A Cross-Reactive Monoclonal Antibody Against Neuraminidases of Both H9N2 and H3N2 Influenza Viruses Shows Protection in Mice Challenging Models

Neuraminidases (NAs) of H9N2 avian influenza virus (AIV) and H3N2 human seasonal influenza virus (HSIV) share similar antigenic structures. However, there are few reports on epitopes shared by these two NAs. We previously reported a monoclonal antibody (mAb) 1G8 against the NA of H9N2 AIV with neuraminidase inhibition (NI) ability. In this study, 1G8 was shown to cross-react with and inhibit the NA of H3N2 HSIV. In a passive transfer experiment, 1G8 provided protection to mice challenged with rescued H1N2 viruses carrying H9N2 NA or H3N2 NA. Mutation at amino acid position 199 was also selected and proved to be crucial for H3N2 HSIV to escape from mAb 1G8. Moreover, we found that residue 199 contributed to inducing broad protective antibodies without the influence of the N-linked glycosylation at amino acid position 200 in NAs. Residues as residue 199, which are not shielded by glycosylation modification, would form ideal epitopes for developing universal vaccine and protective antibodies.

Download Full-text

Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

Scientific Reports ◽

10.1038/s41598-020-79590-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

James D. Allen ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Next Generation ◽

Virus Infections ◽

Wild Type ◽

Influenza Hemagglutinin ◽

H3n2 Influenza

AbstractWhile vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

Download Full-text

Computationally Optimized Broadly Reactive Hemagglutinin Elicits Hemagglutination Inhibition Antibodies against a Panel of H3N2 Influenza Virus Cocirculating Variants

Journal of Virology ◽

10.1128/jvi.01581-17 ◽

2017 ◽

Vol 91 (24) ◽

Cited By ~ 29

Author(s):

Terianne M. Wong ◽

James D. Allen ◽

Anne-Gaelle Bebin-Blackwell ◽

Donald M. Carter ◽

Timothy Alefantis ◽

...

Keyword(s):

Influenza Virus ◽

Influenza Vaccine ◽

Influenza Viruses ◽

Influenza Vaccines ◽

Influenza B ◽

Wild Type ◽

Protective Antibodies ◽

H3n2 Influenza ◽

H3n2 Influenza Virus

ABSTRACT Each influenza season, a set of wild-type viruses, representing one H1N1, one H3N2, and one to two influenza B isolates, are selected for inclusion in the annual seasonal influenza vaccine. In order to develop broadly reactive subtype-specific influenza vaccines, a methodology called computationally optimized broadly reactive antigens (COBRA) was used to design novel hemagglutinin (HA) vaccine immunogens. COBRA technology was effectively used to design HA immunogens that elicited antibodies that neutralized H5N1 and H1N1 isolates. In this report, the development and characterization of 17 prototype H3N2 COBRA HA proteins were screened in mice and ferrets for the elicitation of antibodies with HA inhibition (HAI) activity against human seasonal H3N2 viruses that were isolated over the last 48 years. The most effective COBRA HA vaccine regimens elicited antibodies with broader HAI activity against a panel of H3N2 viruses than wild-type H3 HA vaccines. The top leading COBRA HA candidates were tested against cocirculating variants. These variants were not efficiently detected by antibodies elicited by the wild-type HA from viruses selected as the vaccine candidates. The T-11 COBRA HA vaccine elicited antibodies with HAI and neutralization activity against all cocirculating variants from 2004 to 2007. This is the first report demonstrating broader breadth of vaccine-induced antibodies against cocirculating H3N2 strains compared to the wild-type HA antigens that were represented in commercial influenza vaccines. IMPORTANCE There is a need for an improved influenza vaccine that elicits immune responses that recognize a broader number of influenza virus strains to prevent infection and transmission. Using the COBRA approach, a set of vaccines against influenza viruses in the H3N2 subtype was tested for the ability to elicit antibodies that neutralize virus infection against not only historical vaccine strains of H3N2 but also a set of cocirculating variants that circulated between 2004 and 2007. Three of the H3N2 COBRA vaccines recognized all of the cocirculating strains during this era, but the chosen wild-type vaccine strains were not able to elicit antibodies with HAI activity against these cocirculating strains. Therefore, the COBRA vaccines have the ability to elicit protective antibodies against not only the dominant vaccine strains but also minor circulating strains that can evolve into the dominant vaccine strains in the future.

