Research ArticleVACCINES

Prolonged evolution of the memory B cell response induced by a replicating adenovirus-influenza H5 vaccine

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Science Immunology  19 Apr 2019:
Vol. 4, Issue 34, eaau2710
DOI: 10.1126/sciimmunol.aau2710
  • Fig. 1 Intranasal Ad4-H5-Vtn vaccination induces durable serum neutralizing antibodies with breadth that extends to group 2 viruses.

    (A) Serum neutralization of H5N1 elicited by Ad4-H5-Vtn immunization by the indicated route. Serum samples collected at weeks 8 (W8) and 26 were analyzed in the H5N1 PVEI assay. P values were calculated using t tests. ns, not significant. (B) Serum neutralization of group 1 or 2 influenza A or influenza B strains in the PVEI assay. (C) ELISA binding of serum from six vaccinees to group 1 or 2 influenza A or influenza B HA proteins. AUC, area under curve.

  • Fig. 2 A subset of isolated antibodies are particularly broad and potent.

    (A) Neutralization of multiple group 1 or 2 influenza strains assessed using the PVEI assay. Antibodies isolated from sorted B cells of Ad4-H5-Vtn vaccinees are displayed on the left and controls on the right. IC50 values of <0.1 μg/ml are highlighted in red, between 0.1 and 1 μg/ml in orange, between 1 and 10 μg/ml in yellow, and between 10 and 50 μg/ml in green. Any IC50 values of >50 μg/ml were excluded from median IC50 calculations for each antibody displayed. (B) Breadth and potency curve of four mAbs isolated from sorted B cells. The IC50 against influenza B was excluded from the calculation. (C) SPR binding of 429 B01 to HA1, HA2, or HA0 of the indicated influenza viruses. RU, resonance units. NB, no binding. (D) ELISA binding to H5 HA protein of 429 B01 alone (empty circles) or in competition with F10 (solid circles). CR6261 was used as a positive control (right). Ab, antibody. RLU, relative light units.

  • Fig. 3 Structural basis of HA recognition by the 429 B01 class of antibodies.

    (A) Negative-stain EM of antibody in complex with HA as indicated. Scale bars, 10 nm. (B) Cocrystal structure of 429 B01 in complex with HK68 H3 HA. HA1, HA2, and antibody heavy and light chains are depicted in cartoons colored green, cyan, orange, and slate, respectively. Inset: A 90° view of the interface with interacting CDR loops shown in cartoon and HA as surface representation. (C) Epitope of 429 B01 colored in orange. A black dashed arrow indicates the orientation of the antibody defined by the line connecting Cα atoms of heavy chain Cys22 (orange circle with H inside) and light chain Cys23 (purple circle with L inside). (D) Detailed interactions of 429 B01 with HA. Residues from CDR H3, CDR L2, and L3 of 429 B01 form extensive hydrogen bonds with HA. (E) Epitope of antibody 39.29 (salmon) derived from the same germline gene as 429 B01 overlapped with that of 429 B01 (black lines). (F) Position of the CDR H3 and residues conserved between 429 B01 and 39.29 after superposition over the HA2 region. (G) Sequence alignment of 429 B01 with deduced germline sequences. Paratope residues are colored as in (B). Sequences of 39.29 are shown to highlight the conservation of a Val-Phe-Gly motif in the CDR H3.

  • Fig. 4 Dominant interaction at the tip of CDR H3 encoded by the IGHD3-03 gene.

    (A) Analysis of the junctional sequence of 429 B01. Germline gene-encoded nucleotide and amino acid residues are shown in black with the corresponding junctions colored in light blue. Somatically mutated amino acids and nucleotides are colored red. Nucleotides deleted by exonuclease trimming are crossed out. (B) Sequence alignment of the CDR H3 of 429 B01 in comparison with published stem-specific antibodies. The HD3-3–encoded motif was conserved between antibodies derived from varied immunoglobulin heavy chain variable region (IGHV) genes. (C) Comparison of binding modes of HA stem–specific antibodies sharing the same HD3-3 gene.

  • Fig. 5 Expansion of H5-specific B cells is prolonged after Ad4-H5-Vtn vaccination.

    (A) Expansion of H5- or H1H5-specific B cell populations at sequential time points after Ad4-H5-Vtn vaccination. Vaccinee 849 is presented. B cells were stained with HA probes H5 (A/Vietnam /1203/2004) and H1 (A/New Caledonia/20/1999). (B) Summary of expansion of H5-, H1-, or H1H5-specific peripheral blood memory B cell populations in all six subjects. (C) Last day of Ad4 shedding detected in each of the six participants.

