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Antiviral Research (v.96, #1)

Inside Back Cover: Notes to Authors (pp. ibc).

Prelim II: Editorial Board (pp. ii).

Inhibition of rotavirus infection in cultured cells by N-acetyl-cysteine, PPARγ agonists and NSAIDs by Carlos A. Guererero; Andrea Murillo; Orlando Acosta (pp. 1-12).

► Rotavirus infectivity is inhibited in cultured cells by N-acetylcysteine. ► Rotavirus infectivity is inhibited in cultured cells by ascorbic acid. ► Rotavirus infectivity is inhibited in vitro by pioglitazone and rosiglitazone. ► Ibuprofen, and indomethacin inhibit rotavirus infectivity in cultured cells. ► These drugs have in common that they affect the NF-kB pathway.Although the current rotavirus vaccines have shown good tolerance and significant efficacy, it would be useful to develop alternative or complementary strategies aimed at preventing or treating acute diarrhoeal disease caused by this viral agent. A variety of antiviral strategies other than vaccines have been assayed for rotavirus infection management. The recently demonstrated sensitivity of rotavirus infectivity to thiol/disulfide reagents prompted assays for screening drugs that potentially affect cellular redox reactions. MA104 or Caco-2 cells were inoculated with the rotavirus strains RRV, Wa, Wi or M69 and then incubated with different concentrations of drugs belonging to a selected group of 60 drugs that are currently used in humans for purposes other than rotavirus infection treatment. Eighteen of these drugs were able to inhibit rotavirus infectivity to different extents. A more systematic evaluation was performed with drugs that could be used in children such as N-acetylcysteine and ascorbic acid, in addition to ibuprofen, pioglitazone and rosiglitazone, all of which affecting cellular pathways potentially needed by the rotavirus infection process. Evidence is provided here that rotavirus infectivity is significantly inhibited by NAC in different cell-culture systems. These findings suggest that NAC has the potential to be used as a therapeutic tool for treatment and prevention of rotavirus disease in children.

Keywords: Rotavirus infection; N; -Acetylcysteine; Thiazolidinediones; NSAIDs; Redox reactions

Treatment of oseltamivir-resistant influenza A (H1N1) virus infections in mice with antiviral agents by Donald F. Smee; Justin G. Julander; E. Bart Tarbet; Matthew Gross; Jack Nguyen (pp. 13-20).

Influenza A/Mississippi/03/2001 (H1N1) and A/Hong Kong/2369/2009 (H1N1) viruses containing the neuraminidase gene mutation H275Y (conferring resistance to oseltamivir) were adapted to mice and evaluated for suitability as models for lethal infection and antiviral treatment. The viral neuraminidases were resistant to peramivir and oseltamivir carboxylate but sensitive to zanamivir. Similar pattern of antiviral activity were seen in MDCK cell assays. Lethal infections were achieved in mice with the two viruses. Oral oseltamivir at 100 and 300mg/kg/day bid for 5day starting at −2h gave 30% and 60% protection from death, respectively, due to the A/Mississippi/03/2001 infection. Intraperitoneal treatments with zanamivir at 30 and 100mg/kg/day starting at −2h gave 60% and 90% protection, respectively. Neither compound at <300mg/kg/day protected mice when treatments began at +24h. Amantadine was effective at 10, 30, and 100mg/kg/day, rimantadine was protective at 10 and 30mg/kg/day (highest dose tested), and ribavirin was active at 30 and 75mg/kg/day, with survival ranging from 60–100% for oral treatments initiated at −2h. For treatments begun at +24h, amantadine was protective at 30 and 100mg/kg/day, rimantadine showed efficacy at 10 and 30mg/kg/day, and ribavirin was active at 75mg/kg/day, with 60–100% survival per group. In the A/Hong Kong/2369/2009 infection, oral oseltamivir at 100 and 300mg/kg/day starting at −2h gave 50% and 70% protection from death, respectively. These infection models will be useful to study newly discovered anti-influenza virus agents and to evaluate compounds in combination.

