Virus-Like Particles Containing the Tetrameric Ectodomain of Influenza Matrix Protein 2 and Flagellin Induce Heterosubtypic Protection in Mice

The ectodomain of matrix protein 2 (M2e) is highly conserved among influenza A viruses and can be a promising candidate antigen for a broadly cross-protective vaccine. In this study, a tetrameric M2e (tM2e) and a truncated form of flagellin (tFliC) were coincorporated into virus-like particles (VLPs) to enhance its immunogenicity. Our data showed that the majority of M2e in VLPs was presented as tetramers by introducing a foreign tetramerization motif GCN4. Intranasal immunization with tM2e VLPs significantly enhanced the levels of serum IgG and IgG subclasses compared to soluble M2e (sM2e) in mice. tM2e VLPs also induced higher M2e-specific T-cell and mucosal antibody responses, conferring complete protection against homologous influenza virus infection. The immunogenicity of tM2e VLPs was further enhanced by coincorporation of the membrane-anchored tFliC (tM2e chimeric VLPs) or coadministration with tFliC VLPs as a mixture, but not the soluble flagellin, inducing strong humoral and cellular immune responses conferring cross-protection against lethal challenge with heterotypic influenza viruses. These results support the development of tM2e chimeric VLPs as universal vaccines and warrant further investigation.

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N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus

Viruses ◽

10.3390/v13050815 ◽

2021 ◽

Vol 13 (5) ◽

pp. 815

Author(s):

Cindy M. Spruit ◽

Nikoloz Nemanichvili ◽

Masatoshi Okamatsu ◽

Hiromu Takematsu ◽

Geert-Jan Boons ◽

...

Keyword(s):

Sialic Acid ◽

Animal Models ◽

Influenza A ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Upper Respiratory Tract ◽

Human Influenza ◽

Influenza A Viruses ◽

Sialic Acid Content ◽

Acetylneuraminic Acid

The first step in influenza virus infection is the binding of hemagglutinin to sialic acid-containing glycans present on the cell surface. Over 50 different sialic acid modifications are known, of which N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two main species. Animal models with α2,6 linked Neu5Ac in the upper respiratory tract, similar to humans, are preferred to enable and mimic infection with unadapted human influenza A viruses. Animal models that are currently most often used to study human influenza are mice and ferrets. Additionally, guinea pigs, cotton rats, Syrian hamsters, tree shrews, domestic swine, and non-human primates (macaques and marmosets) are discussed. The presence of NeuGc and the distribution of sialic acid linkages in the most commonly used models is summarized and experimentally determined. We also evaluated the role of Neu5Gc in infection using Neu5Gc binding viruses and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)-/- knockout mice, which lack Neu5Gc and concluded that Neu5Gc is unlikely to be a decoy receptor. This article provides a base for choosing an appropriate animal model. Although mice are one of the most favored models, they are hardly naturally susceptible to infection with human influenza viruses, possibly because they express mainly α2,3 linked sialic acids with both Neu5Ac and Neu5Gc modifications. We suggest using ferrets, which resemble humans closely in the sialic acid content, both in the linkages and the lack of Neu5Gc, lung organization, susceptibility, and disease pathogenesis.

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H5N1 influenza virus-like particles produced by transient expression in mammalian cells induce humoral and cellular immune responses in mice

Canadian Journal of Microbiology ◽

10.1139/w2012-006 ◽

2012 ◽

Vol 58 (4) ◽

pp. 391-401 ◽

Cited By ~ 4

Author(s):

Ling Tao ◽

Jianjun Chen ◽

Zhenhua Zheng ◽

Jin Meng ◽

Zhenfeng Zhang ◽

...

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Cleavage Site ◽

Mammalian Cells ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Virus Like Particles ◽

