Department of Biochemistry and Molecular Pharmacology; Department of Medicine, Division of Infectious Diseases and Immunology; Department of Microbiology and Physiological Systems; Program in Bioinformatics and Integrative Biology; Graduate School of Biomedical Sciences; Schiffer Lab
Biochemistry, Biophysics, and Structural Biology | Bioinformatics | Computational Biology | Immunity | Immunology of Infectious Disease | Immunoprophylaxis and Therapy | Influenza Humans | Influenza Virus Vaccines | Integrative Biology | Therapeutics | Virology | Viruses
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 influenza 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 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 influenza hemagglutinin (HA) can neutralize many influenza 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 with 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.
hemagglutinin and neuraminidase mutants, resistance, broadly neutralizing antibody, influenza virus
Rights and Permissions
Copyright © 2018, American Society for Microbiology. Accepted manuscript posted after 6 months as allowed by the publisher's author rights policy at https://journals.asm.org/content/statement-author-rights.
DOI of Published Version
J Virol. 2018 Oct 31. pii: JVI.01639-18. doi: 10.1128/JVI.01639-18. [Epub ahead of print] Link to article on publisher's site
Journal of virology
Prachanronarong KL, Canale AS, Liu P, Somasundaran M, Hou S, Poh Y, Han T, Zhu Q, Renzette N, Zeldovich KB, Kowalik TF, Yilmaz NK, Jensen JD, Bolon DN, Marasco WA, Finberg RW, Schiffer CA, Wang JP. (2018). Mutations in influenza A virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody. Schiffer Lab Publications. https://doi.org/10.1128/JVI.01639-18. Retrieved from https://escholarship.umassmed.edu/schiffer/32
Biochemistry, Biophysics, and Structural Biology Commons, Bioinformatics Commons, Computational Biology Commons, Immunity Commons, Immunology of Infectious Disease Commons, Immunoprophylaxis and Therapy Commons, Influenza Humans Commons, Influenza Virus Vaccines Commons, Integrative Biology Commons, Therapeutics Commons, Virology Commons, Viruses Commons