The types of interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses are not well-characterized due to the low number of co-infection cases described since the onset of the pandemic. We have evaluated the interactions between SARS-CoV-2 (D614G mutant) and influenza A(H1N1)pdm09 or respiratory syncytial virus (RSV) in the nasal human airway epithelium (HAE) infected simultaneously or sequentially (24 h apart) with virus combinations. The replication kinetics of each virus were determined by RT-qPCR at different post-infection times. Our results showed that during simultaneous infection, SARS-CoV-2 interferes with RSV-A2 but not with A(H1N1)pdm09 replication. The prior infection of nasal HAE with SARS-CoV-2 reduces the replication kinetics of both respiratory viruses. SARS-CoV-2 replication is decreased by a prior infection with A(H1N1)pdm09 but not with RSV-A2. The pretreatment of nasal HAE with BX795, a TANK-binding kinase 1 inhibitor, partially alleviates the reduced replication of SARS-CoV-2 or influenza A(H1N1)pdm09 during sequential infection with both virus combinations. Thus, a prior infection of nasal HAE with SARS-CoV-2 interferes with the replication kinetics of A(H1N1)pdm09 and RSV-A2, whereas only A(H1N1)pdm09 reduces the subsequent infection with SARS-CoV-2. The mechanism involved in the viral interference between SARS-CoV-2 and A(H1N1)pdm09 is mediated by the production of interferon.

Immunosuppressed individuals can shed influenza virus for prolonged periods of time, leading to the frequent emergence of antiviral resistance. We evaluated the benefits of oseltamivir and favipiravir combination therapy compared to single antiviral agents and monitored the emergence of drug-resistant variants in a pharmacologically immunosuppressed mouse model infected with the A(H1N1) pandemic influenza virus. C57BL/6 mice were immunosuppressed with cyclophosphamide and infected with a lethal dose of pandemic influenza A(H1N1) virus. Forty-eight hours post-infection, mice were treated with oseltamivir (20 mg/kg), favipiravir (20 or 50 mg/kg) or both agents BID for 5 or 10 days. Body weight losses, survival rates, lung viral titers, cytokine levels and emergence of resistant viruses were evaluated. Treatment of immunosuppressed mice with high (50 mg/kg) but not low (20 mg/kg) doses of favipiravir in combination with oseltamivir (20 mg/kg) significantly delayed mortality and reduced lung viral titers compared to treatment with a single drug regimen with oseltamivir but did not prevent the emergence of oseltamivir-resistant H275Y neuraminidase variants. Combination therapy with oseltamivir and favipiravir should be considered for evaluation in clinical trials.

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen with currently no specific treatment or licensed vaccine. Different strategies to prevent this infection have been evaluated, including live-attenuated vaccines (LAV) based on SH and/or G protein deletions. This approach showed promising outcomes but has not been evaluated further using different viral strains. In that regard, we previously showed that different HMPV strains harbor distinct in vitro fusogenic and in vivo pathogenic phenotypes, possibly influencing the selection of vaccine strains. In this study, we investigated the putative contribution of the low conserved SH or G accessory proteins in such strain-dependent phenotypes and generated recombinant wild type (WT) and SH- or G-deleted viruses derived from two different patient-derived HMPV strains, A1/C-85473 and B2/CAN98-75. The ΔSH and ΔG deletions led to different strain-specific phenotypes in both LLC-MK2 cell and reconstituted human airway epithelium models. More interestingly, the ΔG-85473 and especially ΔSH-C-85473 recombinant viruses conferred significant protection against HMPV challenge and induced immunogenicity against a heterologous strain. In conclusion, our results show that the viral genetic backbone should be considered in the design of live-attenuated HMPV vaccines, and that a SH-deleted virus based on the A1/C-85473 HMPV strain could be a promising LAV candidate as it is both attenuated and protective in mice while being efficiently produced in a cell-based system.

Human metapneumovirus (HMPV) and human respiratory virus (HRSV) are two leading causes of acute respiratory tract infection in young children. While there is no licensed drug against HMPV, the monoclonal antibody (mAb) Palivizumab is approved against HRSV for prophylaxis use only. Novel therapeutics against both viruses are therefore needed. Here, we describe the identification of human mAbs targeting these viruses by using flow cytometry-based cell sorting. One hundred and two antibodies were initially identified from flow cytometry-based cell sorting as binding to the fusion protein from HRSV, HMPV or both. Of those, 95 were successfully produced in plants, purified and characterized for binding activity by ELISA and neutralization assays as well as by inhibition of virus replication in mice. Twenty-two highly reactive mAbs targeting either HRSV or HMPV were isolated. Of these, three mAbs inhibited replication in vivo of a single virus while one mAb could reduce both HRSV and HMPV titers in the lung. Overall, this study identifies several human mAbs with virus-specific therapeutic potential and a unique mAb with inhibitory activities against both HRSV and HMPV.

The prolonged treatment of immunosuppressed (IS) individuals with anti-influenza monotherapies may lead to the emergence of drug-resistant variants. Herein, we evaluated oseltamivir and polymerase inhibitors combinations against influenza A/H3N2 infections in an IS mouse model. Mice were IS with cyclophosphamide and infected with 3 × 10³ PFU of a mouse-adapted A/Switzerland/9715293/2013 (H3N2) virus. Forty-eight hours post-infection, the animals started oseltamivir, favipiravir or baloxavir marboxil (BXM) as single or combined therapies for 10 days. Weight losses, survival rates and lung viral titers (LVTs) were determined. The neuraminidase (NA) and polymerase genes from lung viral samples were sequenced. All untreated animals died. Oseltamivir and favipiravir monotherapies only delayed mortality (the mean day to death (MDD) of 21.4 and 24 compared to 11.4 days for those untreated) while a synergistic improvement in survival (80%) and LVT reduction was observed in the oseltamivir/favipiravir group compared to the oseltamivir group. BXM alone or in double/triple combination provided a complete protection and significantly reduced LVTs. Oseltamivir and BXM monotherapies induced the E119V (NA) and I38T (PA) substitutions, respectively, while no resistance mutation was detected with combinations. We found that the multiple dose regimen of BXM alone provided superior benefits compared to oseltamivir and favipiravir monotherapies. Moreover, we suggest the potential for drug combinations to reduce the incidence of resistance.

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen with currently no specific treatment or licensed vaccine. Different strategies to prevent this infection have been evaluated, including live-attenuated vaccines (LAV) based on SH and/or G protein deletions. This approach showed promising outcomes but has not been evaluated further using different viral strains. In that regard, we previously showed that different HMPV strains harbor distinct in vitro fusogenic and in vivo pathogenic phenotypes, possibly influencing the selection of vaccine strains. In this study, we investigated the putative contribution of the low conserved SH or G accessory proteins in such strain-dependent phenotypes and generated recombinant wild type (WT) and SH- or G-deleted viruses derived from two different patient-derived HMPV strains, A1/C-85473 and B2/CAN98-75. The ΔSH and ΔG deletions led to different strain-specific phenotypes in both LLC-MK2 cell and reconstituted human airway epithelium models. More interestingly, the ΔG-85473 and especially ΔSH-C-85473 recombinant viruses conferred significant protection against HMPV challenge and induced immunogenicity against a heterologous strain. In conclusion, our results show that the viral genetic backbone should be considered in the design of live-attenuated HMPV vaccines, and that a SH-deleted virus based on the A1/C-85473 HMPV strain could be a promising LAV candidate as it is both attenuated and protective in mice while being efficiently produced in a cell-based system.