scholarly journals Mutations in the Hemagglutinin Stalk Domain Do Not Permit Escape from a Protective, Stalk-Based Vaccine-Induced Immune Response in the Mouse Model

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ericka Kirkpatrick Roubidoux ◽  
Juan Manuel Carreño ◽  
Meagan McMahon ◽  
Kaijun Jiang ◽  
Harm van Bakel ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Mouse Model ◽  
Selection Pressure ◽  
Cross Reactivity ◽  
Antigenic Drift ◽  
Escape Mutant ◽  
Induced Response ◽  
Lethal Challenge ◽  
Stalk Domain

ABSTRACT Current seasonal influenza virus vaccines target regions of the hemagglutinin (HA) head domain that undergo constant antigenic change, forcing the painstaking annual reformulation of vaccines. The development of broadly protective or universal influenza virus vaccines that induce cross-reactive, protective immune responses could circumvent the need to reformulate current seasonal vaccines. Many of these vaccine candidates target the HA stalk domain, which displays epitopes conserved within and across influenza virus subtypes, including those with pandemic potential. While HA head-mediated antigenic drift is well understood, the potential for antigenic drift in the stalk domain is understudied. Using a panel of HA stalk-specific monoclonal antibodies (MAbs), we applied selection pressure to the stalk domain of A/Netherlands/602/2009 (pdmH1N1) to determine fitness and phenotypes of escape mutant viruses (EMVs). We found that HA stalk MAbs with lower cross-reactivity caused single HA stalk escape mutations, whereas MAbs with broader cross-reactivity forced multiple mutations in the HA. Each escape mutant virus greatly decreased mAb neutralizing activity, but escape mutations did not always ablate MAb binding or Fc-Fc receptor-based effector functions. Escape mutant viruses were not attenuated in vitro but showed attenuation in an in vivo mouse model. Importantly, mice vaccinated with a chimeric HA universal vaccine candidate were protected from lethal challenge with EMVs despite these challenge viruses containing escape mutations in the stalk domain. Our study indicates that while the HA stalk domain can mutate under strong MAb selection pressure, mutant viruses may have attenuated phenotypes and do not evade a polyclonal, stalk-based vaccine-induced response. IMPORTANCE Broadly protective or universal influenza virus vaccines target viral epitopes that appear to be conserved. However, it is unclear whether the virus will be able to escape once immunological pressure is applied to these epitopes through vaccination of large proportions of the population. Studies that investigate the fitness and antigenic characteristics of viruses that escape immunological pressure on these conserved epitopes are therefore urgently needed.

scholarly journals Pandemic influenza virus vaccines boost hemagglutinin stalk-specific antibody responses in primed adult and pediatric cohorts

npj Vaccines ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Raffael Nachbagauer ◽  
Bruno Salaun ◽  
Daniel Stadlbauer ◽  
Mohammad A. Behzadi ◽  
Damien Friel ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Vaccine ◽  
Pandemic Influenza ◽  
Influenza Virus Vaccine ◽  
Cross Reactivity ◽  
Antigenic Drift ◽  
Lethal Challenge ◽  
Cell Responses ◽  
Adults And Children ◽  
Stalk Domain

AbstractLicensed influenza virus vaccines target the head domain of the hemagglutinin (HA) glycoprotein which undergoes constant antigenic drift. The highly conserved HA stalk domain is an attractive target to increase immunologic breadth required for universal influenza virus vaccines. We tested the hypothesis that immunization with a pandemic influenza virus vaccine boosts pre-existing anti-stalk antibodies. We used chimeric cH6/1, full length H2 and H18 HA antigens in an ELISA to measure anti-stalk antibodies in recipients participating in clinical trials of A/H1N1, A/H5N1 and A/H9N2 vaccines. The vaccines induced high titers of anti-H1 stalk antibodies in adults and children, with higher titers elicited by AS03-adjuvanted vaccines. We also observed cross-reactivity to H2 and H18 HAs. The A/H9N2 vaccine elicited plasmablast and memory B-cell responses. Post-vaccination serum from vaccinees protected mice against lethal challenge with cH6/1N5 and cH5/3N4 viruses. These findings support the concept of a chimeric HA stalk-based universal influenza virus vaccine. clinicaltrials.gov: NCT02415842.

