The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases

ABSTRACTCleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivityin vitro. Recently, we reported that inactivation of a single HA-activating protease gene,Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection ofTmprss2knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion ofTmprss4alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast,Tmprss2−/−Tmprss4−/−double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virusin vivo.IMPORTANCEInfluenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously thatTmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. Here we show that deletion of two host protease genes,Tmprss2andTmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection. Thus, TMPRSS4 represents another host cell factor that is involved in cleavage activation of H3N2 influenza virusesin vivo.

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Inhibition of A/Human/Hubei/3/2005 (H3N2) influenza virus infection by silver nanoparticles in vitro and in vivo [Corrigendum]

International Journal of Nanomedicine ◽

10.2147/ijn.s57432 ◽

2013 ◽

pp. 4703 ◽

Cited By ~ 1

Author(s):

Dongxi Xiang ◽

Cong-long Zheng ◽

Yang Zheng ◽

Xiujing Li ◽

Jianjian Yin ◽

...

Keyword(s):

Silver Nanoparticles ◽

Influenza Virus ◽

Virus Infection ◽

Influenza Virus Infection ◽

H3n2 Influenza ◽

H3n2 Influenza Virus

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Structure of an H3N2 influenza virus nucleoprotein

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x2100635x ◽

2021 ◽

Vol 77 (7) ◽

Author(s):

Michael L. Knight ◽

Haitian Fan ◽

David L. V. Bauer ◽

Jonathan M. Grimes ◽

Ervin Fodor ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Rna Binding ◽

H3n2 Virus ◽

Influenza A Viruses ◽

Seasonal Epidemic ◽

H3n2 Influenza ◽

Structural Levels ◽

Viral Genomic Rna ◽

H3n2 Influenza Virus

Influenza A viruses of the H1N1 and H3N2 subtypes are responsible for seasonal epidemic events. The influenza nucleoprotein (NP) binds to the viral genomic RNA and is essential for its replication. Efforts are under way to produce therapeutics and vaccines targeting the NP. Despite this, no structure of an NP from an H3N2 virus has previously been determined. Here, the structure of the A/Northern Territory/60/1968 (H3N2) influenza virus NP is presented at 2.2 Å resolution. The structure is highly similar to those of the A/WSN/1933 (H1N1) and A/Hong Kong/483/97 (H5N1) NPs. Nonconserved amino acids are widely dispersed both at the sequence and structural levels. A movement of the 73–90 RNA-binding loop is observed to be the key difference between the structure determined here and previous structures. The data presented here increase the understanding of structural conservation amongst influenza NPs and may aid in the design of universal interventions against influenza.

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Immunodominance of Antigenic Site B in the Hemagglutinin of the Current H3N2 Influenza Virus in Humans and Mice

Journal of Virology ◽

10.1128/jvi.01100-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 13

Author(s):

Felix Broecker ◽

Sean T. H. Liu ◽

Weina Sun ◽

Florian Krammer ◽

Viviana Simon ◽

...

Keyword(s):

Influenza Virus ◽

Hemagglutination Inhibition ◽

Neutralizing Antibodies ◽

Antigenic Site ◽

Influenza Viruses ◽

Antigenic Sites ◽

Head Domain ◽

Hemagglutinin Protein ◽

H3n2 Influenza ◽

H3n2 Influenza Virus

ABSTRACTThe hemagglutinin protein of H3N2 influenza viruses is the major target of neutralizing antibodies induced by infection and vaccination. However, the virus frequently escapes antibody-mediated neutralization due to mutations in the globular head domain. Five topologically distinct antigenic sites in the head domain of H3 hemagglutinin, A to E, have been previously described by mapping the binding sites of monoclonal antibodies, yet little is known about the contribution of each site to the immunogenicity of modern H3 hemagglutinins, as measured by hemagglutination inhibition activity, which is known to correlate with protection. To investigate the hierarchy of antibody immunodominance, five Δ1 recombinant influenza viruses expressing hemagglutinin of the A/Hong Kong/4801/2014 (H3N2) strain with mutations in single antigenic sites were generated. Next, the Δ1 viruses were used to determine the hierarchy of immunodominance by measuring the hemagglutination inhibition reactivity of mouse antisera and plasma from 18 human subjects before and after seasonal influenza vaccination in 2017-2018. In both mice and humans, mutations in antigenic site B caused the most significant decrease in hemagglutination inhibition titers compared to wild-type hemagglutinin. This study revealed that antigenic site B is immunodominant in the H3N2 influenza virus strain included in the current vaccine preparations.IMPORTANCEInfluenza viruses rapidly evade humoral immunity through antigenic drift, making current vaccines poorly effective and antibody-mediated protection short-lived. The majority of neutralizing antibodies target five antigenic sites in the head domain of the hemagglutinin protein that are also the most sequence-variable regions. A better understanding of the contribution of each antigenic site to the overall antibody response to hemagglutinin may help in the design of improved influenza virus vaccines.

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Computationally Optimized Broadly Reactive Hemagglutinin Elicits Hemagglutination Inhibition Antibodies against a Panel of H3N2 Influenza Virus Cocirculating Variants

Journal of Virology ◽

10.1128/jvi.01581-17 ◽

2017 ◽

Vol 91 (24) ◽

Cited By ~ 29

Author(s):

Terianne M. Wong ◽

James D. Allen ◽

Anne-Gaelle Bebin-Blackwell ◽

Donald M. Carter ◽

Timothy Alefantis ◽

...

