Vasiliy P. Mishin

Novel pandemic influenza A(H1N1) viruses are potently inhibited by DAS181, a sialidase fusion protein.

Background The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses. Methods and Findings The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus. Conclusions The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.

A Human Monoclonal Antibody with Neutralizing Activity against Highly Divergent Influenza Subtypes

The interest in broad-range anti-influenza A monoclonal antibodies (mAbs) has recently been strengthened by the identification of anti-hemagglutinin (HA) mAbs endowed with heterosubtypic neutralizing activity to be used in the design of “universal” prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies.

Neuraminidase Inhibitor Susceptibility Testing in Human Influenza Viruses: A Laboratory Surveillance Perspective

Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n = 5540) collected during the 2008–2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC50s were performed for each virus type/subtype and NAI to identify outliers which were characterized based on a statistical cutoff of IC50 >3 interquartile ranges (IQR) from the 75th percentile. Among 1533 seasonal H1N1 viruses tested, 1431 (93.3%) were outliers for oseltamivir; they all harbored the H275Y mutation in the neuraminidase (NA) and were reported as oseltamivir-resistant. Only 15 (0.7%) of pandemic 2009 H1N1 viruses tested (n = 2259) were resistant to oseltamivir. All influenza A(H3N2) (n = 834) and B (n = 914) viruses were sensitive to oseltamivir, except for one A(H3N2) and one B virus, with D151V and D197E (D198E in N2 numbering) mutations in the NA, respectively. All viruses tested were sensitive to zanamivir, except for six seasonal A(H1N1) and several A(H3N2) outliers (n = 22) which exhibited cell culture induced mutations at residue D151 of the NA. A subset of viruses (n = 1058) tested for peramivir were sensitive to the drug, with exception of H275Y variants that exhibited reduced susceptibility to this NAI. This study summarizes baseline susceptibility patterns of seasonal and pandemic influenza viruses, and seeks to contribute towards criteria for defining NAI resistance.

Enhanced Genetic Characterization of Influenza A(H3N2) Viruses and Vaccine Effectiveness by Genetic Group, 2014-2015.

BACKGROUND During the 2014-2015 US influenza season, expanded genetic characterization of circulating influenza A(H3N2) viruses was used to assess the impact of the genetic variability of influenza A(H3N2) viruses on influenza vaccine effectiveness (VE). METHODS A novel pyrosequencing assay was used to determine genetic group, based on hemagglutinin (HA) gene sequences, of influenza A(H3N2) viruses from patients enrolled at US Influenza Vaccine Effectiveness Network sites. VE was estimated using a test-negative design comparing vaccination among patients infected with influenza A(H3N2) viruses and uninfected patients. RESULTS Among 9710 enrollees, 1868 (19%) tested positive for influenza A(H3N2) virus; genetic characterization of 1397 viruses showed that 1134 (81%) belonged to 1 HA genetic group (3C.2a) of antigenically drifted influenza A(H3N2) viruses. Effectiveness of 2014-2015 influenza vaccination varied by influenza A(H3N2) virus genetic group from 1% (95% confidence interval [CI], -14% to 14%) against illness caused by antigenically drifted influenza A(H3N2) virus group 3C.2a viruses versus 44% (95% CI, 16%-63%) against illness caused by vaccine-like influenza A(H3N2) virus group 3C.3b viruses. CONCLUSIONS Effectiveness of 2014-2015 influenza vaccination varied by genetic group of influenza A(H3N2) virus. Changes in HA genes related to antigenic drift were associated with reduced VE.

Characterization of Drug-Resistant Influenza A(H7N9) Variants Isolated From an Oseltamivir-Treated Patient in Taiwan

BACKGROUND Patients contracting influenza A(H7N9) infection often developed severe disease causing respiratory failure. Neuraminidase (NA) inhibitors (NAIs) are the primary option for treatment, but information on drug-resistance markers for influenza A(H7N9) is limited. METHODS Four NA variants of A/Taiwan/1/2013(H7N9) virus containing a single substitution (NA-E119V, NA-I222K, NA-I222R, or NA-R292K) recovered from an oseltamivir-treated patient were tested for NAI susceptibility in vitro; their replicative fitness was evaluated in cell culture, mice, and ferrets. RESULTS NA-R292K led to highly reduced inhibition by oseltamivir and peramivir, while NA-E119V, NA-I222K, and NA-I222R caused reduced inhibition by oseltamivir. Mice infected with any virus showed severe clinical signs with high mortality rates. NA-I222K virus was the most virulent in mice, whereas virus lacking NA change (NA-WT) and NA-R292K virus seemed the least virulent. Sequence analysis suggests that PB2-S714N increased virulence of NA-I222K virus in mice; NS1-K126R, alone or in combination with PB2-V227M, produced contrasting effects in NA-WT and NA-R292K viruses. In ferrets, all viruses replicated to high titers in the upper respiratory tract but produced only mild illness. NA-R292K virus, showed reduced replicative fitness in this animal model. CONCLUSIONS Our data highlight challenges in assessment of the replicative fitness of H7N9 NA variants that emerged in NAI-treated patients.

