Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Virology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Vaccines and Antiviral Agents | Spotlight

Hemagglutinin Stalk-Reactive Antibodies Interfere with Influenza Virus Neuraminidase Activity by Steric Hindrance

Yao-Qing Chen, Linda Yu-Ling Lan, Min Huang, Carole Henry, Patrick C. Wilson
Stacey Schultz-Cherry, Editor
Yao-Qing Chen
aDepartment of Medicine, Section of Rheumatology, Knapp Center for Lupus and Immunology, University of Chicago, Chicago, Illinois, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Linda Yu-Ling Lan
bCommittee on Immunology, University of Chicago, Chicago, Illinois, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Min Huang
aDepartment of Medicine, Section of Rheumatology, Knapp Center for Lupus and Immunology, University of Chicago, Chicago, Illinois, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Carole Henry
aDepartment of Medicine, Section of Rheumatology, Knapp Center for Lupus and Immunology, University of Chicago, Chicago, Illinois, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Patrick C. Wilson
aDepartment of Medicine, Section of Rheumatology, Knapp Center for Lupus and Immunology, University of Chicago, Chicago, Illinois, USA
bCommittee on Immunology, University of Chicago, Chicago, Illinois, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Stacey Schultz-Cherry
St. Jude Children's Research Hospital
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JVI.01526-18
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current “one-shot” universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA stalk-reactive antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) was evident for an attached substrate but not for unattached small-molecule cleavage of sialic acid. This finding suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(ab′)2 fragments that occupied reduced area without loss of avidity or disrupted HA/NA interactions showed significantly reduced NI activity. Notably, HA stalk-binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of protection mediated by HA stalk-reactive antibodies.

IMPORTANCE This study reports a new mechanism of protection mediated by influenza hemagglutinin stalk-reactive antibodies, i.e., inhibition of neuraminidase activity by steric hindrance, blocking access of neuraminidase to sialic acids when it abuts hemagglutinin on whole virions.

INTRODUCTION

Influenza is an acute respiratory illness that causes epidemics and pandemics in the human population, causing up to 640,000 deaths annually worldwide (1). The influenza virus particle contains two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA is a trimer protein that contains a globular head and a stalk domain. The head domain mediates binding to host cellular receptor sialic acids, while the stalk domain fuses the virus and host cell membranes to allow the introduction of the influenza virus genes. The NA protein is essential for cleavage of terminal sialic acid residues present on host glycoproteins (Fig. 1A), allowing the ready dispersal of the newly generated virus (2, 3).

FIG 1
  • Open in new tab
  • Download powerpoint
FIG 1

HA stalk-reactive MAbs inhibit influenza virus NA activity. (A) Model of NA activity to cleave bound sialic acids to release newly made virus particles. Note that the cleaved sialic acid substrates provide the change in signal (reduced) for the ELLA and NA-Star assay readouts. (B) Examples of HA head-reactive MAbs and NA-binding MAbs with NI activity, illustrating inhibition of NA enzymatic activity via ELLAs against A/California/7/2009 (H1N1) virus and A/Switzerland/9715293/2013 (H3N2) virus. (C) Model for how HA head-reactive MAbs and NA-reactive MAbs inhibit NA from cleaving the sialic acid from the host cell receptors. (D) HA stalk-reactive MAbs tested for inhibiting NA enzymatic activity via ELLAs against A/California/7/2009 (H1N1) virus and A/Switzerland/9715293/2013 (H3N2) virus. (E) Model for how HA stalk-reactive MAbs inhibit NA from cleaving the sialic acid from the host cell receptors via steric hindrance. (F) HA stalk-reactive MAbs tested for inhibiting NA enzymatic activity via NA-Star assays against A/California/7/2009 (H1N1) virus and A/Switzerland/9715293/2013 (H3N2) virus. (G) Model for why NA-reactive but not HA stalk-reactive MAbs can inhibit NA from cleaving the sialic acid in the NA-Star assays. A dilution of virus that resulted in 90 to 95% maximum signal was used in the following NI assays. Oseltamivir was used as a positive control. Results are shown as means ± SDs of three independent experiments.

Antibodies are a major means of protection from influenza virus infections. Antibody-mediated immunity is the basis of current vaccines and most efforts to improve immunity to influenza. Influenza virus vaccinations induce antibodies that predominantly target the immunodominant globular head of influenza HA, blocking viral attachment to prevent infection (4, 5). However, immunity to the HA head domain is highly susceptible to influenza antigenic drift or viral mutation, which introduces novel amino acids and glycosylation sites that allow the virus to evade existing immunity. The stalk is a more conserved domain, allowing antibodies that target this region to neutralize a wide spectrum of influenza virus subtypes (6–9). The currently appreciated mechanism of protection by HA stalk-reactive antibodies is to lock the HA trimer in a prefusion conformation, preventing pH-triggered conformational changes upon viral uptake into endocytic compartments. This conformational change exposes the fusion peptide that mediates fusion of the viral membrane to the host cell membrane, with subsequent introduction of the viral genome (10). Because of the highly conserved epitopes within the HA stalk domain, induction of antibodies targeting this domain is the basis of several universal influenza vaccine concepts and ongoing clinical trials. Herein, we describe an additional mechanism of protection that is mediated by HA stalk-binding antibodies, i.e., the inhibition of NA activity through steric hindrance blocking access to HA-bound sialic acid.

