Antigenic properties of the envelope of influenza virus rendered soluble by surfactant-solvent systems

SUMMARYDissociating chemical treatments employing surfactant-solvent systems were applied to purified influenza A and B viruses to obtain viral preparations possessing a significantly higher or lower haemagglutinating activity than the intact virus. All preparations, whether with high or low haemagglutinating activity, with the exception of envelope protein solubilized by Triton X-100, were significantly lacking in the ability to excite the formation of haemagglutination-inhibiting and virus-neutralizing antibodies in inoculated ferrets. In contrast to other treatments, Triton X-100 treatment of virus significantly enhanced the antigenicity of viral protein as judged by virus neutralization and haemaggmtination inhibition tests. Yet the haemagglutinating activity of the envelope protein solubilized with Triton X-100 was about 1 % that of the intact virus. Results suggest that the correlation assumed to exist between the haemagglutinating activity of influenza virus and its ability to excite the formation of humoral antibodies is coincidental. Another important point is that the specific antigenicity of viral protein may be lost or enhanced owing to effects, other than solubilization, by surface-active agents.

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Heterologous viral protein interactions within licensed seasonal influenza virus vaccines

npj Vaccines ◽

10.1038/s41541-019-0153-1 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 5

Author(s):

Marina Koroleva ◽

Frances Batarse ◽

Savannah Moritzky ◽

Carole Henry ◽

Francisco Chaves ◽

...

Keyword(s):

Influenza Virus ◽

Protein Interactions ◽

Viral Protein ◽

Neutralizing Antibodies ◽

Influenza A ◽

Protein Composition ◽

Viral Proteins ◽

Influenza B ◽

Draining Lymph Node ◽

Antibody Specificities

AbstractCurrently, licensed influenza virus vaccines are designed and tested only for their ability to elicit hemagglutinin (HA)-reactive, neutralizing antibodies. Despite this, the purification process in vaccine manufacturing often does not completely remove other virion components. In the studies reported here, we have examined the viral protein composition of a panel of licensed vaccines from different manufacturers and licensed in different years. Using western blotting, we found that, beyond HA proteins, there are detectable quantities of neuraminidase (NA), nucleoprotein (NP), and matrix proteins (M1) from both influenza A and influenza B viruses in the vaccines but that the composition differed by source and method of vaccine preparation. We also found that disparities in viral protein composition were associated with distinct patterns of elicited antibody specificities. Strikingly, our studies also revealed that many viral proteins contained in the vaccine form heterologous complexes. When H1 proteins were isolated by immunoprecipitation, NA (N1), M1 (M1-A), H3, and HA-B proteins were co-isolated with the H1. Further biochemical studies suggest that these interactions persist for at least 4 h at 37 °C and that the membrane/intracytoplasmic domains in the intact HA proteins are important for the intermolecular interactions detected. These studies indicate that, if such interactions persist after vaccines reach the draining lymph node, both dendritic cells and HA-specific B cells may take up multiple viral proteins simultaneously. Whether these interactions are beneficial or harmful to the developing immune response will depend on the functional potential of the elicited virus-specific CD4 T cells.

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Saturation of influenza virus neutralization and antibody consumption can both lead to bistable growth kinetics

10.1101/2020.03.02.973958 ◽

2020 ◽

Author(s):

Shilian Xu ◽

Ada W. C. Yan ◽

Heidi Peck ◽

Leah Gillespie ◽

Ian G. Barr ◽

...

