Cross-Reactive Neuraminidase-Inhibiting Antibodies Elicited by Immunization with Recombinant Neuraminidase Proteins of H5N1 and Pandemic H1N1 Influenza A Viruses

ABSTRACTNeuraminidase (NA), an influenza virus envelope glycoprotein, removes sialic acid from receptors for virus release from infected cells. For this study, we used a baculovirus-insect cell expression system to construct and purify recombinant NA (rNA) proteins of H5N1 (A/Vietnam/1203/2004) and pandemic H1N1 (pH1N1) (A/Texas/05/2009) influenza viruses. BALB/c mice immunized with these proteins had high titers of NA-specific IgG and NA-inhibiting (NI) antibodies against H5N1, pH1N1, H3N2, and H7N9 viruses. H5N1 rNA immunization resulted in higher quantities of NA-specific antibody-secreting B cells against H5N1 and heterologous pH1N1 viruses in the spleen. H5N1 rNA and pH1N1 rNA immunizations both provided complete protection against homologous virus challenges, with H5N1 rNA immunization providing better protection against pH1N1 virus challenges. Cross-reactive NI antibodies were further dissected via pH1N1 rNA protein immunizations with I149V (NA with a change of Ile to Val at position 149), N344Y, and I365T/S366N NA mutations. The I365T/S366N mutation of pH1N1 rNA enhanced cross-reactive NI antibodies against H5N1, H3N2, and H7N9 viruses. It is our hope that these findings provide useful information for the development of an NA-based universal influenza vaccine.IMPORTANCENeuraminidase (NA) is an influenza virus enzymatic protein that cleaves sialic acid linkages on infected cell surfaces, thus facilitating viral release and contributing to viral transmission and mucus infection. In currently available inactivated or live, attenuated influenza vaccines based on the antigenic content of hemagglutinin proteins, vaccine efficacy can be contributed partly through NA-elicited immune responses. We investigated the NA immunity of different recombinant NA (rNA) proteins associated with pH1N1 and H5N1 viruses. Our results indicate that H5N1 rNA immunization induced more potent cross-protective immunity than pH1N1 rNA immunization, and three mutated residues, I149V, I365T, and S366N, near the NA enzyme active site(s) are linked to enhanced cross-reactive NA-inhibiting antibodies against heterologous and heterosubtypic influenza A viruses. These findings provide useful information for the development of an NA-based universal influenza vaccine.

Download Full-text

Swine Influenza Virus PA and Neuraminidase Gene Reassortment into Human H1N1 Influenza Virus Is Associated with an Altered Pathogenic Phenotype Linked to Increased MIP-2 Expression

Journal of Virology ◽

10.1128/jvi.00087-15 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5651-5667 ◽

Cited By ~ 6

Author(s):

Daniel Dlugolenski ◽

Les Jones ◽

Elizabeth Howerth ◽

David Wentworth ◽

S. Mark Tompkins ◽

...

Keyword(s):

Influenza Virus ◽

Sialic Acid ◽

Influenza A ◽

H1n1 Virus ◽

Swine Influenza ◽

Influenza Viruses ◽

Pandemic H1n1 ◽

Human Influenza ◽

Swine Virus ◽

Virus Reassortment

ABSTRACTSwine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression.IMPORTANCEInfluenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

Download Full-text

Continual Reintroduction of Human Pandemic H1N1 Influenza A Viruses into Swine in the United States, 2009 to 2014

Journal of Virology ◽

10.1128/jvi.00459-15 ◽

2015 ◽

Vol 89 (12) ◽

pp. 6218-6226 ◽

Cited By ~ 59

Author(s):

Martha I. Nelson ◽

Jered Stratton ◽

Mary Lea Killian ◽

Alicia Janas-Martindale ◽

Amy L. Vincent

Keyword(s):

