus in vitro and in vivo Antivir. Res. (IF 4.271) Pub Date : 2017-07-11 Jae-Hyoung Song, Seong-Ryeol Kim, Eun-Young Heo, Jae-Young Lee, Dong-eun Kim, Sungchan Cho, Sun-Young Chang, Byung-Il Yoon, Jeongmin Seong, Hyun-Jeong Ko
Rhinovirus, a major causative agent of the common cold, is associated with exacerbation of asthma and chronic obstructive pulmonary disease. Currently, there is no antiviral treatment or vaccine for human rhinovirus (HRV). Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) is a deoxycytidine analog with antiviral activity against rhinovirus, as well as enterovirus 71, in vitro. However, the antiviral effects of gemcitabine in vivo have not been investigated. In the current study, we assessed whether gemcitabine mediated antiviral effects in the murine HRV infection model. Intranasal administration of gemcitabine significantly lowered pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and reduction in the number of lung-infiltrating lymphocytes. Interestingly, we found that the addition of UTP and CTP significantly attenuated the antiviral activity of gemcitabine. Thus the limitation of UTP and CTP by the addition of gemcitabine may inhibit the viral RNA synthesis. These results suggest that gemcitabine, an antineoplastic drug, can be repositioned as an antiviral drug to inhibit HRV infection.
Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector Antivir. Res. (IF 4.271) Pub Date : 2017-07-11 Julie Lucifora, Anna Salvetti, Xavier Marniquet, Laurent Mailly, Barbara Testoni, Floriane Fusil, Aurore Inchauspé, Maud Michelet, Marie-Louise Michel, Massimo Levrero, Pierre Cortez, Thomas F. Baumert, François-Loic Cosset, Cécile Challier, Fabien Zoulim, David Durantel
NgAgo-gDNA system efficiently suppresses hepatitis B virus replication through accelerating decay of pregenomic RNA Antivir. Res. (IF 4.271) Pub Date : 2017-07-12 Zhuanchang Wu, Siyu Tan, Leiqi Xu, Lifen Gao, Haizhen Zhu, Chunhong Ma, Xiaohong Liang
Covalently closed circular DNA (cccDNA) in the hepatocytes nucleus is responsible for persistent infection of Hepatitis B virus (HBV). Current antiviral therapy drugs nucleos(t)ide analogs or interferon fail to eradicate HBV cccDNA. Genome editing technique provides an effective approach for HBV treatment through targeting viral cccDNA. Natronobacterium gregoryi Argonaute (NgAgo)-guide DNA (gDNA) system with powerful genome editing prompts us to explore its application in inhibiting HBV replication. Preliminary function verification indicated that NgAgo/EGFP-gDNA obviously inhibited EGFP expression. To further explore the potential role of NgAgo in restricting HBV replication, 10 of gDNAs targeting the critical region of viral genome were designed, only S-142, P-263 and P-2166 gDNAs led to significant inhibition on HBsAg, HBeAg and pregenomic RNA (pgRNA) level in Huh7 and HepG2 cells transfected with pcDNA-HBV1.1 plasmid. Similar results were also found in HBV infected HLCZ01 cells and Huh7-NTCP cells. However, we failed to detect any DNA editing in S-142, P-263 and P-2166 targeting region through T7E1 assay and Sanger sequencing. Remarkably, we found that NgAgo/P-2166 significantly accelerated the decay of viral pgRNA. Taken together, our results firstly demonstrate the potential of NgAgo/gDNA in inhibiting HBV replication through accelerating pgRNA degradation, but not DNA editing.
Identification of a coumarin-based antihistamine-like small molecule as an anti-filoviral entry inhibitor Antivir. Res. (IF 4.271) Pub Date : 2017-06-20 Han Cheng, Adam Schafer, Veronica Soloveva, Dima Gharaibeh, Tara Kenny, Cary Retterer, Rouzbeh Zamani, Sina Bavari, Norton P. Peet, Lijun Rong
Filoviruses, consisting of Ebola virus, Marburg virus and Cuevavirus, cause severe hemorrhagic fevers in humans with high mortality rates up to 90%. Currently, there is no approved vaccine or therapy available for the prevention and treatment of filovirus infection in humans. The recent 2013–2015 West African Ebola epidemic underscores the urgency to develop antiviral therapeutics against these infectious diseases. Our previous study showed that GPCR antagonists, particularly histamine receptor antagonists (antihistamines) inhibit Ebola and Marburg virus entry. In this study, we screened a library of 1220 small molecules with predicted antihistamine activity, identified multiple compounds with potent inhibitory activity against entry of both Ebola and Marburg viruses in human cancer cell lines, and confirmed their anti-Ebola activity in human primary cells. These small molecules target a late-stage of Ebola virus entry. Further structure-activity relationship studies around one compound (cp19) reveal the importance of the coumarin fused ring structure, especially the hydrophobic substituents at positions 3 and/or 4, for its antiviral activity, and this identified scaffold represents a favorable starting point for the rapid development of anti-filovirus therapeutic agents.
Structure-based discovery of clinically approved drugs as Zika virus NS2B-NS3 protease inhibitors that potently inhibit Zika virus infection in vitro and in vivo Antivir. Res. (IF 4.271) Pub Date : 2017-07-14 Shuofeng Yuan, Jasper Fuk-Woo Chan, Helena den-Haan, Kenn Ka-Heng Chik, Anna Jinxia Zhang, Chris Chung-Sing Chan, Vincent Kwok-Man Poon, Cyril Chik-Yan Yip, Winger Wing-Nga Mak, Zheng Zhu, Zijiao Zou, Kah-Meng Tee, Jian-Piao Cai, Kwok-Hung Chan, Jorge de la Peña, Horacio Pérez-Sánchez, José Pedro Cerón-Carrasco, Kwok-Yung Yuen
Zika virus (ZIKV) infection may be associated with severe complications in fetuses and adults, but treatment options are limited. We performed an in silico structure-based screening of a large chemical library to identify potential ZIKV NS2B-NS3 protease inhibitors. Clinically approved drugs belonging to different drug classes were selected among the 100 primary hit compounds with the highest predicted binding affinities to ZIKV NS2B-NS3-protease for validation studies. ZIKV NS2B-NS3 protease inhibitory activity was validated in most of the selected drugs and in vitro anti-ZIKV activity was identified in two of them (novobiocin and lopinavir-ritonavir). Molecular docking and molecular dynamics simulations predicted that novobiocin bound to ZIKV NS2B-NS3-protease with high stability. Dexamethasone-immunosuppressed mice with disseminated ZIKV infection and novobiocin treatment had significantly (P < 0.05) higher survival rate (100% vs 0%), lower mean blood and tissue viral loads, and less severe histopathological changes than untreated controls. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of ZIKV.
