Construction of a recombinant bacmid DNA containing influenza A virus hemagglutinin gene using a site-specific transposition mechanism

AbstractInfluenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between wild-type IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

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Measuring Site-specific Glycosylation Similarity between Influenza a Virus Variants with Statistical Certainty

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra120.002031 ◽

2020 ◽

Vol 19 (9) ◽

pp. 1533-1545

Author(s):

Deborah Chang ◽

William E. Hackett ◽

Lei Zhong ◽

Xiu-Feng Wan ◽

Joseph Zaia

Keyword(s):

Influenza A Virus ◽

Protein Sequence ◽

Influenza A ◽

Viral Fitness ◽

Antigenic Drift ◽

Viral Escape ◽

Strain Selection ◽

Expression Vectors ◽

Site Specific ◽

Measuring Site

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between WT IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

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5187060 Detection of influenza a virus by polymerase chain reaction (PCR) preceded by reverse transcription of a region of the viral hemagglutinin gene

Biotechnology Advances ◽

10.1016/0734-9750(94)90499-5 ◽

1994 ◽

Vol 12 (1) ◽

pp. 149

Keyword(s):

Polymerase Chain Reaction ◽

Influenza A Virus ◽

Reverse Transcription ◽

Influenza A ◽

Chain Reaction ◽

Viral Hemagglutinin ◽

Hemagglutinin Gene ◽

Polymerase Chain

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Molecular evolution of hemagglutinin gene of Influenza A virus

Frontiers in Bioscience ◽

10.2741/s502 ◽

2018 ◽

Vol 10 (1) ◽

pp. 101-118 ◽

Cited By ~ 3

Author(s):

Antara De

Keyword(s):

Molecular Evolution ◽

Influenza A Virus ◽

Influenza A ◽

Hemagglutinin Gene

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Inclusion of site-specific mutations in the PB1-gene of a virulent A/WSN/33 (H1N1) strain of influenza A virus changes its phenotypic characteristics

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2019-6-21-29 ◽

2019 ◽

pp. 21-29

Author(s):

V. Yu. Kost ◽

O. A. Sukhova ◽

I. I. Akopova ◽

E. O. Gorbacheva ◽

K. V. Lisovskaya ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Crucial Importance ◽

Phenotypic Characteristics ◽

Site Specific ◽

Ann Arbor ◽

H1n1 Strain ◽

New Generation ◽

Ts Mutations ◽

Functional Defects

Aim. Study of changes in the phenotypic characteristics of the virulent A/WSN/33 (H1N1) strain of influenza A virus under the influence of the inclusion of site-specific mutations in the PB1-gene of this strain.Materials and methods. Using a two-step polymerase reaction in the PB1 gene of A/ WSN/33 (H1N1) strain were included ts mutations taken from the genome of attenuated CA donors-strains: A/Ann Arbor/6/60 (H2N2), A/Leningrad 134/17/57 (H2N2) and A/ Krasnodar/101/35/59. Ts-phenotype, att-phenotype, immunogenicity, as well as weight loss in mice infected with these mutants were studied in the obtained site-specific mutants.Results. It was shown that the inclusion of ts mutations from the genome of CA donorsstrains of attenuation in the PB1 gene of the virulent A/WSN/33 (H1N1) strain leads to a change in the phenotypic characteristics of this strain to different degrees.Discussion. Analysis of the genome of CA strains- donors of attenuation of influenza virus indicates the crucial importance of the presence of functional defects in the PB1– protein for the formation of the attenuation phenotype of the virus.Conclusion. The technology of site-specific mutagenesis canbe successfully used to modify the PB1 gene of a virulent influenza A virus strain in order to construct a new generation of live influenza vaccines.

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