COVID-19, Influenza and RSV: Surveillance-informed prevention and treatment - Meeting report from an isirv-WHO virtual conference.

The International Society for Influenza and other Respiratory Virus Diseases (isirv) and the WHO held a joint virtual conference from 19th-21st October 2021. While there was a major focus on the global response to the SARS-CoV-2 pandemic, including antivirals, vaccines and surveillance strategies, papers were also presented on treatment and prevention of influenza and respiratory syncytial virus (RSV). Potential therapeutics for SARS-CoV-2 included host-targeted therapies baricitinib, a JAK inhibitor, tocilizumab, an IL-6R inhibitor, verdinexor and direct acting antivirals ensovibep, S-217622, AT-527, and monoclonal antibodies casirivimab and imdevimab, directed against the spike protein. Data from trials of nirsevimab, a monoclonal antibody with a prolonged half-life which binds to the RSV F-protein, and an Ad26.RSV pre-F vaccine were also presented. The expanded role of the WHO Global Influenza Surveillance and Response System to address the SARS-CoV-2 pandemic was also discussed. This report summarizes the oral presentations given at this meeting for the benefit of the broader medical and scientific community involved in surveillance, treatment and prevention of respiratory virus diseases.

Influenza polymerase inhibitor resistance: Assessment of the current state of the art - A report of the isirv Antiviral group.

It is more than 20 years since the neuraminidase inhibitors, oseltamivir and zanamivir were approved for the treatment and prevention of influenza. Guidelines for global surveillance and methods for evaluating resistance were established initially by the Neuraminidase Inhibitor Susceptibility Network (NISN), which merged 10 years ago with the International Society for influenza and other Respiratory Virus Diseases (isirv) to become the isirv-Antiviral Group (isirv-AVG). With the ongoing development of new influenza polymerase inhibitors and recent approval of baloxavir marboxil, the isirv-AVG held a closed meeting in August 2019 to discuss the impact of resistance to these inhibitors. Following this meeting and review of the current literature, this article is intended to summarize current knowledge regarding the clinical impact of resistance to polymerase inhibitors and approaches for surveillance and methods for laboratory evaluation of resistance, both in vitro and in animal models. We highlight limitations and gaps in current knowledge and suggest some strategies for addressing these gaps, including the need for additional clinical studies of influenza antiviral drug combinations. Lessons learned from influenza resistance monitoring may also be helpful for establishing future drug susceptibility surveillance and testing for SARS-CoV-2.

Substitutions at H134 and in the 430-loop region in influenza B neuraminidases can confer reduced susceptibility to multiple neuraminidase inhibitors.

With the introduction of the influenza specific neuraminidase inhibitors (NAIs) in 1999, there were concerns about the emergence and spread of resistant viruses in the community setting. Surveillance and testing of community isolates for their susceptibility to the NAIs was initially carried out by the Neuraminidase Inhibitor Susceptibility Network (NISN) and has subsequently been taken on by the global WHO influenza network laboratories. During the NISN surveillance, we identified two Yamagata lineage influenza B viruses with amino acid substitutions of H134Y (B/Auckland/2/2001) or W438R (B/Yokohama/12/2005) which had slightly elevated IC50 values for zanamivir and/or oseltamivir, but not sufficiently to be characterized as mild outliers at the time. As it has now been well demonstrated that mixed populations can mask the true magnitude of resistance of a mutant, we re-examined both of these isolates by plaque purification to see if the true susceptibilities were being masked due to mixed populations. Results confirmed that the B/Auckland isolate contained both wild type and H134Y mutant populations, with mutant IC50 values > 250 nM for both oseltamivir and peramivir in the enzyme inhibition assay. The B/Yokohama isolate also contained both wild type and W438R mutant populations, the latter now demonstrating IC50 values > 400 nM for zanamivir, oseltamivir and peramivir. In addition, plaque purification of the B/Yokohama isolate identified viruses with other single neuraminidase substitutions H134Y, H134R, H431R, or T436P. H134R and H431R viruses had IC50 values > 400 nM and >250 nM respectively against all three NAIs. All changes conferred much greater resistance to peramivir than to zanamivir, and less to oseltamivir, and affected the kinetics of binding and dissociation of the NAIs. Most affected affinity (Km) for the MUNANA substrate, but some had decreased while others had increased affinity. Despite resistance in the enzyme assay, no reduced susceptibility was seen in plaque reduction assays in MDCK cells for any of the mutant viruses. None of these substitutions was in the active site. Modelling suggests that these substitutions affect the 150 and 430-loop regions described for influenza A NAs, suggesting they may also be important for substrate and inhibitor binding for influenza B NAs.