Targeting heat shock factor 1 as an antiviral strategy against dengue virus replication in vitro and in vivo Antivir. Res. (IF 4.271) Pub Date : 2017-07-18 Tsung-Ting Tsai, Chia-Ling Chen, Cheng-Chieh Tsai, Chiou-Feng Lin
A heterologous prime-boost Ebola virus vaccine regimen induces durable neutralizing antibody response and prevents Ebola virus-like particle entry in mice Antivir. Res. (IF 4.271) Pub Date : 2017-07-18 Tan Chen, Dapeng Li, Yufeng Song, Xi Yang, Qingwei Liu, Xia Jin, Dongming Zhou, Zhong Huang
Ebola virus (EBOV) is one of the most virulent pathogens known to humans. Neutralizing antibodies play a major role in the protection against EBOV infections. Thus, an EBOV vaccine capable of inducing a long-lasting neutralizing antibody response is highly desirable. We report here that a heterologous prime-boost vaccine regimen can elicit durable EBOV-neutralizing antibody response in mice. A chimpanzee serotype 7 adenovirus expressing EBOV GP (denoted AdC7-GP) was generated and used for priming. A truncated version of EBOV GP1 protein (denoted GP1t) was produced at high levels in Drosophila S2 cells and used for boosting. Mouse immunization studies showed that the AdC7-GP prime/GP1t boost vaccine regimen was more potent in eliciting neutralizing antibodies than either the AdC7-GP or GP1t alone. Neutralizing antibodies induced by the heterologous prime-boost regimen sustained at high titers for at least 18 weeks after immunization. Significantly, in vivo challenge studies revealed that the entry of reporter EBOV-like particles was efficiently blocked in mice receiving the heterologous prime-boost regimen even at 18 weeks after the final dose of immunization. These results suggest that this novel AdC7-GP prime/GP1t boost regimen represents an EBOV vaccine approach capable of establishing long-term protection, and therefore warrants further development.
Identification of an essential virulence gene of cyprinid herpesvirus 3 Antivir. Res. (IF 4.271) Pub Date : 2017-07-06 Maxime Boutier, Yuan Gao, Catherine Vancsok, Nicolás M. Suárez, Andrew J. Davison, Alain Vanderplasschen
p38MAPK plays a critical role in induction of a pro-inflammatory phenotype of retinal Müller cells following Zika virus infection Antivir. Res. (IF 4.271) Pub Date : 2017-07-21 Shuang Zhu, Huanle Luo, Hua Liu, Yonju Ha, Elizabeth R. Mays, Ryan E. Lawrence, Evandro Winkelmann, Alan D. Barrett, Sylvia B. Smith, Min Wang, Tian Wang, Wenbo Zhang
Zika virus (ZIKV) infection has been associated with ocular abnormalities such as chorioretinal atrophy, optic nerve abnormalities, posterior uveitis and idiopathic maculopathy. Yet our knowledge about ZIKV infection in retinal cells and its potential contribution to retinal pathology is still very limited. Here we found that primary Müller cells, the principal glial cells in the retina, expressed a high level of ZIKV entry cofactor AXL gene and were highly permissive to ZIKV infection. In addition, ZIKV-infected Müller cells exhibited a pro-inflammatory phenotype and produced many inflammatory and growth factors. While a number of inflammatory signaling pathways such as ERK, p38MAPK, NF-κB, JAK/STAT3 and endoplasmic reticulum stress were activated after ZIKV infection, inhibition of p38MAPK after ZIKV infection most effectively blocked ZIKV-induced inflammatory and growth molecules. In comparison to ZIKV, Dengue virus (DENV), another Flavivirus infected Müller cells more efficiently but induced much lower pro-inflammatory responses. These data suggest that Müller cells play an important role in ZIKV-induced ocular pathology by induction of inflammatory and growth factors in which the p38MAPK pathway has a central role. Blocking p38MAPK may provide a novel approach to control ZIKV-induced ocular inflammation.
Replication of the Zika virus in different iPSC-derived neuronal cells and implications to assess efficacy of antivirals Antivir. Res. (IF 4.271) Pub Date : 2017-07-20 Kristina Lanko, Kristel Eggermont, Abdulsamie Patel, Suzanne Kaptein, Leen Delang, Catherine M. Verfaillie, Johan Neyts
Infections with the Zika virus (ZIKV) are responsible for congenital abnormalities and neurological disorders. We here demonstrate that ZIKV productively infects three types of human iPSC (induced pluripotent stem cells)-derived cells from the neural lineage, i.e. cortical and motor neurons as well as astrocytes. ZIKV infection results in all three cell types in the production of infectious virus particles and induces cytopathic effects (CPE). In cortical and motor neurons, an Asian isolate (PRVABC59) produced roughly 10-fold more virus than the prototypic African strain (MR766 strain). Viral replication and CPE is efficiently inhibited by the nucleoside polymerase inhibitor 7-deaza-2′-C-methyladenosine (7DMA). However, ribavirin and favipiravir, two molecules that inhibit ZIKV replication in Vero cells, did not inhibit ZIKV replication in the neuronal cells. These results highlight the need to assess the potential antiviral activity of novel ZIKV inhibitors in stem cell derived neuronal cultures.
