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Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals

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Science Translational Medicine  27 Jan 2021:
Vol. 13, Issue 578, eabd6990
DOI: 10.1126/scitranslmed.abd6990
  • Fig. 1 Titrations of serum IgG by ELISAs specific to SARS-CoV-2.

    Plasma samples from 17 patients with SARS-CoV-2 were diluted (1:100) and added to plates coated with recombinant SARS-CoV-2 (A) N, (B) S, (C) S1, (D) S2, which was fused to a polyhistidine (HIS) tag, or (E) RBD protein, which was fused to a human hCκ domain. The amount of bound IgG was determined using anti-human IgG (Fc-specific) antibody, and ABTS (2,2’-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was used as the substrate. All experiments were performed in duplicate, and the data are presented as the means ± SD.

  • Fig. 2 Characteristics of the isolated nAbs, stereotypic IGH clonotypes, and RBD binding–predicted clones.

    (A) Serially diluted IgG2/4 was mixed with an equal volume of SARS-CoV-2 containing 100 TCID50, and the IgG2/4-virus mixture was added to Vero cells with eight repeats and incubated for 5 days. Cells infected with 100 TCID50 of SARS-CoV-2, isotype IgG2/4 control, or without the virus were applied as positive, negative, and uninfected controls, respectively. CPE in each well was observed 5 days after infection. (B) Characteristics of nAbs found in patients A and E. (C) IGH clonotypes that are highly homologous to E-3B1 and reactive against recombinant SARS-CoV-2 S and RBD proteins. The right column shows the results of the phage ELISA. All experiments were performed in quadruplicate, and the data are presented as the means ± SD. (D) List of diverse Ig light chain clonotypes that can be paired with the IGH clonotypes from (B) to achieve reactivity. (E) J and (F) VJ gene usage in the IGH repertoire of patients (top) and the binding-predicted IGH clones (bottom). For the VJ gene usage heatmap, the frequency values for the IGH repertoire of all 17 patients were averaged and are displayed (top) along with those of the predicted RBD-binding IGH clones (bottom). N/A, not applicable.

  • Fig. 3 Deep profiling of the IGH repertoires of patients A and E.

    (A and B) IGH repertoires of (A) patient A and (B) patient E were analyzed 11, 17, and 45 (A_d11, A_d17, and A_d45) days and 23, 44, and 99 (E_d23, E_d44, and E_d99) days after symptom onset, respectively. IGH repertoires were examined according to divergence from the germ line and the isotype composition of the sequences. Values for divergence from the germ line were calculated separately for each isotype and are presented as violin plots, ordered by the class-switching event. The bar graphs on the top of the violin plots represent the proportion of each isotype in the repertoire. (C and D) Mapping of three types of RBD-binding IGH sequences (neutralize, bind, and predicted), derived from either (C) patient A or (D) patient E, against the corresponding IGH repertoire. The positions of the RBD-binding IGH sequences in the divergence value were annotated as dot plots on the same scale used for (A) and (B). Bar graphs on the top of the dot plots indicate the isotype compositions of the sequences in the repertoire.

  • Fig. 4 Reactivity of nAbs against recombinant SARS-CoV-2 spike mutants.

    Recombinant wild-type or mutant (V341I, F342L, N354D, V367F, R408I, A435S, G476S, V483A, and D614G) SARS-CoV-2 S, S1, or RBD protein–coated microtiter plates were incubated with varying concentrations of (A) E-3B1-hFc, (B) A-1H4-hFc, (C) A-2F1-hFc, (D) A-2H4-hFc, (E) E-3G9-hFc, and (F) irrelevant scFv-hFc. HRP-conjugated anti-human IgG antibody was used as the probe, and ABTS was used as the substrate. All experiments were performed in triplicate, and data are presented as the means ± SD.

  • Table 1 The stereotypic VH clonotypes against SARS-CoV-2 RBD in the healthy population and in SARS-CoV-2–infected patients.

    The healthy population samples based on publicly available IGH repertoires or patient identification can be found in the sample column. Clonotypes were mapped according to identical VJ gene usage of IGHV3-53/IGHV3-66 and IGHJ6 and perfectly matched HCDR3 amino acid sequences. Read counts of the mapped sequences in the repertoires of each sample were annotated in the occurrence column. For clonotypes with multiple occurrences, the means and SD of divergence were represented. The proportion of each isotype is indicated for each sample as a percentage.

