Replicative #fitness of seasonal #influenza A viruses with decreased susceptibility to #baloxavir (J Infect Dis., abstract)

[Source: Journal of Infectious Diseases, full page: (LINK). Abstract, edited.]

Replicative fitness of seasonal influenza A viruses with decreased susceptibility to baloxavir

Anton Chesnokov, Mira C Patel, Vasiliy P Mishin, Juan A De La Cruz, Lori Lollis, Ha T Nguyen, Vivien Dugan, David E Wentworth, Larisa V Gubareva

The Journal of Infectious Diseases, jiz472,

Published: 21 September 2019



Susceptibility of influenza A viruses to baloxavir can be affected by changes at amino acid residue 38 in polymerase acidic (PA) protein. Information on replicative fitness of PA-I38-substituted viruses remains sparse. We demonstrated that substitutions I38L/M/S/T not only had a differential effect on baloxavir susceptibility (9- to 116-fold), but also on in vitro replicative fitness. While I38L conferred undiminished growth, other substitutions led to mild attenuation. In a ferret model, control viruses outcompeted those carrying I38M or I38T substitutions, although their advantage was limited. These findings offer insights into the attributes of baloxavir resistant viruses needed for informed risk assessment.

Cap-dependent endonuclease inhibitor, replicative fitness, polymerase acidic protein, influenza, drug resistance, ferret, baloxavir acid, antiviral

Issue Section: Brief Report

This content is only available as a PDF.

Author notes

These authors contributed equally to this article and share first authorship

Published by Oxford University Press for the Infectious Diseases Society of America 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Keywords: Influenza A; Antivirals; Drugs Resistance; Baloxavir.



#Mucosal #CD8+ T cell responses induced by an MCMV based #vaccine #vector confer protection against #influenza challenge (PLoS Pathog., abstract)

[Source: PLoS Pathogens, full page: (LINK). Abstract, edited.]


Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge

Xiaoyan Zheng, Jennifer D. Oduro, Julia D. Boehme, Lisa Borkner, Thomas Ebensen, Ulrike Heise, Marcus Gereke, Marina C. Pils, Astrid Krmpotic, Carlos A. Guzmán, Dunja Bruder, Luka Čičin-Šain

Published: September 16, 2019 / DOI: / This is an uncorrected proof.



Cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.


Author summary

Vaccines against influenza typically induce immune responses based on antibodies, small molecules that recognize the virus particles outside of cells and neutralize them before they infect a cell. However, influenza rapidly evolves, escaping immune recognition, and the fastest evolution is seen in the part of the virus that is recognized by antibodies. Therefore, every year we are confronted with new flu strains that are not recognized by our antibodies against the strains from previous years. The other branch of the immune system is made of killer T cells, which recognize infected cells and target them for killing. Influenza does not rapidly evolve to escape T cell killing; thus, vaccines inducing T-cell responses to influenza might provide long-term protection. We introduced an antigen from influenza into the murine cytomegalovirus (MCMV) and used it as a vaccine vector inducing killer T-cell responses of unparalleled strength. Our vector controls influenza replication and provides relief to infected mice, but only if we administered it through the nose, to activate killer T cells that will persist in the lungs close to the airways. Therefore, our data show that the subset of lung-resident killer T cells is sufficient to protect against influenza.


Citation: Zheng X, Oduro JD, Boehme JD, Borkner L, Ebensen T, Heise U, et al. (2019) Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge. PLoS Pathog 15(9): e1008036.

Editor: Christopher M. Snyder, Thomas Jefferson University, UNITED STATES

Received: July 17, 2019; Accepted: August 21, 2019; Published: September 16, 2019

Copyright: © 2019 Zheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This study was supported by the European Research Council through the ERC Starting Grant 260934 to LCS and the Helmholtz Association through the Helmholtz EU Partnering Grant PIE-008 to LCS. XZ was supported by a scholarship from the Chinese Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Keywords: Influenza A; CMV; Vaccines; Animal models.


#Growth activation of #influenza virus by #trypsin and effect of T-705 (#favipiravir) on trypsin-optimized growth condition (Acta Virol., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Acta Virol. 2019;63(3):309-315. doi: 10.4149/av_2019_311.

Growth activation of influenza virus by trypsin and effect of T-705 (favipiravir) on trypsin-optimized growth condition.

Daikoku T, Okuda T, Kawai M, Morita N, Tanaka T, Takemoto M, Fukuda Y, Takahashi K, Nomura N, Shiraki K.



