Since 1977, two subtypes of influenza A viruses, H1N1 and H3N2, along with influenza B viruses, have co-circulated in the human population. These viruses are responsible for seasonal epidemics and may cause global pandemics [1
]. Influenza epidemics can lead to millions of cases of severe respiratory illnesses and to half a million deaths annually worldwide [2
] representing a major public health challenge. Vaccination still remains the first line of prevention against seasonal influenza infections [3
] but the protection conferred by actual vaccines varies from year to year due to the evolution of the circulating strain and the vaccine match [6
]. Antivirals are recommended for the treatment of seasonal influenza viruses, especially in high risk populations (elderly, immunocompromised patients and subjects with co-morbidities) [8
]. Neuraminidase inhibitors (NAIs), such as oseltamivir, zanamivir, peramivir and lanamivir, constitute the main recommended drugs for the treatment of influenza viruses in many countries [9
]. These drugs inhibit the neuraminidase (a glycoprotein that enables the virus to be released from host cell) which reduces clinical illness [9
]. In addition to this major class of antivirals, RNA-dependent RNA polymerase (RdRp) inhibitors are also approved in a few countries to treat influenza A and B viruses. Favipiravir (also known as T-705) is a purine nucleoside analog that is recognized as an alternative substrate by the viral polymerase and is incorporated into the nascent RNA, leading to RNA viral replication errors [13
]. Recently, baloxavir marboxil (BXM; prodrug of baloxavir acid, BXA), a cap-dependent endonuclease inhibitor, has been licensed in a few countries including USA. This compound targets the endonuclease activity of the polymerase acidic (PA) subunit, resulting in the inhibition of RNA transcription and replication [14
]. Not surprisingly, prolonged antiviral therapy may lead to the emergence of drug resistance. Indeed, H275Y, E119G, I427T or I223R (N1 numbering) substitutions, among others in the neuraminidase (NA) protein, induce single or multi-drug resistance [15
]. Rapidly after licensure, the I38T PA substitution was shown to reduce sensitivity to BXM [18
]. On the other hand, the K229R substitution in motif F of the PB1 subunit confers in vitro resistance to favipiravir [22
]. Several in vitro and in vivo studies have shown that dual antiviral therapies may have a significant positive effect on the treatment of influenza infections [13
] and could further delay the emergence of resistance. Indeed, combination therapy with BXM and oseltamivir was reported to produce synergistic responses against the influenza A/PR/8/34 strain and to reduce virus titers [26
]. However, there is a lack of information about combination therapy between this new polymerase inhibitor and other approved drugs. Moreover, the impact of combining two polymerase inhibitors is still unknown. Therefore, we assessed the inhibitory effects of BXA with three NAIs (oseltamivir, zanamivir and peramivir) or two other polymerase inhibitors (favipiravir and ribavirin) in cell cultures and in human airway epithelia (HAE) infected with influenza A(H1N1)pdm09 and A(H3N2) viruses.
2. Materials and Methods
2.1. Cells, Viruses and Compounds
Madin–Darby canine kidney cells over expressing the α2,6 sialic acid receptor (ST6-GalI-MDCK cells) (kindly provided by Y. Kawaoka from the University of Wisconsin, Madison, WI, USA [27
]) were maintained in Minimum Essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), HEPES (Invitrogen, Carlsbad, CA, USA) and 7.5 µg/mL of puromycin.
MucilAirTM reconstituted HAE, issued from primary cells obtained from pools of nasal biopsies, were provided by Epithelix SARL (Geneva, Switzerland) and maintained in air–liquid interphase with specific culture medium in Costar Transwell inserts (Corning, NY, USA), according to the manufacturer’s instructions.
The A/California/7/2009 (H1N1)pdm09, A/Switzerland/9715293/2013 (H3N2) and A/Texas/50/2012 (H3N2) influenza viruses were obtained from NIBSC (code numbers 15/252 and 14/224, respectively) and from the Centre National de Référence des Virus des Infections Respiratoires, Lyon, France Sud.
Baloxavir acid was synthesized at Shionogi & Co., Ltd. (Osaka, Japan) and diluted in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM. Favipiravir (T-705) was purchased from Adooq Bioscience (Irvine, CA, USA) and diluted in DMSO to a stock concentration of 100 mM. Oseltamivir carboxylate (the active form of oseltamivir) was synthesized by Hoffmann-La Roche (Basel, Switzerland) and diluted in sterile water to a stock concentration of 10 mM. Zanamivir and ribavirin were purchased from Sigma (St-Louis, MO, USA) and diluted in sterile water and phosphate-buffered saline (PBS), respectively, to stock concentrations of 10 mM and 100 mM, respectively. Peramivir was synthesized by Biocryst (Durham, NC, USA) and diluted in sterile water to a stock concentration of 10 mM.
