Two Influenza A Virus-Specific Fabs Neutralize by Inhibiting Virus Attachment to Target Cells, While Neutralization by Their IgGs Is Complex and Occurs Simultaneously through Fusion Inhibition and Attachment Inhibition

H9-D3-4R2 (referred to as H9), a murine monoclonal HA1-specific IgG3, recognizes an epitope within antigenic site Cb of influenza virus A/PR/8/34 (H1N1). At 50% neutralization, inhibition of virus-mediated fusion was responsible for the majority of neutralization but, at higher antibody concentrations, the attachment of virus to target cells was also inhibited and may have contributed to neutralization. H9 FAb was also neutralizing, although the concentration needed was two orders of magnitude greater than for the IgG. Functional affinity of the IgG and affinity of the FAb were almost identical, and it is not clear why the neutralization efficiency of the FAb was so low. Unlike its IgG, H9 FAb had no detectable effect on virus attachment but inhibited virus fusion activity. It thus appears that monovalent binding by this antibody is sufficient to inhibit fusion activity and that this was directly responsible for neutralization of infectivity.

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Influenza A Virus Hemagglutinin and Other Pathogen Glycoprotein Interactions with NK Cell Natural Cytotoxicity Receptors NKp46, NKp44, and NKp30

Viruses ◽

10.3390/v13020156 ◽

2021 ◽

Vol 13 (2) ◽

pp. 156

Author(s):

Jasmina M. Luczo ◽

Sydney L. Ronzulli ◽

Stephen M. Tompkins

Keyword(s):

Influenza Virus ◽

Nk Cells ◽

Influenza A Virus ◽

Influenza A ◽

Nk Cell ◽

Influenza Virus Infection ◽

Target Cells ◽

Natural Cytotoxicity ◽

Activating Receptors ◽

Natural Cytotoxicity Receptors

Natural killer (NK) cells are part of the innate immunity repertoire, and function in the recognition and destruction of tumorigenic and pathogen-infected cells. Engagement of NK cell activating receptors can lead to functional activation of NK cells, resulting in lysis of target cells. NK cell activating receptors specific for non-major histocompatibility complex ligands are NKp46, NKp44, NKp30, NKG2D, and CD16 (also known as FcγRIII). The natural cytotoxicity receptors (NCRs), NKp46, NKp44, and NKp30, have been implicated in functional activation of NK cells following influenza virus infection via binding with influenza virus hemagglutinin (HA). In this review we describe NK cell and influenza A virus biology, and the interactions of influenza A virus HA and other pathogen lectins with NK cell natural cytotoxicity receptors (NCRs). We review concepts which intersect viral immunology, traditional virology and glycobiology to provide insights into the interactions between influenza virus HA and the NCRs. Furthermore, we provide expert opinion on future directions that would provide insights into currently unanswered questions.

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Effects of Basic Amino Acids and Their Derivatives on SARS-CoV-2 and Influenza-A Virus Infection

Viruses ◽

10.3390/v13071301 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1301

Author(s):

Ivonne Melano ◽

Li-Lan Kuo ◽

Yan-Chung Lo ◽

Po-Wei Sung ◽

Ni Tien ◽

...

Keyword(s):

Amino Acids ◽

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

Enveloped Viruses ◽

Virus Attachment ◽

Basic Amino Acids ◽

Ester Derivative ◽

Endosomal Acidification

Amino acids have been implicated with virus infection and replication. Here, we demonstrate the effects of two basic amino acids, arginine and lysine, and their ester derivatives on infection of two enveloped viruses, SARS-CoV-2, and influenza A virus. We found that lysine and its ester derivative can efficiently block infection of both viruses in vitro. Furthermore, the arginine ester derivative caused a significant boost in virus infection. Studies on their mechanism of action revealed that the compounds potentially disturb virus uncoating rather than virus attachment and endosomal acidification. Our findings suggest that lysine supplementation and the reduction of arginine-rich food intake can be considered as prophylactic and therapeutic regimens against these viruses while also providing a paradigm for the development of broad-spectrum antivirals.

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Influenza A virus mimetic nanoparticles trigger selective cell uptake

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902563116 ◽

2019 ◽

Vol 116 (20) ◽

pp. 9831-9836 ◽

Cited By ~ 8

Author(s):

Sara Maslanka Figueroa ◽

Anika Veser ◽

Kathrin Abstiens ◽

Daniel Fleischmann ◽

Sebastian Beck ◽

...