Influenza virus is activated by proteolytic cleavage of hemagglutinin by trypsin. After determining the optimal trypsin concentration, intracellular and extracellular influenza A/PR/8/34 (H1N1) and A/Victoria/361/2011 (H3N2) virus productions were compared in cultures treated with T-705 (favipiravir) and GS 4071 (an active form of oseltamivir). Although both drugs efficiently inhibited extracellular viral RNA release in a dose-dependent manner, T-705 inhibited it to the level of the inoculum without trypsin treatment, while GS 4071 inhibited it to a final level 10 times higher than that without trypsin. T-705 inhibited intracellular viral RNA production to the level of input virus in both trypsin-treated and untreated cells. In contrast, GS 4071 dose-dependently inhibited intracellular viral RNA production in cells treated with trypsin but allowed viral RNA synthesis. The level of maximum inhibition by GS 4071 was 10 times higher than that of cells without trypsin and 1,000 times greater than the inoculum titer in cells without trypsin. T-705 inhibited both intracellular and extracellular virus production 1,000 and 10 times more strongly, respectively, than GS 4071. T-705 has powerful anti-influenza activity in the absence of trypsin and even in the trypsin-optimized growth condition, suggesting the therapeutic advantage in treatment of influenza complicated with bacterial pneumonia.

Keywords: influenza; T-705; Tamiflu; trypsin; bacterial trypsin-like protease.

PMID: 31507197 DOI: 10.4149/av_2019_311

Keywords: Influenza A; H1N1; H3N2; Antivirals; Favipiravir; Oseltamivir.


Type I and type III #interferons differ in their #adjuvant activities for #influenza #vaccines (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

Type I and type III interferons differ in their adjuvant activities for influenza vaccines

Liang Ye, Annette Ohnemus, Li Ching Ong, Hans Henrik Gad, Rune Hartmann, Nils Lycke, Peter Staeheli

DOI: 10.1128/JVI.01262-19



Type I and type III interferons (IFN) can promote adaptive immune responses in mice and improve vaccine-induced resistance to viral infections. The adjuvant effect of type III IFN (IFN-λ) specifically boosts mucosal immunity by an indirect mechanism, involving IFN-λ-induced production of thymic stromal lymphopoietin (TSLP), a cytokine that activates immune cells. To date it remained unclear whether the previously described adjuvant effect of type I IFN (IFN-α/β) would also depend on TSLP and whether type I IFN stimulates different antibody subtypes. Here we show that after infection with a live attenuated influenza virus, mice lacking functional type I IFN receptors failed to produce normal amounts of virus-specific IgG2c and IgA antibodies. In contrast, mice lacking functional IFN-λ receptors contained normal levels of virus-specific IgG2c but had reduced IgG1 and IgA antibody levels. When applied together with protein antigen, IFN-α stimulated the production of antigen-specific IgA and IgG2c to a greater extent than IgG1, irrespective of whether the mice expressed functional TSLP receptors and irrespective of whether the vaccine was applied by the intranasal or the intraperitoneal route. Taken together, these results demonstrate that the adjuvant activities of type I and type III IFNs are mechanistically distinct.



Interferons can shape antiviral immune responses, but it is not understood well how they influence vaccine efficacy. We find that type I IFN preferentially promotes the production of antigen-specific IgG2c and IgA antibodies after infection with a live attenuated influenza virus or after immunization with influenza subunit vaccines. By contrast, type III IFN specifically enhances influenza virus-specific IgG1 and IgA production. The adjuvant effect of type I IFN was not dependent on TSLP which is essential for the adjuvant effect of type III IFN. Type I IFN boosted vaccine-induced antibody production after immunization by the intranasal or the intraperitoneal route, whereas type III IFN exhibited its adjuvant activity only when the vaccine was delivered by the mucosal route. Our findings demonstrate that type I and type III IFNs trigger distinct pathways to enhance the efficacy of vaccines. This knowledge might be used to design more efficient vaccines against infectious diseases.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Influenza A; Vaccines; Interferons; Animal models.


#Influenza Viruses in #Mice: Deep #Sequencing Analysis of Serial Passage and Effects of #Sialic Acid Structural #Variation (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

Influenza Viruses in Mice: Deep Sequencing Analysis of Serial Passage and Effects of Sialic Acid Structural Variation

Brian R. Wasik, Ian E.H. Voorhees, Karen N. Barnard, Brynn K. Alford-Lawrence, Wendy S. Weichert, Grace Hood, Aitor Nogales, Luis Martínez-Sobrido, Edward C. Holmes, Colin R. Parrish

DOI: 10.1128/JVI.01039-19



Influenza A viruses have regularly jumped to new host species to cause epidemics or pandemics, an evolutionary process that involves variation in the viral traits necessary to overcome host barriers and facilitate transmission. Mice are not a natural host for influenza virus, but are frequently used as models in studies of pathogenesis, often after multiple passages to achieve higher viral titers that result in clinical disease such as weight loss or death. Here we examine the processes of influenza A virus infection and evolution in mice by comparing single nucleotide variation of a human H1N1 pandemic virus, a seasonal H3N2 virus, and a H3N2 canine influenza virus during experimental passage. We also compared replication and sequence variation in wild-type mice expressing N-glycolylneuraminic acid (Neu5Gc) with that seen in mice expressing only N-acetylneuraminic acid (Neu5Ac). Viruses derived from plasmids were propagated in MDCK cells and then passaged in mice up to four times. Full genome deep sequencing of the plasmids, cultured viruses, and viruses from mice at various passages revealed only small numbers of mutational changes. The H3N2 canine influenza virus showed increases in frequency of sporadic mutations in the PB2, PA, and NA segments. The H1N1 pandemic virus grew well in mice, and while it exhibited the maintenance of some minority mutations, there was no clear evidence for adaptive evolution. The H3N2 seasonal virus did not establish in the mice. Finally, there were no clear sequence differences associated with the presence or absence of Neu5Gc.