We used a panel of well-characterized HA stalk-reactive monoclonal antibodies (MAbs) to explore how the MAbs interfere with NA enzymatic activity, as measured by an enzyme-linked lectin assay (ELLA) or the NA-Star assay (11). The ELLA uses the sialylated glycoprotein fetuin, which is immobilized as a substrate to measure NA inhibition (NI); there is no obvious relationship between the NI ability of an antibody and its virus-binding range (12). This immobilized substrate identifies antibodies that either directly inhibit NA by binding close to the enzymatic active site or sterically inhibit NA from abutting close enough to HA to cleave the sialic acid (13). In contrast, the NA-Star assay uses a small soluble chemiluminescent substrate and so more explicitly distinguishes antibodies that directly inhibit the enzymatic activity of NA by binding close to the enzymatic site (12, 14). The ELLA detects a larger functional area, compared to the NA-star assay. When NA antigenic drift occurs outside the NA enzymatic active domain, it may affect only the ELLA and not the NA-Star assay. For all the ELLA and NA-Star assays, Tamiflu was used as a positive control. Herein, we show that HA stalk-reactive MAbs inhibit the enzymatic activation of influenza A viruses in the ELLA but not in the NA-Star assay, supporting a steric inhibition model. Moreover, we detected reduced NI activity for F(ab′)2 fragments from the stalk-reactive MAbs, compared to the same whole antibodies, providing direct evidence that anti-HA stalk antibodies sterically limit NA access to sialic acids. The NI activity of HA stalk-binding but not NA-binding antibodies is disrupted by dissociating HA and NA from virions, further supporting a steric hindrance-based mechanism of NI. Notably, only antibodies against the HA stalk with NI activity, including two with high affinity binding, were able to mediate microneutralization (MN). These findings suggest that an important component of protection by anti-HA stalk antibodies is steric inhibition of NI activity.

(This article was submitted to an online preprint archive [15].)

RESULTS

Stalk-reactive MAbs interfere with influenza A virus NA activity.It has been shown that a subset of NA-reactive antibodies bind NA outside the enzymatic site and inhibit sialic acid cleavage only in ELLAs and not NA-Star assays (12). Two examples of these antibodies are indicated in Fig. 1B (MAb 1000-3B06 against H1N1 influenza and MAb 229-2C06 against H3N2 influenza). The mechanism of action of these antibodies is likely steric inhibition of NA accessing sialic acid molecules bound by HA (Fig. 1C, left). Similarly, antibodies to epitopes on the outside periphery of the globular head of HA that do not inhibit hemagglutination, and so do not bind the HA receptor binding domain, can nonetheless mediate protection by inhibiting viral egress, similar to NI (16). Some HA stalk-reactive antibodies have also been shown to have NI activity (34, 35). Thus, we hypothesized that antibodies to the stalk region of HA, which would occupy space on the side of the molecule, could similarly mediate NI activity, expanding the mechanism of protection to include both inhibition of fusion and inhibition of egress. As expected (17), antibodies binding the HA head region of H1N1 and H3N2 influenza virus strains robustly inhibited NA activity in ELLAs (Fig. 1B, MAb EM-4C04 against H1N1 influenza and MAb 3-50008-2A06 against H3N2 influenza), because HA cannot bind the sialic acid on fetuin and so NA is not close enough for enzymatic activity (Fig. 1C, right). To determine whether anti-HA stalk antibodies elicited NI activity, we tested the well-characterized MAb 045-2B06 (18), which binds the HA stalk regions of group 1 and group 2 influenza A HA molecules, and MAb CR9114, which binds the HA stalk regions of both A and B influenza strain HAs (16). Both of these antibodies neutralize influenza in vitro and protect mice in vivo upon lethal challenge with influenza, and they can competitively inhibit the binding of each other (18), demonstrating that they bind similar protective HA stalk epitopes. We found that both 045-2B06 and CR9114 provided NI activity against both A/California/7/2009 (H1N1) and A/Switzerland/9715293/2013 (H3N2) in the ELLA (Fig. 1D). The likely mechanism for this inhibition is steric hindrance, in which the antibodies block the access of NA to the sialic acids bound by HA (Fig. 1E). In contrast, neither 045-2B06 nor CR9114 could inhibit the H1N1 and H3N2 viruses cleaving the small unattached substrate in the NA-Star assay (Fig. 1F), suggesting a mechanism of steric hindrance (compare Fig. 1E and G). Our results demonstrate that human antibodies reactive with the HA stalk inhibit the enzymatic activity of the NA protein on H1N1 and H3N2 influenza virus particles.

Stalk-reactive MAbs interfere with NA activity by steric hindrance.Because the antibodies inhibited NI activity on the attached substrate of the ELLA but not on the free substrate of the NA-Star assay, as depicted in Fig. 1D and F, it appears that the mechanism of inhibition is likely steric hindrance of NA access to the sialic acid when bound by HA. To investigate this possibility directly, we generated F(ab′)2 molecules from the 045-2B06 and CR9114 MAbs to reduce the size of the molecules, and thus the degree of steric hindrance, without affecting the avidity of binding. The F(ab′)2 protein concentrations were increased to ensure binding of the F(ab′)2 molecules equivalent to that of the MAbs (Fig. 2A and B). Comparison via the ELLA showed that indeed the F(ab′)2 molecules had significantly reduced NI activity, in comparison to the whole MAbs, against both H1N1 and H3N2 influenza viruses (Fig. 2C). Thus, while not completely allowing access of NA to the sialic acid, the smaller antibody fragments allowed increased NA access to the substrate (Fig. 2D), supporting a steric hindrance model of NI by HA stalk-reactive antibodies. As a final verification that antibodies to the HA stalk were inhibiting NA through steric hindrance, we dissociated the HA and NA molecules from the virions, thus allowing NA to access the sialic acid substrates completely independent of HA, regardless of the presence of antibody. For this analysis, we performed the ELLA in the presence of 1% Triton X-100 to disrupt the viral envelope lipid bilayer, releasing HA and NA in a way similar to seasonal split influenza virus vaccine production (19). The detergent treatment completely abrogated the NI activity of all of the HA stalk-reactive and HA head-reactive MAbs against both H1N1 and H3N2 virus strains, further supporting the steric hindrance model for NI activity (Fig. 3A and B). However, the NA-reactive antibodies were able to inhibit NI activity, demonstrating the integrity of the assay after detergent treatment. In total, these various results demonstrate that, in addition to the well-appreciated mechanism of action of disrupting viral entry, antibodies to the HA stalk region inhibit the access of NA to sialic acid through steric hindrance and thus reduce viral egress.