Keyword(s):

Influenza Virus ◽

Neutralizing Antibodies ◽

Influenza A ◽

Quantitative Relationship ◽

Virus Neutralization ◽

Antibody Concentration ◽

Virus Antibody ◽

Viral Kinetics ◽

Antigenic Properties ◽

Consumption Rates

ABSTRACTInfluenza virus is a major human health threat. Neutralizing antibodies elicited through prior infection or vaccination play an irreplaceable role in protection from subsequent infection. The efficacy of antibody-dependent vaccines relies on both virus replication and neutralization, but their quantitative relationship was unknown. Here we use mathematical models to quantitatively investigate viral survivability determined by antibody concentration and inocula size. We performed focus reduction assays for 49 seasonal influenza A/H3N2 viruses circulating during 2017–2019 against influenza antisera raised in ferrets, and find that the antibody consumption rates of individual reactions were either small or large, and this was strongly positively correlated with virus saturation. Regardless of antibody consumption rate, virus-antibody interactions always lead to antibody-induced bistable viral kinetics. As a result, at a specific interval of antibody concentration, small viral inocula are eliminated but not large virus inocula, which is triggered by saturated virus neutralization or antibody consumption. Our finding highlights virus-antibody interaction with different antigenic properties, thereby explaining commonly observed influenza re-infection and enhancing vaccine efficiency.

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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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A Host-Restricted Self-Attenuated Influenza Virus Provides Broad Pan-Influenza A Protection in a Mouse Model

Frontiers in Immunology ◽

10.3389/fimmu.2021.779223 ◽

2021 ◽

Vol 12 ◽

Author(s):

Minjin Kim ◽

Yucheol Cheong ◽

Jinhee Lee ◽

Jongkwan Lim ◽

Sanguine Byun ◽

...

Keyword(s):

Influenza Virus ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Influenza A ◽

Protective Efficacy ◽

Influenza Viruses ◽

Cross Protection ◽

Infection Model ◽

Wild Type ◽

Group 1

Influenza virus infections can cause a broad range of symptoms, form mild respiratory problems to severe and fatal complications. While influenza virus poses a global health threat, the frequent antigenic change often significantly compromises the protective efficacy of seasonal vaccines, further increasing the vulnerability to viral infection. Therefore, it is in great need to employ strategies for the development of universal influenza vaccines (UIVs) which can elicit broad protection against diverse influenza viruses. Using a mouse infection model, we examined the breadth of protection of the caspase-triggered live attenuated influenza vaccine (ctLAIV), which was self-attenuated by the host caspase-dependent cleavage of internal viral proteins. A single vaccination in mice induced a broad reactive antibody response against four different influenza viruses, H1 and rH5 (HA group 1) and H3 and rH7 subtypes (HA group 2). Notably, despite the lack of detectable neutralizing antibodies, the vaccination provided heterosubtypic protection against the lethal challenge with the viruses. Sterile protection was confirmed by the complete absence of viral titers in the lungs and nasal turbinates after the challenge. Antibody-dependent cellular cytotoxicity (ADCC) activities of non-neutralizing antibodies contributed to cross-protection. The cross-protection remained robust even after in vivo depletion of T cells or NK cells, reflecting the strength and breadth of the antibody-dependent effector function. The robust mucosal secretion of sIgA reflects an additional level of cross-protection. Our data show that the host-restricted designer vaccine serves an option for developing a UIV, providing pan-influenza A protection against both group 1 and 2 influenza viruses. The present results of potency and breadth of protection from wild type and reassortant viruses addressed in the mouse model by single immunization merits further confirmation and validation, preferably in clinically relevant ferret models with wild type challenges.

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Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609316113 ◽

2016 ◽

Vol 113 (42) ◽

pp. 11931-11936 ◽

Cited By ~ 88

Author(s):

Wenqian He ◽

Gene S. Tan ◽

Caitlin E. Mullarkey ◽

Amanda J. Lee ◽

Mannie Man Wai Lam ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Neutralizing Antibodies ◽

Influenza A ◽

Critical Role ◽

Host Cells ◽

Viral Glycoprotein ◽

Effector Functions ◽

Dependent Cell

The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising “universal” influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

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Bayesian inference of antigenic and non-antigenic variables from haemagglutination inhibition assays for influenza surveillance

Royal Society Open Science ◽

10.1098/rsos.180113 ◽

2018 ◽

Vol 5 (7) ◽

pp. 180113

Author(s):