Keyword(s):

Influenza A Virus ◽

Target Cell ◽

Influenza A ◽

Mesangial Cells ◽

Cell Uptake ◽

Target Cells ◽

Ang Ii ◽

Material Loss ◽

Cell Identity ◽

Diagnostic Applications

Poor target cell specificity is currently a major shortcoming of nanoparticles (NPs) used for biomedical applications. It causes significant material loss to off-target sites and poor availability at the intended delivery site. To overcome this limitation, we designed particles that identify cells in a virus-like manner. As a blueprint, we chose a mechanism typical of influenza A virus particles in which ectoenzymatic hemagglutinin activation by target cells is a mandatory prerequisite for binding to a secondary target structure that finally confirms cell identity and allows for uptake of the virus. We developed NPs that probe mesangial cells for the presence of angiotensin-converting enzyme on their surface using angiotensin I (Ang-I) as a proligand. This initial interaction enzymatically transforms Ang-I to a secondary ligand angiotensin II (Ang-II) that has the potential to bind in a second stage to Ang-II type-1 receptor (AT1R). The presence of the receptor confirms the target cell identity and triggers NP uptake via endocytosis. Our virus-mimetic NPs showed outstanding target-cell affinity with picomolar avidities and were able to selectively identify these cells in the presence of 90% off-target cells that carried only the AT1R. Our results demonstrate that the design of virus-mimetic cell interactive NPs is a valuable strategy to enhance NP specificity for therapeutic and diagnostic applications. Our set of primary and secondary targets is particularly suited for the identification of mesangial cells that play a pivotal role in diabetic nephropathy, one of the leading causes of renal failure, for which currently no treatment exists.

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Hemagglutinin 1-Specific Immunoglobulin G and Fab Molecules Mediate Postattachment Neutralization of Influenza A Virus by Inhibition of an Early Fusion Event

Journal of Virology ◽

10.1128/jvi.75.21.10208-10218.2001 ◽

2001 ◽

Vol 75 (21) ◽

pp. 10208-10218 ◽

Cited By ~ 31

Author(s):

M. J. Edwards ◽

N. J. Dimmock

Keyword(s):

Immunoglobulin G ◽

Influenza A ◽

Contact Point ◽

Neutralizing Antibody ◽

Cell Contact ◽

Target Cells ◽

Dependent Manner ◽

Fusion Event ◽

Virus Attachment ◽

Early Interaction

ABSTRACT In standard neutralization (STAN), virus and antibody are reacted together before inoculation of target cells, and inhibition of almost any of the processes concerned in the early interaction of virus and cell, including inhibition of virus attachment to cell receptors, can be the cause of neutralization by a particular monoclonal antibody (MAb). To simplify the interpretation of antibody action, we carried out a study of postattachment neutralization (PAN), where virus is allowed to attach to target cells before neutralizing antibody is introduced. We used influenza virus A/PR/8/34 (H1N1) and monoclonal immunoglobulin G (IgG) molecules and their Fabs specific to antigenic sites Sb (tip), Ca2 (loop), and Cb (hinge) of the hemagglutinin 1 (HA1) protein. All IgGs and Fabs gave PAN, although with reduced efficiency compared with STAN. Thus, bivalent binding of antibody was not essential for PAN. By definition, none of these MAbs gave PAN by inhibiting virus attachment, and they did not elute attached virus from the target cell or inhibit endocytosis of virus. However, virus-cell fusion, as demonstrated by R18 fluorescence dequenching or hemolysis of red blood cells, was inhibited in direct proportion to neutralization and in a dose-dependent manner and was thus likely to be responsible for the observed neutralization. However, to get PAN, it was necessary to inhibit the activation of the prefusion intermediate, the earliest known form on the fusion pathway that is created when virus is incubated at pH 5 and 4°C. PAN antibodies may act by binding HA trimers in contact with the cell and/or trimers in the immediate vicinity of the virus-cell contact point and so inhibit the recruitment of additional receptor-HA complexes.

