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Antiviral Research (v.77, #1)

IFC Editorial Board (pp. co3).

Publisher's Note (pp. v).

Editorial Board (pp. ii).

Discovery and development of antiviral drugs for biodefense: Experience of a small biotechnology company by Tove C. Bolken; Dennis E. Hruby (pp. 1-5).

The unmet need for effective antivirals against potential agents of bioterrorism and emerging infections is obvious; however, the challenges to develop such drugs are daunting. Even with the passage of Project BioShield and more recently the BARDA legislation, there is still not a clear market for these types of drugs and limited federal funding available to support expensive drug development studies. SIGA Technologies, Inc. is a small biotech company committed to developing novel products for the prevention and treatment of severe infectious diseases, with an emphasis on products for diseases that could result from bioterrorism. Through trials and error SIGA has developed an approach to this problem in order to establish the infrastructure necessary to successfully advance new antiviral drugs from the discovery stage on through to licensure. The approach that we have taken to drug development is biology driven and dependent on a dispersive development model utilizing essential collaborations with academic, federal, and private sector partners. This consortium approach requires success in acquiring grants and contracts as well as iterative communication with the government and regulatory agencies. However, it can work as evidenced by the rapid progress of our lead antiviral against smallpox, ST-246, and should serve as the template for development of new antivirals against important biological pathogens.

Keywords: Antiviral; Drug development; Smallpox; Hemorrhagic fever virus; Category A; Biothreat agents

Genetically delivered antibody protects against West Nile virus by Alexander Pereboev; Viktoriya Borisevich; George Tsuladze; Mikhail Shakhmatov; Deborah Hudman; Elena Kazachinskaia; Ivan Razumov; Viktor Svyatchenko; Valery Loktev; Vladimir Yamshchikov (pp. 6-13).

Gene-based delivery of recombinant antibody genes is a promising therapeutic strategy offering numerous advantages including sustained antibody levels, better safety profile and lower production cost. Here we describe generation of a recombinant antibody Fc-9E2 comprising a fusion protein between human Fc of IgG1 and a single-chain Fv derived from a hybridoma 9E2 secreting a mAb neutralizing West Nile virus (WNV). Fc-9E2 was shown to retain parental mAb's specificity and WNV-neutralizing capacity. Adenovirus-mediated in vivo delivery of the antibody gene resulted in sustained Fc-9E2 serum levels leading to abrogation of lethal WNV infection in an animal model.

Keywords: Abbreviations; WNV; West Nile virus; Ad; adenovirus; recAb; recombinant antibody; scFv; single chain fragment variable; Fc; fragment crystallizable; WNVEC; C-terminal fragment of WNV E protein; PRNT; plaque reduction-neutralization testWNV; Passive immunization; Recombinant antibody; Gene delivery; Adenovirus

Yin Chen Hao Tang, a Chinese prescription, inhibits both herpes simplex virus type-1 and type-2 infections in vitro by Hua-Yew Cheng; Liang-Tzung Lin; Hsin-Hsin Huang; Chien-Min Yang; Chun-Ching Lin (pp. 14-19).

Yin Chen Hao Tang (YCHT) is one of the most frequently used prescriptions in the long history of traditional Chinese medicine practice. The prescription contains three Chinese herbs, namely Artemisia capillaries Thunb. (Compositae), Rheum officinale Baillon (Polygonaceae), and Gardenia jasminoids Ellis (Rubiaceae), and has been widely used to treat acute hepatitis with jaundice. In this study, the in vitro anti-HSV-1 and HSV-2 activities of the water extract of YCHT were investigated. Results showed that YCHT water extract inhibited both HSV-1 and HSV-2 infections. However, the inhibition was more effective against HSV-2 than against HSV-1. The IC₅₀ and IC₉₀ values of YCHT water extract against HSV-1 infection were in the range of 142.5–150.1 and 191.3–393.9μg/ml, and against HSV-2 infection they were in the range of 19.6–29.4 and 42.2–97.7μg/ml, respectively. The water extract of YCHT showed no cytotoxic effect at a concentration of 500μg/ml or below, and had a CC₅₀ value of 850.7±1.7μg/ml. The prescription was found to diminish HSV-2 infectivity in a dose-dependent manner, and the activity was influenced by the incubation periods and the incubation temperatures. Concurrent addition of virus with YCHT or pre-treatment of the virus with the prescription extract both protected the cells from infection. In summary, the water extract of YCHT was concluded to inhibit infections by HSV-1 and HSV-2 and this effect was likely mediated through direct inactivation of the virus infectivity.

