Examining the interactions of Galahad™ compound with viruses to develop a novel inactivated influenza A virus vaccine.

Galahad™ is a proanthocyanidin complexed with polysaccharides that inactivates viruses and indicates potential for an innovative approach to making protective vaccines. The polysaccharide portion of Galahad™ consists mainly of arabinan and arabinogalactan. In a seven-day toxicity study in rats, it was not toxic even when tested undiluted. Galahad™ inactivated a wide range of DNA and RNA viruses including adenoviruses, corona viruses such as SARS-CoV-2, and influenza viruses. Electron microscopy studies showed that exposure to Galahad™ caused extensive clumping of virions followed by lack of detection of virions after longer periods of exposure. Based on the viral inactivation data, the hypotheses tested is that Galahad™ inactivation of virus can be used to formulate a protective inactivated virus vaccine. To evaluate this hypothesis, infectious influenza A virus (H5N1, Duck/MN/1525/81) with a titer of 105.7 CCID50/0.1 ml was exposed for 10 min to Galahad™. This treatment caused the infectious virus titer to be reduced to below detectable limits. The Galahad™ -inactivated influenza preparation without adjuvant or preservative was given to BALB/c mice using a variety of routes of administration and dosing regimens. The most protective route of administration and dosing regimen was when mice were given the vaccine twice intranasally, the second dose coming 14 days after the primary vaccine dose. All the mice receiving this vaccine regimen survived the virus challenge while only 20% of the mice receiving placebo survived. This suggests that a Galahad™-inactivated influenza virus vaccine can elicit a protective immune response even without the use of an adjuvant. This technology should be investigated further for its potential to make effective human vaccines.

Lack of selective resistance of influenza A virus in presence of host-targeted antiviral, UV-4B.

Development of antiviral drug resistance is a continuous concern for viruses with high mutation rates such as influenza. The use of antiviral drugs targeting host proteins required for viral replication is less likely to result in the selection of resistant viruses than treating with direct-acting antivirals. The iminosugar UV-4B is a host-targeted glucomimetic that inhibits endoplasmic reticulum α-glucosidase I and II enzymes resulting in improper glycosylation and misfolding of viral glycoproteins. UV-4B has broad-spectrum antiviral activity against diverse viruses including dengue and influenza. To examine the ability of influenza virus to develop resistance against UV-4B, mouse-adapted influenza virus was passaged in mice in the presence or absence of UV-4B and virus isolated from lungs was used to infect the next cohort of mice, for five successive passages. Deep sequencing was performed to identify changes in the viral genome during passaging in the presence or absence of UV-4B. Relatively few minor variants were identified within each virus and the ratio of nonsynonymous to synonymous (dN/dS) substitutions of minor variants confirmed no apparent positive selection following sustained exposure to UV-4B. Three substitutions (one synonymous in PB2, one nonsynonymous in M and PA each) were specifically enriched (>3%) in UV-4B-treated groups at passage five. Recombinant viruses containing each individual or combinations of these nonsynonymous mutations remained sensitive to UV-4B treatment in mice. Overall, these data provide evidence that there is a high genetic barrier to the generation and selection of escape mutants following exposure to host-targeted iminosugar antivirals.

Inhibition of adenovirus serotype 14 infection by octadecyloxyethyl esters of (S)-[(3-hydroxy-2-phosphonomethoxy)propyl]- nucleosides in vitro.