Viral minority variants in the core promoter and precore region identified by deep sequencing are associated with response to peginterferon and adefovir in HBeAg negative chronic hepatitis B patients Antivir. Res. (IF 4.271) Pub Date : 2017-07-25 Louis Jansen, Matthijs R.A. Welkers, Karel A. van Dort, R. Bart Takkenberg, Uri Lopatin, Hans L. Zaaijer, Menno D. de Jong, Hendrik W. Reesink, Neeltje A. Kootstra
The cholesterol transport inhibitor U18666A inhibits type I feline coronavirus infection Antivir. Res. (IF 4.271) Pub Date : 2017-08-03 Tomomi Takano, Misaki Endoh, Hiroaki Fukatsu, Haruko Sakurada, Tomoyoshi Doki, Tsutomu Hohdatsu
Feline infectious peritonitis (FIP) is a feline coronavirus (FCoV)-induced fatal disease in wild and domestic cats. FCoV exists in two serotypes. Type I FCoV is the dominant serotype worldwide. Therefore, it is necessary to develop antiviral drugs against type I FCoV infection. We previously reported that type I FCoV is closely associated with cholesterol throughout the viral life cycle. In this study, we investigated whether U18666A, the cholesterol synthesis and transport inhibitor, shows antiviral effects against type I FCoV. U18666A induced cholesterol accumulation in cells and inhibited type I FCoV replication. Surprisingly, the antiviral activity of U18666A was suppressed by the histone deacetylase inhibitor (HDACi), Vorinostat. HDACi has been reported to revert U18666A-induced dysfunction of Niemann-Pick C1 (NPC1). In conclusion, these findings demonstrate that NPC1 plays an important role in type I FCoV infection. U18666A or other cholesterol transport inhibitor may be considered as the antiviral drug for the treatment of cats with FIP.
Discovery of dapivirine, a nonnucleoside HIV-1 reverse transcriptase inhibitor, as a broad-spectrum antiviral against both influenza A and B viruses Antivir. Res. (IF 4.271) Pub Date : 2017-08-02 Yanmei Hu, Jiantao Zhang, Rami Ghassan Musharrafieh, Chunlong Ma, Raymond Hau, Jun Wang
Efficacy of a high potency O1 Manisa monovalent vaccine against heterologous challenge with foot-and-mouth disease virus of O/SEA/Mya-98 lineage in sheep Antivir. Res. (IF 4.271) Pub Date : 2017-08-03 N.B. Singanallur, J.M. Pacheco, J. Arzt, C. Stenfeldt, G.T. Fosgate, L. Rodriguez, W. Vosloo
Potency tests for commercial oil-adjuvanted foot-and-mouth disease (FMD) vaccines are usually carried out in cattle, using a full dose (2 ml) of vaccine and homologous virus challenge. However, in sheep the recommended vaccine dose is half of the cattle dose (1 ml) and most vaccines have not been potency tested for this species, especially with heterologous viruses. To determine the efficacy of a high potency (>6PD50) FMD virus (FMDV) O1Manisa vaccine in sheep, we carried out a study using a heterologous FMDV (FMDV O/SKR/2010 - Mya-98 strain) challenge. Groups of seven animals each were vaccinated with 2×, 1×, 1/2× or 1/4× dose (2 ml, 1 ml, 0.5 ml or 0.25 ml respectively) and challenged at 7 days post vaccination (dpv). Only 3 of the 7 sheep in the group vaccinated with 2 ml were protected. With 2 additional groups, receiving double or single doses and challenged at 14 dpv, 4 of 7 sheep were protected in each group. None of the sheep had measurable neutralising antibodies against the vaccine or challenge virus at 7 dpv. However, all vaccinated animals challenged at 14 dpv had a homologous neutralising response against FMDV O1 Manisa on the day of challenge and all but one animal also had a heterologous response to FMDV O/SKR/2010. Infectious FMDV and viral RNA could be found in nasal swabs between 1 and 6 days post challenge (dpc) in most vaccinated sheep, but those vaccinated with higher doses or challenged at 14 dpv showed significant decreases in the level of FMDV detection. Intermittent virus shedding was noticed between 1 and 35 dpc in all vaccinated groups, but persistent infection could be demonstrated only in 4 sheep (20%). This study showed that at the recommended dose, a high potency (>6 PD50) FMDV O1Manisa vaccine does not protect sheep against a heterologous challenge at 7 dpv. However, partial protection was observed when a double dose was used at 7 dpv or when double or single dose vaccinated sheep were challenged at 14 dpv.
Cinnamic acid derivatives inhibit hepatitis C virus replication via the induction of oxidative stress Antivir. Res. (IF 4.271) Pub Date : 2017-08-02 Ryota Amano, Atsuya Yamashita, Hirotake Kasai, Tomoka Hori, Sayoko Miyasato, Setsu Saito, Hiromasa Yokoe, Kazunori Takahashi, Tomohisa Tanaka, Teruhime Otoguro, Shinya Maekawa, Nobuyuki Enomoto, Masayoshi Tsubuki, Kohji Moriishi
Several cinnamic acid derivatives have been reported to exhibit antiviral activity. In this study, we prepared 17 synthetic cinnamic acid derivatives and screened them to identify an effective antiviral compound against hepatitis C virus (HCV). Compound 6, one of two hit compounds, suppressed the viral replications of genotypes 1b, 2a, 3a, and 4a with EC50 values of 1.5–8.1 μM and SI values of 16.2–94.2. The effect of compound 6 on the phosphorylation of Tyr705 in signal transducer and activator of transcription 3 (STAT3) was investigated because a cinnamic acid derivative AG490 was reported to suppress HCV replication and the activity of Janus kinase (JAK) 2. Compound 6 potently suppressed HCV replication, but it did not inhibit the JAK1/2-dependent phosphorylation of STAT3 Tyr705 at the same concentration. Furthermore, a pan-JAK inhibitor tofacitinib potently impaired phosphorylation of STAT3 Tyr 705, but it did not inhibit HCV replication in the replicon cells and HCV-infected cells at the same concentration, supporting the notion that the phosphorylated state of STAT3 Tyr705 is not necessarily correlated with HCV replication. The production of reactive oxygen species (ROS) was induced by treatment with compound 6, whereas N-acetyl-cysteine restored HCV replication and impaired ROS production in the replicon cells treated with compound 6. These data suggest that compound 6 inhibits HCV replication via the induction of oxidative stress.