Keywords: Yin Chen Hao Tang; Traditional Chinese medicine; Anti-HSV activity; Virus inactivation

Inhibition of HSV-1 replication and HSV DNA polymerase by the chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(β-d-ribofuranosyl) quinoline-3-carboxylic acid and its aglycone by Thiago Moreno L. Souza; Maria Cecilia Bastos V. De Souza; Vitor F. Ferreira; Carla Veronica B. Santos Canuto; Isakelly Pereira Marques; Carlos Frederico L. Fontes; Izabel C.P.P. Frugulhetti (pp. 20-27).

We describe in this paper that the synthetic chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(β-d-ribofuranosyl) quinoline-3-carboxylic acid (compoundA) and its free aglycogene base (compoundB) inhibit, with low cytotoxicity, the replication of herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). CompoundA inhibited HSV-1 replication in Vero cells with an EC₅₀ of 1.3 and 1.4μM for an acyclovir (ACV)-sensitive strain and an ACV-resistant strain of this virus, respectively. Additionally, it inhibited HSV-2 replication with an EC₅₀ of 1.1μM. CompoundB also inhibited the ACV-sensitive and -resistant HSV-1 strains, and HSV-2 at EC₅₀ values of 1.7, 1.9 and 1.6μM, respectively. Time-of-addition assays, performed with compoundA, suggested that this molecule at an early time point of the HSV replication cycle. Kinetic assays demonstrated that compoundsA andB inhibit the HSV DNA polymerase activity in a noncompetitive fashion, with a K_i equal to 0.1 and 0.2μM, respectively. Taken together, our results suggest that compoundsA andB represent promising lead molecules for further anti-HSV drug design.

Keywords: Herpes simplex virus type 1; HSV-1 DNA polymerase; Chloroxoquinolinic ribonucleoside; Inhibitor

Porcine plasma ficolin binds and reduces infectivity of porcine reproductive and respiratory syndrome virus (PRRSV) in vitro by Natalie D. Keirstead; Changhee Lee; Dongwan Yoo; Andrew S. Brooks; M. Anthony Hayes (pp. 28-38).

Ficolins are collagenous lectins that bind N-acetylated glycans and participate in innate immune responses, including phagocytosis and complement activation. Related collagenous lectins such as mannan binding lectin (MBL) and surfactant proteins A and D possess antiviral activity, but this activity has not been demonstrated for ficolins. In these studies, we used purified porcine plasma ficolin α and recombinant ficolin α to assess their ability to bind and neutralize porcine reproductive and respiratory virus (PRRSV) in various assays. Recombinant ficolin α was designed with a C-terminal 6-histidine tag using a pcDNA3.1 expression vector system in CHO K1 cells. Plasma-purified and recombinant ficolin α reduced cytopathic effect of PRRSV-infected Marc-145 cells in neutralization assays and inhibited replication of infectious viral particles in a GlcNAc-dependent manner. In vitro replication determined by plaque assay was inhibited in the presence of plasma-purified ficolin α and recombinant ficolin. Immunoreactive plasma ficolin α and recombinant ficolin α also bound PRRSV-coated wells in a GlcNAc-dependent manner. These studies indicate that porcine ficolin can bind and neutralize a common arterivirus that is a major pathogen of swine.

Keywords: Abbreviations; ANOVA; analysis of variance; CHO K1 cells; Chinese hamster ovary K1 cell line; CPE; cytopathic effect; ELISA; enzyme-linked immunosorbent assay; GlcNAc; N; -acetyl-; d; -glucosamine; kDa; kilodaltons; MBL; mannan binding lectin; MALDI; matrix-assisted laser desorption/ionization; Marc-145 cells; African monkey kidney cell line; MS/MS; tandem mass spectrometry; PFU; plaque-forming units; pFCN; plasma ficolin α; p; I; isoelectric point; PLSD; protected least significant difference; PRRSV; porcine reproductive and respiratory syndrome virus; rFCN; recombinant ficolin; SDS-PAGE; sodium dilauryl sulfate-polyacrylamide gel electrophoresisFicolins; Mannan binding lectins; Innate immunity; Pigs; N; -Acetylglucosamine; PRRSV

Efficacy of therapeutic intervention with an oral ether–lipid analogue of cidofovir (CMX001) in a lethal mousepox model by Scott Parker; Erin Touchette; Christina Oberle; Merrick Almond; Alice Robertson; Lawrence C. Trost; Bernhard Lampert; George Painter; R. Mark Buller (pp. 39-49).

