Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies

ABSTRACT Antibody detection is crucial for monitoring host immune responses to specific pathogen antigens (Ags) and evaluating vaccine efficacies. The luciferase immunoprecipitation system (LIPS) was developed for sensitive detection of Ag-specific antibodies in sera from various species. In this study, we describe NanoLIPS, an improved LIPS assay based on NanoLuciferase (NLuc), and employ the assay for monitoring antibody responses following influenza virus infection or vaccination. We generated recombinant influenza virus hemagglutinin (HA) proteins tagged with N-terminal (N-NLuc-HA) or C-terminal (C-NLuc-HA) NLuc reporters. NLuc-HA yielded an at least 20-fold higher signal-to-noise ratio than did a LIPS assay employing a recombinant HA-Gaussia princeps luciferase (GLuc) fusion protein. NanoLIPS-based detection of anti-HA antibodies yielded highly reproducible results with a broad dynamic range. The levels of antibodies against C-NLuc-HA generated by mice vaccinated with recombinant vaccinia virus DIs strain expressing an influenza virus HA protein (rDIs-HA) was significantly correlated with the protective effect elicited by the rDIs-HA vaccine. C-NLuc-HA underwent glycosylation with native conformations and assembly to form a trimeric structure and was detected by monoclonal antibodies that detect conformational epitopes present on the globular head or stalk domain of HA. Therefore, NanoLIPS is applicable for evaluating vaccine efficacy. We also showed that C-NLuc-HA is applicable for detection of HA-specific antibodies in sera from various experimental species, including mouse, cynomolgus macaque, and tree shrew. Thus, NanoLIPS-based detection of HA offers a simple and high-sensitivity method that detects native conformational epitopes and can be used in various experimental animal models. IMPORTANCE Influenza virus HA-specific antibodies can be detected via the hemagglutination inhibition (HI) assay, the neutralization (NT) assay, and the enzyme-linked immunosorbent assay (ELISA). However, these assays have some drawbacks, including narrow dynamic range and the requirement for large amounts of sera. As an alternative to an ELISA-based method, luciferase immunoprecipitation system (LIPS) was developed. We focused on NanoLuciferase (NLuc), which has a small size, higher intensity, and longer stability. In this study, we developed a technically feasible and highly sensitive method for detecting influenza virus-specific antibodies using a NLuc-tagged recombinant HA protein produced in mammalian cells. HA with a C-terminal NLuc extension (C-NLuc-HA) was glycosylated and formed trimeric complexes when expressed in mammalian cells. Furthermore, C-NLuc-HA was recognized not only by monoclonal antibodies that bind to the globular head domain but also by those that bind to the stalk domain. We also demonstrated that the data obtained by this assay correlate with the protection of an experimental vaccine in animal models.

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Plasticity of the Influenza Virus H5 HA Protein

mBio ◽

10.1128/mbio.03324-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Huihui Kong ◽

David F. Burke ◽

Tiago Jose da Silva Lopes ◽

Kosuke Takada ◽

Masaki Imai ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Mammalian Cells ◽

Influenza A ◽

Viral Antigen ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Highly Pathogenic ◽

Antigenic Properties ◽

Ha Protein

ABSTRACT Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants. IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

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Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain

Scientific Reports ◽

10.1038/s41598-017-14931-7 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 36

Author(s):

Christopher S. Anderson ◽

Sandra Ortega ◽

Francisco A. Chaves ◽

Amelia M. Clark ◽

Hongmei Yang ◽

...

Keyword(s):

Influenza Virus ◽

Viral Fitness ◽

Antigenic Drift ◽

Virus Infections ◽

Head Region ◽

Immune Pressure ◽

Stalk Domain ◽

Ha Protein

Abstract The induction of antibodies specific for the influenza HA protein stalk domain is being pursued as a universal strategy against influenza virus infections. However, little work has been done looking at natural or induced antigenic variability in this domain and the effects on viral fitness. We analyzed human H1 HA head and stalk domain sequences and found substantial variability in both, although variability was highest in the head region. Furthermore, using human immune sera from pandemic A/California/04/2009 immune subjects and mAbs specific for the stalk domain, viruses were selected in vitro containing mutations in both domains that partially contributed to immune evasion. Recombinant viruses encoding amino acid changes in the HA stalk domain replicated well in vitro, and viruses incorporating two of the stalk mutations retained pathogenicity in vivo. These findings demonstrate that the HA protein stalk domain can undergo limited drift under immune pressure and the viruses can retain fitness and virulence in vivo, findings which are important to consider in the context of vaccination targeting this domain.