Keywords: Oseltamivir; Zanamivir; Amantadine; Rimantadine; Ribavirin

The viral RNA capping machinery as a target for antiviral drugs by François Ferron; Etienne Decroly; Barbara Selisko; Bruno Canard (pp. 21-31).

► RNA viral capping mechanisms. ► Enzymology of viral RNA capping. ► Structures of viral RNA capping enzymes. ► Structures of viral RNA capping main «lead» inhibitors.Most viruses modify their genomic and mRNA 5′-ends with the addition of an RNA cap, allowing efficient mRNA translation, limiting degradation by cellular 5′–3′ exonucleases, and avoiding its recognition as foreign RNA by the host cell. Viral RNA caps can be synthesized or acquired through the use of a capping machinery which exhibits a significant diversity in organization, structure and mechanism relative to that of their cellular host. Therefore, viral RNA capping has emerged as an interesting field for antiviral drug design. Here, we review the different pathways and mechanisms used to produce viral mRNA 5′-caps, and present current structures, mechanisms, and inhibitors known to act on viral RNA capping.

Keywords: 5′-Triphosphatase; Guanylyltransferase; Methyltransferase; Endonuclease; Cap snatching; Mechanism; Structure; Inhibitor

Dose- and schedule-dependent protective efficacy of celgosivir in a lethal mouse model for dengue virus infection informs dosing regimen for a proof of concept clinical trial by Satoru Watanabe; Abhay P.S. Rathore; Cynthia Sung; Fan Lu; Yok Moi Khoo; John Connolly; Jenny Low; Eng Eong Ooi; How Sung Lee; Subhash G. Vasudevan (pp. 32-35).

► Celgosivir protection against lethal dengue infection in mice is dose dependent. ► Celgosivir protection is 100% at 50mg/kg BID and 0% at 100mg/kg QD. ► Steady-state Cmin (PK parameter) for celgosivir correlates with mice survival. ► Celgosivir is a candidate drug for evaluation as a dengue therapeutic in humans.Celgosivir (6-O-butanoyl castanospermine), a pro-drug of the naturally occurring castanospermine, is an inhibitor of α-glucosidase I and II that is found to be a potent inhibitor of several enveloped viruses including all four serotypes of dengue virus. We showed previously that the compound fully protected AG129 mice from lethal infection with a mouse adapted dengue virus at a dose of 50mg/kg twice daily (BID) for 5days and was effective even after 48h delayed treatment. Here we show that the protection by celgosivir is dose- and schedule-dependent and that a twice-a-day regimen of 50, 25 or 10mg/kg is more protective than a single daily dose of 100mg/kg. Treatment with 50mg/kg BID castanospermine had comparable efficacy as 25mg/kg BID celgosivir, suggesting that celgosivir is approximately twice as potent as castanospermine with respect to in vivo antiviral efficacy. Pharmacokinetics (PK) studies of celgosivir in mice showed that it rapidly metabolized to castanospermine. Simulation of the PK data with the survival data for the various doses of celgosivir tested suggests that the steady-state minimum concentration is a critical parameter to note in choosing dose and schedule. These results influenced the selection of the dose regimen for a proof-of-concept clinical trial of celgosivir as a treatment against dengue fever.

Keywords: Dengue virus; Antiviral; AG129 lethal infection mouse model; Pharmacokinetics

Therapeutic vaccination against chronic hepatitis C virus infection by Peng Peng Ip; Hans W. Nijman; Jan Wilschut; Toos Daemen (pp. 36-50).

