Effect of Oral Oseltamivir on Virological Outcomes in Low-risk Adults With Influenza: A Randomized Clinical Trial.

BACKGROUND: Duration of viral shedding is a determinant of infectivity and transmissibility, but few data exist about oseltamivir's ability to alter viral shedding. METHODS: From January 2012 through October 2017, a randomized, double-blinded multicenter clinical trial was conducted in adults aged 18-64 years at 42 sites in Thailand, the United States, and Argentina. Participants with influenza A or B and without risk factors for complications of influenza were screened for the study. Eligible participants were randomized to receive oseltamivir 75 mg or placebo twice daily for 5 days. The primary endpoint was the percentage of participants with virus detectable by polymerase chain reaction in nasopharyngeal swab at day 3. RESULTS: Of 716 adults screened for the study, 558 were randomized, and 501 were confirmed to have influenza. Forty-six participants in the pilot study were excluded, and 449 of the 455 participants in the population for the primary analysis had day 3 viral shedding results. Ninety-nine (45.0%) of 220 participants in the oseltamivir arm had virus detected at day 3 compared with 131 (57.2%) of 229 participants in the placebo arm (absolute difference of -12.2% [-21.4%, -3.0%], P =; .010). The median time to alleviation of symptoms was 79.0 hours for the oseltamivir arm and 84.0 hours for the placebo arm (P =; .34) in those with confirmed influenza infection. CONCLUSIONS: Oseltamivir decreased viral shedding in this low-risk population. However, in the population enrolled in this study, it did not significantly decrease the time to resolution of clinical symptoms. CLINICAL TRIALS REGISTRATION: NCT01314911.

Oseltamivir, amantadine, and ribavirin combination antiviral therapy versus oseltamivir monotherapy for the treatment of influenza: a multicentre, double-blind, randomised phase 2 trial.

BACKGROUND: Influenza continues to have a substantial socioeconomic and health impact despite a long established vaccination programme and approved antivirals. Preclinical data suggest that combining antivirals might be more effective than administering oseltamivir alone in the treatment of influenza. METHODS: We did a randomised, double-blind, multicentre phase 2 trial of a combination of oseltamivir, amantadine, and ribavirin versus oseltamivir monotherapy with matching placebo for the treatment of influenza in 50 sites, consisting of academic medical centre clinics, emergency rooms, and private physician offices in the USA, Thailand, Mexico, Argentina, and Australia. Participants who were aged at least 18 years with influenza and were at increased risk of complications were randomly assigned (1:1) by an online computer-generated randomisation system to receive either oseltamivir (75 mg), amantadine (100 mg), and ribavirin (600 mg) combination therapy or oseltamivir monotherapy twice daily for 5 days, given orally, and participants were followed up for 28 days. Blinded treatment kits were used to achieve masking of patients and staff. The primary endpoint was the percentage of participants with virus detectable by PCR in nasopharyngeal swab at day 3, and was assessed in participants who were randomised, had influenza infection confirmed by the central laboratory on a baseline nasopharyngeal sample, and had received at least one dose of study drug. Safety assessment was done in all patients in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT01227967. FINDINGS: Between March 1, 2011, and April 29, 2016, 633 participants were randomly assigned to receive combination antiviral therapy (n=316) or monotherapy (n=317). Seven participants were excluded from analysis: three were not properly randomised, three withdrew from the study, and one was lost to follow-up. The primary analysis included 394 participants, excluding 47 in the pilot phase, 172 without confirmed influenza, and 13 without an endpoint sample. 80 (40·0%) of 200 participants in the combination group had detectable virus at day 3 compared with 97 (50·0%) of 194 (mean difference 10·0, 95% CI 0·2-19·8, p=0·046) in the monotherapy group. The most common adverse events were gastrointestinal-related disorders, primarily nausea (65 [12%] of 556 reported adverse events in the combination group vs 63 [11%] of 585 reported adverse events in the monotherapy group), diarrhoea (56 [10%] of 556 vs 64 [11%] of 585), and vomiting (39 [7%] of 556 vs 23 [4%] of 585). There was no benefit in multiple clinical secondary endpoints, such as median duration of symptoms (4·5 days in the combination group vs 4·0 days in the monotherapy group; p=0·21). One death occurred in the study in an elderly participant in the monotherapy group who died of cardiovascular failure 13 days after randomisation, judged by the site investigator as not related to study intervention. INTERPRETATION: Although combination treatment showed a significant decrease in viral shedding at day 3 relative to monotherapy, this difference was not associated with improved clinical benefit. More work is needed to understand why there was no clinical benefit when a difference in virological outcome was identified. FUNDING: National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA.