Download Full-text

Elicitation of Protective Antibodies against 20 Years of Future H3N2 Cocirculating Influenza Virus Variants in Ferrets Preimmune to Historical H3N2 Influenza Viruses

Journal of Virology ◽

10.1128/jvi.00946-18 ◽

2018 ◽

Vol 93 (3) ◽

Cited By ~ 10

Author(s):

James D. Allen ◽

Hyesun Jang ◽

Joshua DiNapoli ◽

Harold Kleanthous ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Neutralizing Antibodies ◽

Influenza Viruses ◽

Human Condition ◽

World Health ◽

Virus Infections ◽

Wild Type ◽

Virus Like Particle ◽

Protective Antibodies ◽

H3n2 Influenza

ABSTRACTThe vast majority of people already have preexisting immune responses to influenza viruses from one or more subtypes. However, almost all preclinical studies evaluate new influenza vaccine candidates in immunologically naive animals. Recently, our group demonstrated that priming naive ferrets with broadly reactive H1 COBRA HA-based vaccines boosted preexisting antibodies induced by wild-type H1N1 virus infections. These H1 COBRA hemagglutinin (HA) antigens induced antibodies with HAI activity against multiple antigenically different H1N1 viral variants. In this study, ferrets, preimmune to historical H3N2 viruses, were vaccinated with virus-like particle (VLP) vaccines expressing either an HA from a wild-type H3 influenza virus or a COBRA H3 HA antigen (T6, T7, T10, or T11). The elicited antisera had the ability to neutralize virus infection against either a panel of viruses representing vaccine strains selected by the World Health Organization or a set of viral variants that cocirculated during the same time period. Preimmune animals vaccinated with H3 COBRA T10 HA antigen elicited sera with higher hemagglutination inhibition (HAI) antibody titers than antisera elicited by VLP vaccines with wild-type HA VLPs in preimmune ferrets. However, while the T11 COBRA vaccine did not elicit HAI activity, the elicited antibodies did neutralize antigenically distinct H3N2 influenza viruses. Overall, H3 COBRA-based HA vaccines were able to neutralize both historical H3 and contemporary, as well as future, H3N2 viruses with higher titers than vaccines with wild-type H3 HA antigens. This is the first report demonstrating the effectiveness of a broadly reactive H3N3 vaccine in a preimmune ferret model.IMPORTANCEAfter exposure to influenza virus, the host generates neutralizing anti-hemagglutinin (anti-HA) antibodies against that specific infecting influenza strain. These antibodies can also neutralize some, but not all, cocirculating strains. The goal of next-generation influenza vaccines, such as HA head-based COBRA, is to stimulate broadly protective neutralizing antibodies against all strains circulating within a subtype, in particular those that persist over multiple influenza seasons, without requiring an update to the vaccine. To mimic the human condition, COBRA HA virus-like particle vaccines were tested in ferrets that were previously exposed to historical H3N2 influenza viruses. In this model, these vaccines elicited broadly protective antibodies that neutralized cocirculating H3N2 influenza viruses isolated over a 20-year period. This is the first study to show the effectiveness of H3N3 COBRA HA vaccines in a host with preexisting immunity to influenza.

Download Full-text

Isolation of novel triple-reassortant swine H3N2 influenza viruses possessing the hemagglutinin and neuraminidase genes of a seasonal influenza virus in Vietnam in 2010

Influenza and Other Respiratory Viruses ◽

10.1111/j.1750-2659.2011.00267.x ◽

2011 ◽

Vol 6 (1) ◽

pp. 6-10 ◽

Cited By ~ 23

Author(s):

Long Thanh Ngo ◽

Yasuaki Hiromoto ◽

Vu Phong Pham ◽

Ha Thi Hong Le ◽

Ha Truc Nguyen ◽

...

Keyword(s):

Influenza Virus ◽

Seasonal Influenza ◽

Influenza Viruses ◽

H3n2 Influenza

Download Full-text

Generation of a recombinant chickenized monoclonal antibody against the neuraminidase of H9N2 avian influenza virus

AMB Express ◽

10.1186/s13568-020-01086-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Fei Wang ◽

Yajuan Wang ◽

Zhimin Wan ◽

Hongxia Shao ◽

Kun Qian ◽

...

Keyword(s):

Monoclonal Antibody ◽

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

H9n2 Avian Influenza Virus

Download Full-text

Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

Journal of Virology ◽

10.1128/jvi.75.17.8127-8136.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 8127-8136 ◽

Cited By ~ 78

Author(s):

Daniel R. Perez ◽

Ruben O. Donis

Keyword(s):

Amino Acids ◽

Influenza Virus ◽

Amino Acid ◽

Viral Replication ◽

Influenza A Virus ◽

Influenza A ◽

Amino Acid Sequences ◽

Influenza Viruses ◽

Virus Growth ◽

Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

Download Full-text

Molecular Characterization of Seasonal Influenza A and B from Hospitalized Patients in Thailand in 2018–2019

Viruses ◽

10.3390/v13060977 ◽

2021 ◽

Vol 13 (6) ◽

pp. 977

Author(s):

Kobporn Boonnak ◽

Chayasin Mansanguan ◽

Dennis Schuerch ◽

Usa Boonyuen ◽

Hatairat Lerdsamran ◽

...