Emmanuel S. Adabor ◽

Wilfred Ndifon

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Bayesian Method ◽

Haemagglutination Inhibition ◽

Virus Evolution ◽

Influenza Viruses ◽

Influenza Surveillance ◽

Antigenic Properties ◽

Analytical Work

Haemagglutination inhibition (HI) assays are typically used for comparing and characterizing influenza viruses. Data obtained from the assays (titres) are used quantitatively to determine antigenic differences between influenza strains. However, the use of these titres has been criticized as they sometimes fail to capture accurate antigenic differences between strains. Our previous analytical work revealed how antigenic and non-antigenic variables contribute to the titres. Building on this previous work, we have developed a Bayesian method for decoupling antigenic and non-antigenic contributions to the titres in this paper. We apply this method to a compendium of HI titres of influenza A (H3N2) viruses curated from 1968 to 2016. Remarkably, the results of this fit indicate that the non-antigenic variable, which is inversely correlated with viral avidity for the red blood cells used in HI assays, oscillates during the course of influenza virus evolution, with a period that corresponds roughly to the timescale on which antigenic variants replace each other. Together, the results suggest that the new Bayesian method is applicable to the analysis of long-term dynamics of both antigenic and non-antigenic properties of influenza virus.

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Divergent Human-Origin Influenza Viruses Detected in Australian Swine Populations

Journal of Virology ◽

10.1128/jvi.00316-18 ◽

2018 ◽

Vol 92 (16) ◽

Cited By ~ 6

Author(s):

Frank Y. K. Wong ◽

Celeste Donato ◽

Yi-Mo Deng ◽

Don Teng ◽

Naomi Komadina ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Swine Influenza ◽

Influenza Viruses ◽

Antigenic Drift ◽

Human Origin ◽

Human Influenza ◽

Influenza A Viruses ◽

Data Set ◽

Antigenic Properties

ABSTRACTGlobal swine populations infected with influenza A viruses pose a persistent pandemic risk. With the exception of a few countries, our understanding of the genetic diversity of swine influenza viruses is limited, hampering control measures and pandemic risk assessment. Here we report the genomic characteristics and evolutionary history of influenza A viruses isolated in Australia from 2012 to 2016 from two geographically isolated swine populations in the states of Queensland and Western Australia. Phylogenetic analysis with an expansive human and swine influenza virus data set comprising >40,000 sequences sampled globally revealed evidence of the pervasive introduction and long-term establishment of gene segments derived from several human influenza viruses of past seasons, including the H1N1/1977, H1N1/1995, H3N2/1968, and H3N2/2003, and the H1N1 2009 pandemic (H1N1pdm09) influenza A viruses, and a genotype that contained gene segments derived from the past three pandemics (1968, reemerged 1977, and 2009). Of the six human-derived gene lineages, only one, comprising two viruses isolated in Queensland during 2012, was closely related to swine viruses detected from other regions, indicating a previously undetected circulation of Australian swine lineages for approximately 3 to 44 years. Although the date of introduction of these lineages into Australian swine populations could not be accurately ascertained, we found evidence of sustained transmission of two lineages in swine from 2012 to 2016. The continued detection of human-origin influenza virus lineages in swine over several decades with little or unpredictable antigenic drift indicates that isolated swine populations can act as antigenic archives of human influenza viruses, raising the risk of reemergence in humans when sufficient susceptible populations arise.IMPORTANCEWe describe the evolutionary origins and antigenic properties of influenza A viruses isolated from two separate Australian swine populations from 2012 to 2016, showing that these viruses are distinct from each other and from those isolated from swine globally. Whole-genome sequencing of virus isolates revealed a high genotypic diversity that had been generated exclusively through the introduction and establishment of human influenza viruses that circulated in past seasons. We detected six reassortants with gene segments derived from human H1N1/H1N1pdm09 and various human H3N2 viruses that circulated during various periods since 1968. We also found that these swine viruses were not related to swine viruses collected elsewhere, indicating independent circulation. The detection of unique lineages and genotypes in Australia suggests that isolated swine populations that are sufficiently large can sustain influenza virus for extensive periods; we show direct evidence of a sustained transmission for at least 4 years between 2012 and 2016.

