Preclinical and clinical developments for combination treatment of influenza.

Antiviral drugs are an important measure of control for influenza in the population, particularly for those that are severely ill or hospitalised. The neuraminidase inhibitor (NAI) class of drugs, including oseltamivir, have been the standard of care (SOC) for severe influenza illness for many years. The approval of drugs with novel mechanisms of action, such as baloxavir marboxil, is important and broadens potential treatment options for combination therapy. The use of antiviral treatments in combination for influenza is of interest; one potential benefit of this treatment strategy is that the combination of drugs with different mechanisms of action may lower the selection of resistance due to treatment. In addition, combination therapy may become an important treatment option to improve patient outcomes in those with severe illness due to influenza or those that are immunocompromised. Clinical trials increasingly evaluate drug combinations in a range of patient cohorts. Here, we summarise preclinical and clinical advances in combination therapy for the treatment of influenza with reference to immunocompromised animal models and clinical data in hospitalised patient cohorts where available. There is a wide array of drug categories in development that have also been tested in combination. Therefore, in this review, we have included polymerase inhibitors, monoclonal antibodies (mAbs), host-targeted therapies, and adjunctive therapies. Combination treatment regimens should be carefully evaluated to determine whether they provide an added benefit relative to effectiveness of monotherapy and in a variety of patient cohorts, particularly, if there is a greater chance of an adverse outcome. Safe and effective treatment of influenza is important not only for seasonal influenza infection, but also if a pandemic strain was to emerge.

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model.

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.

Baloxavir treatment of ferrets infected with influenza A(H1N1)pdm09 virus reduces onward transmission.

Influenza viruses cause seasonal outbreaks and pose a continuous pandemic threat. Although vaccines are available for influenza control, their efficacy varies each season and a vaccine for a novel pandemic virus manufactured using current technology will not be available fast enough to mitigate the effect of the first pandemic wave. Antivirals can be effective against many different influenza viruses but have not thus far been used extensively for outbreak control. Baloxavir, a recently licensed antiviral drug that targets the influenza virus endonuclease, has been shown to reduce virus shedding more effectively than oseltamivir, a widely used neuraminidase inhibitor drug. Thus it is possible that treatment with baloxavir might also interrupt onward virus transmission. To test this, we utilized the ferret model, which is the most commonly used animal model to study influenza virus transmission. We established a subcutaneous baloxavir administration method in ferrets which achieved similar pharmacokinetics to the approved human oral dose. Transmission studies were then conducted in two different locations with different experimental setups to compare the onward transmission of A(H1N1)pdm09 virus from infected ferrets treated with baloxavir, oseltamivir or placebo to naïve sentinel ferrets exposed either indirectly in adjacent cages or directly by co-housing. We found that baloxavir treatment reduced infectious viral shedding in the upper respiratory tract of ferrets compared to placebo, and reduced the frequency of transmission amongst sentinels in both experimental setups, even when treatment was delayed until 2 days post-infection. In contrast, oseltamivir treatment did not substantially affect viral shedding or transmission compared to placebo. We did not detect the emergence of baloxavir-resistant variants in treated animals or in untreated sentinels. Our results support the concept that antivirals which decrease viral shedding could also reduce influenza transmission in the community.

Pre-Clinical Evaluation of the Antiviral Activity of Epigalocatechin-3-Gallate, a Component of Green Tea, against Influenza A(H1N1)pdm Viruses.

Influenza antiviral drugs are important tools in our fight against both annual influenza epidemics and pandemics. Polyphenols are a group of compounds found in plants, some of which have demonstrated promising antiviral activity. Previous in vitro and mouse studies have outlined the anti-influenza virus effectiveness of the polyphenol epigallocatechin-3-gallate (EGCG); however, no study has utilised the ferret model, which is considered the gold-standard for influenza antiviral studies. This study aimed to explore the antiviral efficacy of EGCG in vitro and in ferrets. We first performed studies in Madin-Darby Canine Kidney (MDCK) and human lung carcinoma (Calu-3) cells, which demonstrated antiviral activity. In MDCK cells, we observed a selective index (SI, CC50/IC50) of 77 (290 µM/3.8 µM) and 96 (290 µM/3.0 µM) against A/California/07/2009 and A/Victoria/2570/2019 (H1N1)pdm09 influenza virus, respectively. Calu-3 cells demonstrated a SI of 16 (420 µM/26 µM) and 18 (420 µM/24 µM). Ferrets infected with A/California/07/2009 influenza virus and treated with EGCG (500 mg/kg/day for 4 days) had no change in respiratory tissue viral titres, in contrast to oseltamivir treatment, which significantly reduced viral load in the lungs of treated animals. Therefore, we demonstrated that although EGCG showed antiviral activity in vitro against influenza viruses, the drug failed to impair viral replication in the respiratory tract of ferrets.

