Antiviral agents and therapeutics against respiratory viruses.

INTRODUCTION: Respiratory viruses are responsible for significant worldwide morbidity and mortality. While vaccines are highly effective at reducing the morbidity and mortality associated with viral infections, this protection is incomplete. It requires a high degree of compliance, which is hindered by vaccine hesitancy. To address these gaps, antiviral agents and therapeutics are crucial in combating diseases caused by respiratory viruses. Antiviral agents are broadly classified into two groups: 1) direct-acting antivirals (DAA) and 2) host-directed antivirals (HDA). AREAS COVERED: This review comprehensively examines Phase II FDA-approved antiviral drugs for influenza virus, SARS-CoV-2, and RSV as published in clinicaltrials.gov. It focuses on DAAs and various monoclonal antibodies (mAbs) that have been approved for the prevention and treatment of viral respiratory tract infections. EXPERT OPINION: Antiviral drugs being developed assess different mechanisms of action to combat viruses and other delivery routes (i.e. oral, inhalation, or parenteral). The associated clinical trials address the impact on disease while determining the appropriate dosage levels for further investigation in Phase III. A robust pipeline of agents is necessary to meet the global need for effective antiviral therapeutics.

Probenecid Inhibits Human Metapneumovirus (HMPV) Replication In Vitro and in BALB/c Mice.

Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infection and causes significant morbidity and mortality. There is no specific antiviral drug to treat HMPV or vaccine to prevent HMPV. This study determined if probenecid, a host-targeting antiviral drug, had prophylactic (pre-virus) or therapeutic (post-virus) efficacy to inhibit HMPV replication in LLC-MK2 cells in vitro and in the lungs of BALB/c mice. This study showed that ≥0.5 µM probenecid significantly inhibited HMPV replication in vitro, and 2-200 mg/kg probenecid prophylaxis or treatment reduced HMPV replication in BALB/c mice.

Rapid Detection of SARS-CoV-2 Variants Using an Angiotensin-Converting Enzyme 2-Based Surface-Enhanced Raman Spectroscopy Sensor Enhanced by CoVari Deep Learning Algorithms.

An integrated approach combining surface-enhanced Raman spectroscopy (SERS) with a specialized deep learning algorithm to rapidly and accurately detect and quantify SARS-CoV-2 variants is developed based on an angiotensin-converting enzyme 2 (ACE2)-functionalized AgNR@SiO2 array SERS sensor. SERS spectra with concentrations of different variants were collected using a portable Raman system. After appropriate spectral preprocessing, a deep learning algorithm, CoVari, is developed to predict both the viral variant species and concentrations. Using a 10-fold cross-validation strategy, the model achieves an average accuracy of 99.9% in discriminating between different virus variants and R2 values larger than 0.98 for quantifying viral concentrations of the three viruses, demonstrating the high quality of the detection. The limit of detection of the ACE2 SERS sensor is determined to be 10.472, 11.882, and 21.591 PFU/mL for SARS-CoV-2, SARS-CoV-2 B1, and CoV-NL63, respectively. The feature importance of virus classification and concentration regression in the CoVari algorithm are calculated based on a permutation algorithm, which showed a clear correlation to the biochemical origins of the spectra or spectral changes. In an unknown specimen test, classification accuracy can achieve >90% for concentrations larger than 781 PFU/mL, and the predicted concentrations consistently align with actual values, highlighting the robustness of the proposed algorithm. Based on the CoVari architecture and the output vector, this algorithm can be generalized to predict both viral variant species and concentrations simultaneously for a broader range of viruses. These results demonstrate that the SERS + CoVari strategy has the potential for rapid and quantitative detection of virus variants and potentially point-of-care diagnostic platforms.

A pan-respiratory antiviral chemotype targeting a transient host multi-protein complex.

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

mRNA vaccines encoding influenza virus hemagglutinin (HA) elicits immunity in mice from influenza A virus challenge.