Cellular Immune Responses ◽

H5n1 Influenza ◽

Cellular Immune

Vaccination is an effective way to protect from influenza virus infection. Among the new candidates of influenza vaccines, influenza virus-like particles (VLPs) seem to be promising. Here, we generated 2 types of H5N1 influenza VLPs by co-expressing influenza virus Env (envelope protein) and murine leukemia virus (MLV) Gag–Pol. VLPs generated by co-transfection of pHCMV-wtH5 or pHCMV-mtH5 with pSV-Mo-MLVgagpol and pHCMV-N1 were named as wtH5N1 VLPs or mtH5N1 VLPs. The plasmid of pHCMV-wtH5 encoded the wild-type hemagglutinin (HA) (wtH5) from A/swine/Anhui/ca/2004 (H5N1) with a multibasic cleavage site, while pHCMV-mtH5 encoded the modified mutant-type (mtH5) with a monobasic cleavage site. Influenza virus HA VLPs were characterized and equal amounts of them were used to immunize mice subcutaneously, intraperitoneally, or intramuscularly. The levels of HA-specific IgG1, IFN-γ, and neutralization antibodies were significantly induced in mice immunized with wtH5N1 VLPs or mtH5N1 VLPs via all 3 routes, while HA-specific IgG2a was barely detectable. IL-4 secretion was detected in mice subcutaneously immunized with wtH5N1 VLPs or mtH5N1 VLPs, or intramuscularly immunized with mtH5N1 VLPs. Our results indicated that both H5N1 influenza VLPs could induce specific humoral and cellular immune responses in immunized mice. In conclusion, our study provides helpful information for designing new candidate vaccines against H5N1 influenza viruses.

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Attenuation and immunogenicity in mice of temperature-sensitive influenza viruses expressing truncated NS1 proteins

Journal of General Virology ◽

10.1099/vir.0.80991-0 ◽

2005 ◽

Vol 86 (10) ◽

pp. 2817-2821 ◽

Cited By ~ 36

Author(s):

Ana M. Falcón ◽

Ana Fernandez-Sesma ◽

Yurie Nakaya ◽

Thomas M. Moran ◽

Juan Ortín ◽

...

Keyword(s):

Influenza A ◽

Influenza Viruses ◽

Wild Type Virus ◽

Temperature Sensitive ◽

Ns1 Protein ◽

Wild Type ◽

Influenza A Viruses ◽

Lethal Challenge

It was previously shown that two mutant influenza A viruses expressing C-terminally truncated forms of the NS1 protein (NS1-81 and NS1-110) were temperature sensitive in vitro. These viruses contain HA, NA and M genes derived from influenza A/WSN/33 H1N1 virus (mouse-adapted), and the remaining five genes from human influenza A/Victoria/3/75 virus. Mice intranasally infected with the NS1 mutant viruses showed undetectable levels of virus in lungs at day 3, whereas those infected with the NS1 wild-type control virus still had detectable levels of virus at this time. Nevertheless, the temperature-sensitive mutant viruses induced specific cellular and humoral immune responses similar to those induced by the wild-type virus. Mice immunized with the NS1 mutant viruses were protected against a lethal challenge with influenza A/WSN/33 virus. These results indicate that truncations in the NS1 protein resulting in temperature-sensitive phenotypes in vitro correlate with attenuation in vivo without compromising viral immunogenicity, an ideal characteristic for live attenuated viral vaccines.

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Influenza A Viruses Control Expression of Proviral Human p53 Isoforms p53β and Δ133p53α

Journal of Virology ◽

10.1128/jvi.07143-11 ◽

2012 ◽

Vol 86 (16) ◽

pp. 8452-8460 ◽

Cited By ~ 22

Author(s):

Olivier Terrier ◽

Virginie Marcel ◽

Gaëlle Cartet ◽

David P. Lane ◽

Bruno Lina ◽

...

Keyword(s):

Virus Infection ◽

Cell Fate ◽

Influenza A ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Lung Epithelial Cells ◽

Dependent Manner ◽

Influenza A Viruses ◽

P53 Isoforms

Previous studies have described the role of p53 isoforms, including p53β and Δ133p53α, in the modulation of the activity of full-length p53, which regulates cell fate. In the context of influenza virus infection, an interplay between influenza viruses and p53 has been described, with p53 being involved in the antiviral response. However, the role of physiological p53 isoforms has never been explored in this context. Here, we demonstrate that p53 isoforms play a role in influenza A virus infection by using silencing and transient expression strategies in human lung epithelial cells. In addition, with the help of a panel of different influenza viruses from different subtypes, we also show that infection differentially regulates the expressions of p53β and Δ133p53α. Altogether, our results highlight the role of p53 isoforms in the viral cycle of influenza A viruses, with p53β and Δ133p53α acting as regulators of viral production in a p53-dependent manner.