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Broadly Reactive Human Monoclonal Antibodies Elicited following Pandemic H1N1 Influenza Virus Exposure Protect Mice against Highly Pathogenic H5N1 Challenge

Journal of Virology ◽

10.1128/jvi.00949-18 ◽

2018 ◽

Vol 92 (16) ◽

Cited By ~ 8

Author(s):

Raffael Nachbagauer ◽

David Shore ◽

Hua Yang ◽

Scott K. Johnson ◽

Jon D. Gabbard ◽

...

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Influenza Viruses ◽

Health Concern ◽

Human Monoclonal Antibodies ◽

Lethal Challenge ◽

Highly Pathogenic ◽

Zoonotic Infections ◽

Stalk Domain ◽

Group 1

ABSTRACT Broadly cross-reactive antibodies (Abs) that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes, including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (MAbs) for their binding breadth and affinity, in vitro neutralization capacity, and in vivo protective potential against an highly pathogenic avian influenza virus. The monoclonal antibodies were isolated from individuals shortly following infection with (70-1F02 and 1009-3B05) or vaccination against (05-2G02 and 09-3A01) A(H1N1)pdm09. Three of the MAbs bound HAs from multiple strains of group 1 viruses, and one MAb, 05-2G02, bound to both group 1 and group 2 influenza A virus HAs. All four antibodies prophylactically protected mice against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) strain. Two MAbs, 70-1F02 and 09-3A01, were further tested for their therapeutic efficacy against the same strain and showed good efficacy in this setting as well. One MAb, 70-1F02, cocrystallized with H5 HA and showed heavy-chain-only interactions similar to those seen with the previously described CR6261 anti-stalk antibody. Finally, we show that antibodies that compete with these MAbs are prevalent in serum from an individual recently infected with the A(H1N1)pdm09 virus. The antibodies described here can be developed into broad-spectrum antiviral therapeutics that could be used to combat infections by zoonotic or emerging pandemic influenza viruses. IMPORTANCE The rise in zoonotic infections of humans by emerging influenza viruses is a worldwide public health concern. The majority of recent zoonotic human influenza cases were caused by H7N9 and H5Nx viruses and were associated with high morbidity and mortality. In addition, seasonal influenza viruses are estimated to cause up to 650,000 deaths annually worldwide. Currently available antiviral treatment options include only neuraminidase inhibitors, but some influenza viruses are naturally resistant to these drugs, and others quickly develop resistance-conferring mutations. Alternative therapeutics are urgently needed. Broadly protective antibodies that target the conserved “stalk” domain of the hemagglutinin represent potential potent antiviral prophylactic and therapeutic agents that can assist pandemic preparedness. Here, we describe four human monoclonal antibodies that target conserved regions of influenza HA and characterize their binding spectrum as well as their protective capacity in prophylactic and therapeutic settings against a lethal challenge with a zoonotic influenza virus.

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H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans

PLoS Pathogens ◽

10.1371/journal.ppat.1010098 ◽

2021 ◽

Vol 17 (12) ◽

pp. e1010098

Author(s):

Fangtao Li ◽

Jiyu Liu ◽

Jizhe Yang ◽

Haoran Sun ◽

Zhimin Jiang ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Mammalian Cells ◽

Matrix Protein ◽

H9n2 Virus ◽

Virus Release ◽

Mammalian Cell Lines ◽

M1 Protein ◽

Ha Protein

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection.

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Hemagglutinin Stalk- and Neuraminidase-Specific Monoclonal Antibodies Protect against Lethal H10N8 Influenza Virus Infection in Mice

Journal of Virology ◽

10.1128/jvi.02275-15 ◽

2015 ◽

Vol 90 (2) ◽

pp. 851-861 ◽

Cited By ~ 38

Author(s):

Teddy John Wohlbold ◽

Veronika Chromikova ◽

Gene S. Tan ◽

Philip Meade ◽

Fatima Amanat ◽

...