Influenza viruses cause epidemics and can cause pandemics with substantial morbidity with some mortality every year. Seasonal influenza vaccines have incomplete effectiveness and elicit a narrow antibody response that often does not protect against mutations occurring in influenza viruses. Thus, various vaccine approaches have been investigated to improve safety and efficacy. Here, we evaluate an mRNA influenza vaccine encoding hemagglutinin (HA) proteins in a BALB/c mouse model. The results show that mRNA vaccination elicits neutralizing and serum antibodies to each influenza virus strain contained in the current quadrivalent vaccine that is designed to protect against four different influenza viruses including two influenza A viruses (IAV) and two influenza B (IBV), as well as several antigenically distinct influenza virus strains in both hemagglutination inhibition assay (HAI) and virus neutralization assays. The quadrivalent mRNA vaccines had antibody titers comparable to the antibodies elicited by the monovalent vaccines to each tested virus regardless of dosage following an mRNA booster vaccine. Mice vaccinated with mRNA encoding an H1 HA had decreased weight loss and decreased lung viral titers compared to mice not vaccinated with an mRNA encoding an H1 HA. Overall, this study demonstrates the efficacy of mRNA-based seasonal influenza vaccines are their potential to replace both the currently available split-inactivated, and live-attenuated seasonal influenza vaccines.

Probenecid Inhibits Influenza A(H5N1) and A(H7N9) Viruses In Vitro and in Mice.

Avian influenza (AI) viruses cause infection in birds and humans. Several H5N1 and H7N9 variants are highly pathogenic avian influenza (HPAI) viruses. H5N1 is a highly infectious bird virus infecting primarily poultry, but unlike other AIs, H5N1 also infects mammals and transmits to humans with a case fatality rate above 40%. Similarly, H7N9 can infect humans, with a case fatality rate of over 40%. Since 1996, there have been several HPAI outbreaks affecting humans, emphasizing the need for safe and effective antivirals. We show that probenecid potently inhibits H5N1 and H7N9 replication in prophylactically or therapeutically treated A549 cells and normal human broncho-epithelial (NHBE) cells, and H5N1 replication in VeroE6 cells and mice.

Antiviral Activity of Probenecid and Oseltamivir on Influenza Virus Replication.

Influenza can cause respiratory infections, leading to significant morbidity and mortality in humans. While current influenza vaccines offer varying levels of protection, there remains a pressing need for effective antiviral drugs to supplement vaccine efforts. Currently, the FDA-approved antiviral drugs for influenza include oseltamivir, zanamivir, peramivir, and baloxavir marboxil. These antivirals primarily target the virus, making them vulnerable to drug resistance. In this study, we evaluated the efficacy of the neuraminidase inhibitor, oseltamivir, against probenecid, which targets the host cells and is less likely to engender resistance. Our results show that probenecid has superior antiviral efficacy compared to oseltamivir in both in vitro replication assays and in vivo mouse models of influenza infection.

Screening Drugs for Broad-Spectrum, Host-Directed Antiviral Activity: Lessons from the Development of Probenecid for COVID-19.

In the early stages of drug discovery, researchers develop assays that are compatible with high throughput screening (HTS) and structure activity relationship (SAR) measurements. These assays are designed to evaluate the effectiveness of new and known molecular entities, typically targeting specific features within the virus. Drugs that inhibit virus replication by inhibiting a host gene or pathway are often missed because the goal is to identify active antiviral agents against known viral targets. Screening efforts should be sufficiently robust to identify all potential targets regardless of the antiviral mechanism to avoid misleading conclusions.

Understanding immunity to influenza: implications for future vaccine development.

INTRODUCTION: Influenza virus changes its genotype through antigenic drift or shift making it difficult to develop immunity to infection or vaccination. Zoonotic influenza A virus (IAV) strains can become established in humans. Several impediments to human infection and transmission include sialic acid expression, host anti-viral factors (including interferons), and other elements that govern viral replication. Controlling influenza infection, replication, and transmission is important because IAVs cause annual epidemics and occasional pandemics. Effective seasonal influenza vaccines exist, but these vaccines do not fully protect against novel or pandemic strains. AREAS COVERED: With new vaccine production technology, vaccines can be produced rapidly. Universal IAV vaccines are being developed to protect against seasonal, novel, and zoonotic IAVs. These efforts are being enhanced and accelerated by a better understanding the host immune response to influenza viruses. EXPERT OPINION: This review discusses several implications for future influenza vaccine development. Host immune responses to influenza virus infection or vaccination can guide vaccine development as anti-influenza immunity is affected by responses influenced by the previous immune history including first and subsequent exposures to influenza virus infections and vaccinations.

Interferons-Implications in the Immune Response to Respiratory Viruses.