Enhanced protection against experimental Junin virus infection through the use of a modified favipiravir loading dose strategy Antivir. Res. (IF 4.271) Pub Date : 2017-08-02 Brian B. Gowen, Jonna B. Westover, Eric J. Sefing, Arnaud J. Van Wettere, Kevin W. Bailey, Luci Wandersee, Takashi Komeno, Yousuke Furuta
A collection of Old and New World arenaviruses are etiologic agents of viral hemorrhagic fever, a syndrome that features hematologic abnormalities, vascular leak, hypovolemia, and multi-organ failure. Treatment is limited to ribavirin for Lassa fever and immune plasma for Argentine hemorrhagic fever. Improved therapeutic options that are safe, more effective and widely available are needed. Here, we show that modification of favipiravir treatment to include a high-dose loading period achieves complete protection in a guinea pig model of Argentine hemorrhagic fever when treatment was initiated two days following challenge with Junin virus (JUNV). This loading dose strategy also protected 50% of animals from lethal disease when treatment was delayed until 5 days post-infection and extended the survival time in those that succumbed. Consistent with the survival data, dramatic reductions in serum and tissue virus loads were observed in animals treated with favipiravir. This is the first report demonstrating complete protection against uniformly lethal JUNV infection in guinea pigs by administration of a small molecule antiviral drug.
Inhibitory effects of metachromin A on hepatitis B virus production via impairment of the viral promoter activity Antivir. Res. (IF 4.271) Pub Date : 2017-08-04 Atsuya Yamashita, Mayumi Tamaki, Hirotake Kasai, Tomohisa Tanaka, Teruhime Otoguro, Akihide Ryo, Shinya Maekawa, Nobuyuki Enomoto, Nicole J. de Voogd, Junichi Tanaka, Kohji Moriishi
The currently available antiviral agents for chronic infection with hepatitis B virus (HBV) are pegylated interferon-α and nucleoside/nucleotide analogues, although it has been difficult to completely eliminate covalently closed circular DNA (cccDNA) from patients. To identify an antiviral compound targeting HBV core promoter, 15 terpenes originating from marine organisms were screened using a cell line expressing firefly luciferase under the control of the HBV core promoter. Metachromin A, which is a merosesquiterpene isolated from the marine sponge Dactylospongia metachromia, inhibited the viral promoter activity at the highest level among the tested compounds, and suppressed HBV production with an EC50 value of 0.8 μM regardless of interferon signaling and cytotoxicity. The analysis on the structure-activity relationship revealed that the hydroquinone moiety, and the double bonds at carbon numbers-5 and -9 in metachromin A are crucial for anti-HBV activity. Furthermore, metachromin A reduced the protein level but not the RNA level of hepatic nuclear factor 4α, which mainly upregulates the activities of enhancer I/X promoter and enhancer II/core promoter. These results suggest that metachromin A can inhibit HBV production via impairment of the viral promoter activity. Antiviral agents targeting the viral promoter may ameliorate HBV-related disorders regardless of remaining cccDNA.
Interferon-independent antiviral activity of 25-hydroxycholesterol in a teleost fish Antivir. Res. (IF 4.271) Pub Date : 2017-08-05 Patricia Pereiro, Gabriel Forn-Cuní, Sonia Dios, Julio Coll, Antonio Figueras, Beatriz Novoa
Oxysterols are a family of cholesterol oxygenated derivatives with diverse roles in many biological activities and have recently been linked with the induction of a cellular antiviral state. The antiviral effects of 25-hydroxycholesterol (25HC) extend to several mammalian enveloped and non-enveloped viruses. It has been reported that the expression of the gene encoding cholesterol 25-hydroxylase (CH25H) is induced by interferons (IFNs). In this work, five ch25h genes were identified in the zebrafish (Danio rerio) genome. The ch25h genes showed different tissue expression patterns and differed in their expression after immune stimulation with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (PolyI:C) and Spring Viremia Carp Virus (SVCV). Only one of the 5 genes, ch25hb, was overexpressed after the administration of the treatments. Synteny and phylogenetic analyses revealed that ch25hb is the putative homolog of mammalian Ch25h in zebrafish, while the remaining zebrafish ch25h genes are products of duplications within the teleost lineage. Interestingly, its modulation was not mediated by type I IFNs, contrasting previous reports on mammalian orthologs. Nevertheless, in vivo overexpression of ch25hb in zebrafish larvae significantly reduced mortality after SVCV challenge. Viral replication was also negatively affected by 25HC administration to the zebrafish cell line ZF4. In conclusion, the interferon-independent antiviral role of 25HC was extended to a non-mammalian species for the first time, and dual activity that both protects the cells and interacts with the virus cannot be discarded.