Passaging of an influenza A(H1N1)pdm09 virus in a difluoro sialic acid inhibitor selects for a novel, but unfit I106M neuraminidase mutant.

An influenza A(H1N1)pdm09 and an influenza B virus were passaged in 3-fluoro(eq)-4-guanidino difluoro sialic acid (3Feq4Gu DFSA), an inhibitor of the influenza neuraminidase (NA) to determine whether resistant variants could be selected. 3Feq4Gu DFSA is a mechanism-based inhibitor, forming a covalent link to Y406 in the NA active site. Given its similarity to the natural substrate, sialic acid, we predicted resistant variants would be difficult to select. Yields of both viruses decreased with passaging, so that after 12 passages both viruses were only growing to low titers. Drug concentrations were decreased for another three passages. There was no difference in NA sensitivity in the MUNANA fluorescence-based assay, nor in plaque assays for the passaged virus stocks. All influenza B plaques were still wild type in all assays. There were isolated small diffuse plaques in the P15 pdm09 stock, which after purification had barely detectable NA or hemagglutinin (HA) activity. These had a novel non-active site I106M substitution in the NA gene, but unexpectedly no HA changes. The I106M may impact NA function through steric effects on the movement of the 150 and 430-loops. The I106M viruses had similar replication kinetics in MDCK cells as wild type viruses, but their ability to bind to and infect CHO-K1 cells expressing high levels of cell-bound mucin was compromised. The I106M substitution was unstable, with progeny rapidly reverting to wild type by three different mechanisms. Some had reverted to I106, some had V106, both with wild type NA and HA properties. A third group retained the I106M, but had a compensating R363K substitution, which regained almost wild type NA properties. These viruses now agglutinated chicken red blood cells (CRBCs) but unlike the I/V106, they rebound after elution at 37 °C. There were no mutations in the HA, but each phenotype correlated with the NA sequence. We propose that the activity in the I106M mutant is insufficient to remove carbohydrates from the virion HA and NA, sterically limiting HA access to CRBC receptors, thus resulting in poor HA binding.

Influenza Virus Neuraminidase Structure and Functions.

With the constant threat of emergence of a novel influenza virus pandemic, there must be continued evaluation of the molecular mechanisms that contribute to virulence. Although the influenza A virus surface glycoprotein neuraminidase (NA) has been studied mainly in the context of its role in viral release from cells, accumulating evidence suggests it plays an important, multifunctional role in virus infection and fitness. This review investigates the various structural features of NA, linking these with functional outcomes in viral replication. The contribution of evolving NA activity to viral attachment, entry and release of virions from infected cells, and maintenance of functional balance with the viral hemagglutinin are also discussed. Greater insight into the role of this important antiviral drug target is warranted.

Structure of an Influenza A virus N9 neuraminidase with a tetrabrachion-domain stalk.

The influenza neuraminidase (NA) is a homotetramer with head, stalk, transmembrane and cytoplasmic regions. The structure of the NA head with a stalk has never been determined. The NA head from an N9 subtype influenza A virus, A/tern/Australia/G70C/1975 (H1N9), was expressed with an artificial stalk derived from the tetrabrachion (TB) tetramerization domain from Staphylothermus marinus. The NA was successfully crystallized both with and without the TB stalk, and the structures were determined to 2.6 and 2.3 Šresolution, respectively. Comparisons of the two NAs with the native N9 NA structure from egg-grown virus showed that the artificial TB stalk maintained the native NA head structure, supporting previous biological observations.