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

2017 ◽  
Vol 7 (1) ◽  
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.

scholarly journals Cross-Protection of Chicken Immunoglobulin Y Antibodies against H5N1 and H1N1 Viruses Passively Administered in Mice

2011 ◽  
Vol 18 (7) ◽  
pp. 1083-1090 ◽  
Author(s):  
Michael G. Wallach ◽  
Richard J. Webby ◽  
Fakhrul Islam ◽  
Stephen Walkden-Brown ◽  
Eva Emmoth ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza Pandemic ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Antigenic Drift ◽  
Virus Infectivity ◽  
Lethal Challenge ◽  
H5n1 Influenza

ABSTRACTInfluenza viruses remain a major threat to global health due to their ability to undergo change through antigenic drift and antigenic shift. We postulated that avian IgY antibodies represent a low-cost, effective, and well-tolerated approach that can easily be scaled up to produce enormous quantities of protective antibodies. These IgY antibodies can be administered passively in humans (orally and intranasally) and can be used quickly and safely to help in the fight against an influenza pandemic. In this study, we raised IgY antibodies against H1N1, H3N2, and H5N1 influenza viruses. We demonstrated that, using whole inactivated viruses alone and in combination to immunize hens, we were able to induce a high level of anti-influenza virus IgY in the sera and eggs, which lasted for at least 2 months after two immunizations. Furthermore, we found that by use ofin vitroassays to test for the ability of IgY to inhibit hemagglutination (HI test) and virus infectivity (serum neutralization test), IgYs inhibited the homologous as well as in some cases heterologous clades and strains of viruses. Using anin vivomouse model system, we found that, when administered intranasally 1 h prior to infection, IgY to H5N1 protected 100% of the mice against lethal challenge with H5N1. Of particular interest was the finding that IgY to H5N1 cross-protected against A/Puerto Rico/8/34 (H1N1) bothin vitroandin vivo. Based on our results, we conclude that anti-influenza virus IgY can be used to help prevent influenza virus infection.

scholarly journals Extending the Stalk Enhances Immunogenicity of the Influenza Virus Neuraminidase

2019 ◽  
Vol 93 (18) ◽  
Author(s):  
Felix Broecker ◽  
Allen Zheng ◽  
Nungruthai Suntronwong ◽  
Weina Sun ◽  
Mark J. Bailey ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Mouse Model ◽  
Influenza Viruses ◽  
Antibody Responses ◽  
H3n2 Virus ◽  
Wild Type ◽  
Virus Challenge ◽  
Virus Particles ◽  
Stalk Domain

ABSTRACT Influenza viruses express two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). Anti-NA antibodies protect from lethal influenza virus challenge in the mouse model and correlate inversely with virus shedding and symptoms in humans. Consequently, the NA is a promising target for influenza virus vaccine design. Current seasonal vaccines, however, poorly induce anti-NA antibodies, partly because of the immunodominance of the HA over the NA when the two glycoproteins are closely associated. To address this issue, here we investigated whether extending the stalk domain of the NA could render it more immunogenic on virus particles. Two recombinant influenza viruses based on the H1N1 strain A/Puerto Rico/8/1934 (PR8) were rescued with NA stalk domains extended by 15 or 30 amino acids. Formalin-inactivated viruses expressing wild-type NA or the stalk-extended NA variants were used to vaccinate mice. The virus with the 30-amino-acid stalk extension induced significantly higher anti-NA IgG responses (characterized by increased in vitro antibody-dependent cellular cytotoxicity [ADCC] activity) than the wild-type PR8 virus, while anti-HA IgG levels were unaffected. Similarly, extending the stalk domain of the NA of a recent H3N2 virus enhanced the induction of anti-NA IgGs in mice. On the basis of these results, we hypothesize that the subdominance of the NA can be modulated if the protein is modified such that its height surpasses that of the HA on the viral membrane. Extending the stalk domain of NA may help to enhance its immunogenicity in influenza virus vaccines without compromising antibody responses to HA. IMPORTANCE The efficacy of influenza virus vaccines could be improved by enhancing the immunogenicity of the NA protein. One of the reasons for its poor immunogenicity is the immunodominance of the HA over the NA in many seasonal influenza virus vaccines. Here we demonstrate that, in the mouse model, extending the stalk domain of the NA protein can enhance its immunogenicity on virus particles and overcome the immunodominance of the HA without affecting antibody responses to the HA. The antibody repertoire is broadened by the extended NA and includes additional ADCC-active antibodies. Our findings may assist in the efforts toward more effective influenza virus vaccines.