Influenza Virus ◽

Influenza A ◽

The United States ◽

Influenza Viruses ◽

Pandemic H1n1 ◽

Influenza A Viruses ◽

Virus Diversity ◽

Swine Population ◽

The U.S

ABSTRACTThe diversity of influenza A viruses in swine (swIAVs) presents an important pandemic threat. Knowledge of the human-swine interface is particularly important for understanding how viruses with pandemic potential evolve in swine hosts. Through phylogenetic analysis of contemporary swIAVs in the United States, we demonstrate that human-to-swine transmission of pandemic H1N1 (pH1N1) viruses has occurred continuously in the years following the 2009 H1N1 pandemic and has been an important contributor to the genetic diversity of U.S. swIAVs. Although pandemic H1 and N1 segments had been largely removed from the U.S. swine population by 2013 via reassortment with other swIAVs, these antigens reemerged following multiple human-to-swine transmission events during the 2013-2014 seasonal epidemic. These findings indicate that the six internal gene segments from pH1N1 viruses are likely to be sustained long term in the U.S. swine population, with periodic reemergence of pandemic hemagglutinin (HA) and neuraminidase (NA) segments in association with seasonal pH1N1 epidemics in humans. Vaccinating U.S. swine workers may reduce infection of both humans and swine and in turn limit the role of humans as sources of influenza virus diversity in pigs.IMPORTANCESwine are important hosts in the evolution of influenza A viruses with pandemic potential. Here, we analyze influenza virus sequence data generated by the U.S. Department of Agriculture's national surveillance system to identify the central role of humans in the reemergence of pandemic H1N1 (pH1N1) influenza viruses in U.S. swine herds in 2014. These findings emphasize the important role of humans as continuous sources of influenza virus diversity in swine and indicate that influenza viruses with pandemic HA and NA segments are likely to continue to reemerge in U.S. swine in association with seasonal pH1N1 epidemics in humans.

Download Full-text

Influenza: An Emerging and Re-emerging Public Health Threat in Nepal

Annals of Clinical Chemistry and Laboratory Medicine ◽

10.3126/acclm.v3i2.20737 ◽

2018 ◽

Vol 3 (2) ◽

pp. 1-2

Author(s):

Bishnu Prasad Upadhyay

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Winter Season ◽

Emerging Infectious Diseases ◽

Influenza Viruses ◽

Influenza B ◽

Influenza A Viruses ◽

Influenza A H1n1 ◽

New Variant ◽

Seasonal Epidemics

Influenza virus type A and B are responsible for seasonal epidemics as well as pandemics in human. Influenza A viruses are further divided into two major groups namely, low pathogenic seasonal influenza (A/H1N1, A/H1N1 pdm09, A/H3N2) and highly pathogenic influenza virus (H5N1, H5N6, H7N9) on the basis of two surface antigens: hemagglutinin (HA) and neuraminidase (NA). Mutations, including substitutions, deletions, and insertions, are one of the most important mechanisms for producing new variant of influenza viruses. During the last 30 years; more than 50 viral threat has been evolved in South-East Asian countriesof them influenza is one of the major emerging and re-emerging infectious diseases of global concern. Similar to tropical and sub-tropical countries of Southeast Asia; circulation of A/H1N1 pdm09, A/H3N2 and influenza B has been circulating throughout the year with the peak during July-November in Nepal. However; the rate of infection transmission reach peak during the post-rain and winter season of Nepal.

Download Full-text

N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus

Viruses ◽

10.3390/v13050815 ◽

2021 ◽

Vol 13 (5) ◽

pp. 815

Author(s):

Cindy M. Spruit ◽

Nikoloz Nemanichvili ◽

Masatoshi Okamatsu ◽

Hiromu Takematsu ◽

Geert-Jan Boons ◽

...

Keyword(s):

Sialic Acid ◽

Animal Models ◽

Influenza A ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Upper Respiratory Tract ◽

Human Influenza ◽

Influenza A Viruses ◽

Sialic Acid Content ◽

Acetylneuraminic Acid

The first step in influenza virus infection is the binding of hemagglutinin to sialic acid-containing glycans present on the cell surface. Over 50 different sialic acid modifications are known, of which N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two main species. Animal models with α2,6 linked Neu5Ac in the upper respiratory tract, similar to humans, are preferred to enable and mimic infection with unadapted human influenza A viruses. Animal models that are currently most often used to study human influenza are mice and ferrets. Additionally, guinea pigs, cotton rats, Syrian hamsters, tree shrews, domestic swine, and non-human primates (macaques and marmosets) are discussed. The presence of NeuGc and the distribution of sialic acid linkages in the most commonly used models is summarized and experimentally determined. We also evaluated the role of Neu5Gc in infection using Neu5Gc binding viruses and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)-/- knockout mice, which lack Neu5Gc and concluded that Neu5Gc is unlikely to be a decoy receptor. This article provides a base for choosing an appropriate animal model. Although mice are one of the most favored models, they are hardly naturally susceptible to infection with human influenza viruses, possibly because they express mainly α2,3 linked sialic acids with both Neu5Ac and Neu5Gc modifications. We suggest using ferrets, which resemble humans closely in the sialic acid content, both in the linkages and the lack of Neu5Gc, lung organization, susceptibility, and disease pathogenesis.