On September 22, 2008, a physician on Prince of Wales Island, Alaska, notified the Alaska Department of Health and Social Services (ADHSS) of an unusually high number of adult patients with recently diagnosed pneumonia (n = 10), including three persons who required hospitalization and one who died. ADHSS and CDC conducted an investigation to determine the cause and distribution of the outbreak, identify risk factors for hospitalization, and implement control measures. This report summarizes the results of that investigation, which found that the outbreak was caused by adenovirus 14 (Ad14), an emerging adenovirus serotype in the United States that is associated with a higher rate of severe illness compared with other adenoviruses. Among the 46 cases identified in the outbreak from September 1 through October 27, 2008, the most frequently observed characteristics included the following: male (70%), Alaska Native (61%), underlying pulmonary disease (44%), aged > or = 65 years (26%), and current smoker (48%). Patients aged > or = 65 years had a fivefold increased risk for hospitalization. The most commonly reported symptoms were cough (100%), shortness of breath (87%), and fever (74%). Of the 11 hospitalized patients, three required intensive care, and one required mechanical ventilation. One death was reported. Ad14 isolates obtained during the outbreak were identical genetically to those in recent community-acquired outbreaks in the United States which suggests the emergence of a new, and possibly more virulent Ad14 variant. Clinicians should consider Ad14 infection in the differential diagnosis for patients with community-acquired pneumonia, particularly when unexplained clusters of severe respiratory infections are detected.

Molecular Mechanism Underlying the Action of Influenza A Virus Fusion Inhibitor MBX2546.

Influenza A virus envelop protein hemagglutinin (HA) plays important roles in viral entry. We previously have reported that MBX2546, a novel influenza A virus inhibitor, binds to HA and inhibits HA-mediated membrane fusion. In this report, we show that (i) both binding and stabilization of HA by MBX2546 are required for the inhibition of viral infection and (ii) the binding of HA by MBX2546 represses the low-pH-induced conformational change in the HA, which is a prerequisite for membrane fusion. Mutations in MBX2546-resistant influenza A/PR/8/34 (H1N1) viruses are mapped in the HA stem region near the amino terminus of HA2. Finally, we have modeled the binding site of MBX2546 using molecular dynamics and find that the resulting structure is in good agreement with our results. Together, these studies underscore the importance of the HA stem loop region as a potential target for therapeutic intervention.

Prophylactic and therapeutic intranasal administration with an immunomodulator, Hiltonol® (Poly IC:LC), in a lethal SARS-CoV-infected BALB/c mouse model.

Hiltonol®, (Poly IC:LC), a potent immunomodulator, is a synthetic, double-stranded polyriboinosinic-polyribocytidylic acid (poly IC) stabilized with Poly-L-lysine and carboxymethyl cellulose (LC). Hiltonol® was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. Hiltonol® at 5, 1, 0.5 or 0.25 mg/kg/day by intranasal (i.n.) route resulted in significant survival benefit when administered at selected times 24 h prior to challenge with a lethal dose of mouse-adapted severe acute respiratory syndrome coronavirus (SARS-CoV). The infected BALB/c mice receiving the Hiltonol® treatments were also significantly effective in protecting mice against weight loss due to infection (p < 0.001). Groups of 20 mice were dosed with Hiltonol® at 2.5 or 0.75 mg/kg by intranasal instillation 7, 14, and 21 days before virus exposure and a second dose was given 24 h later, prophylactic Hiltonol® treatments (2.5 mg/kg/day) were completely protective in preventing death, and in causing significant reduction in lung hemorrhage scores, lung weights and lung virus titers. Hiltonol® was also effective as a therapeutic when give up to 8 h post virus exposure; 100% of the-infected mice were protected against death when Hiltonol® was administered at 5 mg/kg/day 8 h after infection. Our data suggest that Hiltonol® treatment of SARS-CoV infection in mice leads to substantial prophylactic and therapeutic effects and could be used for treatment of other virus disease such as those caused by MERS-CoV a related coronavirus. These properties might be therapeutically advantageous if Hiltonol® is considered for possible clinical use.

Activities of JNJ63623872 and oseltamivir against influenza A H1N1pdm and H3N2 virus infections in mice.