► Immune responses against hepatitis C virus play a significant role in viral clearance. ► A prophylactic or therapeutic vaccine against HCV is not yet available. ► Therapeutic HCV vaccines aim to induce both cellular and humoral immune responses. ► Therapeutic HCV vaccines are based on peptides, DNA, virus vectors, yeast and dendritic cells. ► In vaccine trials, robust immune responses correlate with HCV RNA clearance.Approximately 170 million people worldwide are chronic carriers of Hepatitis C virus (HCV). To date, there is no prophylactic vaccine available against HCV. The standard-of-care therapy for HCV infection involves a combination of pegylated interferon-α and ribavirin. This therapy, which is commonly associated with side effects, has a curative rate varying from 43% (HCV genotype 1) to 80% (HCV genotype 2). In 2011, two direct-acting antiviral agents, telaprevir and boceprevir, were approved by the US Food and drug Administration and are now being used in combination with standard-of-care therapy in selected patients infected with HCV genotype 1. Although both drugs are promising, resulting in a shortening of therapy, these drugs also induce additional side effects and have reduced efficacy in patients who did not respond to standard-of-care previously.An alternative approach would be to treat HCV by stimulating the immune system with a therapeutic vaccine ideally aimed at (i) the eradication of HCV-infected cells and (ii) neutralization of infectious HCV particles. The challenge is to develop therapeutic vaccination strategies that are either at least as effective as antiviral drugs but with lower side effects, or vaccines that, when combined with antiviral drugs, can circumvent long-term use of these drugs thereby reducing their side effects.In this review, we summarize and discuss recent preclinical developments in the area of therapeutic vaccination against chronic HCV infection. Although neutralizing antibodies have been described to exert protective immunity, clinical studies on the induction of neutralizing antibodies in therapeutic settings are limited. Therefore, we will primarily discuss therapeutic vaccines which aim to induce effective cellular immune response against HCV.

Keywords: Hepatitis C virus; Hepatocellular carcinoma; Therapeutic vaccines

Anti-retroviral drugs do not facilitate hepatitis C virus (HCV) infection in vitro by Lisa Sandmann; Matthew Wilson; David Back; Heiner Wedemeyer; Michael P. Manns; Eike Steinmann; Thomas Pietschmann; Thomas von Hahn; Sandra Ciesek (pp. 51-58).

► We screened common antiretroviral drugs for direct effects on HCV replication cycle. ► No enhancing effects on HCV infectivity could be observed. ► Maraviroc reduces HCV infectivity if cells are exposed prior to virus infection. ► Raltegravir and ritonavir boosted atazanavir inhibit HCV replication. ► Antiretrovirals are not responsible for the rising incidence of acute HCV infection.An estimated 4 to 5 million people are co-infected with HIV/HCV worldwide. Recently observed outbreaks of acute HCV infection among HIV-positive men who have sex with men (MSM) have been linked to behavioral factors such as high risk sexual practices and recreational drug use. However, at the molecular level, many drugs such as glucocorticoids or cyclosporine A have been found to modulate viral replication. Thus, it is conceivable that drugs used in highly active antiretroviral therapy (HAART) may heighten susceptibility to HCV infection and contribute to the recent outbreaks. We therefore performed a comprehensive screen of antiretroviral drugs covering all available drug classes both individually and in typical combinations used during HAART to probe for direct effects on HCV cell entry, replication, new particle assembly and release. Importantly, no significant enhancement or inhibition of HCV cell entry, replication or new particle production was detected. While raltegravir and ritonavir boosted atazanavir reduce HCV replication, a tenfold reduction of HCVcc entry by the CCR5 antagonist maraviroc was observed.In conclusion, commonly used HAART agents do not specifically enhance HCV replication. Thus recent epidemic outbreaks of acute HCV in HIV-infected MSM are unlikely to be related to enhancing effects of HAART drugs.

Keywords: Hepatitis C virus; HIV; Co-infection; Antiretroviral drugs; MSM; Acute hepatitis C virus infection

Strong and multi-antigen specific immunity by hepatitis B core antigen (HBcAg)-based vaccines in a murine model of chronic hepatitis B: HBcAg is a candidate for a therapeutic vaccine against hepatitis B virus by Sheikh Mohammad Fazle Akbar; Shiyi Chen; Mamun Al-Mahtab; Masanori Abe; Yoichi Hiasa; Morikazu Onji (pp. 59-64).