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

International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE

Clinical and Vaccine Immunology ◽

10.1128/cvi.00278-15 ◽

2015 ◽

Vol 22 (8) ◽

pp. 957-964 ◽

Cited By ~ 28

Author(s):

Karen L. Laurie ◽

Othmar G. Engelhardt ◽

John Wood ◽

Alan Heath ◽

Jacqueline M. Katz ◽

...

Keyword(s):

Influenza Virus ◽

Incubation Time ◽

Influenza A ◽

Influenza Viruses ◽

Enzyme Linked Immunosorbent Assay ◽

Influenza A Viruses ◽

H5n1 Influenza ◽

The World ◽

International Laboratory ◽

The Impact

ABSTRACTThe microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HA MN assay protocols to enable better correlation of these assays in the future.

Download Full-text

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.

Download Full-text

Role of Sialic Acid Binding Specificity of the 1918 Influenza Virus Hemagglutinin Protein in Virulence and Pathogenesis for Mice

Journal of Virology ◽

10.1128/jvi.02596-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3754-3761 ◽

Cited By ~ 50

Author(s):

Li Qi ◽

John C. Kash ◽

Vivien G. Dugan ◽

Ruixue Wang ◽

Guozhong Jin ◽

...

Keyword(s):

Influenza Virus ◽

Sialic Acid ◽

Influenza A ◽

Influenza Pandemic ◽

Binding Specificity ◽

Influenza Viruses ◽

Virulence Determinants ◽

Sialic Acid Binding ◽

1918 Influenza

ABSTRACT The 1918 influenza pandemic caused more than 40 million deaths and likely resulted from the introduction and adaptation of a novel avian-like virus. Influenza A virus hemagglutinins are important in host switching and virulence. Avian-adapted influenza virus hemagglutinins bind sialic acid receptors linked via α2-3 glycosidic bonds, while human-adapted hemagglutinins bind α2-6 receptors. Sequence analysis of 1918 isolates showed hemagglutinin genes with α2-6 or mixed α2-6/α2-3 binding. To characterize the role of the sialic acid binding specificity of the 1918 hemagglutinin, we evaluated in mice chimeric influenza viruses expressing wild-type and mutant hemagglutinin genes from avian and 1918 strains with differing receptor specificities. Viruses expressing 1918 hemagglutinin possessing either α2-6, α2-3, or α2-3/α2-6 sialic acid specificity were fatal to mice, with similar pathology and cellular tropism. Changing α2-3 to α2-6 binding specificity did not increase the lethality of an avian-adapted hemagglutinin. Thus, the 1918 hemagglutinin contains murine virulence determinants independent of receptor binding specificity.

Download Full-text

Resurrected ancestral influenza A viruses recapitulate the avian-to-mammalian adaptation of European avian-like swine influenza virus

10.21203/rs.3.rs-436907/v1 ◽

2021 ◽

Author(s):

Wen Su ◽

Rhodri Harfoot ◽

Yvonne Su ◽

Jennifer DeBeauchamp ◽

Udayan Joseph ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Influenza A ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Polymerase Activity ◽

Influenza Viruses ◽

Mammalian Host ◽

Influenza A Viruses ◽

Sequence Reconstruction

Abstract The emergence of a pandemic influenza virus may be better anticipated if we better understand the evolutionary steps taken by avian influenza viruses as they adapt to mammals. We used ancestral sequence reconstruction to resurrect viruses representing initial adaptive stages of the European avian-like H1N1 virus as it transitioned from avian to swine hosts. We demonstrate that efficient transmissibility in pigs was gained through stepwise adaptation after 1983. These time-dependent adaptations resulted in changes in hemagglutinin receptor binding specificity and increased viral polymerase activity. An NP-R351K mutation under strong positive selection increased the transmissibility of a reconstructed virus. The stepwise-adaptation of a wholly avian influenza virus to a mammalian host suggests a window where targeted intervention may have highest impact. Successful intervention will, however, require strategic coordination of surveillance and risk assessment activities to identify these adapting viruses and guide pandemic preparedness resources.

Download Full-text