JNJ63623872 (formerly known as VX-787) is an inhibitor of influenza A virus polymerases through interaction with the viral PB2 subunit. This interaction blocks the cap-snatching activity of the virus that is essential for virus replication. Previously published work has documented antiviral activity of JNJ63623872 in cell culture and mouse infection studies. In this report, we extend the in vivo observations by comparing the efficacies of JNJ63623872 and oseltamivir in mice infected with influenza A/California/04/2009 (H1N1pdm) and A/Victoria/3/75 (H3N2) viruses. Animals received JNJ63623872 or oseltamivir orally twice daily for 10 days starting 2 h pre-infection. JNJ63623872 (2, 6, and 20 mg/kg/day) and oseltamivir (20 mg/kg/day) completely prevented death in the H1N1pdm virus infection. Weight loss at nadir was only 12% in mice receiving 2 mg/kg/day of JNJ63623872 compared to 23% and 32%, respectively, in oseltamivir-treated (20 mg/kg/day) and placebo groups. Lung hemorrhage scores, lung weights, and lung virus titers on day 6 were reduced in a dose-responsive manner by JNJ63623872 treatments, whereas oseltamivir treatments were not as effective. JNJ63623872 was less active against H3N2 virus infection, with more body weight loss occurring and only 30% survival at the 2-mg/kg/day dose. Lung scores, lung weights, and H3N2 viral titers in lungs of mice were reduced less by JNJ63623872 treatments compared to the H1N1pdm infection. Nevertheless, the 20-mg/kg/day dose of JNJ63623872 was more effective than oseltamivir (20 mg/kg/day) in improving body weight and reducing the severity of lung infection. JNJ63623872 appears to be an important new drug candidate to treat influenza A H1N1pdm and H3N2 virus infections.

Serum amyloid A (SAA) is an early biomarker of influenza virus disease in BALB/c, C57BL/2, Swiss-Webster, and DBA.2 mice.

Assessment of influenza virus disease progression and efficacy of antiviral therapy in the widely used mouse models relies mostly on body weight loss and lung virus titers as markers of disease. However, both parameters have their shortcomings. Therefore, the aim of our study was to find non-invasive markers in the murine model of severe influenza that could detect disease early and predict disease outcome. BALB/c mice were lethally infected with influenza A(H1N1)pdm09 virus and serum samples were collected at various time points. Enzyme-linked immunosorbent assays were performed to quantify amounts of serum amyloid A (SAA), C-reactive protein, complement 3, transferrin, corticosterone, prostaglandin E2, H2O2, and alpha-2,6-sialyltransferase. We found that SAA was the most promising candidate with levels acutely and temporarily elevated by several hundred-fold 3 days post virus inoculation. Upon treatment with oseltamivir phosphate, levels of SAA were significantly decreased. High levels of SAA were associated with poor disease prognosis, whereas body weight loss was not as a reliable predictor of disease outcome. SAA levels were also transiently increased in BALB/c mice infected with influenza A(H3N2) and influenza B virus, as well as in C57BL/2, Swiss-Webster, and DBA.2 mice infected with influenza A(H1N1)pdm09 virus. High levels of SAA often, but not always, were associated with disease outcome in these other influenza virus mouse models. Therefore, SAA represents a valid biomarker for influenza disease detection in all tested mouse strains but its prognostic value is limited to BALB/c mice infected with influenza A(H1N1)pdm09 virus.

The Iminosugar UV-4 is a Broad Inhibitor of Influenza A and B Viruses ex Vivo and in Mice.

Iminosugars that are competitive inhibitors of endoplasmic reticulum (ER) α-glucosidases have been demonstrated to have antiviral activity against a diverse set of viruses. A novel iminosugar, UV-4B, has recently been shown to provide protection against lethal infections with dengue and influenza A (H1N1) viruses in mice. In the current study, the breadth of activity of UV-4B against influenza was examined ex vivo and in vivo. Efficacy of UV-4B against influenza A and B viruses was shown in primary human bronchial epithelial cells, a principal target tissue for influenza. Efficacy of UV-4B against influenza A (H1N1 and H3N2 subtypes) and influenza B was demonstrated using multiple lethal mouse models with readouts including mortality and weight loss. Clinical trials are ongoing to demonstrate safety of UV-4B and future studies to evaluate antiviral activity against influenza in humans are planned.