scholarly journals The Long Road to a Universal Influenza Virus Vaccine

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 125
Author(s):  
Peter Palese
Keyword(s):  
Influenza Virus ◽  
Virus Vaccine ◽  
Influenza A ◽  
Influenza Virus Vaccine ◽  
Antigenic Drift ◽  
Surface Glycoprotein ◽  
Influenza B ◽  
Virus Infections ◽  
Stalk Domain

Seasonal and pandemic influenza virus infections can cause significant disease worldwide. Current vaccines only provide limited, short-lived protection, and antigenic drift/shift in the hemagglutinin (HA) surface glycoprotein necessitates their annual reformulation and re-administration. To overcome these limitations, universal influenza virus vaccine strategies aim at eliciting broadly protective antibodies to conserved epitopes of the HA. We have developed two approaches. (1) The first is based on “chimeric” HA constructs that retain the conserved stalk domain of the HA and have exotic HA heads. Vaccination and boosting with such constructs successfully redirects the immune system in animals and in humans towards the conserved immune sub-dominant domains of the HA stalks; this results in an antigenic silencing of the HA heads and a protective immune response facilitated by the conserved HA stalks. In mice and ferrets, such a strategy protects the animals against homo-subtypic and hetero-subtypic challenge with influenza A strains as well as against influenza B variants. It is hoped that vaccine constructs expressing three components (i.e., conserved group 1 HA stalks, conserved group 2 HA stalks, and conserved influenza B HA stalks) will be protective against all future seasonal and pandemic strains. (2) The “mosaic” HA approach is based on antigenic silencing of the major immunodominant antigenic sites of the HA heads by only replacing those epitopes with corresponding sequences of exotic avian HAs, yielding “mosaic” HAs. In mice, a prime-boost vaccination regime with inactivated viruses expressing “mosaic” HAs elicited highly cross-reactive antibodies against the stalk domain of the HAs that were capable of eliciting Fc-mediated effector functions in vitro. Extensive trials will be necessary in the future in order to identify the optimal vaccination regime (“chimeric” HA-based versus “mosaic” HA-based) in humans.

scholarly journals A Replication-Defective Influenza Virus Vaccine Confers Complete Protection against H7N9 Viral Infection in Mice

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 207 ◽  
Author(s):  
Shelby Landreth ◽  
Yao Lu ◽  
Kannupriya Pandey ◽  
Yan Zhou
Keyword(s):  
Influenza Virus ◽  
Virus Vaccine ◽  
Cleavage Site ◽  
Case Fatality ◽  
Body Weight Loss ◽  
Wild Type Virus ◽  
Mouse Lung ◽  
H7n9 Virus ◽  
Lethal Challenge

Avian influenza H7N9 viruses continue to pose a great threat to public health, which is evident by their high case-fatality rates. Although H7N9 was first isolated in humans in China in 2013, to date, there is no commercial vaccine available against this particular strain. Our previous studies developed a replication-defective influenza virus through mutation of the hemagglutinin (HA) cleavage site from a trypsin-sensitive to an elastase-sensitive motif. In this study, we report the development of a reassortant mutant influenza virus derived from the human isolate A/British Columbia/01/2015 (H7N9) [BC15 (H7N9)], which is the QVT virus. The HA gene of this virus possesses three mutations at the cleavage site, Lys-Gly-Arg were mutated to Gln-Thr-Val at amino acid (aa) positions 337, 338, and 339, respectively. We report this virus to rely on elastase in vitro, possess unaltered replication abilities when elastase was provided compared to the wild type virus in vitro, and to be non-virulent and replication-defective in mice. In addition, we report this virus to induce significant levels of antibodies and IFN-γ and IL-5 secreting cells, and to protect mice against a lethal challenge of the BC15 (H7N9) virus. This protection is demonstrated through the lack of body weight loss, 100% survival rate, and the prevention of BC15 (H7N9) viral replication as well as the reduction of proinflammatory cytokines induced in the mouse lung associated with the influenza disease. Therefore, these results provide strong evidence for the use of this reassortant mutant H7N9 virus as a replication-defective virus vaccine candidate against H7N9 viruses.