In the 21st century we are faced with the potential use of natural or recombinant VARV and MPXV as biological weapons, and the emergence of human MPXV. Such an occurrences would require therapeutic and prophylactic intervention with antivirals. Cidofovir, an antiviral approved for the treatment of cytomegalovirus retinitis in AIDS patients, has activity against poxviruses, but must be administered intravenously and is associated with nephrotoxicity. An ether–lipid analogue of CDV, CMX001 (HDP-CDV), has potent antiviral activity against a range of DNA viruses including poxviruses, excellent oral bioavailability and minimal nephrotoxicity. CMX001 and CDV are equally efficacious at protecting mice from mortality following high ectromelia virus doses (10,000×LD₅₀) introduced by the intra-nasal route or small particle aerosol. Using CMX001 at a 10mg/kg dose followed by 2.5mg/kg doses every other-day for 14 days provided solid protection against mortality and weight loss following an intra-nasal challenge of (100–200)×LD₅₀ of ectromelia virus. Furthermore, complete protection against mortality was achieved when administration was delayed until as late as 5 days post-infection, which is 3–4 days prior to the death of the untreated controls. This therapeutic window would be equivalent to intervening during the rash stage of ordinary smallpox.

Keywords: Antiviral; Bioterrorism; Ectromelia; Monkeypox; Oral drug; Smallpox

Therapeutic potential of dendritic cell-based immunization against HBV in transgenic mice by Wen-Zheng Jiang; Yan Fan; Xia Liu; Ya-Li Zhang; Jie-Jun Wen; Wen-Li Hao; Min Qian (pp. 50-55).

Hepatitis B virus (HBV) transgenic mice that express HBV envelope proteins represent a model of chronic HBV infection suitable for the development of therapeutic immunization strategies. To address immunologically therapeutic effects induced by peptide-pulsed DCs, HBV transgenic mice were immunized with peptide-pulsed DCs, and the mice were killed after three times of immunization and the splenocytes were stimulated in vitro and detected by IFN-γ ELISPOT and cytotoxic T lymphocyte (CTL) activity. The data demonstrated that HBV-specific CD8+ T cell response could be induced and CD8+ T cells had specific CTL activity. Furthermore, ELISA and fluorescent quantitative PCR were performed to detect the level of serum HBsAg and HBV DNA and the results demonstrated that HBV-specific peptide-pulsed DCs could significantly reduce the concentration of serum HBsAg and HBV DNA. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured and no significant differences were observed between the different groups, which indicated that no hepatocellular injury occurred. Taken together, the data strongly demonstrated that CD8+ T cell responses and antiviral immunity were elicited in HBV transgenic mice, suggesting that peptide-pulsed DCs could elicit an effective antiviral immunity.

Keywords: Hepatitis B virus; Dentritic cell; Epitope; Cytotoxic T lymphocyte; Transgenic mice

Nitazoxanide, tizoxanide and other thiazolides are potent inhibitors of hepatitis B virus and hepatitis C virus replication by Brent E. Korba; Abigail B. Montero; Kristine Farrar; Karen Gaye; Sampa Mukerjee; Marc S. Ayers; Jean-François Rossignol (pp. 56-63).

Nitazoxanide (NTZ), a thiazolide anti-infective, is active against anaerobic bacteria, protozoa, and a range of viruses in cell culture models, and is currently in phase II clinical development for treating chronic hepatitis C. In this report, we characterize the activities of NTZ and its active metabolite, tizoxanide (TIZ), along with other thiazolides against hepatitis B virus (HBV) and hepatitis C virus (HCV) replication in standard antiviral assays. NTZ and TIZ exhibited potent inhibition of both HBV and HCV replication. NTZ was equally effective at inhibiting replication of lamivudine (LMV) and adefovir dipovoxil (ADV)-resistant HBV mutants and against 2′-C-methyl cytidine (2′CmeC) and telaprevir (VX-950)-resistant HCV mutants. NTZ displayed synergistic interactions with LMV or ADV against HBV, and with recombinant interferon alpha-2b (IFN) or 2′CmeC against HCV. Pre-treatment of HCV replicon-containing cells with NTZ potentiated the effect of subsequent treatment with NTZ plus IFN, but not NTZ plus 2′CmeC. NTZ induced reductions in several HBV proteins (HBsAg, HBeAg, HBcAg) produced by 2.2.15 cells, but did not affect HBV RNA transcription. NTZ, TIZ, and other thiazolides are promising new antiviral agents that may enhance current or future anti-hepatitis therapies.