2.2. In Vitro Studies of Antiviral Combinations
ST6-GalI-MDCK cells (5000 cells/well) were seeded in 96-well plates and inoculated with 200 Median Tissue Culture Infectious Dose per well (TCID50/well) of each of the influenza strains (A/California/7/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2)). Infected cells were incubated for 60 min at 37 °C in a 5% CO2
atmosphere, followed by the addition of BXA, each NAI, favipiravir and ribavirin in two-fold serial dilutions. For A(H1N1)pdm09 infections, concentrations of 0.0175–10 nmol/L were used for BXA; 2.34–1000 nmol/L for oseltamivir; 2.34–1000 nmol/L for zanamivir; 0.468–400 nmol/L for peramivir; 78.125–80,000 nmol/L for favipiravir; 78.125–40,000 nmol/L for ribavirin, diluted in MEM and TPCK (L-1-Tosylamide-2-phenylethyl chloromethyl ketone) trypsin (1 mg/mL). For A(H3N2) infections, concentrations of 0.39–200 nmol/L were used for BXA; 7.8–4000 nmol/L for oseltamivir; 35.15–16,000 nmol/L for zanamivir; 1.56–800 nmol/L for peramivir; 312.5–160,000 nmol/L for favipiravir; 78.125–40,000 nmol/L for ribavirin, diluted in MEM and TPCK trypsin (1 mg/mL). These dilutions were based on Fukao et al. [26
] and on our preliminary experiments. Forty-eight hours later, cell viability was assessed by adding 10 µL/well of 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) and absorbance was measured at 490 nm with a plate reader. The percentage of cell survival was calculated as described elsewhere [28
]. Then, 50% effective concentration (EC50) values were calculated for each viral strain using Prism software (GraphPad, v7). For two-drug combination studies, the EC50 value of each drug was used to determine an equipotent ratio between the two compounds. The synergism, additivity or antagonism were calculated using the combination index (CI) values, as reported by Chou [30
Briefly, the weighted average CI (CIwt) was calculated for each combination as (CI50 + 2 × CI75 + 3 × CI90 + 4 × CI95)/10 to estimate drug combination effects at high levels of virus inhibition and to increase therapeutic relevance [31
Drug combination effects were defined as CIwt < 0.7, synergism; CIwt > 0.7 and <0.9, moderate synergism; CIwt > 0.9 and <1.2, additivity; CIwt > 1.2 and <1.45, moderate antagonism; CIwt > 1.45, antagonism.
2.3. Evaluation of Antiviral Combinations in HAE
The apical poles of HAE were gently washed twice with warm OptiMEM medium (Gibco, ThermoFisher Scientific, Gaithersburg, MD, USA)) and then infected with a 150 μL dilution of A/California/7/2009 (H1N1)pdm09 or A/Texas/50/2012 (H3N2) in OptiMEM, at a multiplicity of infection (MOI) of 0.1 or 0.01, respectively. For mock infection, the same procedure was followed using OptiMEM as inoculum. After 1 h incubation at 37 °C, the viral inoculum was removed. At 5, 24 and 48 hpi, treatments with specific dilutions of antiviral molecules alone or in combination in MucilAir® culture medium (Epithelix SAS, Saint Julien-en-Genevois, France) were applied through basolateral poles. At 24, 48 (before treatment renewal) and 72 hpi, apical washes with 150 μL OptiMEM were performed and stored in two aliquots at −80 °C for TCID50 viral titration. At 24, 48 and 72 hpi, transepithelial electrical resistance (TEER) between the apical and basal poles of the HAE, considered as a surrogate of epithelium integrity, was measured using a dedicated volt-ohm meter (EVOM2) and compared to baselines values measured before infection (t = 0).
2.4. Statistical Analysis
All experimental assays were performed in triplicate at a minimum, and representative results are shown. HAE viral titers in the drug combination groups were compared against those of both the untreated (*) and most performant single-treated (#) groups using mixed model two-way analysis of variance (ANOVA) with Bonferroni post hoc test. The testing level (α) was 0.05. Statistical analyses were performed on all available data, using GraphPad, Prism 7.
Antivirals play a crucial role in the treatment and control of influenza epidemics and pandemics [33
]. The M2 ion-channel blockers (adamantanes including amantadine and rimantadine) [34
], NAIs (oseltamivir, zanamivir, peramivir or laninamivir) [35
] and the polymerase complex inhibitors (BXM or favipiravir) have demonstrated clinical benefits, but the emergence of viral resistance may limit the clinical efficacy of monotherapies [36
]. Antiviral combinations have been suggested to prevent or delay the emergence of resistance by targeting various steps in the virus replicative process [38
]. Multiple therapies may allow for dosage reduction [33
], in addition to improving therapeutic effect [38
]. Drug combinations already exist to treat cancer [39
] and infectious diseases caused by HIV and hepatitis C [40
]. In a clinical study, the triple combination of oseltamivir, amantadine and ribavirin has been reported to decrease influenza viral shedding compared to monotherapy [24
In this study, we aimed at investigating the potential benefits of combining BXA to most approved antivirals against seasonal influenza A(H1N1) and A(H3N2) strains using both a classic cell line and a physiologically and predictively relevant HAE model.