Characterization of oseltamivir-resistant influenza virus populations in immunosuppressed patients using digital-droplet PCR: Comparison with qPCR and next generation sequencing analysis Antivir. Res. (IF 4.271) Pub Date : 2017-08-03 Maxime Pichon, Alexandre Gaymard, Laurence Josset, Martine Valette, Gilles Millat, Bruno Lina, Vanessa Escuret
IntroductionThe H275Y substitution in neuraminidase (NA) confers oseltamivir-resistance in A(H1N1) influenza viruses (IV). Droplet digital PCR (ddPCR) is a new technique to explore single nucleotide polymorphisms. The aim of this study was to compare the performances of reverse transcriptase (RT)-ddPCR, RT-qPCR and next generation sequencing (NGS). We also analyzed the proportions of H275Y-NA substitution for two immunosuppressed patients with sustained shedding of A(H1N1)pdm09 IV.MethodsRT-qPCR was performed using the ABI7500 platform. RT-ddPCR was carried out using the QX200 ddPCR platform. We strengthened our results by a NGS assay (Ion PGM™ sequencer). Discrimination performance and sensitivity of the RT-ddPCR assay were evaluated using mixes of wild type (WT) and mutated H275Y-NA-coding segments.ResultsThe performance of RT-ddPCR was better than RT-qPCR, using NGS assay as a gold standard. RT-ddPCR was able to detect 0.28% oseltamivir-resistant IV in a WT IV population and 0.55% WT IV in an oseltamivir-resistant IV population. For the first patient, the H275Y-NA substitution was selected by oseltamivir treatment and reached about 50% of the IV population before dropping to less than 2% after treatment discontinuation which was under the lower limit of quantification by RT-qPCR and RT-ddPCR (<2%) after treatment stop. Then, five days after oseltamivir was re-introduced, the H275Y-NA substitution rose up to 100%. For the second patient, the H275Y-NA substitution reached about 30% two days after oseltamivir discontinuation.ConclusionRT-ddPCR demonstrated better performances than classical RT-qPCR to estimate oseltamivir-resistant IV proportions. This technique could be used to detect earlier emergence of H275Y-NA substitution.
Immunogenicity of an influenza virus-vectored vaccine carrying the hepatitis C virus protein epitopes in mice Antivir. Res. (IF 4.271) Pub Date : 2017-08-01 Shaogeng Zhang, Fang Sun, Tianyu Ren, Yueqiang Duan, Hongjing Gu, Chengcai Lai, Zhaohai Wang, Peirui Zhang, Xiliang Wang, Penghui Yang
Hepatitis C virus (HCV) has a devastating impact on human health, and infections can progress into liver fibrosis, cirrhosis, and hepatocellular carcinoma. There is no effective HCV vaccine. In this study, we rescued a recombinant PR8 influenza viral vector, called rgFLU-HCVCE1E2, carrying the core and envelope glycoprotein (C/E1/E2) epitopes of HCV inserted into the influenza nonstructural protein 1 gene. The morphological characteristics of rgFLU-HCVCE1E2 and the expression of the C/E1/E2 epitopes of HCV were examined. rgFLU-HCVCE1E2 replicated in various cell lines, including MDCK, A549, and Huh7.5 cells. More importantly, in BALB/c mice immunized intranasally twice at a 21-day interval with 104, 105, or 106 TCID50 rgFLU-HCVCE1E2, the viral vector induced a robust antibody response to influenza and HCV and potent IFN-γ and IL-4 secretion in response to HCV antigens in a dose-dependent manner. The rgFLU-HCVCE1E2 virus also stimulated IFN-γ production by virus-specific peripheral blood mononuclear cells in patients with chronic HCV infection. The study demonstrated that rgFLU-HCVCE1E2 carrying HCV antigens is immunogenic in vivo and has potential for the development of a HCV vaccine.
Dual-targeted anti-TB/anti-HIV heterodimers Antivir. Res. (IF 4.271) Pub Date : 2017-07-22 Liudmila Alexandrova, Sonia Zicari, Elena Matyugina, Anastasia Khandazhinskaya, Tatiana Smirnova, Sofya Andreevskaya, Larisa Chernousova, Christophe Vanpouille, Sergei Kochetkov, Leonid Margolis
HIV and M. tuberculosis are two intersecting epidemics making the search for new dual action drugs against both pathogens extremely important. Here, we report on the synthesis and suppressive activities of five dual-targeted HIV/TB compounds. These compounds are heterodimers of AZT, as anti-HIV molecules, and 5-substituted uracil derivatives, as anti-TB molecules. We found that these compounds inhibit the growth of M. tuberculosis and suppress the replication of HIV in human cell cultures and human lymphoid tissues ex vivo. We identified one particular heterodimer that inhibited both HIV and the drug-resistant TB strain MS-115 most potently. This compound demonstrated low toxicity and had no cytostatic effect on cells in culture, constituting an ideal candidate for future development and further in vivo testing.
Highlights of the 30th International Conference on Antiviral Research Antivir. Res. (IF 4.271) Pub Date : 2017-08-01 Graciela Andrei, Kara Carter, Zlatko Janeba, Aruna Sampath, Luis M. Schang, E. Bart Tarbet, R. Anthony Vere Hodge, Mike Bray, José A. Esté
The 30th International Conference on Antiviral Research (ICAR) was held in Atlanta, GA, USA from May 18 to 21, 2017. This report provides an account of award lectures, invited keynote addresses and oral presentations during the meeting. The 2017 Gertrude Elion Memorial Lecture Award by Michael Sofia highlighted one of the most important accomplishments in recent drug discovery in antiviral research, the identification of the hepatitis C virus direct-acting antiviral sofosbuvir and new alternatives to combat hepatitis B virus (HBV) infection. The Antonín Holý Lecture Award by David Chu on medicinal chemistry provided an overview of early developments of nucleoside analogs for the treatment of HIV and varicella zoster virus infection and how this knowledge serves to develop new drugs targeting HBV. Priscilla Yang gave the first ISAR Women in Science lecture. She reported on pharmacological validation of new antiviral targets for dengue, Zika and other flaviviruses. The William Prusoff Young Investigator Lecture Award by Maaike Everts described the Alabama Drug Discovery Alliance and the Antiviral Drug Discovery and Development Consortium, and how they are helping to accelerate the development of new antivirals. The 30th ICAR was a success in promoting new discoveries in antiviral drug development and research. The 31st ICAR will be held in Porto, Portugal, June 11–15, 2018.