Keywords: Hepatitis B virus; Hepatitis C virus; Nitazoxanide; Thiazolides; Antiviral therapy

The dolabellane diterpene Dolabelladienetriol is a typical noncompetitive inhibitor of HIV-1 reverse transcriptase enzyme by Claudio Cesar Cirne-Santos; Thiago Moreno L. Souza; Valéria L. Teixeira; Carlos Frederico L. Fontes; Moacyr A. Rebello; Luiz Roberto R. Castello-Branco; Celina M. Abreu; Amílcar Tanuri; Izabel C.P.P. Frugulhetti; Dumith Chequer Bou-Habib (pp. 64-71).

We recently described that a dollabelane diterpene isolated from the marine algae Dictyota pfaffii (Dolabelladienetriol) inhibits the human immunodeficiency virus type 1 (HIV-1) enzyme reverse transcriptase (RT), and HIV-1 replication in primary cells. Based on these findings, we investigated additional antiretroviral properties of Dolabelladienetriol. Here, we describe that Dolabelladienetriol blocked the synthesis and integration of HIV-1 provirus and completely abrogated viral replication in primary cells. Also, studies of kinetic mode of action revealed that the Dolabelladienetriol is a nonnucleoside RT inhibitor (NNRTI), acting as a noncompetitive inhibitor, with a K_i value equal to 7.2μM. To assess whether Dolabelladienetriol could potentiate the anti-HIV-1 effects of other HIV-1 inhibitors, HIV-1-infected cells were treated with Dolabelladienetriol at its EC₅₀ dose plus sub-optimal concentrations of classical antiretrovirals. Dolabelladienetriol provided an additive effect with the nucleoside RT inhibitor AZT, and a synergistic effect with the protease inhibitor atazanavir sulphate. There was no increment of the anti-HIV-1 effect resulting from the combination between Dolabelladienetriol and the NNRTI nevirapine. Using a large panel of HIV-1 isolates harboring NNRTI resistance mutations, we found no cross-resistance between Dolabelladienetriol and clinical available NNRTIs. Thus, Dolabelladienetriol is an NNRTI, with potent activity against HIV-1 isolates carrying common NNRTI-associated resistance mutations. Dolabelladienetriol may be considered as a potential new agent for anti-HIV-1 therapy.

Keywords: AIDS; HIV-1; Reverse transcriptase; Diterpene; Dolabelladienetriol

Amantadine-resistance among H5N1 avian influenza viruses isolated in Northern China by Guimei He; Jian Qiao; Changgui Dong; Cheng He; Lihong Zhao; Yong Tian (pp. 72-76).

We tested the amantadine-resistance among avian influenza A (H5N1) viruses isolated from chicken in Hebei Province of Northern China from 2001 to 2005, and investigated the amantadine use in this area. Plague reduction assay in MDCK cells showed that 83.3% isolates (5/6) were amantadine-resistant strains. The M2 sequence analysis revealed that four of five resistant isolates contained the point mutations (Ser to Asn) at position 31 that could confer resistance to amantadine. These results indicated that the incidence of amantadine-resistant viruses isolated in Northern China was particularly high. In the investigation of amantadine use, we found that amantadine was used extensively in poultry farms in this area, which maybe was one of reasons of the high amantadine-resistance incidence.

Keywords: Amantadine-resistance; H5N1 avian influenza viruses; Northern China; M2 gene

Hydroxyurea enhances the activity of acyclovir and cidofovir against herpes simplex virus type 1 resistant strains harboring mutations in the thymidine kinase and/or the DNA polymerase genes by Yan Sergerie; Guy Boivin (pp. 77-80).

Drug-resistant herpes simplex virus type 1 (HSV-1) recombinant strains harboring mutations in the thymidine kinase and/or the DNA polymerase genes were evaluated for their susceptibility to various antivirals in the presence of 25μg/ml of hydroxyurea (HyU). The latter compound decreased the 50% inhibitory concentrations of acyclovir by 1.5–3.8-fold and that of cidofovir by 2.7–14.4-fold. However, HyU did not affect the susceptibilities of the various recombinant mutants to foscarnet. Hydroxyurea, a ribonucleotide reductase inhibitor, can increase the activity of nucleoside/nucleotide analogues against drug-resistant viruses.

Keywords: Herpes simplex virus; Hydroxyurea; Acyclovir; Cidofovir; Resistance

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Antiviral Research (v.77, #1)