A broadly neutralizing human monoclonal antibody is effective against H7N9.

Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (-24 h) or double-dose (-12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (-12 h) combined with oseltamivir at 50 mg/kg (-12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.

New small molecule entry inhibitors targeting hemagglutinin-mediated influenza a virus fusion.

Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC50] of 0.3 to 5.9 µM); (ii) are selective (50% cytotoxicity concentration [CC(50)] of >100 µM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 µM(2) % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process.

In vitro activity of favipiravir and neuraminidase inhibitor combinations against oseltamivir-sensitive and oseltamivir-resistant pandemic influenza A (H1N1) virus.

Few anti-influenza drugs are licensed in the United States for the prevention and therapy of influenza A and B virus infections. This shortage, coupled with continuously emerging drug resistance, as detected through a global surveillance network, seriously limits our anti-influenza armamentarium. Combination therapy appears to offer several advantages over traditional monotherapy in not only delaying development of resistance but also potentially enhancing single antiviral activity. In the present study, we evaluated the antiviral drug susceptibilities of fourteen pandemic influenza A (H1N1) virus isolates in MDCK cells. In addition, we evaluated favipiravir (T-705), an investigational drug with a broad antiviral spectrum and a unique mode of action, alone and in dual combination with the neuraminidase inhibitors (NAIs) oseltamivir, peramivir, or zanamivir, against oseltamivir-sensitive pandemic influenza A/California/07/2009 (H1N1) and oseltamivir-resistant A/Hong Kong/2369/2009 (H1N1) virus. Mean inhibitory values showed that the tested virus isolates remained sensitive to commonly used antiviral drugs, with the exception of the Hong Kong virus isolate. Drug dose-response curves confirmed complete drug resistance to oseltamivir, partial sensitivity to peramivir, and retained susceptibility to zanamivir and favipiravir against the A/Hong Kong/2369/2009 virus. Three-dimensional analysis of drug interactions using the MacSynergy(TM) II program indicated an overall synergistic interaction when favipiravir was combined with the NAIs against the oseltamivir-sensitive influenza virus, and an additive effect against the oseltamivir-resistant virus. Although the clinical relevance of these drug combinations remains to be evaluated, results obtained from this study support the use of combination therapy with favipiravir and NAIs for treatment of human influenza virus infections.

Favipiravir (T-705), a novel viral RNA polymerase inhibitor.

Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is an antiviral drug that selectively inhibits the RNA-dependent RNA polymerase of influenza virus. It is phosphoribosylated by cellular enzymes to its active form, favipiravir-ribofuranosyl-5'-triphosphate (RTP). Its antiviral effect is attenuated by the addition of purine nucleic acids, indicating the viral RNA polymerase mistakenly recognizes favipiravir-RTP as a purine nucleotide. Favipiravir is active against a broad range of influenza viruses, including A(H1N1)pdm09, A(H5N1) and the recently emerged A(H7N9) avian virus. It also inhibits influenza strains resistant to current antiviral drugs, and shows a synergistic effect in combination with oseltamivir, thereby expanding influenza treatment options. A Phase III clinical evaluation of favipiravir for influenza therapy has been completed in Japan and two Phase II studies have been completed in the United States. In addition to its anti-influenza activity, favipiravir blocks the replication of many other RNA viruses, including arenaviruses (Junin, Machupo and Pichinde); phleboviruses (Rift Valley fever, sandfly fever and Punta Toro); hantaviruses (Maporal, Dobrava, and Prospect Hill); flaviviruses (yellow fever and West Nile); enteroviruses (polio- and rhinoviruses); an alphavirus, Western equine encephalitis virus; a paramyxovirus, respiratory syncytial virus; and noroviruses. With its unique mechanism of action and broad range of antiviral activity, favipiravir is a promising drug candidate for influenza and many other RNA viral diseases for which there are no approved therapies.

Methods for evaluation of antiviral efficacy against influenza virus infections in animal models.