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Virus Infection ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Morbidity And Mortality ◽

Head Domain ◽

Group 2 ◽

Reactive Antibody ◽

Globular Head

ABSTRACTBetween November 2013 and February 2014, China reported three human cases of H10N8 influenza virus infection in the Jiangxi province, two of which were fatal. Using hybridoma technology, we isolated a panel of H10- and N8-directed monoclonal antibodies (MAbs) and further characterized the binding reactivity of these antibodies (via enzyme-linked immunosorbent assay) to a range of purified virus and recombinant protein substrates. The H10-directed MAbs displayed functional hemagglutination inhibition (HI) and neutralization activity, and the N8-directed antibodies displayed functional neuraminidase inhibition (NI) activity against H10N8. Surprisingly, the HI-reactive H10 antibodies, as well as a previously generated, group 2 hemagglutinin (HA) stalk-reactive antibody, demonstrated NI activity against H10N8 and an H10N7 strain; this phenomenon was absent when virus was treated with detergent, suggesting the anti-HA antibodies inhibited neuraminidase enzymatic activity through steric hindrance. We tested the prophylactic efficacy of one representative H10-reactive, N8-reactive, and group 2 HA stalk-reactive antibodyin vivousing a BALB/c challenge model. All three antibodies were protective at a high dose (5 mg/kg). At a low dose (0.5 mg/kg), only the anti-N8 antibody prevented weight loss. Together, these data suggest that antibody targets other than the globular head domain of the HA may be efficacious in preventing influenza virus-induced morbidity and mortality.IMPORTANCEAvian H10N8 and H10N7 viruses have recently crossed the species barrier, causing morbidity and mortality in humans and other mammals. Although these reports are likely isolated incidents, it is possible that more cases may emerge in future winter seasons, similar to H7N9. Furthermore, regular transmission of avian influenza viruses to humans increases the risk of adaptive mutations and reassortment events, which may result in a novel virus with pandemic potential. Currently, no specific therapeutics or vaccines are available against the H10N8 influenza virus subtype. We generated a panel of H10- and N8-reactive MAbs. Although these antibodies may practically be developed into therapeutic agents, characterizing the protective potential of MAbs that have targets other than the HA globular head domain will provide insight into novel antibody-mediated mechanisms of protection and help to better understand correlates of protection for influenza A virus infection.

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Influenza hemagglutinin-specific IgA Fc-effector functionality is restricted to stalk epitopes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2018102118 ◽

2021 ◽

Vol 118 (8) ◽

pp. e2018102118

Author(s):

Alec W. Freyn ◽

Julianna Han ◽

Jenna J. Guthmiller ◽

Mark J. Bailey ◽

Karlynn Neu ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Secretory Iga ◽

Polymorphonuclear Cell ◽

Specific Antibodies ◽

High Affinity ◽

Influenza Hemagglutinin ◽

Specific Subset ◽

Hemagglutinin Protein ◽

Stalk Domain ◽

Effector Activity

In this study, we utilized a panel of human immunoglobulin (Ig) IgA monoclonal antibodies isolated from the plasmablasts of eight donors after 2014/2015 influenza virus vaccination (Fluarix) to study the binding and functional specificities of this isotype. In this cohort, isolated IgA monoclonal antibodies were primarily elicited against the hemagglutinin protein of the H1N1 component of the vaccine. To compare effector functionalities, an H1-specific subset of antibodies targeting distinct epitopes were expressed as monomeric, dimeric, or secretory IgA, as well as in an IgG1 backbone. When expressed with an IgG Fc domain, all antibodies elicited Fc-effector activity in a primary polymorphonuclear cell-based assay which differs from previous observations that found only stalk-specific antibodies activate the low-affinity FcγRIIIa. However, when expressed with IgA Fc domains, only antibodies targeting the stalk domain showed Fc-effector activity in line with these previous findings. To identify the cause of this discrepancy, we then confirmed that IgG signaling through the high-affinity FcγI receptor was not restricted to stalk epitopes. Since no corresponding high-affinity Fcα receptor exists, the IgA repertoire may therefore be limited to stalk-specific epitopes in the context of Fc receptor signaling.