Triple combination antiviral drug (TCAD) composed of amantadine, oseltamivir, and ribavirin impedes the selection of drug-resistant influenza A virus.

Widespread resistance among circulating influenza A strains to at least one of the anti-influenza drugs is a major public health concern. A triple combination antiviral drug (TCAD) regimen comprised of amantadine, oseltamivir, and ribavirin has been shown to have synergistic and broad spectrum activity against influenza A strains, including drug resistant strains. Here, we used mathematical modeling along with three different experimental approaches to understand the effects of single agents, double combinations, and the TCAD regimen on resistance in influenza in vitro, including: 1) serial passage at constant drug concentrations, 2) serial passage at escalating drug concentrations, and 3) evaluation of the contribution of each component of the TCAD regimen to the suppression of resistance. Consistent with the modeling which demonstrated that three drugs were required to suppress the emergence of resistance in influenza A, treatment with the TCAD regimen resulted in the sustained suppression of drug resistant viruses, whereas treatment with amantadine alone or the amantadine-oseltamivir double combination led to the rapid selection of resistant variants which comprised ∼100% of the population. Furthermore, the TCAD regimen imposed a high genetic barrier to resistance, requiring multiple mutations in order to escape the effects of all the drugs in the regimen. Finally, we demonstrate that each drug in the TCAD regimen made a significant contribution to the suppression of virus breakthrough and resistance at clinically achievable concentrations. Taken together, these data demonstrate that the TCAD regimen was superior to double combinations and single agents at suppressing resistance, and that three drugs at a minimum were required to impede the selection of drug resistant variants in influenza A virus. The use of mathematical modeling with multiple experimental designs and molecular readouts to evaluate and optimize combination drug regimens for the suppression of resistance may be broadly applicable to other infectious diseases.

Induction of interferon-gamma-inducible protein 10 by SARS-CoV infection, interferon alfacon 1 and interferon inducer in human bronchial epithelial Calu-3 cells and BALB/c mice.

BACKGROUND: The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is poorly understood. Several mechanisms involving both direct effects on target cells and indirect effects via the immune system might exist. SARS-CoV has been shown in vitro to induce changes of cytokines and chemokines in various human and animal cells. We previously reported that interferon (IFN) alfacon-1 was more active against SARS-CoV infection in human bronchial epithelial Calu-3 cells than in African green monkey kidney epithelial cells on day 3 post-infection. METHODS: In the current study, we first evaluated the efficacy of IFN-alfacon 1 in Calu-3 cells during the first 7 days of virus infection. We then used the two-antibody sandwich ELISA method to detect IFN-gamma-inducible protein 10 (IP-10). We further evaluated the efficacy of antivirals directed against SARS-CoV infection in BALB/c mice. RESULTS: A potent, prolonged inhibition of SARS-CoV replication in Calu-3 cells with IFN-alfacon 1 was observed. Furthermore, IP-10, an IFN-inducible leukocyte chemoattractant, was detected in Calu-3 cells after SARS-CoV infection. Interestingly, IP-10 expression was shown to be significantly increased when SARS-CoV-infected Calu-3 cells were treated with IFN alfacon-1. IP-10 expression was detected in the lungs of SARS-CoV-infected BALB/c mice. Significantly high levels of mouse IP-10 in BALB/c mice was also detected when SARS-CoV-infected mice were treated with the interferon inducer, polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose (poly IC:LC). Treatment with poly IC:LC by intranasal route were effective in protecting mice against a lethal infection with mouse-adapted SARS-CoV and reduced the viral lung titres. CONCLUSIONS: Our data might provide an important insight into the mechanism of pathogenesis of SARS-CoV and these properties might be therapeutically advantageous.

Triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant influenza virus strains in vitro.

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza.

Triple combination of oseltamivir, amantadine, and ribavirin displays synergistic activity against multiple influenza virus strains in vitro.

The recurring emergence of influenza virus strains that are resistant to available antiviral medications has become a global health concern, especially in light of the potential for a new influenza virus pandemic. Currently, virtually all circulating strains of influenza A virus in the United States are resistant to either of the two major classes of anti-influenza drugs (adamantanes and neuraminidase inhibitors). Thus, new therapeutic approaches that can be rapidly deployed and that will address the issue of recurring resistance should be developed. We have tested double and triple combinations of the approved anti-influenza drugs oseltamivir and amantadine together with ribavirin against three influenza virus strains using cytopathic effect inhibition assays in MDCK cells. We selected A/New Caledonia/20/99 (H1N1) and A/Sydney/05/97 (H3N2) as representatives of the wild-type versions of the predominant circulating seasonal influenza virus strains and A/Duck/MN/1525/81 (H5N1) as a representative of avian influenza virus strains. Dose-response curves were generated for all drug combinations, and the degree of drug interaction was quantified using a model that calculates the synergy (or antagonism) between the drugs in double and triple combinations. This report demonstrates that a triple combination of antivirals was highly synergistic against influenza A virus. Importantly, the synergy of the triple combination was 2- to 13-fold greater than the synergy of any double combination depending on the influenza virus subtype. These data support the investigation of a novel combination of oseltamivir, amantadine, and ribavirin as an effective treatment for both seasonal and pandemic influenza virus, allowing the efficient use of the existing drug supplies.