Compounds undergoing preclinical development for anti-influenza virus activity require evaluation in small animal models. Laboratory mice are most commonly used for initial studies because of size, cost, and availability. Cotton rats, guinea pigs, and ferrets (particularly) have been used for more advanced studies. Each animal infection model has certain limitations relative to human influenza infections. For example, the fever response that is evident in humans only occurs with consistency in ferrets. Mice infected with mouse-adapted viruses and ferrets infected with highly pathogenic avian influenza viruses suffer severe disease, whereas cotton rats and guinea pigs manifest few symptoms. Thus, for each animal model there is a certain set of disease parameters that can be measured. Here we describe methods for assessing the efficacy of anti-influenza virus compounds in each of these animal species.

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-ß) expression.

The human parainfluenza virus type 3 (HPIV3) phosphoprotein (P) gene is unusual as it contains an editing site where nontemplated ribonucleotide residues can be inserted. This RNA editing can lead to the expression of the viral P, PD, putative W, and theoretical V protein from a single gene. Although the HPIV3 PD protein has been detected, its function and those of the W and V proteins are poorly understood. Therefore, we first used reverse genetics techniques to construct and rescue a recombinant (r)HPIV3 clone with a polyhistidine sequence at the 5' end of the P gene for tagged protein detection. Western blot analysis demonstrated the presence of the P, PD, and W proteins, but no V protein was detected. Then, we functionally studied the D domain of the PD protein by constructing two rHPIV3 knockout clones that are deficient in the expression of the D domain. Results from growth kinetic studies with infected MA-104 and A596 cells showed that viral replication of the two knockout viruses (rHPIV3-ΔES and rHPIV3-ΔD) was comparable to that of the parental virus in both cell lines. However, viral mRNA transcription and genomic replication was significantly reduced. Furthermore, cytokine/chemokine profiles of A549 cells infected with either knockout virus were unchanged or showed lower levels compared to those from cells infected with the parental virus. These data suggest that the D domain of the PD protein may play a luxury role in HPIV3 RNA synthesis and may also be involved in disrupting the expression of beta interferon.

Prophylactic and therapeutic testing of Nicotiana-derived RSV-neutralizing human monoclonal antibodies in the cotton rat model.

Severe lower respiratory tract infection in infants and small children is commonly caused by respiratory syncytial virus (RSV). Palivizumab (Synagis(®)), a humanized IgG1 monoclonal antibody (mAb) approved for RSV immunoprophylaxis in at-risk neonates, is highly effective, but pharmacoeconomic analyses suggest its use may not be cost-effective. Previously described potent RSV neutralizers (human Fab R19 and F2-5; human IgG RF-1 and RF-2) were produced in IgG format in a rapid and inexpensive Nicotiana-based manufacturing system for comparison with palivizumab. Both plant-derived (palivizumab-N) and commercial palivizumab, which is produced in a mouse myeloma cell line, showed protection in prophylactic (p < 0.001 for both mAbs) and therapeutic protocols (p < 0.001 and p < 0.05 respectively). The additional plant-derived human mAbs directed against alternative epitopes displayed neutralizing activity, but conferred less protection in vivo than palivizumab-N or palivizumab. Palivizumab remains one of the most efficacious RSV mAbs described to date. Production in plants may reduce manufacturing costs and improve the pharmacoeconomics of RSV immunoprophylaxis and therapy.

Synergistic combinations of favipiravir and oseltamivir against wild-type pandemic and oseltamivir-resistant influenza A virus infections in mice.