Effect of Baloxavir and Oseltamivir in Combination on Infection with Influenza Viruses with PA/I38T or PA/E23K Substitutions in the Ferret Model.

Amino acid substitutions I38T and E23K in the influenza polymerase acidic (PA) protein lead to reduced susceptibility to the influenza antiviral drug baloxavir. The in vivo effectiveness of baloxavir and oseltamivir for treatment of these viruses is currently unknown. Using patient-derived influenza isolates, combination therapy was equally effective as monotherapy in reducing viral titers in the upper respiratory tract of ferrets infected with A(H1N1pdm09)-PA/E23K or A(H3N2)-PA/I38T. When treated with baloxavir plus oseltamivir, infection with a mixture of PA/I38T or PA/E23K and corresponding wild-type virus was characterized by a lower selection of viruses with reduced baloxavir susceptibility over the course of infection compared to baloxavir monotherapy. De novo emergence of the oseltamivir resistance mutation NA/H275Y occurred in ferrets treated with oseltamivir alone but not in ferrets treated with baloxavir plus oseltamivir. Our data suggest that combination therapy with influenza drugs with different mechanisms of action decreased the selection pressure for viruses with reduced drug susceptibility. IMPORTANCE Influenza viruses cause significant morbidity and mortality worldwide but can be treated with antiviral drugs. In 2018, a highly effective antiviral drug, baloxavir marboxil, was licensed. However, the selection of viruses with baloxavir resistance was relatively high following treatment, which may compromise the effectiveness of the drug. Here, we took two different influenza viruses that are resistant to baloxavir and tested the effectiveness alone and in combination with oseltamivir (a second influenza antiviral drug) in the ferret model. Our findings suggest that combination treatment may be a more effective method than monotherapy to reduce the selection of resistant viruses. These results may have important clinical implications for the treatment of influenza.

Assessing the fitness of a dual-antiviral drug resistant human influenza virus in the ferret model.

Influenza antivirals are important tools in our fight against annual influenza epidemics and future influenza pandemics. Combinations of antivirals may reduce the likelihood of drug resistance and improve clinical outcomes. Previously, two hospitalised immunocompromised influenza patients, who received a combination of a neuraminidase inhibitor and baloxavir marboxil, shed influenza viruses resistant to both drugs. Here-in, the replicative fitness of one of these A(H1N1)pdm09 virus isolates with dual resistance mutations (NA-H275Y and PA-I38T) was similar to wild type virus (WT) in vitro, but reduced in the upper respiratory tracts of challenged ferrets. The dual-mutant virus transmitted well between ferrets in an airborne transmission model, but was outcompeted by the WT when the two viruses were co-administered. These results indicate the dual-mutant virus had a moderate loss of viral fitness compared to the WT virus, suggesting that while person-to-person transmission of the dual-resistant virus may be possible, widespread community transmission is unlikely.

A rapid pyrosequencing assay for the molecular detection of influenza viruses with reduced baloxavir susceptibility due to PA/I38X amino acid substitutions.

Baloxavir marboxil is a novel endonuclease inhibitor licensed for treatment of otherwise healthy or high-risk individuals infected with influenza. Viruses with reduced baloxavir susceptibility due to amino acid substitutions at residue 38 of the PA have been detected in some individuals following treatment. Here, we describe a genotypic pyrosequencing method that can be used to rapidly screen circulating influenza A and B viruses for substitutions in the PA/I38 codon and to quantify mixed viral populations. This method is suitable for surveillance of baloxavir susceptibility and to analyse samples from hospitalised patients undergoing baloxavir treatment to aid in clinical decision making.

Host-targeted nitazoxanide has a high barrier to resistance but does not reduce the emergence or proliferation of oseltamivir-resistant influenza viruses in vitro or in vivo when used in combination with oseltamivir.