Mice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals, but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not affect influenza evolution in this model. Overall, our results show that while mice provide a useful animal model for influenza pathology, host passage evolution will vary depending on the specific virus tested.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Influenza A; H1N1pdm09; H3N2; Animal models.


#Circadian control of #lung #inflammation in #influenza #infection (Nat Commun., abstract)

[Source: Nature Communications, full page: (LINK). Abstract, edited.]

Article / Open Access / Published: 11 September 2019

Circadian control of lung inflammation in influenza infection

Shaon Sengupta, Soon Y. Tang, Jill C. Devine, Seán T. Anderson, Soumyashant Nayak, Shirley L. Zhang, Alex Valenzuela, Devin G. Fisher, Gregory R. Grant, Carolina B. López & Garret A. FitzGerald

Nature Communications, volume 10, Article number: 4107 (2019)



Influenza is a leading cause of respiratory mortality and morbidity. While inflammation is essential for fighting infection, a balance of anti-viral defense and host tolerance is necessary for recovery. Circadian rhythms have been shown to modulate inflammation. However, the importance of diurnal variability in the timing of influenza infection is not well understood. Here we demonstrate that endogenous rhythms affect survival in influenza infection. Circadian control of influenza infection is mediated by enhanced inflammation as proven by increased cellularity in bronchoalveolar lavage (BAL), pulmonary transcriptomic profile and histology and is not attributable to viral burden. Better survival is associated with a time dependent preponderance of NK and NKT cells and lower proportion of inflammatory monocytes in the lung. Further, using a series of genetic mouse mutants, we elucidate cellular mechanisms underlying circadian gating of influenza infection.

Keywords: Influenza A; Immunology; Animal models.


#Vaccination With Viral Vectors Expressing Chimeric #Hemagglutinin, NP and M1 #Antigens Protects Ferrets Against #Influenza Virus Challenge (Front Immunol., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Front Immunol. 2019 Aug 21;10:2005. doi: 10.3389/fimmu.2019.02005. eCollection 2019.

Vaccination With Viral Vectors Expressing Chimeric Hemagglutinin, NP and M1 Antigens Protects Ferrets Against Influenza Virus Challenge.

McMahon M1, Asthagiri Arunkumar G1,2, Liu WC1,3, Stadlbauer D1,4, Albrecht RA1,3, Pavot V5, Aramouni M5, Lambe T5, Gilbert SC5, Krammer F1.

Author information: 1 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, United States. 2 Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, United States. 3 Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States. 4 Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria. 5 The Jenner Institute, University of Oxford, Oxford, United Kingdom.



Seasonal influenza viruses cause significant morbidity and mortality in the global population every year. Although seasonal vaccination limits disease, mismatches between the circulating strain and the vaccine strain can severely impair vaccine effectiveness. Because of this, there is an urgent need for a universal vaccine that induces broad protection against drifted seasonal and emerging pandemic influenza viruses. Targeting the conserved stalk region of the influenza virus hemagglutinin (HA), the major glycoprotein on the surface of the virus, results in the production of broadly protective antibody responses. Furthermore, replication deficient viral vectors based on Chimpanzee Adenovirus Oxford 1 (ChAdOx1) and modified vaccinia Ankara (MVA) virus expressing the influenza virus internal antigens, the nucleoprotein (NP) and matrix 1 (M1) protein, can induce strong heterosubtypic influenza virus-specific T cell responses in vaccinated individuals. Here, we combine these two platforms to evaluate the efficacy of a viral vectored vaccination regimen in protecting ferrets from H3N2 influenza virus infection. We observed that viral vectored vaccines expressing both stalk-targeting, chimeric HA constructs, and the NP+M1 fusion protein, in a prime-boost regimen resulted in the production of antibodies toward group 2 HAs, the HA stalk, NP and M1, as well as in induction of influenza virus-specific-IFNγ responses. The immune response induced by this vaccination regime ultimately reduced viral titers in the respiratory tract of influenza virus infected ferrets. Overall, these results improve our understanding of vaccination platforms capable of harnessing both cellular and humoral immunity with the goal of developing a universal influenza virus vaccine.

KEYWORDS: CD8 T-cells; influenza; stalk antibodies; universal influenza virus vaccine; vectored vaccine

PMID: 31497029 PMCID: PMC6712942 DOI: 10.3389/fimmu.2019.02005

Keywords: Influenza A; Vaccines; Animal models.