Immune responses in DAA treated chronic hepatitis C patients with and without prior RG-101 dosing Antivir. Res. (IF 4.271) Pub Date : 2017-08-24 Meike H. van der Ree, Femke Stelma, Sophie B. Willemse, Anthony Brown, Leo Swadling, Marc van der Valk, M.J. Sinnige, Ad C. van Nuenen, J. Marleen L. de Vree, Paul Klenerman, Eleanor Barnes, Neeltje A. Kootstra, Hendrik W. Reesink
Background&aimsWith the introduction of DAA's, the majority of treated chronic hepatitis C patients (CHC) achieve a viral cure. The exact mechanisms by which the virus is cleared after successful therapy, is still unknown. The aim was to assess the role of the immune system and miRNA levels in acquiring a sustained virological response after DAA treatment in CHC patients with and without prior RG-101 (anti-miR-122) dosing.MethodsIn this multicenter, investigator-initiated study, 29 patients with hepatitis C virus (HCV) genotype 1 (n = 11), 3 (n = 17), or 4 (n = 1) infection were treated with sofosbuvir and daclatasvir ± ribavirin. 18 patients were previously treated with RG-101. IP-10 levels were measured by ELISA. Ex vivo HCV-specific T cell responses were quantified in IFN-γ-ELISpot assays. Plasma levels of miR-122 were measured by qPCR.ResultsAll patients had an SVR12. IP-10 levels rapidly declined during treatment, but were still elevated 24 weeks after treatment as compared to healthy controls (median 53.82 and 39.4 pg/mL, p = 0.02). Functional IFN-γ HCV-specific T cell responses did not change by week 12 of follow-up (77.5 versus 125 SFU/106 PBMC, p = 0.46). At follow-up week 12, there was no difference in plasma miR-122 levels between healthy controls and patients with and without prior RG-101 dosing.ConclusionsOur data shows that successful treatment of CHC patients with and without prior RG-101 dosing results in reduction of broad immune activation, and normalization of miR-122 levels (EudraCT: 2014-002808-25).Trial registrationEudraCT: 2014-002808-25.
Role of hepatitis B core protein in HBV transcription and recruitment of histone acetyltransferases to cccDNA minichromosome Antivir. Res. (IF 4.271) Pub Date : 2017-05-10 Chun Kong Chong, Ching Yan Serene Cheng, Sin Yi Jasmine Tsoi, Fung-Yu Huang, Fen Liu, Wai-Kay Seto, Ching-Lung Lai, Man-Fung Yuen, Danny Ka-Ho Wong
The hepatitis B core protein (HBc) has been suggested to interact with covalently closed circular DNA (cccDNA) and regulate hepatitis B virus (HBV) transcription. However, direct evidence is lacking. We aimed to identify the specific HBc region(s) responsible for transcription regulation and its interaction with cccDNA. Seventeen mutants with mutations at the four arginine-rich clusters of the HBc carboxyl-terminal domain (CTD) were created. The effect of HBc mutations on the levels of HBV DNA, RNA, and hepatitis B surface antigen (HBsAg) were measured. The association of cccDNA with mutant HBc and histone acetyltransferases (HATs) was assessed by chromatin immunoprecipitation (ChIP). Compared with wild-type HBc, HBc mutants with mutations in clusters III and IV resulted in a significant reduction in HBV RNA levels (all P < 0.05). HBc arginine clusters III and IV mutants also had a significantly lower levels of intracellular HBV DNA (<5% of wild-type; P < 0.001) and HBsAg (<10% of wild-type; P < 0.0001). cccDNA-ChIP assay demonstrated that HBc clusters III and IV mutants had a smaller degree of association with cccDNA (P < 0.001). In the HBc mutants, the association between HATs with cccDNA were reduced. In conclusion, HBc-CTD arginine residues at clusters III and IV play an important role in the regulation of HBV transcription as well as subsequent replication steps, likely through the reduced interaction of HBc with cccDNA and reduced acetylation of cccDNA-bound histones. These findings may provide clues to the identification of novel therapeutic targets against HBV.
Development of an animal model of progressive vaccinia in nu/nu mice and the use of bioluminescence imaging for assessment of the efficacy of monoclonal antibodies against vaccinial B5 and L1 proteins Antivir. Res. (IF 4.271) Pub Date : 2017-05-08 Marina Zaitseva, Antonia Thomas, Clement A. Meseda, Charles Y.K. Cheung, Claudia G. Diaz, Yan Xiang, Shane Crotty, Hana Golding
Bioluminescence imaging (BLI) was used to follow dissemination of recombinant vaccinia virus (VACV) expressing luciferase (IHD-J-Luc) in BALB/c nu/nu mice treated post-challenge with monoclonal antibodies (MAbs) against L1 and B5 VACV proteins in a model of Progressive Vaccinia (PV). Areas Under the flux Curve (AUC) were calculated for viral loads in multiple organs in individual mice. Following scarification with 105 pfu, IHD-J-Luc VACV undergoes fast replication at the injection site and disseminates rapidly to the inguinal lymph nodes followed by spleen, liver, and axillary lymph nodes within 2–3 days and before primary lesions are visible at the site of scarification. Extension of survival in nude mice treated with a combination of anti-B5 and anti-L1 MAbs 24 h post challenge correlated with a significant reduction in viral load at the site of scarification and delayed systemic dissemination. Nude mice reconstituted with 104 T cells prior to challenge with IHD-J-Luc, and treated with MAbs post-challenge, survived infection, cleared the virus from all organs and scarification site, and developed anti-VACV IgG and VACV-specific polyfunctional CD8+ T cells that co-expressed the degranulation marker CD107a, and IFNγ and TNFα cytokines. All T cell reconstituted mice survived intranasal re-challenge with IHD-J-Luc (104 pfu) two months after the primary infection. Thus, using BLI to monitor VACV replication in a PV model, we showed that anti-VACV MAbs administered post challenge extended survival of nude mice and protected T cell reconstituted nude mice from lethality by reducing replication at the site of scarification and systemic dissemination of VACV.