Firstly, we observed that BXA had the lowest EC50 (Table 1
) against A(H1N1) and A(H3N2) viruses. These effective concentrations of BXA were followed by those of NAIs and other polymerase inhibitors. Of note, EC50 values were higher for A(H3N2) than for A(H1N1). Depending on the antiviral, effective concentrations could differ by 2.5- to 39-fold between strains. Additionally, BXA susceptibilities of influenza viruses have been shown to vary depending on viral strain or subtype [20
]. Therefore, determinations of EC50 value for each drug were the first step in the systematic evaluation of drug combinations by using an equipotent ratio between BXA and other antivirals.
Secondly, our results show that combinations of BXA with NAIs (oseltamivir, peramivir or zanamivir) have higher potency compared to individual drugs, against A(H1N1)pdm09 and A(H3N2) viruses. Such synergistic effects were confirmed in the HAE model, for which the physiological relevance and predictive value have been previously described by our group and others [33
]. These findings are in agreement with those reported by Fukao et al., where in vitro combinations of BXA and approved NAIs also resulted in a synergistic effect against the A/PR/8/34 (H1N1) virus [26
]. In that study, the authors further showed that the combination of suboptimal doses of BXM and oseltamivir enhanced therapeutic effects in mice, including reduced lung viral titers.
Combinations of other polymerase inhibitors with NAIs, as well as combinations of two polymerase inhibitors, have also been studied. Ormond and colleagues demonstrated that the combination of oseltamivir with favipiravir negatively impacted viral replication after several passages [45
]. These results are in accordance with different animal studies where peramivir–favipiravir [46
] or oseltamivir–favipiravir [13
] combinations provided various benefits, including improving survival, controlling drug-resistant A(H1N1) strains or delaying mortality in immunocompromised animals.
In our study, we also assessed two polymerase inhibitors: favipiravir and ribavirin (a transcriptase inhibitor of Polymerase Basic 1—PB1) [47
] in combination with BXA. We obtained synergistic effects for the BXA-favipiravir combination against the two influenza A strains in cell culture, which were further observed in HAE mainly in the case of A(H3N2). In contrast, the BXA-ribavirin combination showed mixed results compared to single-drug treatment, depending on the infection model and viral subtype used. In fact, considering that BXA and ribavirin compounds inhibit two polymerase subunits (PA and PB1, respectively), we suggest that these two agents targeting similar but non-identical functions can interfere or compete in their mechanism of action, resulting in antagonism. Indeed, the PA and PB1 subunits, which interact extensively [49
], could be in close proximity during the transcription/replication process, which may limit their accessibility to the two inhibitors simultaneously. In a modeling drug action experiment, Yin et al. [50
] demonstrated that, when used in combination, drugs interact in many unexpected ways and show a plethora of different outcomes, leading to drug synergy or antagonism. Biological functions involve many molecules that interact in a network manner. Thus, the final effects of drug combinations also depend on the interactions of their targets in a network manner [50
], especially in physiological models, such as HAE, which is constituted by primary differentiated human cells mimicking in vivo epithelia. Indeed, a household study showed that the combination of oseltamivir and zanamivir, administrated within 24 h of onset of symptoms, was more effective in reducing the transmission of influenza compared to monotherapy [51
], whereas we reported that the same combination was not more protective than zanamivir monotherapy against A(H1N1)pdm09 and A(H3N2) infections in mice [52
]. This difference could be explained by the severity of influenza infections (uncomplicated versus severe infections). Moreover, the optimal dose of each component greatly affects drug interactions [53
]. Our combination experiments were based on a fixed ratio calculation. We hypothesize that this method could not apply to the combination of BXA and ribavirin and that a different ratio may lead to another type of drug response. Indeed, different dose combinations may lead to different effects [54
]. Interestingly, in some cases, drugs with antagonist effects could also be beneficial against resistant viral strains [50
Additional in vitro studies using resistant strains (I38T or H275Y variants) should be done to better understand drug activities and to evaluate the impact of combination therapy on mutant viral replication. Additionally, further animal studies should be conducted to evaluate the effectiveness of our synergistic combinations in a complete system.
In summary, our results suggest that BXM stands as a potential good candidate for combination therapy with several existing drugs to improve antiviral activity and eventually delay drug resistance of influenza type A viruses. Combination therapies with BXM should be further considered for animal studies and clinical investigations.