Keyword(s):

Amino Acid ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Surface Proteins ◽

Antigenic Drift ◽

Influenza B ◽

Receptor Binding Site ◽

Antigenic Sites ◽

Ha Protein

Influenza viruses continue to be a major public health threat due to the possible emergence of more virulent influenza virus strains resulting from dynamic changes in virus adaptability, consequent of functional mutations and antigenic drift in surface proteins, especially hemagglutinin (HA) and neuraminidase (NA). In this study, we describe the genetic and evolutionary characteristics of H1N1, H3N2, and influenza B strains detected in severe cases of seasonal influenza in Thailand from 2018 to 2019. We genetically characterized seven A/H1N1 isolates, seven A/H3N2 isolates, and six influenza B isolates. Five of the seven A/H1N1 viruses were found to belong to clade 6B.1 and were antigenically similar to A/Switzerland/3330/2017 (H1N1), whereas two isolates belonged to clade 6B.1A1 and clustered with A/Brisbane/02/2018 (H1N1). Interestingly, we observed additional mutations at antigenic sites (S91R, S181T, T202I) as well as a unique mutation at a receptor binding site (S200P). Three-dimensional (3D) protein structure analysis of hemagglutinin protein reveals that this unique mutation may lead to the altered binding of the HA protein to a sialic acid receptor. A/H3N2 isolates were found to belong to clade 3C.2a2 and 3C.2a1b, clustering with A/Switzerland/8060/2017 (H3N2) and A/South Australia/34/2019 (H3N2), respectively. Amino acid sequence analysis revealed 10 mutations at antigenic sites including T144A/I, T151K, Q213R, S214P, T176K, D69N, Q277R, N137K, N187K, and E78K/G. All influenza B isolates in this study belong to the Victoria lineage. Five out of six isolates belong to clade 1A3-DEL, which relate closely to B/Washington/02/2009, with one isolate lacking the three amino acid deletion on the HA segment at position K162, N163, and D164. In comparison to the B/Colorado/06/2017, which is the representative of influenza B Victoria lineage vaccine strain, these substitutions include G129D, G133R, K136E, and V180R for HA protein. Importantly, the susceptibility to oseltamivir of influenza B isolates, but not A/H1N1 and A/H3N2 isolates, were reduced as assessed by the phenotypic assay. This study demonstrates the importance of monitoring genetic variation in influenza viruses regarding how acquired mutations could be associated with an improved adaptability for efficient transmission.

Download Full-text

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.

Download Full-text

Broadly Reactive H2 Hemagglutinin Vaccines Elicit Cross-Reactive Antibodies in Ferrets Preimmune to Seasonal Influenza A Viruses

mSphere ◽

10.1128/msphere.00052-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Z. Beau Reneer ◽

Amanda L. Skarlupka ◽

Parker J. Jamieson ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Recombinant Proteins ◽

Human Population ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Wild Type ◽

Influenza A Viruses ◽

Global Pandemic

ABSTRACT Influenza vaccines have traditionally been tested in naive mice and ferrets. However, humans are first exposed to influenza viruses within the first few years of their lives. Therefore, there is a pressing need to test influenza virus vaccines in animal models that have been previously exposed to influenza viruses before being vaccinated. In this study, previously described H2 computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) vaccines (Z1 and Z5) were tested in influenza virus “preimmune” ferret models. Ferrets were infected with historical, seasonal influenza viruses to establish preimmunity. These preimmune ferrets were then vaccinated with either COBRA H2 HA recombinant proteins or wild-type H2 HA recombinant proteins in a prime-boost regimen. A set of naive preimmune or nonpreimmune ferrets were also vaccinated to control for the effects of the multiple different preimmunities. All of the ferrets were then challenged with a swine H2N3 influenza virus. Ferrets with preexisting immune responses influenced recombinant H2 HA-elicited antibodies following vaccination, as measured by hemagglutination inhibition (HAI) and classical neutralization assays. Having both H3N2 and H1N1 immunological memory regardless of the order of exposure significantly decreased viral nasal wash titers and completely protected all ferrets from both morbidity and mortality, including the mock-vaccinated ferrets in the group. While the vast majority of the preimmune ferrets were protected from both morbidity and mortality across all of the different preimmunities, the Z1 COBRA HA-vaccinated ferrets had significantly higher antibody titers and recognized the highest number of H2 influenza viruses in a classical neutralization assay compared to the other H2 HA vaccines. IMPORTANCE H1N1 and H3N2 influenza viruses have cocirculated in the human population since 1977. Nearly every human alive today has antibodies and memory B and T cells against these two subtypes of influenza viruses. H2N2 influenza viruses caused the 1957 global pandemic and people born after 1968 have never been exposed to H2 influenza viruses. It is quite likely that a future H2 influenza virus could transmit within the human population and start a new global pandemic, since the majority of people alive today are immunologically naive to viruses of this subtype. Therefore, an effective vaccine for H2 influenza viruses should be tested in an animal model with previous exposure to influenza viruses that have circulated in humans. Ferrets were infected with historical influenza A viruses to more accurately mimic the immune responses in people who have preexisting immune responses to seasonal influenza viruses. In this study, preimmune ferrets were vaccinated with wild-type (WT) and COBRA H2 recombinant HA proteins in order to examine the effects that preexisting immunity to seasonal human influenza viruses have on the elicitation of broadly cross-reactive antibodies from heterologous vaccination.

Download Full-text