AIM: Favipiravir and oseltamivir are antiviral compounds used for the treatment of influenza infections. We have aimed to investigate the efficacy of the compounds in combination to treat influenza H1N1 virus infections in mice. MATERIALS & METHODS: Mice infected with pandemic influenza A/California/04/2009 (H1N1pdm) virus or an oseltamivir-resistant (H275Y neuraminidase mutation) influenza A/Mississippi/ 3/2001 (H1N1) virus were treated orally with inhibitors twice a day for 5 days starting 4 h after infection. RESULTS: Complete protection from death was afforded by favipiravir treatments of 100 mg/kg/day, but lower doses were less effective. Combinations of oseltamivir (1 and 3 mg/kg/day) with favipiravir (3, 10 and 30 mg/kg/day) resulted in a synergistic improvement in survival rates against H1N1pdm infections. Significant reductions in lung virus titers also occurred. Against the H275Y virus infection, oseltamivir alone was only 30% protective from death at 100 mg/kg/day, but combinations of the two compounds produced a synergistic improvement in survival rate. CONCLUSION: The utility of treating H1N1 influenza virus infections with oseltamivir and favipiravir in combination has been established.

Antiviral agents derived from novel 1-adamantyl singlet nitrenes.

BACKGROUND: Amantadine constitutes an interesting, diamond crystal lattice-shaped, antivirally active amine with an inhibitory effect on influenza A viruses causing common 'flu' in humans. Unfortunately, amantadine forfeited most of its therapeutic potential because of resistance development in recent influenza A virus isolates. The antiviral efficacy of amantadine congeners can be chemically modified, resulting in re-constitution, improvement and/or extension of antiviral activities mediated by amino-adamantyls. METHODS: Newly synthesized compounds were evaluated towards HIV type-1 (HIV-1) replication in primary human lymphocytes. One N-phenacyl amantadine derivative was investigated for inhibiting the in vitro replication of respiratory viruses (influenza A viruses, influenza B virus, human parainfluenza virus type 3 and severe acute respiratory syndrome coronavirus). RESULTS: Two ketone-stabilized 1-adamantyl singlet nitrenes were discovered serendipitously. To our best knowledge these are the first persistently stable nitrenes to be reported. Their structure was proved by determining the X-ray single crystal structure of one hydrolytic elaboration product. This salt adduct revealed an incommensurately modulated crystal structure, which was solved by extensive computational refinement. We could show that ketone-stabilized 1-adamantyl singlet nitrenes are versatile synthons for the synthesis of antiviral drug candidates. An amantadine-folate conjugate was inhibitory on HIV-1 replication in primary human lymphocytes, and one N-phenacyl amantadine derivative was inhibitory towards low pathogenic avian influenza A virus (H5N1) replication in vitro. CONCLUSIONS: These results indicate that the aromatic-aliphatic ketone-stabilized 1-adamantyl singlet nitrenes, beyond being of fundamental interest in organic chemistry, represent versatile synthons for the synthesis of new amantadine-related potentially antiviral drugs.

Efficacy of combined therapy with amantadine, oseltamivir, and ribavirin in vivo against susceptible and amantadine-resistant influenza A viruses.

The limited efficacy of existing antiviral therapies for influenza--coupled with widespread baseline antiviral resistance--highlights the urgent need for more effective therapy. We describe a triple combination antiviral drug (TCAD) regimen composed of amantadine, oseltamivir, and ribavirin that is highly efficacious at reducing mortality and weight loss in mouse models of influenza infection. TCAD therapy was superior to dual and single drug regimens in mice infected with drug-susceptible, low pathogenic A/H5N1 (A/Duck/MN/1525/81) and amantadine-resistant 2009 A/H1N1 influenza (A/California/04/09). Treatment with TCAD afforded >90% survival in mice infected with both viruses, whereas treatment with dual and single drug regimens resulted in 0% to 60% survival. Importantly, amantadine had no activity as monotherapy against the amantadine-resistant virus, but demonstrated dose-dependent protection in combination with oseltamivir and ribavirin, indicative that amantadine's activity had been restored in the context of TCAD therapy. Furthermore, TCAD therapy provided survival benefit when treatment was delayed until 72 hours post-infection, whereas oseltamivir monotherapy was not protective after 24 hours post-infection. These findings demonstrate in vivo efficacy of TCAD therapy and confirm previous reports of the synergy and broad spectrum activity of TCAD therapy against susceptible and resistant influenza strains in vitro.