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UBIQUITOUS REASSORTMENTS IN INFLUENZA A VIRUSES

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720008003813 ◽

2008 ◽

Vol 06 (05) ◽

pp. 981-999 ◽

Cited By ~ 3

Author(s):

XIU-FENG WAN ◽

MUFIT OZDEN ◽

GUOHUI LIN

Keyword(s):

Influenza A ◽

Matrix Protein ◽

Minimum Spanning Tree ◽

Migratory Birds ◽

Rna Virus ◽

Influenza Pandemic ◽

Influenza Viruses ◽

Influenza A Viruses ◽

H5n1 Avian Influenza Virus ◽

Pandemic Strains

The influenza A virus is a negative-stranded RNA virus composed of eight segmented RNA molecules, including polymerases (PB2, PB1, PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (MP), and nonstructure gene (NS). The influenza A viruses are notorious for rapid mutations, frequent reassortments, and possible recombinations. Among these evolutionary events, reassortments refer to exchanges of discrete RNA segments between co-infected influenza viruses, and they have facilitated the generation of pandemic and epidemic strains. Thus, identification of reassortments will be critical for pandemic and epidemic prevention and control. This paper presents a reassortment identification method based on distance measurement using complete composition vector (CCV) and segment clustering using a minimum spanning tree (MST) algorithm. By applying this method, we identified 34 potential reassortment clusters among 2,641 PB2 segments of influenza A viruses. Among the 83 serotypes tested, at least 56 (67.46%) exchanged their fragments with another serotype of influenza A viruses. These identified reassortments involve 1,957 H2N1 and 1,968 H3N2 influenza pandemic strains as well as H5N1 avian influenza virus isolates, which have generated the potential for a future pandemic threat. More frequent reassortments were found to occur in wild birds, especially migratory birds. This MST clustering program is written in Java and will be available upon request.

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Hemagglutinin Compatibility between Avian and Human Influenza A viruses using Human Matrix Protein: Based on Scholtissek et al.’s (2002) Article

Journal of Student Science and Technology ◽

10.13034/jsst.v8i2.72 ◽

2015 ◽

Vol 8 (2) ◽

Author(s):

Katherine Lien

Keyword(s):

Experimental Model ◽

Influenza A ◽

Matrix Protein ◽

Influenza Viruses ◽

M Protein ◽

Human Influenza ◽

Influenza A Viruses ◽

Large Segment ◽

A Cell ◽

Different Strains

Through the review of Scholtissek et al.(1), evolution between different strains of influenza A viruses were examined to enable better preparation for future pandemics. Pandemics are the result of antigenic shifts, cumulative reassortants between circulating viruses that form novel gene sequences. The process may produce a virus which a large segment of the population has no immunological memory of, and consequently, are susceptible to the strain.The pandemics in 1918, 1967, 1968 and 2009 were caused by influenza A viruses with hemagglutinin (HA) proteins of 1, 2, or 3 - three out of sixteen known HA subtypes. This raises the question whether pandemics can contain other HA subtypes. Since influenza viruses have segmented genomes, it may require at least two different strains to swap their gene segments in order to co-infect a cell; the better viral compatibility between the parent viruses, the more virulent the reassortant is. A collection of HA subtypes in avian strains and Matrix (M) protein in human strains were used in the experimental model by Scholtissek et al. to examine the recombinants’ survivability and virulence. Although the results conclude that it is not possible for future pandemics to contain other HA subtypes, the work of Scholtissek et al. leads to further research on influenza A reservoirs. Ce document est un résumé au sujet de l'article de Christoph Scholtissek (1) publié en 2002. J’examinerai son modèle expérimental, en mettant en évidence les résultats et donnant un aperçu des recherches plus élaborées. En étudiant des modèles de la coopération entre les virus, ceci permet d’aider à se préparer face aux futures pandémies et épidémies. De tels évènements sont causés par des changements antigéniques produits par l’accumulation de réassortiments entre les virus en circulation et divers éléments. Les virus grippaux A sont en constante évolution, et nécessitent une surveillance constante en anticipation à une pandémie. Les pandémies antérieures, soient celles en 1918, 1957, 1968 et 2009, ont démontré à avoir les hémagglutinines (HA) 1, 2 et 3 – trois des seize sous-types HA possibles. Ceci remet en question la possibilité que les pandémies puissent contenir d’autres sous-types HA. Afin que les virus puissent former des virus réassortis potentiellement nouveaux ils doivent bien coopérer, ce qui est précisément ce que Scholtissek tente d'enquêter. Son modèle expérimental implique des réassortiments entre les différents sous-types d’HA dans des souches aviaires et des souches humaines détenant des M-protéines, afin de déterminer la compatibilité virale. Bien que les résultats concluent qu'il est très peu probable que de futures pandémies détiennent d'autres sous-types HA, ils fournissent des indices du potentiel pandémique. En outre, son article incite la recherche plus à fond sur d’autres réservoirs de la grippe A, les méthodes pour surmonter les barrières entre espèces et le réassortiment efficaces.