FIG 2
  • Open in new tab
  • Download powerpoint
FIG 2

F(ab′)2 molecules from stalk-reactive MAbs have reduced interference of virus NA activity by steric hindrance. (A) Binding avidity of 045-2B06 and its F(ab′)2 fragments against A/California/7/2009 recombinant HA. (B) Binding avidity of CR9114 and its F(ab′)2 fragments against A/California/7/2009 recombinant HA. (C) HA stalk-reactive MAbs and their F(ab′)2 fragments tested for inhibiting NA enzymatic activity via ELLAs against A/California/7/2009 (H1N1) virus and A/Switzerland/9715293/2013 (H3N2) virus. (D) Model for why F(ab′)2 fragments of HA stalk-reactive MAbs have reduced inhibition of NI in ELLAs. Results are shown as means ± SDs of three independent experiments. Statistical significance is indicated as follows: **, P ≤ 0.05; ***, P ≤ 0.01.

FIG 3
  • Open in new tab
  • Download powerpoint
FIG 3

HA stalk-reactive MAbs cannot interfere with influenza virus NA activity if HA and NA are dissociated from the viral envelope. (A) A/California/7/2009 (H1N1) virus and A/Switzerland/9715293/2013 (H3N2) virus were treated with 1% Triton X-100 for 1 h at 37°C, and HA stalk-reactive and HA head-reactive MAbs, as well as NA-reactive MAbs, were tested for NI activity via ELLAs against Triton X-100-treated A/California/7/2009 (H1N1) virus and A/Switzerland/9715293/2013 (H3N2) virus. (B) Model for why HA reactive MAbs cannot disrupt NA activity, whereas NA-reactive MAbs can, when the HA and NA glycoproteins are dissociated from the viral envelope. Results are shown as means ± SDs of two independent experiments.

Stalk-reactive MAbs have different capacities for NI activity, which appears important for potency.Various HA stalk-reactive antibodies have been shown to have distinct amino acids involved in binding, various angles of interaction, and differences in specificity, as indicated by the spectrum of influenza strains bound (10). Thus, it was predicted that not all HA stalk-binding antibodies would elicit NI activity to the same degree. We tested a panel of 13 human MAbs that had been verified to bind HA stalk epitopes (11) for NI activity by ELLA. Criteria for being classified as HA stalk reactive included competitive binding with structure-verified HA stalk-reactive antibodies, including CR9114 (16) and SC70-F02 (20), and binding in a pH-dependent fashion, because the HA stalk epitope is disrupted at low pH (11). The majority, but not all, of the anti-HA stalk antibodies tested could inhibit NA activity, including 69% (9 of 13 antibodies) of those that are reactive with an H1N1 influenza virus (Fig. 4A and C) and 3 of 4 that bind an H3N2 influenza virus (Fig. 4B and D). Notably, none of those antibodies had NI activity using the NA-Star assay (Fig. 4E to H), suggesting that, like CR9114 and 045-2B06, they all inhibit NI via steric interference between NA and sialic acid. Importantly, after testing the panel of MAbs using a MN assay, we noted that only the antibodies with NI activity were able neutralize viral infection in vitro (Fig. 5A and B). While 2 of the antibodies that lacked MN capacity had low avidity, possibly explaining the lack of both MN and NI activities, the other 2 were of equal avidity, compared to the set of HA stalk-binding antibodies that did inhibit NI and neutralize infection (Fig. 5C). The lack of NI and MN activities of 2 of the HA stalk-binding antibodies with high avidity suggests that the epitope is bound in an atypical fashion. Thus, we considered whether these antibodies were of the stereotypical variety with restricted variable gene repertoires, biased for VH1-69 and VH1-18 immunoglobulin heavy chain gene usage. Notably, while most of the NI-positive antibodies appeared to be stereotypical (Fig. 6A), none of the NI-negative anti-HA stalk antibodies used the most typical anti-HA stalk VH gene, VH1-69 (Fig. 6B). One of the high-avidity NI-negative antibodies used VH3-23, which is indeed atypical, while the other high-avidity NI-negative antibody used VH1-18, which is a common stalk-related gene that is highly similar to VH1-69. There were no other features of the immunoglobulin gene repertoire that were distinct between NI-positive and NI-negative antibodies (Fig. 6C to F). These studies demonstrate that, while the majority of HA stalk-reactive antibodies inhibit NA activity, a subset do not and the capacity for NI appears to be important for potency.

FIG 4
  • Open in new tab
  • Download powerpoint
FIG 4

Stalk-reactive MAbs affect virus NA activity differently. (A and C) HA stalk-reactive MAbs were tested for NI activity via ELLAs against A/California/7/2009 (H1N1) virus. (B and D) HA stalk-reactive MAbs were tested for NI activity via ELLAs against A/Switzerland/9715293/2013 (H3N2) virus. (E and G) HA stalk-reactive MAbs were tested for NI activity via NA-Star assays against A/California/7/2009 (H1N1) virus. (F and H) HA stalk-reactive MAbs were tested for NI activity via NA-Star assays against A/Switzerland/9715293/2013 (H3N2) virus. Oseltamivir was used as a positive control for all assays. Data are representative of two independent experiments.

FIG 5
  • Open in new tab
  • Download powerpoint
FIG 5

NI activity of influenza HA stalk-reactive antibodies correlates with MN capacity. (A and B) HA stalk-reactive MAbs with or without NI activity were tested for neutralization in in vitro MN assays using A/California/7/2009 (H1N1) virus (A) and A/Switzerland/9715293/2013 (H3N2) virus (B). Positive-control HA reactive MAbs EM-4C04 (anti-H1N1) and 229-1C01 (anti-H3N2) bind HA and neutralize these influenza virus strains. Influenza-nonreactive MAb 003-15D3 was used as a negative control in the assays. (C) HA stalk-reactive MAbs were tested for binding affinity against recombinant HA protein. Kd values for antibody binding were determined by Scatchard analysis of ELISA data using nonlinear regression (one-site binding model) with GraphPad Prism software. Bars indicate median values. Data are representative of two independent experiments. n.s., not significant.