    Healthy population
    SampleV geneJ geneCDR3 amino acidDivergenceIsotypeOccurrence
    326650IGHV3-53/3-66IGHJ6DLYYYGMDV0.007 ± 0.003M (100%)12
    326713IGHV3-53/3-66IGHJ6DLYYYGMDV0.005 ± 0.010M (92.3%), G
    (7.7%)
    13
    326780IGHV3-53/3-66IGHJ6DLYYYGMDV0.014 ± 0.010M (97.4%), G
    (2.6%)
    38
    326797IGHV3-53IGHJ6DLYYYGMDV0.004M (100%)1
    327059IGHV3-53/3-66IGHJ6DLYYYGMDV0.003 ± 0.005M (100%)8
    D103IGHV3-53IGHJ6DLYYYGMDV0.008 ± 0.020M (100%)9
    326650IGHV3-53/3-66IGHJ6DLDYYGMDV0.006 ± 0.002M (75%), G (25%)4
    326713IGHV3-53/3-66IGHJ6DLDYYGMDV0.012 ± 0.018M (100%)4
    326797IGHV3-66IGHJ6DLDYYGMDV0.055M (100%)1
    327059IGHV3-53/3-66IGHJ6DLDYYGMDV0.001 ± 0.002M (100%)4
    D103IGHV3-53IGHJ6DLDYYGMDV0.053M (100%)1
    326713IGHV3-53/3-66IGHJ6DLVAYGMDV0.008 ± 0.011M (100%)2
    326713IGHV3-53IGHJ6DLVYYGDMV0.001 ± 0.002M (100%)3
    326797IGHV3-53IGHJ6DLVYYGMDV0.089 ± 0.008M (100%)2
    326713IGHV3-53IGHJ6DLVVYGMDV0.024 ± 0.052M (100%)5
    326780IGHV3-53/3-66IGHJ6DLSYYGMDV0.024 ± 0.024M (98.44%), D
    (0.78%), G (0.78%)
    128
    D103IGHV3-53IGHJ6DLSYYGMDV0.022 ± 0.003M (100%)2
    327059IGHV3-53IGHJ6DLGDYGMDV0.000M (100%)1
    326713IGHV3-66IGHJ6DAVSYGMDV0.000 ± 0.000M (100%)2
    SARS-CoV-2–infected patients
    SampleV geneJ geneCDR3 amino acidDivergenceIsotypeOccurrence
    AIGHV3-53IGHJ6DLYYYGMDV0.002 ± 0.004M (5.1%), G1
    (94.9%)
    59
    BIGHV3-53IGHJ6DLYYYGMDV0.000 ± 0.000M (33.3%), G1
    (66.7%)
    3
    GIGHV3-53/3-66IGHJ6DLYYYGMDV0.005 ± 0.003G1 (84.6%), A1
    (15.4%)
    14
    IIGHV3-53IGHJ6DLYYYGMDV0.000 ± 0.000M (100%)4
    KIGHV3-53IGHJ6DLYYYGMDV0.009 ± 0.000G1 (100%)2
    AIGHV3-53IGHJ6DLAVYGMDV0.004 ± 0.000G1 (100%)2
    EIGHV3-66IGHJ6DLAVYGMDV0.018 ± 0.000G1 (100%)6
    AIGHV3-53IGHJ6DLDYYGMDV0.000 ± 0.000G1 (100%)3
    EIGHV3-53IGHJ6DLDYYGMDV0.004 ± 0.000A1 (100%)4
    IIGHV3-66IGHJ6DLDYYGMDV0.002 ± 0.003G1 (100%)5
    KIGHV3-53IGHJ6DLDYYGMDV0.007 ± 0.005G1 (100%)107
    MIGHV3-53IGHJ6DLDYYGMDV0.018G1 (100%)1
    AIGHV3-53IGHJ6DLVAYGMDV0.008 ± 0.017G1 (100%)14
    BIGHV3-53IGHJ6DLVAYGMDV0.009G1 (100%)1
    EIGHV3-53IGHJ6DLVAYGMDV0.005 ± 0.002G1 (100%)6
    DIGHV3-53IGHJ6DLVYYGMDV0.004G1 (100%)1
    EIGHV3-53IGHJ6DLVYYGMDV0.013A1 (100%)1
    FIGHV3-53IGHJ6DLVYYGDMV0.001 ± 0.003M (75%), G1 (25%)16
    BIGHV3-53IGHJ6DLVVYGMDV0.002 ± 0.002M (27.3%), G1
    (72.7%)
    11
    EIGHV3-53IGHJ6DLVVYGMDV0.013 ± 0.000A2 (100%)4
    HIGHV3-53IGHJ6DLVVYGMDV0.009 ± 0.000G1 (100%)7
    AIGHV3-53IGHJ6DLSYYGMDV0.013 ± 0.016G1 (100%)5
    FIGHV3-53IGHJ6DLSYYGMDV0.018G1 (100%)1
    OIGHV3-53IGHJ6DLSYYGMDV0.000G1 (100%)1
    AIGHV3-53IGHJ6DLGDYGMDV0.009 ± 0.000G1 (100%)3
    EIGHV3-53IGHJ6DLGDYGMDV0.018 ± 0.019G1 (85.7%), A1
    (14.3%)
    7
    FIGHV3-53IGHJ6DLGDYGMDV0.003 ± 0.002M (92.0%), G1
    (8.0%)
    163
    HIGHV3-53IGHJ6DLGDYGDMV0.004 ± 0.000G1 (100%)8
    GIGHV3-53IGHJ6DAVSYGMDV0.004 ± 0.004M (7.0%), G1
    (93.0%)
    57
    IIGHV3-53IGHJ6DAVSYGMDV0.007 ± 0.003G1 (100%)9
    PIGHV3-53IGHJ6DAVSYGMDV0.000 ± 0.000G1 (100%)3
    EIGHV3-53IGHJ6DLGPYGMDV0.009G1 (100%)1
    IIGHV3-53/3-66IGHJ6DLGPYGMDV0.010 ± 0.003G3 (40%), G1 (40%),
    A1 (20%)
    4
    AIGHV3-53IGHJ6DLVIYGMDV0.003 ± 0.004M (5.9%), G1
    (94.1%)
    17
    IIGHV3-66IGHJ6DLVIYGMDV0.007 ± 0.004G1 (100%)8
    EIGHV3-53/3-66IGHJ6DLVVLGMDV0.009 ± 0.000A2 (100%)20
    IIGHV3-53IGHJ6DLVVLGMDV0.000G1 (100%)1