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Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function

Journal of Virology ◽

10.1128/jvi.01441-18 ◽

2018 ◽

Vol 92 (24) ◽

Cited By ~ 12

Author(s):

Yejin Jang ◽

Jin Soo Shin ◽

Yi-Seul Yoon ◽

Yun Young Go ◽

Hye Won Lee ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Matrix Protein ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Proton Channel ◽

Therapeutic Effects ◽

Monovalent Ions ◽

Endosomal Acidification ◽

Oseltamivir Phosphate

ABSTRACT Screening of chemical libraries with 2,000 synthetic compounds identified salinomycin as a hit against influenza A and B viruses, with 50% effective concentrations ranging from 0.4 to 4.3 μM in cells. This compound is a carboxylic polyether ionophore that exchanges monovalent ions for protons across lipid bilayer membranes. Monitoring the time course of viral infection showed that salinomycin blocked nuclear migration of viral nuclear protein (NP), the most abundant component of the viral ribonucleoprotein (vRNP) complex. It caused cytoplasmic accumulation of NP, particularly within perinuclear endosomes, during virus entry. This was primarily associated with failure to acidify the endosomal-lysosomal compartments. Similar to the case with amantadine (AMT), proton channel activity of viral matrix protein 2 (M2) was blocked by salinomycin. Using purified retroviral Gag-based virus-like particles (VLPs) with M2, it was proved that salinomycin directly affects the kinetics of a proton influx into the particles but in a manner different from that of AMT. Notably, oral administration of salinomycin together with the neuraminidase inhibitor oseltamivir phosphate (OSV-P) led to enhanced antiviral effect over that with either compound used alone in influenza A virus-infected mouse models. These results provide a new paradigm for developing antivirals and their combination therapy that control both host and viral factors.IMPORTANCE Influenza virus is a main cause of viral respiratory infection in humans as well as animals, occasionally with high mortality. Circulation of influenza viruses resistant to the matrix protein 2 (M2) inhibitor, amantadine, is highly prevalent. Moreover, the frequency of detection of viruses resistant to the neuraminidase inhibitors, including oseltamivir phosphate (OSV-P) or zanamivir, is also increasing. These issues highlight the need for discovery of new antiviral agents with different mechanisms. Salinomycin as the monovalent cation-proton antiporter exhibited consistent inhibitory effects against influenza A and B viruses. It plays multifunctional roles by blocking endosomal acidification and by inactivating the proton transport function of M2, the key steps for influenza virus uncoating. Notably, salinomycin resulted in marked therapeutic effects in influenza virus-infected mice when combined with OSV-P, suggesting that its chemical derivatives could be developed as an adjuvant antiviral therapy to treat influenza infections resistant or less sensitive to existing drugs.

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Alterations of membrane curvature during influenza virus budding

Biochemical Society Transactions ◽

10.1042/bst20140136 ◽

2014 ◽

Vol 42 (5) ◽

pp. 1425-1428 ◽

Cited By ~ 8

Author(s):

Agnieszka Martyna ◽

Jeremy Rossman

Keyword(s):