FIG 6
  • Open in new tab
  • Download powerpoint
FIG 6

Heavy chain gene features of HA stalk-reactive MAbs with or without NI activity. (A and B) Usage of VH immunoglobulin genes by HA stalk-reactive B cells with NI activity (A) and HA stalk-reactive B cells without NI activity (B). (C and D) Usage of JH immunoglobulin genes by HA stalk-reactive B cells with NI activity (C) and HA stalk-reactive B cells without NI activity (D). (E) Complementarity-determining region 3 (CDR3) length of HA stalk-reactive MAbs with and without NI activity. Data are represented as means ± SDs. (F) Total heavy chain amino acid mutations for HA stalk-reactive MAbs with or without NI activity, based on analysis using the NCBI IgBlast tool (https://www.ncbi.nlm.nih.gov/igblast). Data are represented as means ± SDs. The antibodies represented in this figure were cloned from six different subjects (subjects 13001, 030, 045, 051, SFV005, and SFV009).

DISCUSSION

The development of a “one-shot” universal influenza vaccine that will provide long-term or improved immunity to influenza epidemics and pandemics is currently a major focus of the biomedical community (21, 22). The HA stalk is an important target for the ongoing design of universal influenza vaccines and for recent and ongoing clinical trials, because the protective epitopes in this portion of HA are highly conserved across many influenza subtypes (10, 23, 24). While the appreciated mechanism of action of HA stalk-binding antibodies is to inhibit viral entry by disrupting the HA conformational change required for fusion of the viral envelope to the host cell membrane, one recent study using a microscopy-based assay suggested that HA stalk-reactive antibodies also block influenza virus particle release (25). Herein, we show that antibodies to the HA stalk region inhibit the activity of NA through steric hindrance on whole virions, adding to the mechanisms of protection mediated by this broadly reactive class of antibodies and providing a mechanism for the previously observed inhibition of viral release.

It is notable that not all antibodies that bind the HA stalk region can inhibit NI activity, although, for the collection of antistalk antibodies tested for this study, NI activity correlated perfectly with MN activity. Thus, NI activity is at least an important correlate of neutralization capacity and at most a critical component of the activity of HA stalk-binding antibodies. It is known that preexisting NA-reactive antibodies can reduce the number of cases of infection and decrease disease severity (26, 27). Moreover, adult influenza virus challenge studies showed that the NI titer is more predictive of protection and reduced disease (28). In future studies, it will be important to distinguish the relative contributions of inhibition of viral entry versus viral egress in protection for HA stalk-reactive antibodies.

A second interesting observation that should be evaluated is that there is a potential correlation between the capacity for NI activity and the type of anti-HA stalk antibody elicited, in that none of the NI-negative antibodies binding the HA stalk was encoded by the stereotypical VH1-69 gene. A more substantial evaluation of many anti-HA stalk antibodies for various properties and structural studies to evaluate the particular contact residues and angle of binding for stalk-reactive antibodies with NI activity will provide insight into this issue. Identifying stereotypical features of anti-HA stalk antibodies with NI activity could inform universal influenza vaccine design, by, for example, providing an impetus to develop germline-boosting prime-boost strategies to elicit a particular class of anti-HA stalk antibodies preferentially. In conclusion, this study identifies NI inhibition as a new and additional mechanism of action for the important class of anti-influenza antibodies that bind highly conserved epitopes on the HA stalk.

MATERIALS AND METHODS

Cells and viruses.Both human embryonic kidney (HEK) 293T cells and Madin-Darby canine kidney (MDCK) cells were obtained from the ATCC. The 293T cells were maintained at 37°C with 5% CO2 in advanced Dulbecco’s modified Eagle’s medium (DMEM) with 2% Ultra-Low IgG fetal bovine serum (FBS) (Gibco), 2 mM GlutaMAX (Gibco), and penicillin and streptomycin (100 mg/ml; Gibco). The MDCK cells were maintained at 37°C with 5% CO2 in DMEM with 10% FBS (Gibco), 1% l-glutamine (Gibco), and 1% antibiotic-antimycotic (Gibco). All influenza virus stocks (A/California/7/2009 H1N1 and A/Switzerland/9715293/2013 H3N2) used for the assays were freshly grown in specific-pathogen-free (SPF) eggs, harvested, purified, tittered, and stored at −80°C.

Recombinant MAb expression and purification.Antibodies were generated as described previously (11, 29–31). Briefly, the VH, Vκ, or Vλ genes amplified from each single cell were cloned into IgG1, Igκ, or Igλ expression vectors as described previously. Nine micrograms of each paired heavy and light chain plasmid DNA was transfected into the 293 cells using polyethylenimine (PEI) 25 K (catalog no. 23966-2; Polysciences), and the cells were incubated overnight. The next day, the transfection medium was aspirated from each plate and replaced with 25 ml of protein-free hybridoma medium (PFHM) II (Gibco). Four to 5 days later, secreted MAbs were purified from the supernatant using protein A beads. The MAbs was further concentrated and the buffer was exchanged with phosphate-buffered saline (PBS) (pH 7.4) using Amicon Ultra centrifugal filter units (30-kDa cutoff; Millipore). The final protein concentrations were determined using a NanoDrop device (Thermo Scientific).