A major limitation of the currently available influenza antivirals is the potential development of drug resistance. The adamantanes, neuraminidase inhibitors, and more recently polymerase inhibitors, have all been associated with the emergence of viral resistance in preclinical, clinical studies or in clinical use. As a result, host-targeted drugs that act on cellular proteins or functions have become an attractive option for influenza treatment as they are less likely to select for resistance. Nitazoxanide (NTZ) is a host-targeted antiviral that is currently in Phase III clinical trials for the treatment of influenza. In this study, we investigated the propensity for circulating influenza viruses to develop resistance to nitazoxanide in vitro by serially passaging viruses under selective pressure. Phenotypic and genotypic analysis of viruses passaged ten times in the presence of up to 20 µM tizoxanide (TIZ; the active metabolite of nitazoxanide) showed that none had a significant change in TIZ susceptibility, and amino acid substitutions arising that were unique to TIZ passaged viruses, did not alter TIZ susceptibility. Combination therapy, particularly utilising drugs with different mechanisms of action, is another option for combatting antiviral resistance, and while combination therapy has been shown to improve antiviral effects, the effect of reducing the emergence and selection of drug-resistant virus has been less widely investigated. Here we examined the use of TIZ in combination with oseltamivir, both in vitro and using the ferret model for influenza infection and found that the combination of the two drugs did not provide significant benefit in reducing the emergence or selection of oseltamivir-resistant virus. These in vitro findings suggest that clinical use of NTZ may be significantly less likely to select for resistance in circulating influenza viruses compared to virus-targeted antivirals, and although the combination of NTZ with oseltamivir did not reduce the emergence of oseltamivir-resistant virus in vitro or in vivo, combination therapy with NTZ and other newer classes of influenza antiviral drugs should be considered due to NTZ's higher host-based barrier to resistance.

Baloxavir marboxil susceptibility of influenza viruses from the Asia-Pacific, 2012-2018.

Baloxavir Marboxil (BXM) is an influenza polymerase inhibitor antiviral that binds to the endonuclease region in the PA subunit of influenza A and B viruses. To establish the baseline susceptibility of viruses circulating prior to licensure of BXM and to monitor for susceptibility post-BXM use, a cell culture-based focus reduction assay was developed to determine the susceptibility of 286 circulating seasonal influenza viruses, A(H1N1)pdm09, A(H3N2), B (Yamagata/Victoria) lineage viruses, including neuraminidase inhibitor (NAI) resistant viruses, to Baloxavir Acid (BXA), the active metabolic form of BXM. BXA was effective against all influenza subtypes tested with mean EC50 values (minimum-maximum) of 0.7 ±â€¯0.5 nM (0.1-2.1 nM), 1.2 ±â€¯0.6 nM (0.1-2.4), 7.2 ±â€¯3.5 nM (0.7-14.8), and 5.8 ±â€¯4.5 nM (1.8-15.5) obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses, respectively. Using reverse genetics, amino acid substitutions known to alter BXA susceptibility were introduced into the PA protein resulting in EC50 fold change increases that ranged from 2 to 65. Our study demonstrates that currently circulating viruses are susceptible to BXA and that the newly developed focus reduction assay is well suited to susceptibility monitoring in reference laboratories.

Influenza antivirals currently in late-phase clinical trial.

Influenza antiviral drugs are important for the control of influenza, most specifically for the treatment of influenza patients with severe disease following infection with a seasonal influenza virus, a newly emerging influenza strain, or in the event of a pandemic. Many influenza antivirals that are currently under investigation in late-stage clinical trials differ in their mechanism of action compared to drugs currently licensed for the treatment of influenza. Nitazoxanide and DAS181 target components of the host cell and alter the ability of the virus to replicate efficiently, while small molecule drugs such as T705, JNJ63623872 and S-033188 bind to the viral polymerase complex and restrict viral replication. Monoclonal antibodies that are currently in clinical trial for the treatment of influenza most commonly are targeted to the stem region of the haemagglutinin molecule. Early findings from animal models and in vitro studies suggest that many of the new antiviral drugs when tested in combination with oseltamivir have improved effectiveness over monotherapy. Clinical trials assessing both monotherapy and combination therapy are currently under investigation. It is hoped that as new antivirals are licensed, they will improve the standard of care and outcomes for influenza patients with severe disease.