title: Rapid development of broadly influenza neutralizing antibodies through redundant mutations.
abstract: The neutralizing antibody response to influenza virus is dominated by antibodies that bind to the globular head of haemagglutinin, which undergoes a continuous antigenic drift, necessitating the re-formulation of influenza vaccines on an annual basis. Recently, several laboratories have described a new class of rare influenza-neutralizing antibodies that target a conserved site in the haemagglutinin stem. Most of these antibodies use the heavy-chain variable region VH1-69 gene, and structural data demonstrate that they bind to the haemagglutinin stem through conserved heavy-chain complementarity determining region (HCDR) residues. However, the VH1-69 antibodies are highly mutated and are produced by some but not all individuals, suggesting that several somatic mutations may be required for their development. To address this, here we characterize 197 anti-stem antibodies from a single donor, reconstruct the developmental pathways of several VH1-69 clones and identify two key elements that are required for the initial development of most VH1-69 antibodies: a polymorphic germline-encoded phenylalanine at position 54 and a conserved tyrosine at position 98 in HCDR3. Strikingly, in most cases a single proline to alanine mutation at position 52a in HCDR2 is sufficient to confer high affinity binding to the selecting H1 antigen, consistent with rapid affinity maturation. Surprisingly, additional favourable mutations continue to accumulate, increasing the breadth of reactivity and making both the initial mutations and phenylalanine at position 54 functionally redundant. These results define VH1-69 allele polymorphism, rearrangement of the VDJ gene segments and single somatic mutations as the three requirements for generating broadly neutralizing VH1-69 antibodies and reveal an unexpected redundancy in the affinity maturation process.
authors.affiliation: Insitute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland.
authors.forename: Leontios
authors.initials: L
authors.lastname: Pappas
authors.affiliation: Insitute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Foglierini
authors.affiliation: Insitute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland.
authors.forename: Luca
authors.initials: L
authors.lastname: Piccoli
authors.affiliation: Department of Infectious Diseases and Vaccines MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland 20878, USA.
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Kallewaard
authors.affiliation: Viral Pathogens and Biosafety Unit, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: Turrini
authors.affiliation: Insitute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland.
authors.forename: Chiara
authors.initials: C
authors.lastname: Silacci
authors.affiliation: Insitute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland.
authors.forename: Blanca
authors.initials: B
authors.lastname: Fernandez-Rodriguez
authors.affiliation: Humabs BioMed SA, Via Mirasole 1, 6500 Bellinzona, Switzerland.
authors.forename: Gloria
authors.initials: G
authors.lastname: Agatic
authors.affiliation: Insitute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland.
authors.forename: Isabella
authors.initials: I
authors.lastname: Giacchetto-Sasselli
authors.affiliation: Unit of Preventive Medicine, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pellicciotta
authors.affiliation: Insitute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland.
authors.forename: Federica
authors.initials: F
authors.lastname: Sallusto
authors.affiliation: Department of Infectious Diseases and Vaccines MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland 20878, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: Viral Pathogens and Biosafety Unit, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Vicenzi
authors.affiliation: 1] Insitute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland [2] Humabs BioMed SA, Via Mirasole 1, 6500 Bellinzona, Switzerland [3].
authors.forename: Davide
authors.initials: D
authors.lastname: Corti
authors.affiliation: 1] Insitute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland [2] Insitute for Microbiology, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland [3].
authors.forename: Antonio
authors.initials: A
authors.lastname: Lanzavecchia
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D057134:Antibodies, Neutralizing / Q000235:genetics*; D002478:Cells, Cultured; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics*; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007251:Influenza, Human / Q000276:immunology* / Q000821:virology; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D009975:Orthomyxoviridae / Q000276:immunology* / Q000378:metabolism; D011110:Polymorphism, Genetic; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D055815:Young Adult
chemical_list: D057134:Antibodies, Neutralizing; D022801:Complementarity Determining Regions; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007143:Immunoglobulin Heavy Chains; C483468:hemagglutinin, human influenza A virus