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In Vitro Characterization of Influenza A Virus Attachment in the Upper and Lower Respiratory Tracts of Pigs

Veterinary Pathology ◽

10.1177/0300985812467469 ◽

2012 ◽

Vol 50 (4) ◽

pp. 648-658 ◽

Cited By ~ 8

Author(s):

S. E. Detmer ◽

M. R. Gramer ◽

S. M. Goyal ◽

M. Torremorell

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Virus Attachment ◽

In Vitro Characterization

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Key amino acid residues of neuraminidase involved in influenza A virus entry

Pathogens and Disease ◽

10.1093/femspd/ftz063 ◽

2019 ◽

Vol 77 (6) ◽

Author(s):

Fangzhao Chen ◽

Teng Liu ◽

Jiagui Xu ◽

Yingna Huang ◽

Shuwen Liu ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Influenza A Virus ◽

Viral Entry ◽

Influenza A ◽

Mutational Analysis ◽

Critical Role ◽

Target Cells ◽

Amino Acid Residues ◽

Influenza Virus Neuraminidase

ABSTRACT Generally, influenza virus neuraminidase (NA) plays a critical role in the release stage of influenza virus. Recently, it has been found that NA may promote influenza virus to access the target cells. However, the mechanism remain unclear. Here, we reported that peramivir indeed possessed anti-influenza A virus (IAV) activity in the stage of viral entry. Importantly, we verified the critical residues of influenza NA involved in the viral entry. As a result, peramivir as an efficient NA inhibitor could suppress the initiation of IAV infection. Furthermore, mutational analysis showed NA might be associated with viral entry via amino acids residues R118, E119, D151, R152, W178, I222, E227, E276, R292 and R371. Our results demonstrated NA must contain the key amino acid residues can involve in IAV entry.

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Cross-Reactivity between Hepatitis C Virus and Influenza A Virus Determinant-Specific Cytotoxic T Cells

Journal of Virology ◽

10.1128/jvi.75.23.11392-11400.2001 ◽

2001 ◽

Vol 75 (23) ◽

pp. 11392-11400 ◽

Cited By ~ 170

Author(s):

Heiner Wedemeyer ◽

Eishiro Mizukoshi ◽

Anthony R. Davis ◽

Jack R. Bennink ◽

Barbara Rehermann

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

Influenza A Virus ◽

Influenza A ◽

Blood Donors ◽

Cytotoxic T Cells ◽

Target Cells ◽

Memory Cells

ABSTRACT The cellular immune response contributes to viral clearance as well as to liver injury in acute and chronic hepatitis C virus (HCV) infection. An immunodominant determinant frequently recognized by liver-infiltrating and circulating CD8+ T cells of HCV-infected patients is the HCVNS3-1073 peptide CVNGVCWTV. Using a sensitive in vitro technique with HCV peptides and multiple cytokines, we were able to expand cytotoxic T cells specific for this determinant not only from the blood of 11 of 20 HCV-infected patients (55%) but also from the blood of 9 of 15 HCV-negative blood donors (60%), while a second HCV NS3 determinant was recognized only by HCV-infected patients and not by seronegative controls. The T-cell response of these healthy blood donors was mediated by memory T cells, which cross-reacted with a novel T-cell determinant of the A/PR/8/34 influenza A virus (IV) that is endogenously processed from the neuraminidase (NA) protein. Both the HCV NS3 and the IV NA peptide displayed a high degree of sequence homology, bound to the HLA-A2 molecule with high affinity, and were recognized by cytotoxic T lymphocytes with similar affinity (10−8 M). Using the HLA-A2-transgenic mouse model, we then demonstrated directly that HCV-specific T cells could be induced in vivo by IV infection. Splenocytes harvested from IV-infected mice at the peak of the primary response (day 7 effector cells) or following complete recovery (day 21 memory cells) recognized the HCV NS3 peptide, lysed peptide-pulsed target cells, and produced gamma interferon. These results exemplify that host responses to an infectious agent are influenced by cross-reactive memory cells induced by past exposure to heterologous viruses, which could have important consequences for vaccine development.

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RNA-seq Analysis of Influenza A Virus-Induced Transcriptional Changes in Mice Lung and Its Possible Implications for the Virus Pathogenicity in Mice

Viruses ◽

10.3390/v13102031 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2031

Author(s):

Tianxin Ma ◽

Abdou Nagy ◽

Guanlong Xu ◽

Lingxiang Xin ◽

Danqi Bao ◽

...