RO0504985 is an inhibitor of CMGC kinase proteins and has anti-human cytomegalovirus activity Antivir. Res. (IF 4.271) Pub Date : 2017-05-10 Blair L. Strang
Public-private partnerships allow many previously unavailable compounds to be screened for antiviral activity. Here a screening method was used to identify an oxindole compound, RO0504985, from a Roche kinase inhibitor library that inhibited human cytomegalovirus (HCMV) protein production. RO0504985 was previously described as an inhibitor of cyclin-dependent kinase 2 (CDK2). However, using kinase selectivity assays it was found that RO0504985 was an inhibitor of several CMGC group kinase proteins, including CDK2. Using virus yield reduction assays it was observed that RO0504985 inhibited replication of different HCMV strains at low micromolar concentrations. Western blotting was used to investigate how RO0504985 inhibited HCMV replication. Treatment of HCMV infected cells with RO0504985 inhibited production of the immediate early viral IE2 proteins and the late viral protein pp28. Thus, RO0504985 inhibited HCMV replication by preventing production of specific HCMV proteins necessary for virus replication.
Study of rubella candidate vaccine based on a structurally modified plant virus Antivir. Res. (IF 4.271) Pub Date : 2017-05-13 Ekaterina A. Trifonova, Vladimir A. Zenin, Nikolai A. Nikitin, Maria S. Yurkova, Ekaterina M. Ryabchevskaya, Egor V. Putlyaev, Ekaterina K. Donchenko, Olga A. Kondakova, Alexey N. Fedorov, Joseph G. Atabekov, Olga V. Karpova
Anti-inflammatory effects of rosmarinic acid-4-O-β-D-glucoside in reducing acute lung injury in mice infected with influenza virus Antivir. Res. (IF 4.271) Pub Date : 2017-04-29 Jian-xing Liu, Ying Zhang, Qiu-Ping Hu, Ji-qiang Li, Yun-tao Liu, Qing-guang Wu, Jian-guo Wu, Xiao-ping Lai, Zhong-de Zhang, Xiong Li, Geng Li
Rosmarinic acid-4-O-β-D-glucoside (RAG) is a dicaffeoyl phenolic compound isolated from Sarcandra glabra (Thunb.) Nakai. Preliminary studies show that RAG has significant anti-inflammatory properties and can alleviate ear swelling in mice and the paw swelling in rats. Here, the anti-influenza effects of RAG were investigated in mice infected with A/FM/1/47 H1N1 virus. The survival rate and body weight were observed, the lung edema, virus copies, inflammatory cytokines (including IL-4, IL-5, TNF-α and IFN-γ) and oxidative damage indexes (including SOD, MDA, NO, and CAT) were measured. Moreover, immune cell recruitment in alveoli was measured with white blood cells and differential counts. Therapeutic RAG concentrations substantially improve the symptoms, mitigate body weight loss and alleviate lung edema induced by virus, thus improve survival protection effects. Furthermore, RAG was shown to regulate influenza virus-induced inflammatory cytokine expression, specifically by downregulating the Th1 cell cytokines IFN-γ, TNF-α and upregulating the Th2 cell cytokines IL-4, IL-5. Cell migration and infiltration were also diminished after RAG administration.
Preclinical evaluation of VIS513, a therapeutic antibody against dengue virus, in non-human primates Antivir. Res. (IF 4.271) Pub Date : 2017-05-18 Eugenia Z. Ong, Yadunanda Budigi, Hwee Cheng Tan, Luke N. Robinson, Kirk J. Rowley, Alexander Winnett, Sven Hobbie, Zachary Shriver, Gregory J. Babcock, Eng Eong Ooi
Despite useful in vivo activity, no therapeutic against dengue virus (DENV) has demonstrated efficacy in clinical trials. Herein, we explored dosing and virological endpoints to guide the design of human trials of VIS513, a pan-serotype anti-DENV IgG1 antibody, in non-human primates (NHPs). Dosing VIS513 pre- or post-peak viremia in NHPs neutralized infectious DENV although RNAemia remained detectable post-treatment; differential interaction of human IgGs with macaque Fc-gamma receptors may delay clearance of neutralized DENV. Our findings suggest useful antiviral utility of VIS513 and highlight an important consideration when evaluating virological endpoints of trials for anti-DENV biologics.
