title: Biogenesis of influenza a virus hemagglutinin cross-protective stem epitopes.
abstract: Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Javier G
authors.initials: JG
authors.lastname: Magadán
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Meghan O
authors.initials: MO
authors.lastname: Altman
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: William L
authors.initials: WL
authors.lastname: Ince
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Heather D
authors.initials: HD
authors.lastname: Hickman
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: James
authors.initials: J
authors.lastname: Stevens
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Aaron
authors.initials: A
authors.lastname: Chevalier
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Department of Medicine, Section of Rheumatology, Committee on Immunology, Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois, United States of America.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Wilson
authors.affiliation: Emory Vaccine Center, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Rafi
authors.initials: R
authors.lastname: Ahmed
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Jack R
authors.initials: JR
authors.lastname: Bennink
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Yewdell
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000627:therapeutic use; D000914:Antibodies, Viral / Q000276:immunology; D000915:Antibody Affinity; D000940:Antigenic Variation / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D004285:Dogs; D000939:Epitopes / Q000276:immunology*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000276:immunology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology*; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000276:immunology*; D007252:Influenza Vaccines / Q000276:immunology; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000276:immunology; D014611:Vaccination
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D011994:Recombinant Proteins; C483468:hemagglutinin, human influenza A virus