Interferons (IFN) are an assemblage of signaling proteins made and released by various host cells in response to stimuli, including viruses. Respiratory syncytial virus (RSV), influenza virus, and SARS-CoV-2 are major causes of respiratory disease that induce or antagonize IFN responses depending on various factors. In this review, the role and function of type I, II, and III IFN responses to respiratory virus infections are considered. In addition, the role of the viral proteins in modifying anti-viral immunity is noted, as are the specific IFN responses that underly the correlates of immunity and protection from disease.

Immunogenicity and protective efficacy of an RSV G S177Q central conserved domain nanoparticle vaccine.

Introduction: Respiratory syncytial virus (RSV) can cause lower respiratory tract disease in infants and elderly populations. Despite decades of research, there remains no safe and approved RSV vaccine. Previously, we showed that an RSV G glycoprotein subunit vaccine candidate with a single point mutation within the central conserved domain (CCD), i.e. S177Q, considerably improved immunogenicity. Methods: Here, we examine the development of nanoparticle (NP) vaccines having either an RSV G protein CCD with wild-type sequence (NPWT) or an S177Q mutation (NP-S177Q). The NP vaccine immunogens were adjuvanted with monophosphoryl lipid A (MPLA), a TLR4 agonist to improve Th1- type responses. BALB/c mice were primed with 10 µg of NP-WT vaccine, NPS177Q, or vehicle, rested, and then boosted with a high (25 µg) or low (10 µg) dose of the NP-WT or NP-S177Q homologous candidate and subsequently challenged with RSV A2. Results: The results showed that mice boosted with NP-S177Q developed superior immunogenicity and neutralizing antibodies compared to NP-WT boosting. IgG from either NP-S177Q or NP-WT vaccinated mice did not interfere with fractalkine (CX3CL1) binding to CX3CR1 and effectively blocked G protein CX3C-CX3CR1 binding. Both NP-WT and NP-S177Q vaccination induced similar neutralizing antibodies to RSV in challenged mice compared to vehicle control. NP-S177Q boosting improved correlates of protection including reduced BAL cell infiltration following RSV challenge. However, the NP vaccine platform will require improvement due to the poor solubility and the unexpectedly weaker Th1-type IgG2a response. Discussion: The results from this study support further NP-S177Q vaccine candidate development.

Oral Probenecid for Nonhospitalized Adults with Symptomatic Mild-to-Moderate COVID-19.

Probenecid is an orally bioavailable, uricosuric agent that was first approved in 1951 for the treatment of gout, but was later found to have potent, broad-spectrum antiviral activity against several respiratory viruses including SARS-CoV-2. We conducted a phase 2 randomized, placebo-controlled, single-blind, dose-range finding study in non-hospitalized patients with symptomatic, mild-to-moderate COVID-19. Patients were randomly assigned in a 1:1:1 ratio to receive either 500 mg of probenecid, 1000 mg of probenecid, or a matching placebo every 12 h for five days. The patients' COVID-19 viral load hospitalization, or death from any cause through day 28, as well as safety, were evaluated. COVID-19-related symptoms were assessed at baseline, and on days 3, 5, 10, 15, and 28. The primary endpoints of the study were time to first negative SARS-CoV-2 viral test (or viral clearance) and the proportion of patients that were symptom-free at day 5. A total of 75 patients were randomized, with 25 patients in each group. All of the patients completed the study as planned with no hospitalizations or deaths being reported. The median time to viral clearance was significantly shorter for the probenecid 1000 mg group than for placebo (7 days vs. 11 days, respectively; p < 0.0001), and for the probenecid 500 mg group versus placebo (9 days vs. 11 days, respectively; p < 0.0001). In addition, the median time to viral clearance was significantly shorter for the probenecid 1000 mg group than for the probenecid 500 mg group (7 days vs. 9 days, respectively; p < 0.0001). All patients reported at least one COVID-19-related symptom on days 3 and 5; however, on day 10, a significantly greater proportion of patients receiving probenecid 1000 mg reported the complete resolution of symptoms versus placebo (68% vs. 20%, respectively; p = 0.0006), as well as for those receiving probenecid 500 mg versus placebo (56% vs. 20%, respectively, p = 0.0087). The incidence of adverse events during treatment was similar across all groups for any adverse event, and was 12%. All events were mild with no serious adverse events reported and no discontinuations due to an adverse event. The treatment of patients with symptomatic, mild-to-moderate COVID-19 with probenecid resulted in a significant, dose-dependent decrease in the time to viral clearance and a significantly higher proportion of patients reporting complete symptom resolution by day 10. (Supported by TrippBio; ClinicalTrials.gov number, NCT05442983 and Clinical Trials Registry India number CTRI/2022/07/043726).

