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Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits

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Science Translational Medicine  01 Jul 2020:
Vol. 12, Issue 550, eabc3539
DOI: 10.1126/scitranslmed.abc3539
  • Fig. 1 SARS-CoV-2 spike binding and SARS-CoV-2 neutralization by serum antibodies generated after rabbit immunization with spike antigens.

    (A) Schematic representation of the SARS-CoV-2 spike protein and subdomains. Spike S1+S2 ectodomain (amino acids 16 to 1213) lacking the cytoplasmic and transmembrane domains (CT-TM), S1 domain (amino acids 16 to 685), receptor binding domain (RBD) (amino acids 319 to 541), and S2 domain (amino acids 686 to 1213), all containing 6× His tag at C terminus, were commercially produced in either HEK 293 mammalian cells (S1 and RBD) or insect cells (S1+S2 ectodomain and S2 domain). The receptor binding motif (RBM) encompasses residues 437 to 508. (B) Binding of purified proteins to human ACE2 (hACE2) proteins in SPR. Sensorgrams represent binding of purified spike proteins on low-density His-captured chips to hACE2 protein (5 μg/ml). (C) SPR binding of antibodies from two rabbits each immunized twice with SARS-CoV-2 antigens to spike protein and domains from SARS-CoV-2 (S1+S2, black; S1, blue; RBD, red; and S2, purple). Total antibody binding is represented in maximum resonance units (RU) in this figure for 10-fold serum dilution. All SPR experiments were performed twice, and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR were <5%. The data shown are average values of two experimental runs. (D) Antibody off-rate constants were directly determined from the serum sample interaction with SARS-CoV-2 spike ectodomain (S1+S2), S1, S2, and RBD using SPR in the dissociation phase only for the sensorgrams with Max RU in the range of 20 to 100 RU. (E) RBD-hACE2 competition assay. Percent inhibition of hACE2 binding to RBD in the presence of 1:50 dilutions of post-second vaccination rabbit serum was measured by SPR. (F) End-point virus neutralization titers for one rabbit from each group using wild-type (wt) SARS-CoV-2 virus in a classical Biosafety Level 3 neutralization assay based on CPE (cytopathic effect) was performed as described in Materials and Methods. (G) Anti-spike ectodomain (S1+S2) binding antibody affinity as measured by antibody dissociation rates (off rates) of post-vaccinated rabbit polyclonal antibodies correlated with the wild-type (wt) SARS-CoV-2 virus end-point neutralization titers (r = −0.9975; P < 0.005). Pearson two-tailed correlations are reported for the calculation of correlations between anti-S1+S2 antibody affinity and end-point titers for one rabbit per immunogen. The color scheme in (G) is the same as in (D) or (F).

  • Fig. 2 Antibody epitope repertoires generated by different SARS-CoV-2 spike antigens.

    (A) Number of IgG-bound SARS-CoV-2 GFPDL phage clones using the post-second vaccination rabbit polyclonal sera. (B to E) Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, and their alignment to the spike protein of SARS-CoV-2 are shown for the four vaccine groups: S1+S2 ectodomain (B), S1 (C), S2 domain (D), and S1-RBD (RBD) (E). The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown enclosed in a red box in the respective alignments in each panel. (F) Elucidation of the antibody epitope profile in SARS-CoV-2 spike after rabbit vaccination. Antigenic sites within the SARS-CoV-2 spike protein recognized by serum antibodies after rabbit vaccination [based on data presented in Fig. 1 (B to E)]. The amino acid designation is based on the SARS-CoV-2 spike protein sequence (fig. S4). The antigenic regions/sites are depicted below the spike schematic and are color coded. Epitopes of each protein are numbered in a sequential fashion indicated in black. The antigenic epitopes are color coded unique to this study (red bars) or if they were previously predicted by algorithms (black bars) by Grifoni et al. (19). Sequence residues for each antigenic site and their sequence conservation with other human coronaviruses are shown in table S1. The GFPDL affinity selection was performed twice. Similar numbers of phage clones and epitope repertoire were observed in both phage display analyses. aa, amino acids.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/550/eabc3539/DC1

    Fig. S1. Purified SARS-CoV-2 proteins analyzed by SDS–polyacrylamide gel electrophoresis under reducing and nonreducing conditions.

    Fig. S2. Anti-spike reactivity of post-vaccination rabbit sera measured by ELISA.

    Fig. S3. Isotyping of rabbit serum binding to the SARS-CoV-2 spike protein after immunization.

    Fig. S4. Steady-state equilibrium analysis of serum antibody binding by SPR.

    Fig. S5. Neutralizing activity of post-vaccination rabbit serum antibodies in a pseudovirion neutralization assay.

    Fig. S6. Sequence alignment of spike protein from diverse CoV strains.

    Fig. S7. Structural representation of antigenic sites identified in SARS-CoV-2 using GFPDL.

    Table S1. Sequence conservation of antigenic sites among different CoV strains.

    Data file S1. Primary data.

  • The PDF file includes:

    • Figure S1: Purified SARS-CoV-2 proteins analyzed by SDS-PAGE under reducing and non-reducing conditions.
    • Figure S2: Anti-spike reactivity of post-vaccination rabbit sera measured by ELISA.
    • Figure S3: Isotyping of rabbit serum binding to the SARS-CoV-2 spike protein following immunization.
    • Figure S4: Steady-state equilibrium analysis of serum antibody binding by SPR.
    • Figure S5: Neutralizing activity of post-vaccination rabbit serum antibodies in a pseudovirion neutralization assay.
    • Figure S6. Sequence alignment of spike protein from diverse CoV strains.
    • Figure S7. Structural representation of antigenic sites identified in SARS-CoV-2 using GFDPL.
    • Table S1. Sequence conservation of antigenic sites among different CoV strains

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    Other Supplementary Material for this manuscript includes the following:

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