Preparation of F(ab′)2 fragments.The whole antibodies CR9114 and 045-2B06 were reduced to F(ab′)2 fragments using the Pierce F(ab′)2 digestion kit (catalog no. 44988; Thermo Fisher), according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay.High-protein-binding microtiter plates (Costar) were coated overnight at 4°C with recombinant HAs or NAs at 1 μg/ml in PBS. After blocking, serially diluted (3-fold) antibodies starting at 10 μg/ml were incubated for 1 h at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody diluted 1:1,000 (Jackson Immuno Research) was used to detect binding of MAbs, and the result was developed with Super AquaBlue enzyme-linked immunosorbent assay (ELISA) substrate (eBiosciences). Absorbance was measured at 405 nm on a microplate spectrophotometer (Bio-Rad). To standardize the assays, antibodies with known binding characteristics were included on each plate, and the plates were developed when the absorbance of the control reached an optical density at 405 nm (OD405) of 3.0 (32). To determine the binding of F(ab′)2 fragments by ELISA, a HRP-conjugated goat anti-kappa antibody (SouthernBiotech) was used as a secondary antibody. Affinity constant (Kd) values for antibody binding were determined by Scatchard plot analysis of ELISA data using nonlinear regression (one-site binding model) with GraphPad Prism software.

NA ELLA.ELLAs were performed as described previously (13, 33). Flat-bottom 96-well plates (Thermo Scientific) were coated overnight at 4°C with 100 μl of fetuin (Sigma) at 25 μg/ml. To determine the amount of virus to use in the ELLA, serial dilutions (2-fold) of the targeted virus were made in Dulbecco’s PBS (DPBS) with 0.05% Tween 20 and 1% bovine serum albumin (BSA) (DPBSTBSA), into fetuin-coated plates containing an equal volume of DPBS. The plates were incubated for 18 h at 37°C and washed six times with PBS with 0.05% Tween 20, and 100 μl/well of HRP-conjugated peanut agglutinin (PNA) lectin (Sigma-Aldrich) in DPBSTBSA was added for 2 h at room temperature in the dark. The plates were washed six times and developed with Super AquaBlue ELISA substrate (eBiosciences). Absorbance was read at 405 nm on a microplate spectrophotometer (Bio-Rad). The dilution of virus that resulted in 90 to 95% of the maximum signal was chosen for use in the subsequent ELLAs. To measure the NI titers of the antibodies, the antibodies were serially diluted in DPBSTBSA and then incubated for 18 h at 37°C with an equal volume (50 μl) of the selected virus dilution in duplicate wells of a fetuin-coated plate. Eight wells containing diluent without antibody served as the positive (virus-only) control. Data were analyzed using Prism software, and the 50% inhibitory concentration (IC50) was defined as the concentration at which 50% of the NA activity was inhibited, compared to the negative control.

NA-Star assay.The NA-Star assay was performed according to the manufacturer's instructions for the resistance detection kit (Applied Biosystems, Darmstadt, Germany) (14). In brief, 25 μl of test MAbs in serial 2-fold dilutions in NA-Star assay buffer [26 mM 2-(N-morpholino)ethanesulfonic acid [MES], 4 mM calcium chloride [pH 6.0]) were mixed with 25 μl of 1.58 × 103 50% tissue culture infectious doses (TCID50)/ml of A/California/7/2009 virus or 2.5 × 104 TCID50/ml of A/Switzerland/9715293/2013 virus, and the plates were incubated at 37°C for 30 min. After addition of 10 μl of 1,000-fold diluted NA-Star substrate, the plates were incubated at room temperature for another 30 min. The reaction was stopped by the addition of 60 μl of NA-Star accelerator. The chemiluminescence was determined by using a DTX 880 plate reader (Beckman Coulter). Data points were analyzed using Prism software, and the IC50 was defined as the concentration at which 50% of the NA activity was inhibited, compared to the negative control. The final concentration of antibody (IC50) was determined using Prism software (GraphPad).

NI assay in the presence of detergent.To perform the NI assay in the presence of detergent, all steps remained identical to those listed above except as follows. First, a final concentration of 1% Triton X-100 (Fisher Bioreagents) was added directly to the virus particles, and they were shaken gently at 37°C for 1 h. During that time, antibodies were diluted using PBS with 1% Triton X-100. Prior to incubation with MAbs, the virus preparation was diluted in PBS containing 1% BSA and 1% Triton X-100. The NI assay was then performed as detailed above.

MN assay.The MN assay for antibody characterization was carried out as described previously (18). Briefly, MDCK cells were maintained at 37°C with 5% CO2 in DMEM supplemented with 10% FBS. On the day before the experiment, confluent MDCK cells in a 96-well format were washed twice with PBS and incubated in minimal essential medium (MEM) supplemented with 1 μg/ml tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin. Serial 2-fold dilutions (starting concentration, 64 μg/ml) of MAb were mixed with an equal volume of 100 TCID50 of virus, and the mixture was incubated for 1 h at 37°C. The mixture was removed, and cells were cultured for 20 h at 37°C with 1× MEM supplemented with 1 μg/ml TPCK-treated trypsin and an appropriate MAb concentration. Cells were washed twice with PBS, fixed with 80% acetone at −20°C for 1 h or overnight, washed three times with PBS with 0.05% Tween 20, blocked for 30 min with 10% FBS, and then treated for 30 min with 2% H2O2 at room temperature. A biotinylated anti-nucleoside phosphorylase antibody (1:3,000) in 3% BSA-PBS was added for 1 h at room temperature. The plates were developed with Super AquaBlue ELISA substrate and read at 405 nm. The signals from uninfected wells were averaged to represent 100% inhibition, and the signals from virus-infected wells without MAb were averaged to represent 0% inhibition. Duplicate wells were used to calculate the mean and standard deviation (SD) of neutralization, and IC50 values were determined from a sigmoidal dose-response curve. The inhibition ratio was calculated as follows: [(OD405 for positive control − OD405 for sample)/(OD405 for positive control − OD405 for negative control)] × 100%. The final concentration of antibody that reduced infection to 50% (IC50) was determined using Prism software (GraphPad).

Data analysis and statistics.GraphPad Prism 7 was used to perform all statistical analyses. Data are presented as means and SDs of triplicates.

ACKNOWLEDGMENTS

We thank Nai-Ying Zheng for purifying influenza viruses and for experimental assistance.

This project was funded in part by the National Institute of Allergy and Infectious Diseases, through grants U19AI082724 (P.C.W.), 5U19AI109946 (P.C.W.), and U19AI057266 (P.C.W.) and NIAID Centers of Excellence for Influenza Research and Surveillance grant HHSN272201400005C.