Identification of Indonesian clade 2.1 highly pathogenic influenza A(H5N1) viruses with N294S and S246N neuraminidase substitutions which further reduce oseltamivir susceptibility.

We have tested the in vitro susceptibility to the neuraminidase (NA) inhibitors of 96 highly pathogenic clade 2.1 A(H5N1) viruses from Indonesia, isolated between 2008 and 2011. HPAI virus samples obtained through the Influenza Virus Monitoring (IVM) surveillance program in Indonesia were tested for susceptibility to oseltamivir and zanamivir. The NAs of four viruses were identified as extreme outliers to oseltamivir, based on statistical analysis by box plots, with IC50 values ranging from 46 to 62 nM. The NAs of two of these viruses from Sumatra and Aceh, had an N294S substitution, while one virus from Sulawesi had an S246N NA substitution. The NAs of all four viruses showed a specific loss of slow binding to oseltamivir in an IC50 kinetics assay. As observed in our previous surveillance, there was only a minimal effect on the sensitivity to zanamivir or peramivir for these mutants or any of the other isolates tested. The continued circulation of subtype H5N1 viruses in avian species poses an on-going zoonotic threat. The fact that we continue to identify avian isolates with naturally occurring mutations conferring reduced oseltamivir susceptibility remains a concern, given oseltamivir will be a key antiviral in the event of a new pandemic emerging.

Structural and Functional Analysis of Anti-Influenza Activity of 4-, 7-, 8- and 9-Deoxygenated 2,3-Difluoro- N-acetylneuraminic Acid Derivatives.

Competitive inhibitors of the influenza neuraminidase (NA) were discovered almost 20 years ago, with zanamivir and oseltamivir licensed globally. These compounds are based on a transition state analogue of the sialic acid substrate. We recently showed that 5- N-(acetylamino)-2,3,5-trideoxy-2,3-difluoro-d-erythro-ß-l-manno-2-nonulopyranosonic acid (DFSA) and its derivatives are also potent inhibitors of the influenza NA. They are mechanism based inhibitors, forming a covalent bond between the C2 of the sugar ring and Y406 in the NA active site, thus inactivating the enzyme. We have now synthesized a series of deoxygenated DFSA derivatives in order to understand the contribution of each hydroxyl in DFSA to binding and inhibition of the influenza NA. We have investigated their relative efficacy in enzyme assays in vitro, in cell culture, and by X-ray crystallography. We found loss of the 8- and 9-OH had the biggest impact on the affinity of binding and antiviral potency.

Prevention and treatment of respiratory viral infections: Presentations on antivirals, traditional therapies and host-directed interventions at the 5th ISIRV Antiviral Group conference.

The International Society for Influenza and other Respiratory Virus Diseases held its 5th Antiviral Group (isirv-AVG) Conference in Shanghai, China, in conjunction with the Shanghai Public Health Center and Fudan University from 14-16 June 2017. The three-day programme encompassed presentations on some of the clinical features, management, immune responses and virology of respiratory infections, including influenza A(H1N1)pdm09 and A(H7N9) viruses, MERS-CoV, SARS-CoV, adenovirus Type 80, enterovirus D68, metapneumovirus and respiratory syncytial virus (RSV). Updates were presented on several therapeutics currently in clinical trials, including influenza polymerase inhibitors pimodivir/JNJ6362387, S033188, favipiravir, monoclonal antibodies MHAA45449A and VIS410, and host directed strategies for influenza including nitazoxanide, and polymerase ALS-008112 and fusion inhibitors AK0529, GS-5806 for RSV. Updates were also given on the use of the currently licensed neuraminidase inhibitors. Given the location in China, there were also presentations on the use of Traditional Chinese Medicines. Following on from the previous conference, there were ongoing discussions on appropriate endpoints for severe influenza in clinical trials from regulators and clinicians, an issue which remains unresolved. The aim of this conference summary is to provide information for not only conference participants, but a detailed referenced review of the current status of clinical trials, and pre-clinical development of therapeutics and vaccines for influenza and other respiratory diseases for a broader audience.