Anti-inflammatory effects of adjunctive macrolide treatment in adults hospitalized with influenza: A randomized controlled trial Antivir. Res. (IF 4.271) Pub Date : 2017-05-20 Nelson Lee, Chun-Kwok Wong, Martin C.W. Chan, Esther S.L. Yeung, Wilson W.S. Tam, Owen T.Y. Tsang, Kin-Wing Choi, Paul K.S. Chan, Angela Kwok, Grace C.Y. Lui, Wai-Shing Leung, Irene M.H. Yung, Rity Y.K. Wong, Catherine S.K. Cheung, David S.C. Hui
Introduction− Macrolides can ameliorate inflammation in respiratory diseases, providing clinical benefits. Data in influenza is lacking.Method− A randomized, open-label, multicenter trial among adults hospitalized for laboratory-confirmed influenza was conducted. Study treatments of oseltamivir and azithromycin (500 mg/day), or oseltamivir alone, both for 5 days, were allocated at 1:1 ratio. The primary outcome was plasma cytokine/chemokine concentration change over time (Day 0–10); secondary outcomes were viral load and symptom score changes. Generalized Estimating Equation (GEE) models were used to analyze longitudinal data.Results− Fifty patients were randomized to the oseltamivir-azithromycin or oseltamivir groups, with comparable baseline characteristics (age, 57 ± 18 years; A/H3N2, 70%), complications (72%), and viral load. Pro-inflammatory cytokines IL-6 (GEE: β −0.037, 95%CI-0.067,-0.007, P = 0.016; reduction from baseline −83.4% vs −59.5%), CXCL8/IL-8 (β −0.018, 95%CI-0.037,0.000, P = 0.056; −80.5% vs −58.0%), IL-17 (β −0.064, 95%CI-0.117,-0.012, P = 0.015; −74.0% vs −34.3%), CXCL9/MIG (β −0.010, 95%CI-0.020,0.000, P = 0.043; −71.3% vs −56.0%), sTNFR-1, IL-18, and CRP declined faster in the oseltamivir-azithromycin group. There was a trend toward faster symptom resolution (β −0.463, 95%CI-1.297,0.371). Viral RNA decline (P = 0.777) and culture-negativity rates were unaffected. Additional ex vivo studies confirmed reduced induction of IL-6 (P = 0.017) and CXCL8/IL-8 (P = 0.005) with azithromycin.Conclusion− We found significant anti-inflammatory effects with adjunctive macrolide treatment in adults with severe influenza infections. Virus control was unimpaired. Clinical benefits of a macrolide-containing regimen deserve further study.[ClinicalTrials.gov NCT01779570]
A single intranasal administration of virus-like particle vaccine induces an efficient protection for mice against human respiratory syncytial virus Antivir. Res. (IF 4.271) Pub Date : 2017-05-18 Yue-Ying Jiao, Yuan-Hui Fu, Yi-Fei Yan, Ying Hua, Yao Ma, Xiu-Juan Zhang, Jing-Dong Song, Xiang-Lei Peng, Jiaqiang Huang, Tao Hong, Jin-Sheng He
Human respiratory syncytial virus (RSV) is an important pediatric pathogen causing acute viral respiratory disease in infants and young children. However, no licensed vaccines are currently available. Virus-like particles (VLPs) may bring new hope to producing RSV VLP vaccine with high immunogenicity and safety. Here, we constructed the recombinants of matrix protein (M) and fusion glycoprotein (F) of RSV, respectively into a replication-deficient first-generation adenoviral vector (FGAd), which were used to co-infect Vero cells to assemble RSV VLPs successfully. The resulting VLPs showed similar immunoreactivity and function to RSV virion in vitro. Moreover, Th1 polarized response, and effective mucosal virus-neutralizing antibody and CD8+ T-cell responses were induced by a single intranasal (i.n.) administration of RSV VLPs rather than intramuscular (i.m.) inoculation, although the comparable RSV F-specific serum IgG and long-lasting RSV-specific neutralizing antibody were detected in the mice immunized by both routes. Upon RSV challenge, VLP-immunized mice showed increased viral clearance but decreased signs of enhanced lung pathology and fewer eosinophils compared to mice immunized with formalin-inactivated RSV (FI-RSV). In addition, a single i.n. RSV VLP vaccine has the capability to induce RSV-specific long-lasting neutralizing antibody responses observable up to 15 months. Our results demonstrate that the long-term and memory immune responses in mice against RSV were induced by a single i.n. administration of RSV VLP vaccine, suggesting a successful approach of RSV VLPs as an effective and safe mucosal vaccine against RSV infection, and an applicable and qualified platform of FGAd-infected Vero cells for VLP production.
Distinct patterns of cellular immune response elicited by influenza non-adjuvanted and AS03-adjuvanted monovalent H1N1(pdm09) vaccine Antivir. Res. (IF 4.271) Pub Date : 2017-05-23 Sarah Giarola-Silva, Jordana G.A. Coelho-dos-Reis, Marina Moraes Mourão, Ana Carolina Campi-Azevedo, Erick E. Nakagaki Silva, Maria Luiza-Silva, Marina Angela Martins, Amanda Cardoso de Oliveira Silveira-Cassette, Maurício Azevedo Batista, Vanessa Peruhype-Magalhães, Lis Ribeiro do Valle Antonelli, José Geraldo Leite Ribeiro, Silvana Maria Elói-Santos, Alexandre Vieira Machado, Andréa Teixeira-Carvalho, Olindo Assis Martins-Filho, Márcio Sobreira Silva Araújo
The study aimed at identifying biomarkers of immune response elicited by non-adjuvanted-(NAV) and adjuvanted-(AV) H1N1(pdm09) vaccines. The results showed that despite both vaccines elicited similar levels of anti-H1N1 antibodies at day30 after vaccination, higher reactivity was observed in AV at day180. While AV induced early changes in cell-surface molecules on monocytes, CD4+, CD8+ T-cells and B-cells, NAV triggered minor changes, starting later on at day3. Furthermore, AV induced a late and persistent increase in TLR gene expression after day3, except for tlr4, while NAV displayed earlier but transient tlr3/4/7/9 up-regulation. Contrasting with NAV, prominent chemokine gene expression (cxcl8,cxcl9,ccl5) and a broad spectrum up-regulation of plasmatic biomarkers (CXCL8,IL-6,IL-1β,IL-12,IL-10) was evident in AV, which showed a major involvement of TNF and IL-10. Similarly, AV induced a robust IL-10-modulated proinflammatory storm, with early and persistent involvement of TNF-α/IL-12/IFN-γ axis derived from NK-cells, CD4+ and CD8+ T-cells along with promiscuous production of IL-4/IL-5/IL-13. Conversely, NAV promotes a concise and restricted intracytoplasmic chemokine/cytokine response, essentially mediated by TNF-α and IL-4, with late IL-10 production by CD8+ T-cells. Systems biology approach underscored that AV guided the formation of an imbricate network characterized by a progressive increase in the number of neighborhood connections amongst innate and adaptive immunity. In AV, the early cross-talk between innate and adaptive immunity, followed by the triad NK/CD4+/CD8+ T-cells at day3, sponsored a later/robust biomarker network. These findings indicate the relevance of adjuvanted vaccination to orchestrate broad, balanced and multifactorial cellular immune events that lead ultimately to a stronger H1N1 humoral immunity.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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