Immune Prophylaxis Targeting the Respiratory Syncytial Virus (RSV) G Protein.

The respiratory syncytial virus (RSV) causes significant respiratory disease in young infants and the elderly. Immune prophylaxis in infants is currently limited to palivizumab, an anti-RSV fusion (F) protein monoclonal antibody (mAb). While anti-F protein mAbs neutralize RSV, they are unable to prevent aberrant pathogenic responses provoked by the RSV attachment (G) protein. Recently, the co-crystal structures of two high-affinity anti-G protein mAbs that bind the central conserved domain (CCD) at distinct non-overlapping epitopes were solved. mAbs 3D3 and 2D10 are broadly neutralizing and block G protein CX3C-mediated chemotaxis by binding antigenic sites γ1 and γ2, respectively, which is known to reduce RSV disease. Previous studies have established 3D3 as a potential immunoprophylactic and therapeutic; however, there has been no similar evaluation of 2D10 available. Here, we sought to determine the differences in neutralization and immunity to RSV Line19F infection which recapitulates human RSV infection in mouse models making it useful for therapeutic antibody studies. Prophylactic (24 h prior to infection) or therapeutic (72 h post-infection) treatment of mice with 3D3, 2D10, or palivizumab were compared to isotype control antibody treatment. The results show that 2D10 can neutralize RSV Line19F both prophylactically and therapeutically, and can reduce disease-causing immune responses in a prophylactic but not therapeutic context. In contrast, 3D3 was able to significantly (p < 0.05) reduce lung virus titers and IL-13 in a prophylactic and therapeutic regimen suggesting subtle but important differences in immune responses to RSV infection with mAbs that bind distinct epitopes.

Anti-G protein antibodies targeting the RSV G protein CX3C chemokine region improve the interferon response.

Background: Respiratory syncytial virus (RSV) is a poor inducer of antiviral interferon (IFN) responses which result in incomplete immunity and RSV disease. Several RSV proteins alter antiviral responses, including the non-structural proteins (NS1, NS2) and the major viral surface proteins, that is, fusion (F) and attachment (G) proteins. The G protein modifies the host immune response to infection linked in part through a CX3 C chemokine motif. Anti-G protein monoclonal antibodies (mAbs), that is, clones 3D3 and 2D10 that target the G protein CX3C chemokine motif can neutralize RSV and inhibit G protein-CX3CR1 mediated chemotaxis. Objectives: Determine how monoclonal antibodies against the RSV F and G proteins modify the type I and III IFN responses to RSV infection. Design: As the G protein CX3 C motif is implicated in IFN antagonism, we evaluated two mAbs that block G protein CX3C-CX3CR1 interaction and compared responses to isotype mAb control using a functional cellular assay and mouse model. Methods: Mouse lung epithelial cells (MLE-15 cells) and BALB/c mice were infected with RSV Line19 F following prophylactic mAb treatment. Cell supernatant or bronchoalveolar lavage fluid (BALF) were assayed for types I and III IFNs. Cells were interrogated for changes in IFN-related gene expression. Results: Treatment with an anti-G protein mAb (3D3) resulted in improved IFN responses compared with isotype control following infection with RSV, partially independently of neutralization, and this was linked to upregulated SOCS1 expression. Conclusions: These findings show that anti-G protein antibodies improve the protective early antiviral response, which has important implications for vaccine and therapeutic design. Plain Language Summary: RSV is a leading cause of respiratory disease in infants and the elderly. The only Food and Drug Administration-approved prophylactic treatment is limited to an anti-F protein monoclonal antibody (mAb), that is, palivizumab which has modest efficacy against RSV disease. Accumulating evidence suggests that targeting the RSV attachment (G) protein may provide improved protection from RSV disease. It is known that the G protein is an IFN antagonist, and IFN has been shown to be protective against RSV disease. In this study, we compared IFN responses in mouse lung epithelial (MLE-15) cells and in mice infected with RSV Line19 F treated with anti-G protein or anti-F protein mAbs. The levels of type I and III IFNs were determined. Anti-G protein mAbs improved the levels of IFNs compared with isotype-treated controls. These findings support the concept that anti-G protein mAbs mediate improved IFN responses against RSV disease, which may enable improved treatment of RSV infections.