Y.-Q.C. designed and performed experiments, analyzed data, and wrote the manuscript, L.Y.-L.L., M.H., and C.H. performed experiments and revised the manuscript, and P.C.W. designed and directed the project, analyzed data, and wrote the manuscript.

We declare no financial or commercial conflicts of interest.

FOOTNOTES

    • Received 1 September 2018.
    • Accepted 27 November 2018.
    • Accepted manuscript posted online 5 December 2018.
  • Copyright © 2019 American Society for Microbiology.

All Rights Reserved.

REFERENCES

  1. 1.↵
    1. Iuliano AD,
    2. Roguski KM,
    3. Chang HH,
    4. Muscatello DJ,
    5. Palekar R,
    6. Tempia S,
    7. Cohen C,
    8. Gran JM,
    9. Schanzer D,
    10. Cowling BJ,
    11. Wu P,
    12. Kyncl J,
    13. Ang LW,
    14. Park M,
    15. Redlberger-Fritz M,
    16. Yu H,
    17. Espenhain L,
    18. Krishnan A,
    19. Emukule G,
    20. van Asten L,
    21. Pereira da Silva S,
    22. Aungkulanon S,
    23. Buchholz U,
    24. Widdowson MA,
    25. Bresee JS
    . 2018. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet 391:1285–1300. doi:10.1016/S0140-6736(17)33293-2.
    OpenUrlCrossRef
  2. 2.↵
    1. Matrosovich MN,
    2. Matrosovich TY,
    3. Gray T,
    4. Roberts NA,
    5. Klenk HD
    . 2004. Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J Virol 78:12665–12667. doi:10.1128/JVI.78.22.12665-12667.2004.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Palese P,
    2. Compans R
    . 1976. Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action. J Gen Virol 33:159–163. doi:10.1099/0022-1317-33-1-159.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    1. Wrammert J,
    2. Koutsonanos D,
    3. Li GM,
    4. Edupuganti S,
    5. Sui J,
    6. Morrissey M,
    7. McCausland M,
    8. Skountzou I,
    9. Hornig M,
    10. Lipkin WI,
    11. Mehta A,
    12. Razavi B,
    13. Del Rio C,
    14. Zheng NY,
    15. Lee JH,
    16. Huang M,
    17. Ali Z,
    18. Kaur K,
    19. Andrews S,
    20. Amara RR,
    21. Wang Y,
    22. Das SR,
    23. O'Donnell CD,
    24. Yewdell JW,
    25. Subbarao K,
    26. Marasco WA,
    27. Mulligan MJ,
    28. Compans R,
    29. Ahmed R,
    30. Wilson PC
    . 2011. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med 208:181–193. doi:10.1084/jem.20101352.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    1. Corti D,
    2. Suguitan AL, Jr,
    3. Pinna D,
    4. Silacci C,
    5. Fernandez-Rodriguez BM,
    6. Vanzetta F,
    7. Santos C,
    8. Luke CJ,
    9. Torres-Velez FJ,
    10. Temperton NJ,
    11. Weiss RA,
    12. Sallusto F,
    13. Subbarao K,
    14. Lanzavecchia A
    . 2010. Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine. J Clin Invest 120:1663–1673. doi:10.1172/JCI41902.
    OpenUrlCrossRefPubMedWeb of Science
  6. 6.↵
    1. Ekiert DC,
    2. Bhabha G,
    3. Elsliger MA,
    4. Friesen RH,
    5. Jongeneelen M,
    6. Throsby M,
    7. Goudsmit J,
    8. Wilson IA
    . 2009. Antibody recognition of a highly conserved influenza virus epitope. Science 324:246–251. doi:10.1126/science.1171491.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Neu KE,
    2. Henry Dunand CJ,
    3. Wilson PC
    . 2016. Heads, stalks and everything else: how can antibodies eradicate influenza as a human disease? Curr Opin Immunol 42:48–55. doi:10.1016/j.coi.2016.05.012.
    OpenUrlCrossRef
  8. 8.↵
    1. Okuno Y,
    2. Isegawa Y,
    3. Sasao F,
    4. Ueda S
    . 1993. A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains. J Virol 67:2552–2558.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Corti D,
    2. Voss J,
    3. Gamblin SJ,
    4. Codoni G,
    5. Macagno A,
    6. Jarrossay D,
    7. Vachieri SG,
    8. Pinna D,
    9. Minola A,
    10. Vanzetta F,
    11. Silacci C,
    12. Fernandez-Rodriguez BM,
    13. Agatic G,
    14. Bianchi S,
    15. Giacchetto-Sasselli I,
    16. Calder L,
    17. Sallusto F,
    18. Collins P,
    19. Haire LF,
    20. Temperton N,
    21. Langedijk JP,
    22. Skehel JJ,
    23. Lanzavecchia A
    . 2011. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science 333:850–856. doi:10.1126/science.1205669.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Wu NC,
    2. Wilson IA
    . 2018. Structural insights into the design of novel anti-influenza therapies. Nat Struct Mol Biol 25:115–121. doi:10.1038/s41594-018-0025-9.
    OpenUrlCrossRef
  11. 11.↵
    1. Andrews SF,
    2. Huang Y,
    3. Kaur K,
    4. Popova LI,
    5. Ho IY,
    6. Pauli NT,
    7. Henry Dunand CJ,
    8. Taylor WM,
    9. Lim S,
    10. Huang M,
    11. Qu X,
    12. Lee JH,
    13. Salgado-Ferrer M,
    14. Krammer F,
    15. Palese P,
    16. Wrammert J,
    17. Ahmed R,
    18. Wilson PC
    . 2015. Immune history profoundly affects broadly protective B cell responses to influenza. Sci Transl Med 7:316ra192. doi:10.1126/scitranslmed.aad0522.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Chen YQ,
    2. Wohlbold TJ,
    3. Zheng NY,
    4. Huang M,