Meeting report: 4th ISIRV antiviral group conference: Novel antiviral therapies for influenza and other respiratory viruses.

The International Society for Influenza and other Respiratory Virus Diseases (isirv) held its 4th Antiviral Group Conference at the University of Texas on 2-4 June, 2015. With emerging resistance to the drugs currently licensed for treatment and prophylaxis of influenza viruses, primarily the neuraminidase inhibitor oseltamivir phosphate (Tamiflu) and the M2 inhibitors amantadine and rimantadine, and the lack of effective interventions against other respiratory viruses, the 3-day programme focused on the discovery and development of inhibitors of several virus targets and key host cell factors involved in virus replication or mediating the inflammatory response. Virus targets included the influenza haemagglutinin, neuraminidase and M2 proteins, and both the respiratory syncytial virus and influenza polymerases and nucleoproteins. Therapies for rhinoviruses and MERS and SARS coronaviruses were also discussed. With the emerging development of monoclonal antibodies as therapeutics, the potential implications of antibody-dependent enhancement of disease were also addressed. Topics covered all aspects from structural and molecular biology to preclinical and clinical studies. The importance of suitable clinical trial endpoints and regulatory issues were also discussed from the perspectives of both industry and government. This meeting summary provides an overview, not only for the conference participants, but also for those interested in the current status of antivirals for respiratory viruses.

Catalytic mechanism and novel receptor binding sites of human parainfluenza virus type 3 hemagglutinin-neuraminidase (hPIV3 HN).

The human parainfluenza virus type 3 (hPIV3) hemagglutinin-neuraminidase (HN) has opposing functions of binding sialic acid receptors and cleaving them, facilitating virus release. The crystal structure of hPIV3 HN complexed with the substrate analogue difluorosialic acid (DFSA) revealed that catalysis by HN involves the formation of a covalently linked sialosyl-enzyme intermediate which was trapped along with a transition-state analogue resembling an oxocarbenium ion. This mechanism of enzyme catalysis was also confirmed in the crystal structure of the influenza N9 neuraminidase complexed with DFSA. Additionally, novel secondary receptor binding sites were identified in the hPIV3 HN-DFSA complex including one near the catalytic cavity which upon binding DFSA imposes subtle changes and may help the HN balance the opposing functions. Multiple receptor binding sites may increase avidity to facilitate cell binding and fusion promotion. The secondary receptor binding sites in the paramyxoviruses are so far unique to each virus type.

The neuraminidases of MDCK grown human influenza A(H3N2) viruses isolated since 1994 can demonstrate receptor binding.

BACKGROUND: The neuraminidases (NAs) of MDCK passaged human influenza A(H3N2) strains isolated since 2005 are reported to have dual functions of cleavage of sialic acid and receptor binding. NA agglutination of red blood cells (RBCs) can be inhibited by neuraminidase inhibitors (NAIs), thus distinguishing it from haemagglutinin (HA) binding. We wanted to know if viruses prior to 2005 can demonstrate this property. METHODS: Pairs of influenza A(H3N2) isolates ranging from 1993-2008 passaged in parallel only in eggs or in MDCK cells were tested for inhibition of haemagglutination by various NAIs. RESULTS: Only viruses isolated since 1994 and cultured in MDCK cells bound chicken RBCs solely through their NA. NAI inhibition of agglutination of turkey RBCs was seen for some, but not all of these same MDCK grown viruses. Efficacy of inhibition of enzyme activity and haemagglutination differed between NAIs. For many viruses lower concentrations of oseltamivir could inhibit agglutination compared to zanamivir, although they could both inhibit enzyme activity at comparable concentrations. An E119V mutation reduced sensitivity to oseltamivir and 4-aminoDANA for both the enzyme assay and inhibition of agglutination. Sequence analysis of the NAs and HAs of some paired viruses revealed mutations in the haemagglutinin of all egg passaged viruses. For many of the paired egg and MDCK cultured viruses we found no differences in their NA sequences by Sanger sequencing. However, deep sequencing of MDCK grown isolates revealed low levels of variant populations with mutations at either D151 or T148 in the NA, suggesting mutations at either site may be able to confer this property. CONCLUSIONS: The NA active site of MDCK cultured human influenza A(H3N2) viruses isolated since 1994 can express dual enzyme and receptor binding functions. Binding correlated with either D151 or T148 mutations. The catalytic and receptor binding sites do not appear to be structurally identical since relative concentrations of the NAIs to inhibit enzyme activity and agglutination differ.