The small molecule AZD6244 inhibits dengue virus replication in vitro and protects against lethal challenge in a mouse model

2020 ◽  
Vol 165 (3) ◽  
pp. 671-681 ◽  
Author(s):  
Leonardo C. de Oliveira ◽  
Aryádina M. Ribeiro ◽  
Jonas D. Albarnaz ◽  
Alice A. Torres ◽  
Luís F. Z. Guimarães ◽  
...  
Keyword(s):  
Mouse Model ◽  
Dengue Virus ◽  
Virus Replication ◽  
Small Molecule ◽  
Lethal Challenge

scholarly journals Publisher Correction: Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Christopher S. Anderson ◽  
Sandra Ortega ◽  
Francisco A. Chaves ◽  
Amelia M. Clark ◽  
Hongmei Yang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Antigenic Drift ◽  
Influenza Virus Hemagglutinin ◽  
Stalk Domain

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

2018 ◽  
Vol 92 (16) ◽  
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.

scholarly journals Protein Microarray Analysis of the Specificity and Cross-Reactivity of Influenza Virus Hemagglutinin-Specific Antibodies

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Rie Nakajima ◽  
Medalyn Supnet ◽  
Algis Jasinskas ◽  
Aarti Jain ◽  
Omid Taghavian ◽  
...  
Keyword(s):  
Influenza Virus ◽  
High Throughput ◽  
Seasonal Influenza ◽  
Vaccine Development ◽  
Public Health Problem ◽  
Cross Reactivity ◽  
Full Length ◽  
Antigenic Drift ◽  
Influenza B Virus ◽  
Influenza B

ABSTRACTCurrent seasonal influenza virus vaccines engender antibody-mediated protection that is hemagglutinin (HA) subtype specific and relatively short-lived. Coverage for other subtypes or even variants within a subtype could be improved from a better understanding of the factors that promote HA-specific antibody cross-reactivity. Current assays to evaluate cross-reactivity, such as the ELISA, require a separate test for each antigen and are neither high-throughput nor sample-sparing. To address this need, we produced an array of 283 purified HA proteins from influenza A virus subtypes H1 to H16 and H18 and influenza B virus. To evaluate performance, arrays were probed with sera from individuals before and after a booster dose of inactivated heterologous H5N1 vaccine and naturally infected cases at presentation and follow-up during the 2010 to 2011 influenza season, when H3N2 was prevalent. The response to the H5 vaccine boost was IgG only and confined to H5 variants. The response to natural H3N2 infection consisted of IgG and IgA and was reactive with all H3 variants displayed, as well as against other group 2 HA subtypes. In both groups, responses to HA1 proteins were subtype specific. In contrast, baseline signals were higher, and responses broader, against full-length HA proteins (HA1+HA2) compared to HA1 alone. We propose that these elevated baseline signals and breadth come from the recognition of conserved epitopes in the stalk domain by cross-reactive antibodies accumulated from previous exposure(s) to seasonal influenza virus. This array is a valuable high-throughput alternative to the ELISA for monitoring specificity and cross-reactivity of HA antibodies and has many applications in vaccine development.IMPORTANCESeasonal influenza is a serious public health problem because the viral infection spreads easily from person to person and because of antigenic drift in neutralizing epitopes. Influenza vaccination is the most effective way to prevent the disease, although challenging because of the constant evolution of influenza virus subtypes. Our high-throughput protein microarrays allow for interrogation of subunit-specific IgG and IgA responses to 283 different HA proteins comprised of HA1 and HA2 domains as well as full-length HA proteins. This provides a tool that allows for novel insights into the response to exposure to influenza virus antigens. Data generated with our technology will enhance our understanding of the factors that improve the strength, breadth, and durability of vaccine-mediated immune responses and develop more effective vaccines.

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