    5. Huang Y,
    6. Neu KE,
    7. Lee J,
    8. Wan H,
    9. Rojas KT,
    10. Kirkpatrick E,
    11. Henry C,
    12. Palm AE,
    13. Stamper CT,
    14. Lan LY,
    15. Topham DJ,
    16. Treanor J,
    17. Wrammert J,
    18. Ahmed R,
    19. Eichelberger MC,
    20. Georgiou G,
    21. Krammer F,
    22. Wilson PC
    . 2018. Influenza infection in humans induces broadly cross-reactive and protective neuraminidase-reactive antibodies. Cell 173:417–429.e410. doi:10.1016/j.cell.2018.03.030.
    OpenUrlCrossRef
  13. 13.↵
    1. Couzens L,
    2. Gao J,
    3. Westgeest K,
    4. Sandbulte M,
    5. Lugovtsev V,
    6. Fouchier R,
    7. Eichelberger M
    . 2014. An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera. J Virol Methods 210C:7–14. doi:10.1016/j.jviromet.2014.09.003.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Nguyen HT,
    2. Sheu TG,
    3. Mishin VP,
    4. Klimov AI,
    5. Gubareva LV
    . 2010. Assessment of pandemic and seasonal influenza A (H1N1) virus susceptibility to neuraminidase inhibitors in three enzyme activity inhibition assays. Antimicrob Agents Chemother 54:3671–3677. doi:10.1128/AAC.00581-10.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Chen Y-Q,
    2. Lan LY-L,
    3. Huang M,
    4. Henry C,
    5. Wilson PC
    . 2018. Hemagglutinin stalk-reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance. bioRxiv https://doi.org/10.1101/400036.
  16. 16.↵
    1. Dreyfus C,
    2. Laursen NS,
    3. Kwaks T,
    4. Zuijdgeest D,
    5. Khayat R,
    6. Ekiert DC,
    7. Lee JH,
    8. Metlagel Z,
    9. Bujny MV,
    10. Jongeneelen M,
    11. van der Vlugt R,
    12. Lamrani M,
    13. Korse HJ,
    14. Geelen E,
    15. Sahin O,
    16. Sieuwerts M,
    17. Brakenhoff JP,
    18. Vogels R,
    19. Li OT,
    20. Poon LL,
    21. Peiris M,
    22. Koudstaal W,
    23. Ward AB,
    24. Wilson IA,
    25. Goudsmit J,
    26. Friesen RH
    . 2012. Highly conserved protective epitopes on influenza B viruses. Science 337:1343–1348. doi:10.1126/science.1222908.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Kosik I,
    2. Yewdell JW
    . 2017. Influenza A virus hemagglutinin specific antibodies interfere with virion neuraminidase activity via two distinct mechanisms. Virology 500:178–183. doi:10.1016/j.virol.2016.10.024.
    OpenUrlCrossRef
  18. 18.↵
    1. Henry Dunand CJ,
    2. Leon PE,
    3. Kaur K,
    4. Tan GS,
    5. Zheng NY,
    6. Andrews S,
    7. Huang M,
    8. Qu X,
    9. Huang Y,
    10. Salgado-Ferrer M,
    11. Ho IY,
    12. Taylor W,
    13. Hai R,
    14. Wrammert J,
    15. Ahmed R,
    16. Garcia-Sastre A,
    17. Palese P,
    18. Krammer F,
    19. Wilson PC
    . 2015. Preexisting human antibodies neutralize recently emerged H7N9 influenza strains. J Clin Invest 125:1255–1268. doi:10.1172/JCI74374.
    OpenUrlCrossRefPubMed
  19. 19.↵
    1. Gross PA,
    2. Ennis FA,
    3. Gaerlan PF,
    4. Denning CR,
    5. Setia U,
    6. Davis WJ,
    7. Bisberg DS
    . 1981. Comparison of new Triton X-100- and Tween-ether-treated split-treated vaccines in children. J Clin Microbiol 14:534–538.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    1. Nachbagauer R,
    2. Shore D,
    3. Yang H,
    4. Johnson SK,
    5. Gabbard JD,
    6. Tompkins SM,
    7. Wrammert J,
    8. Wilson PC,
    9. Stevens J,
    10. Ahmed R,
    11. Krammer F,
    12. Ellebedy AH
    . 2018. Broadly reactive human monoclonal antibodies elicited following pandemic H1N1 influenza virus exposure protect mice against highly pathogenic H5N1 challenge. J Virol 92:e00949-18.
  21. 21.↵
    1. Paules CI,
    2. Sullivan SG,
    3. Subbarao K,
    4. Fauci AS
    . 2018. Chasing seasonal influenza: the need for a universal influenza vaccine. N Engl J Med 378:7–9. doi:10.1056/NEJMp1714916.
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Paules CI,
    2. Marston HD,
    3. Eisinger RW,
    4. Baltimore D,
    5. Fauci AS
    . 2017. The pathway to a universal influenza vaccine. Immunity 47:599–603. doi:10.1016/j.immuni.2017.09.007.
    OpenUrlCrossRef
  23. 23.↵
    1. Krammer F,
    2. Garcia-Sastre A,
    3. Palese P
    . 2018. Is it possible to develop a “universal” influenza virus vaccine? Potential target antigens and critical aspects for a universal influenza vaccine. Cold Spring Harb Perspect Biol 10:a028845.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Henry C,
    2. Palm AE,
    3. Krammer F,
    4. Wilson PC
    . 2018. From original antigenic sin to the universal influenza virus vaccine. Trends Immunol 39:70–79. doi:10.1016/j.it.2017.08.003.
    OpenUrlCrossRef
  25. 25.↵
    1. Yamayoshi S,
    2. Uraki R,
    3. Ito M,
    4. Kiso M,
    5. Nakatsu S,
    6. Yasuhara A,
    7. Oishi K,
    8. Sasaki T,
    9. Ikuta K,
    10. Kawaoka Y
    . 2017. A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine 17:182–191. doi:10.1016/j.ebiom.2017.03.007.
    OpenUrlCrossRef
  26. 26.↵
    1. Monto AS,
    2. Kendal AP