Neuraminidase mutations conferring resistance to laninamivir lead to faster drug binding and dissociation.

The neuraminidase (NA) inhibitors oseltamivir and zanamivir are administered twice daily for 5days for treatment of influenza. Laninamivir is a 7-methoxy derivative of zanamivir, but a single dose is effective when taken as the laninamivir octanoate prodrug. We show here in IC50 kinetics assays and a solid phase reactivation assay that compared to zanamivir laninamivir also demonstrates slow binding to but slower dissociation from multiple wild type NAs. A D197E mutation in an influenza B and an E119G in an N9 neuraminidase which confer 15- and 150-fold resistance to laninamivir result in faster binding and dissociation. Despite similar IC50s our assays demonstrate more rapid dissociation of laninamivir from clade 1 compared to 2 H5N1 NAs.

Solid phase assay for comparing reactivation rates of neuraminidases of influenza wild type and resistant mutants after inhibitor removal.

The influenza virus neuraminidase inhibitors are normally slow binding inhibitors, but many mutations leading to resistance, also result in the loss of the slow binding phenotype. Mutations can also affect the rate of dissociation of the inhibitors from the neuraminidase, but the assays to measure this require large amounts of virus and are time consuming. To more fully understand the impacts of mutations on the binding and dissociation of the neuraminidase inhibitors we have developed a solid phase reactivation assay, which can use small amounts of crude virus sample bound to an ELISA plate. Multiple viruses can be assayed simultaneously against multiple inhibitors. Using this assay we have demonstrated differences in the relative rates of dissociation of the inhibitors and reactivation of enzyme activity among different influenza A and B viruses for zanamivir, oseltamivir and peramivir. In general oseltamivir dissociated the fastest, and dissociation of peramivir was much slower than both the other inhibitors. Viruses with H274Y, E119V and E119G mutations demonstrated faster dissociation of the inhibitor to which they were resistant. Dissociation of zanamivir and oseltamivir were faster from the D197E mutant, but not of peramivir.

I222 Neuraminidase mutations further reduce oseltamivir susceptibility of Indonesian Clade 2.1 highly pathogenic Avian Influenza A(H5N1) viruses.

We have tested the susceptibility to neuraminidase inhibitors of 155 clade 2.1 H5N1 viruses from Indonesia, isolated between 2006-2008 as well as 12 clade 1 isolates from Thailand and Cambodia from 2004-2007 using a fluorometric MUNANA-based enzyme inhibition assay. The Thailand and Cambodian clade 1 isolates tested here were all susceptible to oseltamivir and zanamivir, and sequence comparison indicated that reduced oseltamivir susceptibility we observed previously with clade 1 Cambodian isolates correlated with an S246G neuraminidase mutation. Eight Indonesian viruses (5%), all bearing I222 neuraminidase mutations, were identified as mild to extreme outliers for oseltamivir based on statistical analysis by box plots. IC50s were from 50 to 500-fold higher than the reference clade 1 virus from Viet Nam, ranging from 43-75 nM for I222T/V mutants and from 268-349 nM for I222M mutants. All eight viruses were from different geographic locales; all I222M variants were from central Sumatra. None of the H5N1 isolates tested demonstrated reduced susceptibility to zanamivir (IC50s all <5 nM). All I222 mutants showed loss of slow binding specifically for oseltamivir in an IC50 kinetics assay. We identified four other Indonesian isolates with higher IC50s which also demonstrated loss of slow binding, including one virus with an I117V mutation. There was a minimal effect on the binding of zanamivir and peramivir for all isolates tested. As H5N1 remains a potential pandemic threat, the incidence of mutations conferring reduced oseltamivir susceptibility is concerning and emphasizes the need for greater surveillance of drug susceptibility.