Keyword(s):

Influenza Virus ◽

Influenza Vaccine ◽

Influenza Viruses ◽

Influenza Vaccines ◽

Influenza B ◽

Wild Type ◽

Protective Antibodies ◽

H3n2 Influenza ◽

H3n2 Influenza Virus

ABSTRACT Each influenza season, a set of wild-type viruses, representing one H1N1, one H3N2, and one to two influenza B isolates, are selected for inclusion in the annual seasonal influenza vaccine. In order to develop broadly reactive subtype-specific influenza vaccines, a methodology called computationally optimized broadly reactive antigens (COBRA) was used to design novel hemagglutinin (HA) vaccine immunogens. COBRA technology was effectively used to design HA immunogens that elicited antibodies that neutralized H5N1 and H1N1 isolates. In this report, the development and characterization of 17 prototype H3N2 COBRA HA proteins were screened in mice and ferrets for the elicitation of antibodies with HA inhibition (HAI) activity against human seasonal H3N2 viruses that were isolated over the last 48 years. The most effective COBRA HA vaccine regimens elicited antibodies with broader HAI activity against a panel of H3N2 viruses than wild-type H3 HA vaccines. The top leading COBRA HA candidates were tested against cocirculating variants. These variants were not efficiently detected by antibodies elicited by the wild-type HA from viruses selected as the vaccine candidates. The T-11 COBRA HA vaccine elicited antibodies with HAI and neutralization activity against all cocirculating variants from 2004 to 2007. This is the first report demonstrating broader breadth of vaccine-induced antibodies against cocirculating H3N2 strains compared to the wild-type HA antigens that were represented in commercial influenza vaccines. IMPORTANCE There is a need for an improved influenza vaccine that elicits immune responses that recognize a broader number of influenza virus strains to prevent infection and transmission. Using the COBRA approach, a set of vaccines against influenza viruses in the H3N2 subtype was tested for the ability to elicit antibodies that neutralize virus infection against not only historical vaccine strains of H3N2 but also a set of cocirculating variants that circulated between 2004 and 2007. Three of the H3N2 COBRA vaccines recognized all of the cocirculating strains during this era, but the chosen wild-type vaccine strains were not able to elicit antibodies with HAI activity against these cocirculating strains. Therefore, the COBRA vaccines have the ability to elicit protective antibodies against not only the dominant vaccine strains but also minor circulating strains that can evolve into the dominant vaccine strains in the future.

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Synergistic PA and HA mutations confer mouse adaptation of a contemporary A/H3N2 influenza virus

Scientific Reports ◽

10.1038/s41598-019-51877-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Mariana Baz ◽

Zeineb M’hamdi ◽

Julie Carbonneau ◽

Sophie Lavigne ◽

Christian Couture ◽

...

Keyword(s):

Animal Model ◽

Influenza Virus ◽

Reverse Genetics ◽

Polymerase Activity ◽

Influenza Viruses ◽

H3n2 Virus ◽

Wild Type Virus ◽

Virus Research ◽

H3n2 Influenza

Abstract The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.

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Antibody-dependent enhancement of influenza disease promoted by increase in hemagglutinin stem flexibility and virus fusion kinetics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821317116 ◽

2019 ◽

Vol 116 (30) ◽

pp. 15194-15199 ◽

Cited By ~ 20

Author(s):

Katie L. Winarski ◽

Juanjie Tang ◽

Laura Klenow ◽

Jeehyun Lee ◽

Elizabeth M. Coyle ◽

...

Keyword(s):

Influenza Virus ◽

Residence Time ◽

Endocytic Pathway ◽

Influenza Vaccines ◽

H3n2 Virus ◽

Dependent Manner ◽

Lung Pathology ◽

Next Generation ◽

Virus Fusion

Several next-generation (universal) influenza vaccines and broadly neutralizing antibodies (bNAbs) are in clinical development. Some of these mediate inhibitions of virus replication at the postentry stage or use Fc-dependent mechanisms. Nonneutralizing antibodies have the potential to mediate enhancement of viral infection or disease. In the current study, two monoclonal antibodies (MAbs) 72/8 and 69/1, enhanced respiratory disease (ERD) in mice following H3N2 virus challenge by demonstrating increased lung pathology and changes in lung cytokine/chemokine levels. MAb 78/2 caused changes in the lung viral loads in a dose-dependent manner. Both MAbs increased HA sensitivity to trypsin cleavage at a higher pH range, suggesting MAb-induced conformational changes. pHrodo-labeled virus particles’ entry and residence time in the endocytic compartment were tracked during infection of Madin-Darby canine kidney (MDCK) cells. Both MAbs reduced H3N2 virus residence time in the endocytic pathway, suggesting faster virus fusion kinetics. Structurally, 78/2 and 69/1 Fabs bound the globular head or base of the head domain of influenza hemagglutinin (HA), respectively, and induced destabilization of the HA stem domain. Together, this study describes Mab-induced destabilization of the influenza HA stem domain, faster kinetics of influenza virus fusion, and ERD in vivo. The in vivo animal model and in vitro assays described could augment preclinical safety evaluation of antibodies and next-generation influenza vaccines that generate antibodies which do not block influenza virus–receptor interaction.

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