Mutations in influenza A virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody

Kristina L. Prachanronarong, Aneth S. Canale, Ping Liu, Mohan Somasundaran, Shurong Hou, Yu Ping Poh, Thomas Han, Quan Zhu, Nicholas Renzette, Konstantin B. Zeldovich, Timothy F. Kowalik, Nese Kurt-Yilmaz, Jeffrey Jensen, Daniel N.A. Bolon, Wayne A. Marasco, Robert W. Finberg, Celia A. Schiffer, Jennifer P. Wang

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure. IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

Original languageEnglish (US)
Article numbere01639-18
JournalJournal of Virology
Volume93
Issue number2
DOIs
StatePublished - Jan 1 2019

Fingerprint

sialidase
Influenza A virus
Hemagglutinins
hemagglutinins
neutralization
Neuraminidase
mutation
Mutation
antibodies
stems
Antibodies
Orthomyxoviridae
Viruses
Neutralizing Antibodies
neutralizing antibodies
viruses
epitopes
Epitopes
influenza A virus NA protein
Genome

Keywords

  • Broadly neutralizing antibody
  • Hemagglutinin
  • Influenza virus
  • Mutants
  • Neuraminidase
  • Resistance

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Prachanronarong, K. L., Canale, A. S., Liu, P., Somasundaran, M., Hou, S., Poh, Y. P., ... Wang, J. P. (2019). Mutations in influenza A virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody. Journal of Virology, 93(2), [e01639-18]. https://doi.org/10.1128/JVI.01639-18

Mutations in influenza A virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody. / Prachanronarong, Kristina L.; Canale, Aneth S.; Liu, Ping; Somasundaran, Mohan; Hou, Shurong; Poh, Yu Ping; Han, Thomas; Zhu, Quan; Renzette, Nicholas; Zeldovich, Konstantin B.; Kowalik, Timothy F.; Kurt-Yilmaz, Nese; Jensen, Jeffrey; Bolon, Daniel N.A.; Marasco, Wayne A.; Finberg, Robert W.; Schiffer, Celia A.; Wang, Jennifer P.

In: Journal of Virology, Vol. 93, No. 2, e01639-18, 01.01.2019.

Research output: Contribution to journalArticle

Prachanronarong, KL, Canale, AS, Liu, P, Somasundaran, M, Hou, S, Poh, YP, Han, T, Zhu, Q, Renzette, N, Zeldovich, KB, Kowalik, TF, Kurt-Yilmaz, N, Jensen, J, Bolon, DNA, Marasco, WA, Finberg, RW, Schiffer, CA & Wang, JP 2019, 'Mutations in influenza A virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody', Journal of Virology, vol. 93, no. 2, e01639-18. https://doi.org/10.1128/JVI.01639-18
Prachanronarong, Kristina L. ; Canale, Aneth S. ; Liu, Ping ; Somasundaran, Mohan ; Hou, Shurong ; Poh, Yu Ping ; Han, Thomas ; Zhu, Quan ; Renzette, Nicholas ; Zeldovich, Konstantin B. ; Kowalik, Timothy F. ; Kurt-Yilmaz, Nese ; Jensen, Jeffrey ; Bolon, Daniel N.A. ; Marasco, Wayne A. ; Finberg, Robert W. ; Schiffer, Celia A. ; Wang, Jennifer P. / Mutations in influenza A virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody. In: Journal of Virology. 2019 ; Vol. 93, No. 2.
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AU - Somasundaran, Mohan

AU - Hou, Shurong

AU - Poh, Yu Ping

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AU - Kowalik, Timothy F.

AU - Kurt-Yilmaz, Nese

AU - Jensen, Jeffrey

AU - Bolon, Daniel N.A.

AU - Marasco, Wayne A.

AU - Finberg, Robert W.

AU - Schiffer, Celia A.

AU - Wang, Jennifer P.

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N2 - Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure. IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

AB - Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure. IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

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