Reduced susceptibility to all neuraminidase inhibitors of influenza H1N1 viruses with haemagglutinin mutations and mutations in non-conserved residues of the neuraminidase.

OBJECTIVES: We characterized human H1N1 influenza isolate A/Hokkaido/15/02, which has haemagglutinin and neuraminidase mutations that reduce drug susceptibility to oseltamivir, zanamivir and peramivir. METHODS: One wild-type and three mutant viruses were isolated by plaque purification. Viruses were tested in MUNANA-based enzyme assays, cell culture and receptor binding assays. RESULTS: Two viruses had a neuraminidase Y155H mutation that reduced susceptibility in the enzyme inhibition assay to all inhibitors by 30-fold to >100-fold. The Y155H mutation reduced plaque size and affected the stability, Km and pH activity profile of the enzyme. In contrast to previous mutants, this neuraminidase demonstrated a slower rate of inhibitor binding in the IC50 kinetics assay. One virus had both the Y155H mutation and a haemagglutinin D225G mutation that rescued the small-plaque phenotype of the Y155H virus and affected receptor binding and drug susceptibility in cell culture and binding assays. We also isolated a third mutant virus, with both neuraminidase V114I and haemagglutinin D225N mutations, which affected susceptibility in the enzyme inhibition assay and receptor binding, respectively, but to lesser extents than the Y155H and D225G mutations. CONCLUSIONS: Neither Y155 nor V114 is conserved across neuraminidase subtypes. Furthermore, Y155 is not conserved even among avian and swine N1 viruses. Structurally, both residues reside far from the neuraminidase active site. D225 forms part of the receptor binding site of the haemagglutinin. We believe this is the first demonstration of a specific haemagglutinin mutation correlating with reduced drug susceptibility in plaque assays in both Madin Darby Canine Kidney and SIAT cells.

Mechanism-based covalent neuraminidase inhibitors with broad-spectrum influenza antiviral activity.

Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here on a new class of specific, mechanism-based anti-influenza drugs that function through the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and we confirm this mode of action with structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad-spectrum activity against drug-resistant strains in vitro. The similarity of their structure to that of the natural substrate and their mechanism-based design make these attractive antiviral candidates.

Influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance.

There are two major classes of antivirals available for the treatment and prevention of influenza, the M2 inhibitors and the neuraminidase inhibitors (NAIs). The M2 inhibitors are cheap, but they are only effective against influenza A viruses, and resistance arises rapidly. The current influenza A H3N2 and pandemic A(H1N1)pdm09 viruses are already resistant to the M2 inhibitors as are many H5N1 viruses. There are four NAIs licensed in some parts of the world, zanamivir, oseltamivir, peramivir, and a long-acting NAI, laninamivir. This review focuses on resistance to the NAIs. Because of differences in their chemistry and subtle differences in NA structures, resistance can be both NAI- and subtype specific. This results in different drug resistance profiles, for example, the H274Y mutation confers resistance to oseltamivir and peramivir, but not to zanamivir, and only in N1 NAs. Mutations at E119, D198, I222, R292, and N294 can also reduce NAI sensitivity. In the winter of 2007-2008, an oseltamivir-resistant seasonal influenza A(H1N1) strain with an H274Y mutation emerged in the northern hemisphere and spread rapidly around the world. In contrast to earlier evidence of such resistant viruses being unfit, this mutant virus remained fully transmissible and pathogenic and became the major seasonal A(H1N1) virus globally within a year. This resistant A(H1N1) virus was displaced by the sensitive A(H1N1)pdm09 virus. Approximately 0.5-1.0% of community A(H1N1)pdm09 isolates are currently resistant to oseltamivir. It is now apparent that variation in non-active site amino acids can affect the fitness of the enzyme and compensate for mutations that confer high-level oseltamivir resistance resulting in minimal impact on enzyme function.

Taking down the FLAG! How insect cell expression challenges an established tag-system.

In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system.