Lipid Raft ◽

Influenza A ◽

Matrix Protein ◽

Membrane Curvature ◽

Influenza Viruses ◽

Apical Plasma Membrane ◽

Lipid Phase ◽

Influenza A Viruses ◽

Enveloped Virus ◽

Virion Release

Influenza A virus belongs to the Orthomyxoviridae family. It is an enveloped virus that contains a segmented and negative-sense RNA genome. Influenza A viruses cause annual epidemics and occasional major pandemics, are a major cause of morbidity and mortality worldwide, and have a significant financial impact on society. Assembly and budding of new viral particles are a complex and multi-step process involving several host and viral factors. Influenza viruses use lipid raft domains in the apical plasma membrane of polarized epithelial cells as sites of budding. Two viral glycoproteins, haemagglutinin and neuraminidase, concentrate in lipid rafts, causing alterations in membrane curvature and initiation of the budding process. Matrix protein 1 (M1), which forms the inner structure of the virion, is then recruited to the site followed by incorporation of the viral ribonucleoproteins and matrix protein 2 (M2). M1 can alter membrane curvature and progress budding, whereas lipid raft-associated M2 stabilizes the site of budding, allowing for proper assembly of the virion. In the later stages of budding, M2 is localized to the neck of the budding virion at the lipid phase boundary, where it causes negative membrane curvature, leading to scission and virion release.

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Efficacy of recombinant NP-M1 and NP-M1-CRT DNA vaccines against Influenza A viruses in mice C57/BL6

10.1101/2021.06.24.449692 ◽

2021 ◽

Author(s):

Hamidreza Attaran ◽

Wen He ◽

wei wang

Keyword(s):

Influenza Virus ◽

Murine Model ◽

Mammalian Cells ◽

Influenza A ◽

Matrix Protein ◽

Protective Efficacy ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Universal Vaccine

Effective vaccination against the influenza virus remains a challenge because of antigenic shift and drift in influenza viruses. Conservation is an important feature of the Nucleoprotein (NP)and Matrix protein 1(M1) qualifying them as potential candidates for developing a universal vaccine against the influenza A virus. Carliticulin (CRT), a member of heat shock protein (HSP) family, are conserved and widely distributed in many microorganisms and mammalian cells. In this study, a plasmid vector encoding the NP-M1-CRT sequence was constructed and compared with the NP-M1 sequence with respect to immunogenicity and protective efficacy in a murine model. The potency of the created construct for provoking humoral, cellular immune responses, and its protective immunity against the lethal influenza virus infection were then compared with commercial split vaccine and then evaluated in a murine model system. NP-M1-CRT as a DNA vaccine combined with in vivo electroporation could significantly improve the immunogenicity of constructed vectors. Serological evaluations demonstrated the potency of our approach to provoke strong anti-NP specific antibody responses. Furthermore, our strategy of immunization in prime-boost groups were able to provide protection against lethal viral challenge using H1N1 subtype. The ease of production of these types of vectors and the fact that they would not require annual updating and manufacturing may provide an alternative cost-effective approach to limit the spread of potential pandemic influenza viruses.

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Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals

Journal of Virology ◽

10.1128/jvi.00742-20 ◽

2020 ◽

Vol 94 (16) ◽

Cited By ~ 1

Author(s):

Allen Zheng ◽

Weina Sun ◽

Xiaoli Xiong ◽

Alec W. Freyn ◽

Julia Peukes ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Influenza Virus Infection ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Virus Infections ◽

Influenza A Viruses ◽

Virus Surface ◽

Virus Challenge ◽

Packaging Signals

ABSTRACT Humoral immune protection against influenza virus infection is mediated largely by antibodies against hemagglutinin (HA) and neuraminidase (NA), the two major glycoproteins on the virus surface. While influenza virus vaccination efforts have focused mainly on HA, NA-based immunity has been shown to reduce disease severity and provide heterologous protection. Current seasonal vaccines do not elicit strong anti-NA responses—in part due to the immunodominance of the HA protein. Here, we demonstrate that by swapping the 5′ and 3′ terminal packaging signals of the HA and NA genomic segments, which contain the RNA promoters, we are able to rescue influenza viruses that express more NA and less HA. Vaccination with formalin-inactivated “rewired” viruses significantly enhances the anti-NA antibody response compared to vaccination with unmodified viruses. Passive transfer of sera from mice immunized with rewired virus vaccines shows better protection against influenza virus challenge. Our results provide evidence that the immunodominance of HA stems in part from its abundance on the viral surface, and that rewiring viral packaging signals—thereby increasing the NA content on viral particles—is a viable strategy for improving the immunogenicity of NA in an influenza virus vaccine. IMPORTANCE Influenza virus infections are a major source of morbidity and mortality worldwide. Increasing evidence highlights neuraminidase as a potential vaccination target. This report demonstrates the efficacy of rewiring influenza virus packaging signals for creating vaccines with more neuraminidase content which provide better neuraminidase (NA)-based protection.

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