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/scitranslmed.abd6990/DC1

    Fig. S1. Titrations of serum IgG by ELISAs specific to MERS-CoV.

    Fig. S2. Reactivity of anti–SARS-CoV-2 scFv Abs against recombinant SARS-CoV-2 RBD.

    Fig. S3. Inhibition of recombinant SARS-CoV-2 S glycoprotein binding to ACE2-expressing cells.

    Fig. S4. In vitro neutralization of SARS-COV-2.

    Fig. S5. Mapping of 11 nAbs to the overlapping IGH repertoire.

    Fig. S6. Existence of Vλ that can be paired with the stereotypic VH.

    Fig. S7. VJ gene usage among the Igκ light chain repertoire of patients.

    Fig. S8. VJ gene usage among the Igλ light chain repertoire of patients.

    Fig. S9. Reactivity of phage-displayed scFv clones in phage ELISA.

    Fig. S10. Deep profiling of the IGH repertoire of patients B, C, D, F, and G.

    Fig. S11. The nearest-neighbor distance histogram for HCDR3 amino acid sequences in the IGH repertoires of patients.

    Fig. S12. Frequency scatter plots for NGS data from four libraries after each round of biopanning.

    Fig. S13. The results of PCA applied to the NGS data of four libraries after each round of biopanning.

    Table S1. Demographic and clinical characteristics.

    Table S2. SARS-CoV-2 RBD-reactive scFv clones.

    Table S3. Class-switched IGH clonotypes homologous to E-3B1.

    Table S4. Human mAbs reactive against MERS-CoV RBD.

    Table S5. Statistics for the preprocessing of the IGH NGS data.

    Table S6. Statistics for the preprocessing of the Igκ and Igλ NGS data.

    Table S7. The RBD-binding prediction clones.

    Table S8. Primers used in the study.

    Data file S1.

  • The PDF file includes:

    • Fig. S1. Titrations of serum IgG by ELISAs specific to MERS-CoV.
    • Fig. S2. Reactivity of anti-SARS-CoV-2 scFv antibodies against recombinant SARS-CoV-2 RBD.
    • Fig. S3. Inhibition of recombinant SARS-CoV-2 S glycoprotein binding to ACE2expressing cells.
    • Fig. S4. In vitro neutralization of SARS-COV-2.
    • Fig. S5. Mapping of 11 nAbs to the overlapping IGH repertoire.
    • Fig. S6. Existence of VL that can be paired with the stereotypic VH.
    • Fig. S7. VJ gene usage among the IG kappa light chain repertoire of patients.
    • Fig. S8. VJ gene usage among the IG lambda light chain repertoire of patients.
    • Fig. S9. Reactivity of phage-displayed scFv clones in phage ELISA.
    • Fig. S11. The nearest-neighbor distance histogram for HCDR3 amino acid sequences in the IGH repertoires of patients.
    • Fig. S12. Frequency scatter plots for NGS data from four libraries after each round of biopanning.
    • Fig. S13. The results of principal component analysis (PCA) applied to the NGS data of four libraries after each round of biopanning.
    • Table S1. Demographic and clinical characteristics.
    • Table S2. SARS-CoV-2 RBD-reactive scFv clones.
    • Table S3. Class-switched IGH clonotypes homologous to E-3B1.
    • Table S4. Human mAbs reactive against MERS-CoV RBD.
    • Table S5. Statistics for the pre-processing of the IGH NGS data.
    • Table S6. Statistics for the pre-processing of the IGκ and IGλ NGS data.
    • Table S7. The RBD-binding prediction clones.
    • Table S8. Primers used in the study.

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    Other Supplementary Material for this manuscript includes the following:

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