► HBsAg and HBcAg-pulsed dendritic cells (DC) were prepared as therapeutic vaccine. ► HBsAg induced HBsAg-specific, but not HBcAg-specific, immune responses in HBV TM. ► HBcAg induced strong HBcAg and HBsAg-specific humoral and cellular immunity in HBV TM. ► HBcAg-based vaccine activated endogenous dendritic cells of HBV TM. ► HBcAg should be incorporated in therapeutic vaccine for treatment of chronic HBV.Experimental evidence suggests that hepatitis B core antigen (HBcAg)-specific cytotoxic T lymphocytes (CTL) are essential for the control of hepatitis B virus (HBV) replication and prevention of liver damage in patients with chronic hepatitis B (CHB). However, most immune therapeutic approaches in CHB patients have been accomplished with hepatitis B surface antigen (HBsAg)-based prophylactic vaccines with unsatisfactory clinical outcomes. In this study, we prepared HBsAg-pulsed dendritic cells (DC) and HBcAg-pulsed DC by culturing spleen DC from HBV transgenic mice (HBV TM) and evaluated the immunomodulatory capabilities of these antigens, which may serve as a better therapy for CHB. The kinetics of HBsAg, antibody levels against HBsAg (anti-HBs), proliferation of HBsAg- and HBcAg-specific lymphocytes, production of antigen-specific CTL, and activation of endogenous DC were compared between HBV TM vaccinated with either HBsAg- or HBcAg-pulsed DC. Vaccination with HBsAg-pulsed DC induced HBsAg-specific immunity, but failed to induce HBcAg-specific immunity in HBV TM. However, immunization of HBV TM with HBcAg-pulsed DC resulted in: (1) HBsAg negativity, (2) production of anti-HBs, and (3) development of HBsAg- and HBcAg-specific T cells and CTL in the spleen and the liver. Additionally, significantly higher levels of activated endogenous DC were detected in HBV TM immunized with HBcAg-pulsed DC compared to HBsAg-pulsed DC ( p<0.05). The capacity of HBcAg to modulate both HBsAg- and HBcAg-specific immunity in HBV TM, and activation of endogenous DC in HBV TM without inducing liver damage suggests that HBcAg should be an integral component of the therapeutic vaccine against CHB.

Keywords: Chronic hepatitis B; Immune therapy; Hepatitis B core antigen; Dendritic cells

High rate of reversibility of renal damage in a cohort of HIV-infected patients receiving tenofovir-containing antiretroviral therapy by Anna Bonjoch; Patricia Echeverría; Núria Perez-Alvarez; Jordi Puig; Carla Estany; Bonaventura Clotet; Eugènia Negredo (pp. 65-69).

► Scare data are available about the recovery of TDF-related renal toxicity. ► Evolution of renal parameters after tenofovir discontinuation was evaluated. ► Renal recovery was usually complete and associated with high CD4 T-cell counts.We assessed the progress of renal damage after discontinuation of tenofovir (TDF) in patients who started therapy with normal renal parameters. Normal local reference values were as follows: estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Disease equation (MDRD), ⩾60mL/min/1.73m²; creatinine, ⩽1.20mg/dL; serum phosphate: ⩾2.69mg/dL; proteinuria: <30mg/dL, and glycosuria: <20mg/dL in nondiabetic patients. A logistic regression analysis was used to evaluate factors related to normalization of renal function.We included 183 patients; 85% were male, and median (IQR) age was 44 (40–50)years. Time on TDF was 39 (22–63)months. After 22 (13–49.5)months from TDF discontinuation, renal parameters returned to normal values in 59% of patients, improved (without reaching normal values) in 9.8%, and did not improve in 31%. Median time until normalization was 4 (2–15.75)months, and time to maximum improvement in patients whose values did not return to normal was 14 (8.75–27.75)months. Follow-up was <12months in 30% of the patients who did not improve. The only factors significantly associated with normalization of renal parameters were nadir CD4 T-cell count ( p=0.034; OR=1.002, per 1 cell of increase) and CD4 T-cell count at the end of therapy with TDF ( p=0.030; OR=1.033, per 1 cell of increase). Reversibility of renal damage was prompt and complete in 59% of patients receiving TDF-containing regimens and was associated with a higher nadir and current CD4+ T-cell count, suggesting a role of preserved cellular immunity in renal recovery in this population.

