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Rapid development of broadly influenza neutralizing antibodies through redundant mutations


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 stem1,2,3,4,5,6. 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 individuals6,7, suggesting that several somatic mutations may be required for their development8,9. 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.

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Figure 1: Rapid affinity maturation and accumulation of redundant somatic mutations in the VH1-69 clone 9.
Figure 2: Rapid affinity maturation through an alternative pathway of redundant mutations in the VH1-69 clone 5.
Figure 3: Genetic requirements and mutational constraints in the HCDR1 and HCDR2 of VH1-69 anti-stem antibodies and the redundant role of F54.
Figure 4: Constraints in the HCDR3 of VH1-69 anti-stem antibodies.


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This work was partly supported by the European Research Council (grant number 250348) IMMUNExplore, the Swiss National Science Foundation (grant number 141254), Fondazione Cariplo Vaccine Program (grant number 2009-3594), the Human Frontiers Science Program (grant number RGP0009/2007-C), the European Commission (grant number FP7-HEALTH-2011-280873) ADITEC and the National Institutes of Health (U19 grant number AI-057266). A.L. is supported by the Helmut Horten Foundation.

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Authors and Affiliations



L.Pa. designed and performed experiments, analysed the data and wrote the manuscript. L.Pa. and M.F. performed phylogenetic analysis. M.F. performed bioinformatic analysis. L.Pi. performed SPR and IF experiments. C.S., B.F.-R., G.A. and I.G.-S. provided technical support for antibody isolation and purification. F.T. and E.V. performed genotypic analysis. N.L.K. and Q.Z. designed, performed and analysed viral neutralization experiments. G.P. provided blood samples from healthy volunteers. F.S. wrote the manuscript. D.C. and A.L. designed the experiments, provided overall supervision and wrote the manuscript.

Corresponding author

Correspondence to Antonio Lanzavecchia.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 VH1-69 antibodies dominate the response to the HA stem and are highly mutated.

a, Frequency of VH gene usage in 197 anti-stem antibodies isolated from the donor analysed in this study. The VH genes are listed in descending order according to their frequency of usage in normal adult human PBMCs as described in ref. 38. b, Load of somatic mutations in the VH of anti-stem antibodies using VH1-69 or other VH genes, compared with anti-head antibodies specific for the H1 CA09 HA. Two-tailed P value was calculated with an unpaired Student’s t-test. ***P ≤ 0.001; *P ≤ 0.05. c, Binding of the UCA IgG antibody (left) and the corresponding mutated antibody (right) to H1 CA09 HA from three representative clones as measured by SPR (complete data set in Supplementary Figs 2 and 3). RU, resonance units.

Extended Data Figure 2 Light chains do not contribute to the binding of VH1-69 antibodies to the HA stem.

Recombinant antibodies were produced using different combinations of VH and VL including a light chain from an antibody of a different specificity, SAC290. The binding of mutated, branchpoint, UCA and VH/VL shuffled antibodies to H1 CA09 HA as measured by ELISA (mean binding titre value of a 1 mg ml−1 antibody solution ± s.e.m. in at least two independent experiments) is shown.

Extended Data Figure 3 Rapid affinity maturation and accumulation of redundant somatic mutations in the VH1-69 clone 1.

a, Alignment of VH amino-acid sequences of mutated antibodies with their UCA and branchpoint configurations. The mutations are highlighted in red. Dots indicate identical residues. Residue positions are annotated according to Kabat numbering. b, Genealogy tree of clone 1 VH nucleotide sequences. c, Binding of mutated, UCA, branchpoint and variant antibodies to H1 CA09 and H5 VN04 HAs. The mean titre value ( ± s.e.m.) (EC50 values of a 1 mg ml−1 antibody solution) of three independent experiments is shown. Mutated residues are shown in parentheses. d, Neutralization of influenza A viruses. IC50 values above 50 µg ml−1 were scored as negative (dotted line). Data are representative of two independent experiments.

Extended Data Figure 4 F29 is not required for high affinity binding of VH1-69 antibodies to the HA stem.

Binding of mutated and variant antibodies to H1 CA09 HA as measured by ELISA. The mean binding titre value ( ± s.e.m.) (EC50 values of a 1 mg ml−1 antibody solution) of at least two independent experiments is shown. Mutated residues are shown in parentheses. Data are representative of two independent experiments.

Extended Data Figure 5 Genetic and mutational requirements in the HCDR1 and HCDR2 of VH1-69 anti-stem antibodies.

a, HCDR1; b, HCDR2. Fully annotated pie charts from Fig. 3a, b. Pie charts indicate the frequency of UCA (white) and mutated residues (IMGT colours), as well as codon usage (underlined nucleotides indicate somatic mutations in that position). UCA codons and amino acids are in red.

Extended Data Figure 6 Frequency of silent and replacing mutations at each codon in the HCDR1 and HCDR2 of VH1-69 anti-stem antibodies.

The replacement to silent mutation ratio (R/S) values calculated at each codon are shown. Values above 2.9, indicative of positive selection, are highlighted in red.

Extended Data Figure 7 Allelic polymorphism in the VH1-69 gene.

a, Nucleotide and b, amino-acid alignments of the 14 VH1-69 alleles. Silent mutations are highlighted in green, replacing mutations in red. Dots indicate identical residues, dashes indicate missing residues in the deposited sequences. c, Summary table of the different VH1-69 allele nomenclatures according to their residue at position 54. See also ref. 39.

Extended Data Figure 8 F54 becomes redundant in the context of highly mutated antibodies.

Binding of branchpoint and mutated antibodies in their original F54 version (grey), as well as in their A54 (black) or L54 (white) versions, to H1 CA09 and H5 VN04 HA. The mean ELISA binding titre value ( ± s.e.m.) (EC50 values of a 1 mg ml−1 antibody solution) of at least two independent experiments is shown.

Extended Data Figure 9 Structural superimposition of the HCDR2 and HCDR3 of antibodies F10, CR6261 and CR9114.

Superimposition of the HCDR2 and HCDR3 of antibodies F10 (pink, PDB 3FKU), CR6261 (red, PDB 3GBM) and CR9114 (yellow, PDB 4FQI) relative to H5 VN04 HA derived by the structural alignment of HA2 atoms on PyMOL, viewed from a different angle compared with Fig. 4c. The antibody F54 and Y98 residues and the conserved HA aromatic residues H18, H38 and W21 are shown as sticks. HCDR2 and HCDR3 amino-acid structural alignments are shown.

Extended Data Figure 10 Pathways for the development of broadly neutralizing anti-stem VH1-69 antibodies.

a, The pie charts outline the fraction of antibodies that follow a major pathway characterized by Y98 and a P52aA/G mutation (exemplified by clones 1, 2, 9, 14 and 19) and alternative pathways found in other clones (exemplified by clone 5). b, Schematic representation of the affinity maturation process leading to broadly neutralizing VH1-69 anti-HA stem antibodies.

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Pappas, L., Foglierini, M., Piccoli, L. et al. Rapid development of broadly influenza neutralizing antibodies through redundant mutations. Nature 516, 418–422 (2014).

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