Rapid Detection of SARS-CoV-2 RNA in Human Nasopharyngeal Specimens Using Surface-Enhanced Raman Spectroscopy and Deep Learning Algorithms.

A rapid and cost-effective method to detect the infection of SARS-CoV-2 is fundamental to mitigating the current COVID-19 pandemic. Herein, a surface-enhanced Raman spectroscopy (SERS) sensor with a deep learning algorithm has been developed for the rapid detection of SARS-CoV-2 RNA in human nasopharyngeal swab (HNS) specimens. The SERS sensor was prepared using a silver nanorod array (AgNR) substrate by assembling DNA probes to capture SARS-CoV-2 RNA. The SERS spectra of HNS specimens were collected after RNA hybridization, and the corresponding SERS peaks were identified. The RNA detection range was determined to be 103-109 copies/mL in saline sodium citrate buffer. A recurrent neural network (RNN)-based deep learning model was developed to classify 40 positive and 120 negative specimens with an overall accuracy of 98.9%. For the blind test of 72 specimens, the RNN model gave a 97.2% accuracy prediction for positive specimens and a 100% accuracy for negative specimens. All the detections were performed in 25 min. These results suggest that the DNA-functionalized AgNR array SERS sensor combined with a deep learning algorithm could serve as a potential rapid point-of-care COVID-19 diagnostic platform.

Microparticle RSV Vaccines Presenting the G Protein CX3C Chemokine Motif in the Context of TLR Signaling Induce Protective Th1 Immune Responses and Prevent Pulmonary Eosinophilia Post-Challenge.

Layer-by-layer microparticle (LbL-MP) fabrication was used to produce synthetic vaccines presenting a fusion peptide containing RSV G protein CX3C chemokine motif and a CD8 epitope of the RSV matrix protein 2 (GM2) with or without a covalently linked TLR2 agonist (Pam3.GM2). Immunization of BALB/c mice with either GM2 or Pam3.GM2 LbL-MP in the absence of adjuvant elicited G-specific antibody responses and M2-specific CD8+ T-cell responses. Following challenge with RSV, mice immunized with the GM2 LbL-MP vaccine developed a Th2-biased immune response in the lungs with elevated levels of IL-4, IL-5, IL-13, and eotaxin in the bronchoalveolar lavage (BAL) fluid and a pulmonary influx of eosinophils. By comparison, mice immunized with the Pam3.GM2 LbL-MP vaccine had considerably lower to non-detectable levels of the Th2 cytokines and chemokines and very low numbers of eosinophils in the BAL fluid post-RSV challenge. In addition, mice immunized with the Pam3.GM2 LbL-MP also had higher levels of RSV G-specific IgG2a and IgG2b in the post-challenge BAL fluid compared to those immunized with the GM2 LbL-MP vaccine. While both candidates protected mice from infection following challenge, as evidenced by the reduction or elimination of RSV plaques, the inclusion of the TLR2 agonist yielded a more potent antibody response, greater protection, and a clear shift away from Th2/eosinophil responses. Since the failure of formalin-inactivated RSV (FI-RSV) vaccines tested in the 1960s has been hypothesized to be partly due to the ablation of host TLR engagement by the vaccine and inappropriate Th2 responses upon subsequent viral infection, these findings stress the importance of appropriate engagement of the innate immune response during initial exposure to RSV G CX3C.

RSV Replication, Transmission, and Disease Are Influenced by the RSV G Protein.

It is important to understand the features affecting virus replication, fitness, and transmissibility as they contribute to the outcome of infection and affect disease intervention approaches. Respiratory syncytial virus (RSV) is a major contributor to respiratory disease, particularly in the infant and elderly populations. Although first described over 60 years ago, there are no approved vaccines and there are limited specific antiviral treatments due in part to our incomplete understanding of the features affecting RSV replication, immunity, and disease. RSV studies have typically focused on using continuous cell lines and conventional RSV strains to establish vaccine development and various antiviral countermeasures. This review outlines how the RSV G protein influences viral features, including replication, transmission, and disease, and how understanding the role of the G protein can improve the understanding of preclinical studies.