Keyword(s):

Cell Cycle ◽

Influenza A Virus ◽

Influenza A ◽

Enrichment Analysis ◽

Alveolar Epithelial Cells ◽

Gene Set Enrichment Analysis ◽

Target Cells ◽

Alveolar Epithelial ◽

Raw264.7 Macrophages ◽

Immune Related Genes

The influenza A virus (IAV) is an important cause of respiratory disease worldwide. It is well known that alveolar epithelial cells are the target cells for the IAV, but there is relatively limited knowledge regarding the role of macrophages during IAV infection. Here, we aimed to analyze transcriptome differences in mouse lungs and macrophage (RAW264.7) cell lines infected with either A/California/04/2009 H1N1 (CA09) or A/chicken/SD/56/2015 H9N2 (SD56) using deep sequencing. The uniquely differentially expressed genes (UDEGs) were analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; the results showed that the lungs infected with the two different viruses had different enrichments of pathways and terms. Interestingly, CA09 virus infection in mice was mostly involved with genes related to the extracellular matrix (ECM), while the most significant differences after SD56 infection in mice were in immune-related genes. Gene set enrichment analysis (GSEA) of RAW264.7 cells revealed that regulation of the cell cycle was of great significance after CA09 infection, whereas the regulation of the immune response was most enriched after SD56 infection, which was consistent with analysis results in the lung. Similar results were obtained from weighted gene co-expression network analysis (WGCNA)，where cell cycle regulation was extensively activated in RAW264.7 macrophages infected with the CA09 virus. Disorder of the cell cycle is likely to affect their normal immune regulation, which may be an important factor leading to their different prognoses. These results provide insight into the mechanism of the CA09 virus that caused a pandemic and explain the different reactivities of monocytes/macrophages infected by H9N2 and H1N1 IAV subtypes.

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Heterosubtypic Antibodies to Influenza A Virus Have Limited Activity against Cell-Bound Virus but Are Not Impaired by Strain-Specific Serum Antibodies

Journal of Virology ◽

10.1128/jvi.03069-14 ◽

2014 ◽

Vol 89 (6) ◽

pp. 3136-3144 ◽

Cited By ~ 15

Author(s):

Arkadiusz Wyrzucki ◽

Matteo Bianchi ◽

Ines Kohler ◽

Marco Steck ◽

Lars Hangartner

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Influenza A Virus ◽

Influenza A ◽

Natural Infection ◽

Influenza Virus Vaccine ◽

Serum Antibodies ◽

Virus Attachment ◽

Endosomal Membrane ◽

Multiple Strains

ABSTRACTThe majority of influenza virus-specific antibodies elicited by vaccination or natural infection are effective only against the eliciting or closely related viruses. Rare stem-specific heterosubtypic monoclonal antibodies (hMAbs) can neutralize multiple strains and subtypes by preventing hemagglutinin (HA)-mediated fusion of the viral membrane with the endosomal membrane. The epitopes recognized by these hMAbs are therefore considered promising targets for the development of pan-influenza virus vaccines. Here, we report the isolation of a novel human HA stem-reactive monoclonal antibody, hMAb 1.12, with exceptionally broad neutralizing activity encompassing viruses from 15 distinct HA subtypes. Using MAb 1.12 and two other monoclonal antibodies, we demonstrate that neutralization by hMAbs is virtually irreversible but becomes severely impaired following virus attachment to cells. In contrast, no interference by human anti-influenza virus serum antibodies was found, indicating that apically binding antibodies do not impair access to the membrane-proximal heterosubtypic epitopes. Our findings therefore encourage development of new vaccine concepts aiming at the induction of stem-specific heterosubtypic antibodies, as we provide support for their effectiveness in individuals previously exposed to influenza virus.IMPORTANCEThe influenza A virus hemagglutinin (HA) can easily accommodate changes in its antigenic structures to escape preexisting immunity. This variability restricts the breadth and long-term efficacy of influenza vaccines. Only a few heterosubtypic antibodies (hMAbs), i.e., antibodies that can neutralize more than one subtype of influenza A virus, have been identified. The molecular interactions between these heterosubtypic antibodies and hemagglutinin are well characterized, yet little is known about the functional properties of these antibodies. Using a new, extraordinarily broad hMAb, we show that virus neutralization by hMAbs is virtually irreversible and that efficient neutralization is possible only if stem-specific hMAbs bind to HA before the virus attaches to the cell surface. No interference between strain-specific human serum immunoglobulin and hMAbs was found, indicating that preexisting humoral immunity to influenza virus does not limit the efficacy of stem-reactive heterosubtypic antibodies. This knowledge supports the development of a pan-influenza virus vaccine.

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