Keywords: Renal impairment; Toxicity; Reversibility; Tenofovir; HIV

A screen for modulators of large T antigen’s ATPase activity uncovers novel inhibitors of Simian Virus 40 and BK virus replication by Sandlin P. Seguin; Alex W. Ireland; Tushar Gupta; Christine M. Wright; Yoshinari Miyata; Peter Wipf; James M. Pipas; Jason E. Gestwicki; Jeffrey L. Brodsky (pp. 70-81).

► Polyomaviruses are linked to an increasing number of diseases. ► To date, specific therapeutics that target this class of viruses are not known. ► A high-throughput screen was performed with a polyomavirus-encoded target. ► Two FDA-approved compounds were identified that inhibit polyomavirus growth. ► A structure activity relationship establishes key features of the compounds.New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg’s ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.

Keywords: Polyomavirus; Bithionol; Hexachlorophene; T antigen; Molecular chaperone; High throughput screen

Anti-hepatitis C virus E2 (HCV/E2) glycoprotein monoclonal antibodies and neutralization interference by Giuseppe Sautto; Nicasio Mancini; Roberta A. Diotti; Laura Solforosi; Massimo Clementi; Roberto Burioni (pp. 82-89).

► We describe an interfering anti-HCV/E2 human monoclonal antibody (mAb) named e509. ► e509 Epitope is centered on an interfering region (epitope II) of HCV/E2 protein. ► e509 Negatively influenced the neutralizing activity of the anti-HCV/E2 mAb AP33. ► No interference was observed on the activity of two anti-HCV/E2 mAbs (e20 and e137). ► e20, e137, e509 Epitopes partially overlap explaining this absence of interference.The suggested HCV escape mechanism consisting in the elicitation of antibody (Ab) subpopulations interfering with the neutralizing activity of other Abs has recently been questioned. In particular, it was originally reported that Abs directed against the 436–447 region (epitope II) of HCV/E2 glycoprotein may interfere with the neutralizing Abs directed against the 412–423 region (epitope I) involved in the binding to CD81. In this paper, we investigate on the molecular features of this phenomenon describing an anti-HCV/E2 monoclonal Ab (mAb) (e509) endowed with a weak neutralizing activity, and whose epitope is centered on epitope II. Interestingly, e509 influenced the potent neutralizing activity of AP33, one of the best characterized anti-HCV/E2 mAb, whereas it did not show any interfering activity against two other broadly neutralizing mAbs (e20 and e137), whose epitopes partially overlap with that of e509 and which possibly displace it from the antigen.These data may give a possible clue to interpret the conflicting studies published to date on the mechanism of interference, suggesting the existence of at least two groups of broadly neutralizing anti-HCV/E2 Abs: (i) those whose epitope is focused on the 412–423 CD81-binding region and whose activity may be hampered by other Abs directed against the 436–447 region, and (ii) those directed against CD81-binding regions but whose epitope contains also residues within the 436–447 region recognized by interfering mAbs, thus competing with them for binding. The conflicting results of previous studies may therefore depend on the relative amount of each of these two populations in the polyclonal preparations used. Overall, a better comprehension of this phenomenon may be of importance in the set up of novel mAb-based anti-HCV therapeutic strategies.

Keywords: Abbreviations; HCV; hepatitis C virus; HCV/E2; HCV E2 glycoprotein; aa; aminoacid; Ab; antibody; mAb; monoclonal antibodyHepatitis C virus; Neutralizing antibody; Interfering antibody; Monoclonal antibody

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Antiviral Research (v.96, #1)

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Antiviral Research (v.96, #1)