Research ArticleINFECTIOUS DISEASE

Zika virus activates de novo and cross-reactive memory B cell responses in dengue-experienced donors

See allHide authors and affiliations

Science Immunology  18 Aug 2017:
Vol. 2, Issue 14, eaan6809
DOI: 10.1126/sciimmunol.aan6809
  • Fig. 1 Analysis of plasmablast responses in one naïve and three DENV-experienced donors.

    (A) Flow cytometric analysis of plasmablast responses in one naïve and three DENV-experienced donors during ongoing ZIKV infection. Plasmablasts are defined herein as CD19+CD3CD20−/loCD38hiCD27hi cells. Plots shown are gated for CD3CD20−/lo cells, with the CD27hiCD38hi plasmablasts marked by a box. The frequency of plasmablasts within the CD3CD20−/lo population is shown next to the gate. The numbers in parentheses show the percentage of peripheral B cells that are plasmablasts. Flow cytometry data were analyzed using FlowJo software (version 10.0.8). (B) Load of somatic mutations [expressed as the number of nucleotide (NT) substitutions] in VH and VL in plasmablast-derived mAbs isolated from a DENV-naïve (donor 1) and three DENV-experienced donors (donors 2 to 4). All VH and VL chains are shown, regardless of whether the paired antibody chain was recovered. (C) Clonal lineage analysis. Heavy-chain sequences were assigned to lineages using Clonify (52). Each lineage is represented as a segment proportional to the lineage size. The total number of recovered heavy chains is shown in the center of each pie. All lineages that contain only a single sequence are combined and shown as a single gray segment.

  • Fig. 2 Binding characteristics of mAbs isolated from plasmablasts during acute ZIKV infection.

    (A) Specificities of the mAbs isolated from one DENV naïve donor and three DENV-experienced donors. Distribution of mAbs that bind to NS1, recombinant E, epitopes expressed only on whole ZIKV, and those for which no specificity could be assigned are shown. The number at the top of each bar represents the total number of mAbs cloned from each donor. (B) Epitope mapping of recombinant E–specific antibodies isolated from three DENV-experienced donors. Pie charts show the distribution of antibodies that bind to epitopes within DIII, within or proximal to the FL on DII, and defined by mAbs ADI-24314 and ADI-24247. 4G2-like FL: Competition with 4G2, WNV E–reactive, and E-FL–sensitive; non-WNV FL: 4G2-competitive, E-FL–sensitive, and WNV E–nonreactive; FL proximal: 4G2 competitive, E-FL–reactive, and WNV E–nonreactive; DIII: Recombinant DIII–reactive; ADI-24314 competitor: 4G2-noncompetitive, ADI-24314–competitive, and E-FL–reactive; ADI-24247 competitor: 4G2-noncompetitive, ADI-24247–competitive, and E-FL–reactive. NC, not characterized because of low binding affinity. (C) Heat map of mAb binding reactivity to ZIKV and DENV1–4 E proteins or whole viral particles (left) and ZIKV and DENV1–4 NS1 proteins (right). The E-specific mAbs shown were isolated from DENV-experienced donors. Apparent binding affinities for recombinant E and NS1 proteins were determined by BLI measurements, and apparent binding affinities for whole virus were determined by ELISA.

  • Fig. 3 Neutralizing activity of mAbs isolated from plasmablasts during acute ZIKV infection in DENV-experienced donors.

    (A) Heat map showing mAb neutralization of ZIKV-Para, DENV1, DENV2, DENV3, and DENV4 at a concentration of 1.0 μg/ml. (B) Heat map showing neutralization IC50s for selected mAbs against ZIKV-Para, DENV1, DENV2, DENV3, and DENV4. Epitope assignments are indicated on the left. (C) Neutralization IC50s for selected mAbs against ZIKV-Para. IC50 values represent the concentration of IgG required to reduce viral infectivity by 50%. Neutralization assays were performed using a live virus plaque reduction assay. N.N., non-neutralizing.

  • Fig. 4 Longitudinal analysis of ZIKV E–specific memory B cell responses in the DENV-experienced donors.

    (A) ZIKV E–specific memory B cell sorting. Fluorescence-activated cell sorting (FACS) plots show ZIKV E reactivity of IgG+ B cells from the three convalescent DENV-experienced donors and a ZIKV seronegative healthy donor control. B cells in quadrant 2 (Q2) were single cell–sorted for mAb cloning. ZIKV E was labeled with two different colors to reduce background binding. (B) Apparent binding affinities of plasmablast and memory B cell–derived mAbs to recombinant ZIKV E. Red bars indicate the median IC50s. (C) Clonal lineage analysis of memory B cell–derived mAbs. Heavy-chain sequences were assigned to lineages using Clonify (52). Each lineage is represented as a segment proportional to the lineage size. The total number of recovered heavy chains is shown in the center of each pie. All lineages that contain only a single sequence are combined and shown as a single gray segment. (D) Heat map showing apparent binding affinities of the memory B cell–derived mAbs to recombinant ZIKV E and DENV1–4 E proteins. Top: mAbs cloned from donor 3. Bottom: mAbs cloned from donor 4. 023, ADI-30023 competitor; 056, ADI-30056 competitor; 314, ADI-24314 competitor; 247, ADI-24247 competitor; FL, 4G2 competitor; DIII, recombinant DIII binder; UK, unknown. (E) Load of somatic mutations (expressed as the number of nucleotide substitutions in the VH) in mAbs isolated from plasmablasts and memory B cells in DENV-experienced donors. Each point represents an individual mAb. Red bars indicate the median number of nucleotide substitutions. (F) Binding of plasmablast and memory B cell–derived mAbs to recombinant ZIKV E. Apparent binding affinities are shown in the plot. Red bars indicate median apparent IC50s. Statistical comparisons were made using Mann-Whitney test (***P < 0.001, **P < 0.01; n.s., not significant).