    . 1973. Effect of neuraminidase antibody on Hong Kong influenza. Lancet 1:623–625.
    OpenUrlPubMedWeb of Science
  27. 27.↵
    1. Murphy BR,
    2. Kasel JA,
    3. Chanock RM
    . 1972. Association of serum anti-neuraminidase antibody with resistance to influenza in man. N Engl J Med 286:1329–1332. doi:10.1056/NEJM197206222862502.
    OpenUrlCrossRefPubMedWeb of Science
  28. 28.↵
    1. Memoli MJ,
    2. Shaw PA,
    3. Han A,
    4. Czajkowski L,
    5. Reed S,
    6. Athota R,
    7. Bristol T,
    8. Fargis S,
    9. Risos K,
    10. Powers JH,
    11. Davey RT, Jr,
    12. Taubenberger JK
    . 2016. Evaluation of antihemagglutinin and antineuraminidase antibodies as correlates of protection in an influenza A/H1N1 virus healthy human challenge model. mBio 7:e00417-16.
  29. 29.↵
    1. Wrammert J,
    2. Smith K,
    3. Miller J,
    4. Langley WA,
    5. Kokko K,
    6. Larsen C,
    7. Zheng NY,
    8. Mays I,
    9. Garman L,
    10. Helms C,
    11. James J,
    12. Air GM,
    13. Capra JD,
    14. Ahmed R,
    15. Wilson PC
    . 2008. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453:667–671. doi:10.1038/nature06890.
    OpenUrlCrossRefPubMedWeb of Science
  30. 30.↵
    1. Smith K,
    2. Garman L,
    3. Wrammert J,
    4. Zheng NY,
    5. Capra JD,
    6. Ahmed R,
    7. Wilson PC
    . 2009. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 4:372–384. doi:10.1038/nprot.2009.3.
    OpenUrlCrossRefPubMed
  31. 31.↵
    1. Henry Dunand CJ,
    2. Leon PE,
    3. Huang M,
    4. Choi A,
    5. Chromikova V,
    6. Ho IY,
    7. Tan GS,
    8. Cruz J,
    9. Hirsh A,
    10. Zheng N-Y,
    11. Mullarkey CE,
    12. Ennis FA,
    13. Terajima M,
    14. Treanor JJ,
    15. Topham DJ,
    16. Subbarao K,
    17. Palese P,
    18. Krammer F,
    19. Wilson PC
    . 2016. Both neutralizing and non-neutralizing human H7N9 influenza vaccine-induced monoclonal antibodies confer protection. Cell Host Microbe 19:800–813. doi:10.1016/j.chom.2016.05.014.
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Lau D,
    2. Lan LY,
    3. Andrews SF,
    4. Henry C,
    5. Rojas KT,
    6. Neu KE,
    7. Huang M,
    8. Huang Y,
    9. DeKosky B,
    10. Palm AE,
    11. Ippolito GC,
    12. Georgiou G,
    13. Wilson PC
    . 2017. Low CD21 expression defines a population of recent germinal center graduates primed for plasma cell differentiation. Sci Immunol 2:eaai8153.
  33. 33.↵
    1. Westgeest KB,
    2. Bestebroer TM,
    3. Spronken MI,
    4. Gao J,
    5. Couzens L,
    6. Osterhaus AD,
    7. Eichelberger M,
    8. Fouchier RA,
    9. de Graaf M
    . 2015. Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses. J Virol Methods 217:55–63. doi:10.1016/j.jviromet.2015.02.014.
    OpenUrlCrossRef
  34. 34.↵
    1. Wohlbold TJ,
    2. Chromikova V,
    3. Tan GS,
    4. Meade P,
    5. Amanat F,
    6. Comella P,
    7. Hirsh A,
    8. Krammer F
    . 2016. Hemagglutinin stalk- and neuraminidase-specific monoclonal antibodies protect against lethal H10N8 influenza virus infection in mice. J Virol 90:851–861. doi:10.1128/JVI.02275-15.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Rajendran M,
    2. Nachbagauer R,
    3. Ermler ME,
    4. Bunduc P,
    5. Amanat F,
    6. Izikson R,
    7. Cox M,
    8. Palese P,
    9. Eichelberger M,
    10. Krammer F
    . 2017. Analysis of anti-influenza virus neuraminidase antibodies in children, adults, and the elderly by ELISA and enzyme inhibition: evidence for original antigenic sin. mBio 8:e02281-16. https://mbio.asm.org/content/8/2/e02281-16.
PreviousNext
Back to top
Download PDF
Citation Tools
Hemagglutinin Stalk-Reactive Antibodies Interfere with Influenza Virus Neuraminidase Activity by Steric Hindrance
Yao-Qing Chen, Linda Yu-Ling Lan, Min Huang, Carole Henry, Patrick C. Wilson
Journal of Virology Feb 2019, 93 (4) e01526-18; DOI: 10.1128/JVI.01526-18

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Virology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Hemagglutinin Stalk-Reactive Antibodies Interfere with Influenza Virus Neuraminidase Activity by Steric Hindrance
(Your Name) has forwarded a page to you from Journal of Virology
(Your Name) thought you would be interested in this article in Journal of Virology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Hemagglutinin Stalk-Reactive Antibodies Interfere with Influenza Virus Neuraminidase Activity by Steric Hindrance
Yao-Qing Chen, Linda Yu-Ling Lan, Min Huang, Carole Henry, Patrick C. Wilson
Journal of Virology Feb 2019, 93 (4) e01526-18; DOI: 10.1128/JVI.01526-18
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

hemagglutinin
influenza
monoclonal antibodies
neuraminidase
stalk-reactive antibody

Related Articles

Cited By...

About

  • About JVI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jvirology

@ASMicrobiology

       

 

JVI in collaboration with

American Society for Virology

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0022-538X; Online ISSN: 1098-5514