C3H/HeN mouse model for the evaluation of antiviral agents for the treatment of Venezuelan equine encephalitis virus infection.

The TC-83 vaccine strain of Venezuelan equine encephalitis virus (VEEV) causes encephalitis and death in C3H/HeN mice infected by intranasal (i.n.) instillation. Since TC-83 is exempt as a select agent, this mouse model was used in the evaluation of antiviral therapies. Virus titers in the brains of infected mice peaked on 4 dpi and persisted at high levels until death at 9.4+/-0.5 dpi. Mouse brains appeared histologically normal on 2 dpi, but developed meningoencephalitis, neuropil vacuolation, and gliosis by 8 dpi. Results from a protein cytokine array showed significant elevations over time in interleukin (IL)-1alpha, IL-1beta, IL-6, IL-12, MCP-1, IFNgamma, TNFalpha, MIP-1alpha, and RANTES in homogenized brain samples of infected mice. Immunohistochemical staining showed a colocalization of viral antigen with neuron markers. Treatment with interferon-alpha B/D or ampligen significantly improved survival, brain virus titer and cytokine levels, mean day-to-death, and weight change in infected mice. The time-course of infection and disease parameters of mice infected with TC-83 VEEV were similar in many ways to disease parameters in mice infected with other VEEV strains. Thus, infection of C3H/HeN mice with TC-83 VEEV may serve as a suitable model for the evaluation of antiviral compounds for the treatment of this viral disease.

Differential pathogenesis of cowpox virus intranasal infections in mice induced by low and high inoculum volumes and effects of cidofovir treatment.

The causes of death from intranasal cowpox virus infections in mice remain unclear. Hypotheses include severe pneumonitis, hepatitis and/or hyperproduction of cytokines and chemokines. This work explores these hypotheses by studying the influence of low- and high-volume virus inocula on viral pathogenesis. BALB/c mice were infected intranasally with a syncytium-forming variant of cowpox virus in 5 microL or 50 microL volumes containing the same infectious virus challenge dose. The 50 microL infection produced a more rapidly lethal disease associated with severe pneumonitis, high lung and nasal virus titres and increased cytokine and chemokine levels in the lungs and nasal tissue, whilst liver infection was minimal. The 5 microL inoculum infection was also lethal, but the infection was primarily confined to the upper respiratory tract and included elevated nasal cytokine and chemokine levels. Levels of the pro-inflammatory cytokine interleukin-6 were particularly high in both infections. Treatment of the infections with cidofovir (100mg/kg/day for 2 days starting 24h after virus exposure) led to survival and suppression of tissue virus titres. Treatment reduced pneumonitis in the 50 microL infection and lessened cytokine hyperproduction in both infections. We conclude that a 5 microL volume inoculum of cowpox virus causes a lethal upper respiratory tract infection, whilst the 50 microL inoculum targets both upper and lower respiratory tracts, with excessive release of systemic pro-inflammatory factors. Cidofovir effectively treated both infections and slowed viral replication sufficiently to subdue the exaggerated release of pro-inflammatory mediators.

A 22-plex chemiluminescent microarray for pneumococcal antibodies.

We developed a chemiluminescent multiplexed microarray that simultaneously determines IgG antibody concentrations to 22 pneumococcal polysaccharide (PnPs) serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 23F, and 33F). We compared the microarray with an enzyme-linked immunosorbent assay (ELISA) for 9 of the 22 serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F). Correlation coefficients (r2) for the comparison of the microarray with ELISA ranged from 0.91 to 0.97 for the 9 serotypes. The microarray detected more than 4-fold increases in antibody concentrations in serum samples from before and 1 month after administration of pneumococcal vaccine for all 22 serotypes tested. The mean interassay and intra-assay coefficients of variation for 12 serum samples for the 22 serotypes were 7.6% and 6.0%, respectively. Inhibition-of-binding studies showed more than 90% inhibition by homologous serotypes and, with few exceptions, less than 25% inhibition by heterologous serotypes. The microarray multiplexing technology is an attractive alternative to ELISA for antibody responses to 23-valent PnPs vaccines.