A Non-VH1-69 Heterosubtypic Neutralizing Human Monoclonal Antibody Protects Mice against H1N1 and H5N1 Viruses

Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections.

An Investigational Antiviral Drug, DAS181, Effectively Inhibits Replication of Zoonotic Influenza A Virus Subtype H7N9 and Protects Mice From Lethality

Human infections caused by avian influenza A virus type subtype H7N9 have been associated with substantial morbidity and mortality. Emergence of virus variants carrying markers of decreased susceptibility to neuraminidase inhibitors was reported. Here we show that DAS181 (Fludase), an antiviral drug with sialidase activity, potently inhibited replication of wild-type influenza A(H7N9) and its oseltamivir-resistant R292K variants in mice. A once-daily administration initiated early after lethal infection hampered body weight loss and completely protected mice from lethality. We observed a time-dependent effect for 24–72-hour delayed DAS181 treatments on morbidity and mortality. The results warrant further investigation of DAS181 for influenza treatment.

H1N1pdm Influenza Infection in Hospitalized Cancer Patients: Clinical Evolution and Viral Analysis

Background The novel influenza A pandemic virus (H1N1pdm) caused considerable morbidity and mortality worldwide in 2009. The aim of the present study was to evaluate the clinical course, duration of viral shedding, H1N1pdm evolution and emergence of antiviral resistance in hospitalized cancer patients with severe H1N1pdm infections during the winter of 2009 in Brazil. Methods We performed a prospective single-center cohort study in a cancer center in Rio de Janeiro, Brazil. Hospitalized patients with cancer and a confirmed diagnosis of influenza A H1N1pdm were evaluated. The main outcome measures in this study were in-hospital mortality, duration of viral shedding, viral persistence and both functional and molecular analyses of H1N1pdm susceptibility to oseltamivir. Results A total of 44 hospitalized patients with suspected influenza-like illness were screened. A total of 24 had diagnosed H1N1pdm infections. The overall hospital mortality in our cohort was 21%. Thirteen (54%) patients required intensive care. The median age of the studied cohort was 14.5 years (3–69 years). Eighteen (75%) patients had received chemotherapy in the previous month, and 14 were neutropenic at the onset of influenza. A total of 10 patients were evaluated for their duration of viral shedding, and 5 (50%) displayed prolonged viral shedding (median 23, range = 11–63 days); however, this was not associated with the emergence of a resistant H1N1pdm virus. Viral evolution was observed in sequentially collected samples. Conclusions Prolonged influenza A H1N1pdm shedding was observed in cancer patients. However, oseltamivir resistance was not detected. Taken together, our data suggest that severely ill cancer patients may constitute a pandemic virus reservoir with major implications for viral propagation.

Neuraminidase mutations conferring resistance to oseltamivir in influenza A(H7N9) viruses

ABSTRACT Human infections by avian influenza A(H7N9) virus entail substantial morbidity and mortality. Treatment of infected patients with the neuraminidase (NA) inhibitor oseltamivir was associated with emergence of viruses carrying NA substitutions. In the NA inhibition (NI) assay, R292K conferred highly reduced inhibition by oseltamivir, while E119V and I222K each caused reduced inhibition. To facilitate establishment of laboratory correlates of clinically relevant resistance, experiments were conducted in ferrets infected with virus carrying wild-type or variant NA genes recovered from the A/Taiwan/1/2013 isolate. Oseltamivir treatment (5 or 25 mg/kg of body weight/dose) was given 4 h postinfection, followed by twice-daily treatment for 5 days. Treatment of ferrets infected with wild-type virus resulted in a modest dose-dependent reduction (0.7 to 1.5 log10 50% tissue culture infectious dose [TCID50]) in nasal wash viral titers and inflammation response. Conversely, treatment failed to significantly inhibit the replication of R292K or E119V virus. A small reduction of viral titers was detected on day 5 in ferrets infected with the I222K virus. The propensity for oseltamivir resistance emergence was assessed in oseltamivir-treated animals infected with wild-type virus; emergence of R292K virus was detected in 3 of 6 ferrets within 5 to 7 days postinfection. Collectively, we demonstrate that R292K, E119V, and I222K reduced the inhibitory activity of oseltamivir, not only in the NI assay, but also in infected ferrets, judged particularly by viral loads in nasal washes, and may signal the need for alternative therapeutics. Thus, these clinical outcomes measured in the ferret model may correlate with clinically relevant oseltamivir resistance in humans. IMPORTANCE This report provides more evidence for using the ferret model to assess the susceptibility of influenza A(H7N9) viruses to oseltamivir, the most prescribed anti-influenza virus drug. The information gained can be used to assist in the establishment of laboratory correlates of human disease and drug therapy. The rapid emergence of viruses with R292K in treated ferrets correlates well with the multiple reports on this NA variant in treated human patients. Our findings highlight the importance of the discovery and characterization of new antiviral drugs with different mechanisms of action and the use of combination treatment strategies against emerging viruses with pandemic potential, such as avian H7N9 virus, particularly against those carrying drug resistance markers.

Cell Culture-Selected Substitutions in Influenza A(H3N2) Neuraminidase Affect Drug Susceptibility Assessment

ABSTRACT Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC50) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n = 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.