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Guiding the Immune Response against Influenza Virus Hemagglutinin toward the Conserved Stalk Domain by Hyperglycosylation of the Globular Head Domain

Journal of Virology ◽

10.1128/jvi.02608-13 ◽

2013 ◽

Vol 88 (1) ◽

pp. 699-704 ◽

Cited By ~ 82

Author(s):

D. Eggink ◽

P. H. Goff ◽

P. Palese

Keyword(s):

Immune Response ◽

Influenza Virus ◽

Head Domain ◽

Influenza Virus Hemagglutinin ◽

Stalk Domain ◽

Globular Head

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Influenza Virus Vaccine Based on the Conserved Hemagglutinin Stalk Domain

mBio ◽

10.1128/mbio.00018-10 ◽

2010 ◽

Vol 1 (1) ◽

Cited By ~ 348

Author(s):

John Steel ◽

Anice C. Lowen ◽

Taia T. Wang ◽

Mark Yondola ◽

Qinshan Gao ◽

...

Keyword(s):

Influenza Virus ◽

Virus Vaccine ◽

Neutralizing Antibodies ◽

Influenza Virus Vaccine ◽

Influenza Vaccines ◽

Host Immune System ◽

Stalk Domain ◽

Further Development ◽

Virus Strains ◽

Globular Head

ABSTRACTAlthough highly effective in the general population when well matched to circulating influenza virus strains, current influenza vaccines are limited in their utility due to the narrow breadth of protection they provide. The strain specificity of vaccines presently in use mirrors the exquisite specificity of the neutralizing antibodies that they induce, that is, antibodies which bind to the highly variable globular head domain of hemagglutinin (HA). Herein, we describe the construction of a novel immunogen comprising the conserved influenza HA stalk domain and lacking the globular head. Vaccination of mice with this headless HA construct elicited immune sera with broader reactivity than those obtained from mice immunized with a full-length HA. Furthermore, the headless HA vaccine provided full protection against death and partial protection against disease following lethal viral challenge. Our results suggest that the response induced by headless HA vaccines is sufficiently potent to warrant their further development toward a universal influenza virus vaccine.IMPORTANCECurrent influenza vaccines are effective against only a narrow range of influenza virus strains. It is for this reason that new vaccines must be generated and administered each year. We now report progress toward the goal of an influenza virus vaccine which would protect against multiple strains. Our approach is based on presentation to the host immune system of a region of the influenza virus—called a “headless hemagglutinin” (headless HA)—which is similar among a multitude of diverse strains. We show that vaccination of mice with a headless HA confers protection to these animals against a lethal influenza virus challenge, thereby demonstrating the viability of the approach. Through further development and testing, we predict that a single immunization with a headless HA vaccine will offer effective protection through several influenza epidemics.

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Identification of antibodies targeting the H3N2 hemagglutinin receptor binding site following vaccination of humans

10.1101/675272 ◽

2019 ◽

Author(s):

Seth J. Zost ◽

Juhye Lee ◽

Megan E. Gumina ◽

Kaela Parkhouse ◽

Carole Henry ◽

...

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Binding Site ◽

Receptor Binding ◽

Receptor Binding Site ◽

Antigenic Sites ◽

Influenza Virus Hemagglutinin ◽

Ha Protein

SUMMARYAntibodies targeting the receptor binding site (RBS) of the influenza virus hemagglutinin (HA) protein are usually not broadly-reactive because their footprints are typically large and extend to nearby variable HA residues. Here, we identified several human H3N2 HA RBS-targeting monoclonal antibodies (mAbs) that were sensitive to substitutions in conventional antigenic sites and were not broadly-reactive. However, we also identified one H3N2 HA RBS-targeting mAb that was exceptionally broadly reactive despite being sensitive to substitutions in residues outside of the RBS. We determined that similar antibodies are present at measurable levels in the sera of some individuals but that they are inefficiently elicited by conventional vaccines. Our data indicate that some HA RBS-targeting antibodies can be surprisingly effective against variable viral strains even if they are somewhat sensitive to substitutions in HA residues adjacent to the RBS.

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