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Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

Journal of Virology ◽

10.1128/jvi.03099-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3610-3618 ◽

Cited By ~ 50

Author(s):

Wenqian He ◽

Caitlin E. Mullarkey ◽

J. Andrew Duty ◽

Thomas M. Moran ◽

Peter Palese ◽

...

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Virus Vaccine ◽

Neutralizing Antibodies ◽

Influenza A ◽

Human Peripheral Blood ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Broadly Neutralizing Antibodies ◽

Variable Regions

ABSTRACTCurrent influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferiorin vitrobut was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design.IMPORTANCEInfluenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the emergence of pandemic strains, a new class of broadly neutralizing antibodies has been recently discovered and may be the key to developing a “universal” influenza virus vaccine. While much has been learned about the biology of these antibodies, most studies have focused only on monoclonal antibodies of IgG subtypes. However, the study of monoclonal antibodies often fails to capture the complexity of antibody functions that occur during natural polyclonal responses. Here, we provide the first detailed analyses of the biological activity of these antibodies in polyclonal contexts, comparing both IgG and IgA isotypes isolated from human donors. The striking differences observed in the functional properties of broadly neutralizing antibodies in polyclonal contexts will be essential for guiding design of “universal” influenza virus vaccines and therapeutics.

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A small-molecule fragment that emulates binding of receptor and broadly neutralizing antibodies to influenza A hemagglutinin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801999115 ◽

2018 ◽

Vol 115 (16) ◽

pp. 4240-4245 ◽

Cited By ~ 12

Author(s):

Rameshwar U. Kadam ◽

Ian A. Wilson

Keyword(s):

Influenza Virus ◽

Small Molecule ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Influenza A ◽

Binding Mode ◽

Host Cells ◽

Viral Pathogen ◽

Group 2 ◽

Group 1

The influenza virus hemagglutinin (HA) glycoprotein mediates receptor binding and membrane fusion during viral entry in host cells. Blocking these key steps in viral infection has applications for development of novel antiinfluenza therapeutics as well as vaccines. However, the lack of structural information on how small molecules can gain a foothold in the small, shallow receptor-binding site (RBS) has hindered drug design against this important target on the viral pathogen. Here, we report on the serendipitous crystallization-based discovery of a small-molecule N-cyclohexyltaurine, commonly known as the buffering agent CHES, that is able to bind to both group-1 and group-2 HAs of influenza A viruses. X-ray structural characterization of group-1 H5N1 A/Vietnam/1203/2004 (H5/Viet) and group-2 H3N2 A/Hong Kong/1/1968 (H3/HK68) HAs at 2.0-Å and 2.57-Å resolution, respectively, revealed that N-cyclohexyltaurine binds to the heart of the conserved HA RBS. N-cyclohexyltaurine mimics the binding mode of the natural receptor sialic acid and RBS-targeting bnAbs through formation of similar hydrogen bonds and CH-π interactions with the HA. In H3/HK68, N-cyclohexyltaurine also binds to a conserved pocket in the stem region, thereby exhibiting a dual-binding mode in group-2 HAs. These long-awaited structural insights into RBS recognition by a noncarbohydrate-based small molecule enhance our knowledge of how to target this important functional site and can serve as a template to guide the development of novel broad-spectrum small-molecule therapeutics against influenza virus.

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Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin

Science ◽

10.1126/science.aaq0620 ◽

2018 ◽

Vol 362 (6414) ◽

pp. 598-602 ◽

Cited By ~ 73

Author(s):

Nick S. Laursen ◽

Robert H. E. Friesen ◽

Xueyong Zhu ◽

Mandy Jongeneelen ◽

Sven Blokland ◽

...

Keyword(s):

Influenza Virus ◽

Neutralizing Antibodies ◽

Influenza A ◽

Influenza Infection ◽

Cross Reactivity ◽

Broadly Neutralizing Antibodies ◽

Adeno Associated Virus ◽

Influenza Hemagglutinin ◽

Influenza Virus Hemagglutinin ◽

Single Domain Antibodies

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus–mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.

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