Figure 1.
S-HBsAg–specific memory antibodies cloned from HBV vaccinees and controllers. (A) Average S-HBsAg reactivity of serum IgGs from HBV vaccinees (HBVv, n = 6, black gate, top) and controllers (HBVc, n = 8, blue gate, bottom) as shown in Fig. S1 A. Shaded regions indicate value ranges. Representative flow-cytometry plots showing S-HBsAg-binding IgG+ memory B cells in HBV vaccinees and controllers as in Fig. S1 B; Bv4 and Bc3 are shown). nS-HBsAg and rS-HBsAg are human-derived nS-HBsAg and rS-HBsAg particles, respectively. (B) S-HBsAg-ELISA reactivities of S-HBsAg–captured IgG+ memory B cell antibodies. capt-rS-HBsAg, rS-HBsAg capture ELISA. Means of triplicate values are shown as measured in Fig. S2 A. (C) Percentage of S-HBsAg–specific monoclonal antibodies (% S-HBsAg+) isolated from HBVv and HBVc (right). % S-HBsAg+ according to anti-HBs antibody titers in HBVc (<150 and >900 IU/ml) are indicated. Groups were compared using the Mann–Whitney test. (D) ELISA reactivity of anti-HBs antibodies against TM domains–deleted S-HBsAg protein (ΔTM-rS-HBsAg). HB1 and mGO53 are positive and negative controls, respectively. The dotted line indicates the cutoff OD405nm for positive reactivity. Means of assay triplicates from three independent experiments are shown. (E) Infrared immunoblot shows anti-HBs memory IgGs reactive against denatured S-HBsAg proteins. The immunoreactive bands correspond to HBV p24 and gp27 proteins. (F) Same as in D but for the cyclic peptides corresponding to putative S-HBsAg loops 122–137 and 139–148. (G) Heat-mapped reactivity of ΔTM-rS-HBsAg–binding antibodies against S-HBsAg overlapping linear peptides. Means of triplicate OD values from one representative experiment (n = 3) are shown. HB1 and mGO53 are positive and negative controls, respectively. Amino acid sequences (left) and values of grand average of hydropathy (GRAVY; right) of S-HBsAg peptides are indicated. Blue arrows indicate peptide-reactive human anti-HBs antibodies. (H) ELISA binding curves of peptide-reactive anti-HBs antibodies are shown (means of assay quadruplicates ± SD).

S-HBsAg–specific memory antibodies cloned from HBV vaccinees and controllers. (A) Average S-HBsAg reactivity of serum IgGs from HBV vaccinees (HBVv, n = 6, black gate, top) and controllers (HBVc, n = 8, blue gate, bottom) as shown in Fig. S1 A. Shaded regions indicate value ranges. Representative flow-cytometry plots showing S-HBsAg-binding IgG+ memory B cells in HBV vaccinees and controllers as in Fig. S1 B; Bv4 and Bc3 are shown). nS-HBsAg and rS-HBsAg are human-derived nS-HBsAg and rS-HBsAg particles, respectively. (B) S-HBsAg-ELISA reactivities of S-HBsAg–captured IgG+ memory B cell antibodies. capt-rS-HBsAg, rS-HBsAg capture ELISA. Means of triplicate values are shown as measured in Fig. S2 A. (C) Percentage of S-HBsAg–specific monoclonal antibodies (% S-HBsAg+) isolated from HBVv and HBVc (right). % S-HBsAg+ according to anti-HBs antibody titers in HBVc (<150 and >900 IU/ml) are indicated. Groups were compared using the Mann–Whitney test. (D) ELISA reactivity of anti-HBs antibodies against TM domains–deleted S-HBsAg protein (ΔTM-rS-HBsAg). HB1 and mGO53 are positive and negative controls, respectively. The dotted line indicates the cutoff OD405nm for positive reactivity. Means of assay triplicates from three independent experiments are shown. (E) Infrared immunoblot shows anti-HBs memory IgGs reactive against denatured S-HBsAg proteins. The immunoreactive bands correspond to HBV p24 and gp27 proteins. (F) Same as in D but for the cyclic peptides corresponding to putative S-HBsAg loops 122–137 and 139–148. (G) Heat-mapped reactivity of ΔTM-rS-HBsAg–binding antibodies against S-HBsAg overlapping linear peptides. Means of triplicate OD values from one representative experiment (n = 3) are shown. HB1 and mGO53 are positive and negative controls, respectively. Amino acid sequences (left) and values of grand average of hydropathy (GRAVY; right) of S-HBsAg peptides are indicated. Blue arrows indicate peptide-reactive human anti-HBs antibodies. (H) ELISA binding curves of peptide-reactive anti-HBs antibodies are shown (means of assay quadruplicates ± SD).

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