  • Fig. 6 Increase in neutralization potency of H5-specific antibodies parallels increases in SHM and affinity.

    (A) Neutralization by H5- or H1H5-specific mAbs against H5-Vtn in the PVEI assay over time. The median IC50 for each time point is displayed. P values were calculated using t tests. (B) Affinity of H5-specific or H1H5-specific mAbs against H5N1 Thailand HA over time. The median affinity for each time point is shown. (C) Antibody pairs isolated from week 4 and later time points with the same heavy and light chain alleles, CDR3 lengths, and junction sequences. H5-specific antibodies are indicated in red, and H1H5-specific antibodies are indicated in blue. (D) Mutation rate (left), affinity toward H5 HA protein calculated on the basis of biolayer interferometry analysis (middle), and neutralization of H5-Vtn assessed by the PVEI assay (right) of antibody pairs. KD, dissociation constant.

  • Fig. 7 Evolution of SHM and clonal expansions is prolonged after Ad4-H5-Vtn vaccination.

    (A) Mutation frequency of H5- or H1H5-specific antibody heavy chain sequences isolated from participants at various time points after vaccination by single-cell sorting. Red horizontal bars indicate the median. (B) Mutation rate of light chain sequences. (C) Dynamics of clonal size of H5- or H1H5-specific antibody sequences. Clonal family members were identified from the NGS dataset of each time point. The size of each clonal family at a time point was normalized by the number of NGS reads. Each color represents a clonal family with the width of the stacked ribbon showing the number of members.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/34/eaau2710/DC1

    Materials and Methods

    Fig. S1. Influence of vaccination route and dose on H5 antibody responses.

    Fig. S2. H1H5- or H5-specific antibody responses in PVEI, MN, and HAI assays.

    Fig. S3. ELISA binding of H1H5- or H5-specific monoclonal antibodies to group 1 or 2 influenza A or influenza B (Massachusetts/2/12) HA proteins.

    Fig. S4. Recognition of HA by IGHV3-30–derived antibodies.

    Fig. S5. ELISA binding of H1H5- or H5-specific monoclonal antibodies isolated before vaccination to H5 Vietnam or H1 New Caledonia HA proteins.

    Fig. S6. Increase in affinity of H5-specific antibodies and avidity maturation of sera against HA1 or HA2 subunits.

    Fig. S7. Alignment of antibody clones that are members of three previously described multidonor classes of HA-specific broadly neutralizing antibodies.

    Fig. S8. SHM of H1-specific antibodies over time.

    Fig. S9. Gating strategy for analyzing and sorting.

    Table S1. Serum neutralization of multiple influenza strains in the PVEI assay after Ad4-H5-Vtn vaccination.

    Table S2. Neutralizing activity of monoclonal antibodies isolated from vaccinees against a five-virus panel.

    Table S3. Crystallographic data collection and refinement statistics.

    Table S4. Composition of 429 B01 epitope and paratope.

    Table S5. Hydrogen bonds between HA and 429 B01 antibody.

    Table S6. Virus strain abbreviations.

    Data file S1.

    References (3746)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Influence of vaccination route and dose on H5 antibody responses.
    • Fig. S2. H1H5- or H5-specific antibody responses in PVEI, MN, and HAI assays.
    • Fig. S3. ELISA binding of H1H5- or H5-specific monoclonal antibodies to group 1 or 2 influenza A or influenza B (Massachusetts/2/12) HA proteins.
    • Fig. S4. Recognition of HA by IGHV3-30–derived antibodies.
    • Fig. S5. ELISA binding of H1H5- or H5-specific monoclonal antibodies isolated before vaccination to H5 Vietnam or H1 New Caledonia HA proteins.
    • Fig. S6. Increase in affinity of H5-specific antibodies and avidity maturation of sera against HA1 or HA2 subunits.
    • Fig. S7. Alignment of antibody clones that are members of three previously described multidonor classes of HA-specific broadly neutralizing antibodies.
    • Fig. S8. SHM of H1-specific antibodies over time.
    • Fig. S9. Gating strategy for analyzing and sorting.
    • Table S1. Serum neutralization of multiple influenza strains in the PVEI assay after Ad4-H5-Vtn vaccination.
    • Table S2. Neutralizing activity of monoclonal antibodies isolated from vaccinees against a five-virus panel.
    • Table S3. Crystallographic data collection and refinement statistics.
    • Table S4. Composition of 429 B01 epitope and paratope.
    • Table S5. Hydrogen bonds between HA and 429 B01 antibody.
    • Table S6. Virus strain abbreviations.
    • References (3746)

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