Influenza B Virus (IBV) Immune-Mediated Disease in C57BL/6 Mice.

Influenza B viruses (IBV) primarily infect humans, causing seasonal epidemics. The absence of an animal reservoir limits pandemic concern, but IBV infections may cause severe respiratory disease, predominantly in young children and the elderly. The IBV disease burden is largely controlled by seasonal influenza vaccination; however, immunity due to vaccination is sometimes incomplete, a feature linked to antigenic mismatches. Thus, understanding the features that contribute to disease pathogenesis is important, particularly immune-mediated versus virus-mediated outcomes. Unexpectedly, C57BL/6 (B6) mice intranasally infected with a low multiplicity of infection of B/Florida/04/2006 developed substantial morbidity and mortality. To address the cause, B6 mice were treated daily with dexamethasone to dampen the immune and pro-inflammatory response to IBV infection, allowing the determination of whether the responses were immune- and/or virus-associated. As expected, dexamethasone (DEX)-treated mice had a lower pro-inflammatory response and reduced lung pathology despite the presence of high viral lung titers, but mortality was comparable to PBS-treated mice, indicating that mortality may be linked to lung virus replication. The results showed that the immune response to IBV is the major cause of morbidity, mortality, lung pathology, and viral clearance. Importantly, the results suggest that a robust lung CTL response and associated leukocyte influx contribute to disease.

Rapid and quantitative detection of respiratory viruses using surface-enhanced Raman spectroscopy and machine learning.

Rapid and sensitive pathogen detection is important for prevention and control of disease. Here, we report a label-free diagnostic platform that combines surface-enhanced Raman scattering (SERS) and machine learning for the rapid and accurate detection of thirteen respiratory virus species including SARS-CoV-2, common human coronaviruses, influenza viruses, and others. Virus detection and measurement have been performed using highly sensitive SiO2 coated silver nanorod array substrates, allowing for detection and identification of their characteristic SERS peaks. Using appropriate spectral processing procedures and machine learning algorithms (MLAs) including support vector machine (SVM), k-nearest neighbor, and random forest, the virus species as well as strains and variants have been differentiated and classified and a differentiation accuracy of >99% has been obtained. Utilizing SVM-based regression, quantitative calibration curves have been constructed to accurately estimate the unknown virus concentrations in buffer and saliva. This study shows that using a combination of SERS, MLA, and regression, it is possible to classify and quantify the virus in saliva, which could aid medical diagnosis and therapeutic intervention.

The Hypothiocyanite and Amantadine Combination Treatment Prevents Lethal Influenza A Virus Infection in Mice.

The influenza virus has a large clinical burden and is associated with significant mortality and morbidity. The development of effective drugs for the treatment or prevention of influenza is important in order to reduce its impact. Adamantanes and neuraminidase inhibitors are two classes of anti-influenza drugs in which resistance has developed; thus, there is an urgent need to explore new therapeutic options. Boosting antiviral innate immune mechanisms in the airways represents an attractive approach. Hypothiocyanite (OSCN-) is produced by the airway epithelium and is effective in reducing the replication of several influenza A virus strains in vitro. It remains, however, largely unexplored whether OSCN- has such an antiviral effect in vivo. Here we determined the therapeutic potential of OSCN-, alone or in combination with amantadine (AMT), in preventing lethal influenza A virus replication in mice and in vitro. Mice intranasally infected with a lethal dose of A/Puerto Rico/8/1934 (H1N1) or A/Hong Kong/8/1968 (H3N2) were cured by the combination treatment of OSCN- and AMT. Monotherapy with OSCN- or AMT alone did not substantially improve survival outcomes. However, AMT+OSCN- treatment significantly inhibited viral replication, and in vitro treatment inhibited viral entry and nuclear transport of different influenza A virus strains (H1N1 and H3N2) including the AMT-resistant strain A/WSN/33 (H1N1). A triple combination treatment consisting of AMT, oseltamivir, and OSCN- was also tested and further inhibited in vitro viral replication of the AMT-resistant A/WSN/33 strain. These results suggest that OSCN- is a promising anti-influenza treatment option when combined with other antiviral drugs.