Effect of statin treatments on highly pathogenic avian influenza H5N1, seasonal and H1N1pdm09 virus infections in BALB/c mice.

Statins are used to control elevated cholesterol or hypercholesterolemia, but have previously been reported to have antiviral properties. AIMS: To show efficacy of statins in various influenza virus mouse models. MATERIALS & METHODS: BALB/c mice were treated intraperitoneally or orally with several types of statins (simvastatin, lovastatin, mevastatin, pitavastatin, atorvastatin or rosuvastatin) at various concentrations before or after infection with either influenza A/Duck/ MN/1525/81 H5N1 virus, influenza A/Vietnam/1203/2004 H5N1 virus, influenza A/ Victoria/3/75 H3N2 virus, influenza A/NWS/33 H1N1 virus or influenza A/CA/04/09 H1N1pdm09 virus. RESULTS: The statins administered intraperitoneally or orally at any dose did not significantly enhance the total survivors relative to untreated controls. In addition, infected mice receiving any concentration of statin were not protected against weight loss due to the infection. None of the statins significantly increased the mean day of death relative to mice in the placebo treatment group. Furthermore, the statins had relatively few ameliorative effects on lung pathology or lung weights at day 3 and 6 after virus exposure, although mice treated with simvastatin did have improved lung function as measured by arterial saturated oxygen levels in one experiment. CONCLUSION: Statins showed relatively little efficacy in any mouse model used by any parameter tested.

In vitro and in vivo efficacy of fluorodeoxycytidine analogs against highly pathogenic avian influenza H5N1, seasonal, and pandemic H1N1 virus infections.

Various fluorodeoxyribonucleosides were evaluated for their antiviral activities against influenza virus infections in vitro and in vivo. Among the most potent inhibitors was 2'-deoxy-2'-fluorocytidine (2'-FdC). It inhibited various strains of low and highly pathogenic avian influenza H5N1 viruses, pandemic H1N1 viruses, an oseltamivir-resistant pandemic H1N1 virus, and seasonal influenza viruses (H3N2, H1N1, influenza B) in MDCK cells, with the 90% inhibitory concentrations ranging from 0.13 to 4.6 µM, as determined by a virus yield reduction assay. 2'-FdC was then tested for efficacy in BALB/c mice infected with a lethal dose of highly pathogenic influenza A/Vietnam/1203/2004 H5N1 virus. 2'FdC (60 mg/kg/d) administered intraperitoneally (i.p.) twice a day beginning 24 h after virus exposure significantly promoted survival (80% survival) of infected mice (p=0.0001). Equally efficacious were the treatment regimens in which mice were treated with 2'-FdC at 30 or 60 mg/kg/day (bid X 8) beginning 24 h before virus exposure. At these doses, 70-80% of the mice were protected from death due to virus infection (p=0.0005, p=0.0001; respectively). The lungs harvested from treated mice at day four of the infection displayed little surface pathology or histopathology, lung weights were lower, and the 60 mg/kg dose reduced lung virus titers, although not significantly compared to the placebo controls. All doses were well tolerated in uninfected mice. 2'-FdC could also be administered as late as 72 h post virus exposure and still significantly protect 60% mice from the lethal effects of the H5N1 virus infection (p=0.019). Other fluorodeoxyribonucleosides tested in the H5N1 mouse model, 2'-deoxy-5-fluorocytidine and 2'-deoxy-2',2'-difluorocytidine, were very toxic at higher doses and not inhibitory at lower doses. Finally, 2'-FdC, which was active in the H5N1 mouse model, was also active in a pandemic H1N1 influenza A infection model in mice. When given at 30 mg/kg/d (bid X 5) beginning 24h before virus exposure), 2'-FdC also significantly enhanced survival of H1N1-infected mice (50%, p=0.038) similar to the results obtained in the H5N1 infection model using a similar dosing regimen (50%, p<0.05). Given the demonstrated in vitro and in vivo inhibition of avian influenza virus replication, 2'FdC may qualify as a lead compound for the development of agents treating influenza virus infections.