  • Fig. 5 Neutralizing activity of memory B cell–derived mAbs.

    (A) Percentage of cross-reactive and ZIKV-specific mAbs that reached neutralization IC50 or IC80 at a concentration of 1.0 μg/ml. (B) Neutralization IC50 values of selected mAbs from each competition group. IC50 values represent the concentration of IgG required to reduce viral infectivity by 50%. Red bars indicate median IC50s. (C) Heat map showing neutralization IC50s for selected cross-reactive mAbs against ZIKV-Para, DENV1, DENV2, DENV3, and DENV4. Neutralization assays were performed using a live virus plaque reduction assay.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/14/eaan6809/DC1

    Fig. S1. Binding of acute serum to ZIKV and DENV1–4 viral particles and NS1 proteins.

    Fig. S2. Plasmablast sorting.

    Fig. S3. Binding of plasmablast-derived mAbs to ZIKV-Para by ELISA.

    Fig. S4. Western blot analysis of mAb binding to ZIKV lysate.

    Fig. S5. Heat map of mAb binding reactivity to recombinant ZIKV E, WNV E, DIII, and E-FL.

    Fig. S6. mAb binding to recombinant ZIKV and DENV E proteins.

    Fig. S7. ELISA binding of whole virus–specific clones to ZIKV and DENV.

    Fig. S8. SHM of NS1-specific mAbs.

    Fig. S9. Representative neutralization curves.

    Fig. S10. Enhancement of ZIKV infection by plasmablast-derived mAbs isolated from DENV-experienced donors.

    Fig. S11. Memory B cell sorting.

    Fig. S12. Distribution of clonal lineage members across plasmablast and memory B cell subsets.

    Fig. S13. Epitope mapping of ZIKV-specific mAbs derived from memory B cells.

    Table S1. Clinical characteristics of ZIKV-infected donors.

    Table S2. Binding properties of ZIKV E–specific mAbs isolated from plasmablasts.

    Table S3. Binding characteristics of mAb 4G2.

    Table S4. Sequences of ZV-67 competitor mAbs.

    Table S5. Competition of ADI-24247 and ADI-24314 with each other and with control mAbs.

    Table S6. Binding properties of NS1-specific mAbs isolated from plasmablasts.

    Table S7. Neutralizing activity of selected plasmablast-derived mAbs.

    Table S8. Binding properties of ZIKV E–specific mAbs isolated from memory B cells.

    Table S9. Clonal lineages shared between plasmablast and memory B cell–derived antibodies.

    Table S10. Neutralizing activity of selected memory B cell–derived mAbs.

  • Supplementary Materials

    Supplementary Material for:

    Zika virus activates de novo and cross-reactive memory B cell responses in dengue-experienced donors

    Thomas F. Rogers, Eileen C. Goodwin, Bryan Briney, Devin Sok, Nathan Beutler, Alexander Strubel, Rebecca Nedellec, Khoa Le, Michael E. Brown, Dennis R. Burton,* Laura M. Walker*

    *Corresponding authors. Email: laura.walker{at}adimab.com (L.M.W.); burton{at}scripps.edu (D.R.B.)

    Published 18 August 2017, Sci. Immunol. 2, eaan6809 (2017)
    DOI: 10.1126/sciimmunol.aan6809

    This PDF file includes:

    • Fig. S1. Binding of acute serum to ZIKV and DENV1–4 viral particles and NS1 proteins.
    • Fig. S2. Plasmablast sorting.
    • Fig. S3. Binding of plasmablast-derived mAbs to ZIKV-Para by ELISA.
    • Fig. S4. Western blot analysis of mAb binding to ZIKV lysate.
    • Fig. S5. Heat map of mAb binding reactivity to recombinant ZIKV E, WNV E, DIII, and E-FL.
    • Fig. S6. mAb binding to recombinant ZIKV and DENV E proteins.
    • Fig. S7. ELISA binding of whole virus–specific clones to ZIKV and DENV.
    • Fig. S8. SHM of NS1-specific mAbs.
    • Fig. S9. Representative neutralization curves.
    • Fig. S10. Enhancement of ZIKV infection by plasmablast-derived mAbs isolated from DENV-experienced donors.
    • Fig. S11. Memory B cell sorting.
    • Fig. S12. Distribution of clonal lineage members across plasmablast and memory B cell subsets.
    • Fig. S13. Epitope mapping of ZIKV-specific mAbs derived from memory B cells.
    • Table S1. Clinical characteristics of ZIKV-infected donors.
    • Table S2. Binding properties of ZIKV E?specific mAbs isolated from plasmablasts.
    • Table S3. Binding characteristics of mAb 4G2.
    • Table S4. Sequences of ZV-67 competitor mAbs.
    • Table S5. Competition of ADI-24247 and ADI-24314 with each other and with control mAbs.
    • Table S6. Binding properties of NS1-specific mAbs isolated from plasmablasts.
    • Table S7. Neutralizing activity of selected plasmablast-derived mAbs.
    • Table S8. Binding properties of ZIKV E–specific mAbs isolated from memory B cells.
    • Table S9. Clonal lineages shared between plasmablast and memory B cell– derived antibodies.
    • Table S10. Neutralizing activity of selected memory B cell–derived mAbs.

    Download PDF

    Files in this Data Supplement:

Navigate This Article