Validating a custom multiplex ELISA against individual commercial immunoassays using clinical samples.

The measurement of multiple antigens in a single sample poses clinical and methodological challenges. Here we describe the validation of a multiplexed sandwich enzyme-linked immunosorbent assay (ELISA) array (microELISA) of nine antigens. The antigens tested simultaneously were: alpha-fetoprotein (AFP), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), cancer antigen 125 (CA 125), CA 15-3, CA 19-9, beta-human chorionic gonadotropin (beta-hCG), luteinizing hormone (LH), and follicle stimulating hormone (FSH). At least 44 clinical samples were tested for each antigen. microELISA results for the nine antigens were then compared with clinical laboratory results obtained for the same antigens in individual chemiluminescent immunoassays. The microELISA had a coefficient of variation (cv) of 7.3% within an assay and 12.6% for assays run at different times. A statistical comparison of results from the microELISA with results from the clinical laboratory showed that the assays had correlation coefficients ranging from 0.99 to 0.76, and Deming regression demonstrated that four of the nine assays were high-quality assays and not statistically different to the individual assays. To determine if the differences in the assays were due to methodology, the microELISA was also compared with conventional ELISAs using identical antibodies and reagents. Deming regression demonstrated that five of the eight assays were high-quality, indicating that a poor correlation between a microELISA and an individual immunoassay are partly due to antibody differences.

TLR3 deletion limits mortality and disease severity due to Phlebovirus infection.

TLR3 was the first member of the TLR family of pattern recognition receptors found to detect a conserved viral molecular pattern, dsRNA, yet supporting evidence for a major role in host defense against viral pathogens is limited. Punta Toro virus (PTV) has been shown to produce severe infection in mice, modeling disease caused by the related highly pathogenic Rift Valley fever phlebovirus in humans and domesticated ungulates. Using TLR3-deficient mice, we investigated the involvement of TLR3 in host defense against PTV infection. Compared with wild-type, TLR3(-/-) mice demonstrate increased resistance to lethal infection and have reduced liver disease associated with hepatotropic PTV infection. Infectious challenge produced comparable peak liver and serum viral loads; however, TLR3(-/-) mice were able to clear systemic virus at a slightly faster rate. Cytokine profiling suggests that TLR3 plays an important role in PTV pathogenesis through the overproduction of inflammatory mediators, which may be central to the observed differences in survival and disease severity. Compared with TLR3-deficient mice, IL-6, MCP-1, IFN-gamma, and RANTES were all present at higher levels in wild-type animals. Most dramatic was the exaggerated levels of IL-6 found systemically and in liver tissue of infected wild-type mice; however, IL-6-deficient animals were found to be more susceptible to lethal PTV infection. Taken together, we conclude that the TLR3-mediated response to PTV infection is detrimental to disease outcome and propose that IL-6, although critical to establishing antiviral defense, contributes to pathogenesis when released in excess, necessitating its controlled production as is seen with TLR3(-/-) mice.

Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin.

Because of the conflicting data concerning the SARS-CoV inhibitory efficacy of ribavirin, an inosine monophosphate (IMP) dehydrogenase inhibitor, studies were done to evaluate the efficacy of ribavirin and other IMP dehydrogenase inhibitors (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), mizoribine, and mycophenolic acid) in preventing viral replication in the lungs of BALB/c mice, a replication model for severe acute respiratory syndrome (SARS) infections (Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., Murphy, B., 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in the respiratory tract of mice. J. Virol. 78, 3572-3577). Ribavirin given at 75 mg/kg 4 h prior to virus exposure and then given twice daily for 3 days beginning at day 0 was found to increase virus lung titers and extend the length of time that virus could be detected in the lungs of mice. Other IMP dehydrogenase inhibitors administered near maximum tolerated doses using the same dosing regimen as for ribavirin were found to slightly enhance virus replication in the lungs. In addition, ribavirin treatment seemed also to promote the production of pro-inflammatory cytokines 4 days after cessation of treatment, although after 3 days of treatment ribavirin inhibited pro-inflammatory cytokine production in infected mice, significantly reducing the levels of the cytokines IL-1alpha, interleukin-5 (IL-5), monocyte chemotactic protein-1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF). These findings suggest that ribavirin may actually contribute to the pathogenesis of SARS-CoV by prolonging and/or enhancing viral replication in the lungs. By not inhibiting viral replication in the lungs of infected mice, ribavirin treatment may have provided a continual source of stimulation for the inflammatory response thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.