B cell populations circulating after vaccination. (A) Plasma cells were quantitated as a fraction of total B cells in three subjects at four available time points. No difference between preimmunization and postimmunization time points was observed (values are mean ± standard error; day 0, 1.2% ± 0.4%, day 42, 1.0% ± 0.3%; day 182, 0.7% ± 0.3%; day 672, 1.0% ± 0.1%). (B) Antigen-specific memory B cells quantitated as a fraction of memory B cells in six subjects. Preimmunization and convalescent samples showed few circulating antigen-specific memory B cells (day 0, 0.007% ± 0.004%; day 672, 0.010% ± 0.005%) while samples taken 2 weeks after immunization had a significantly larger fraction of circulating antigen-specific memory B cells (day 42, 0.10% ± 0.03%; day 182, 0.23% ± 0.10%; P = 0.001 (asterisks); Kruskal-Wallis statistic, 16.3). (C) Quantitation of antigen-specific memory B cells by flow cytometry. B cells stained with fluorescently labeled gp120_W6.1D were gated for memory B cells. Antigen-specific B cells appear as a diagonal in the shown gate; low frequencies were observed at early and late time points. Elevated numbers of circulating antigen-specific B cells were found 2 weeks after the second and fourth immunizations.

Increased maturation of recovered antibodies following repeat immunization. (A) HC mutation frequencies of Env-reactive antibodies after two immunizations (day 42) was 1.8% ± 0.6%; antibodies recovered after four immunizations (day 182 and 672) had a mutation frequency of 3.8% ± 0.5% (P = 0.0095; Mann-Whitney U = 40). (B) HC mutation frequencies of antibodies not reactive with HIV-1 gp120 or gp140 in any assay are shown. The number of antibodies shown appears below each column. Mean mutation frequency ± standard error for each time point was as follows: day 0, 7.7% ± 0.6%; day 42, 6.4% ± 0.3%; day 182, 7.7% ± 0.5%; day 672, 4.6% ± 0.4%. Antibodies recovered at day 672 had a lower mean mutation frequency than those recovered at the other three time points (P < 0.001; Kruskal-Wallis statistic, 20.5). (C) Clonal lineage 15.4.31 consisted of two rMAbs, one isolated at day 42 and one at day 182. Antibody 5148 was nearly unmutated (see Fig. S1 in the supplemental material). (D) CDR H3 length of Env-reactive rMAbs shown as a histogram. Median CDR H3 length was 16.5 amino acids; the mean was 17.2 amino acids. (E) Reactivity of clonal lineage 15.4.31 in binding antibody multiplex (Luminex) assay and ELISA, tested at 1 μg/ml. Antibody 3830 had a 4.7% HC mutation frequency and reacted strongly to gp120_W6.1D in both assays. Antibody 5148 had a 0% HC mutation frequency, reacted weakly in the multiplex binding assay, and did not react in ELISA. MFI, mean fluorescence intensity. (F) CDR H3 lengths of all recovered rMAbs, regardless of reactivity, shown as a histogram. Median CDR H3 length was 14 amino acids; the mean was 14.7.

HEp-2 cell reactivities of recovered rMAbs. All images were taken with a 40× objective; all antibodies were tested at 25 μg/ml, except 13D5, which was tested at 50 μg/ml. The exposure was 10 s unless otherwise noted. Scale bar, 25 μm. Images show results with the following antibodies: 3491, 12-s exposure, diffuse staining and dividing cells (A); 3797, 8-s exposure, dividing cells (B); 4364, 7-s exposure, nuclear and cytoplasmic fibrillar pattern (C); 2F5 (positive control), diffuse (D); 17b (negative control), no staining (E); 13D5 (murine MAb), 15s exposure, fibrillar pattern (F); 3491_UA, diffuse (G); 4364, 7-s exposure, magnified to show fibrillar staining; 13D5, 15-s exposure, magnified to show fibrillar staining (I).

Plasma antibody activity induced by GSK PRO HIV-002. (A to C) Vaccine-induced reactivity by isotype. Serial serum samples from vaccine recipients were analyzed for vaccine-specific antibody. Binding antibody multiplex assay data are plotted as estimated antibody concentrations for IgG subclasses; asterisks indicate that antibodies at day 0 were below the limit of detection. IgG subclass responses to HIV-1 gp120 appeared at day 42, with IgG1 responses peaking at day 98 (A). Antibody responses to Nef (B) and Tat (C) also appeared at day 42. The overall concentrations of HIV-1-specific antibody responses mirror the relative concentrations present in blood. (D) Neutralization activity of d182 serum. Serum samples were tested for activity against a panel of Env-pseudotyped viruses in the TZM-bl assay. No neutralizing activity was detected at a 1:20 dilution of preimmunization samples. In contrast, serum samples from day 182 showed neutralization of autologous W6.1D-TCLA.7 Env-pseudotyped virus (geometric mean titer, 1:1645) and neutralization against HIV-1 MN.3 (geometric mean titer, 1:448) in all vaccine recipients. Most vaccine recipients (26/30, or 87%) developed neutralizing activity against HIV-1 SF162.LS (geometric mean titer, 1:75) while a minority developed activity against HIV-1 BaL.26 (9/30, or 30% of subjects with neutralizing activity). (E) ADCC activity of serum IgG. Serum IgG from the four subjects (identified by GSK numbers) from whom rMAbs were recovered was tested for ADCC activity as described. No activity was detected in samples prior to immunization. In contrast, ADCC activity was detected in all samples from day 182 with a peak in granzyme B (GzB) activity comparable to activity levels of the recovered rMAbs (Fig. 4).

Recovery of Env-specific antibodies from vaccine recipients. (A) Plasma cells yielded 402 rMAbs distributed across all four time points sorted. Env-reactive rMAbs were isolated only from postvaccination time points; 5/402 (1.2%) of rMAbs reacted with gp120 (orange wedge). (B) Antigen-specific memory B cells yielded 51 rMAbs across three postimmunization time points; no antibodies were isolated from the day 0 samples. Env-reactive rMAbs predominated; 29/51 (57%) of isolated rMAbs reacted with gp120. Antigen-specific memory B cell sorting was more efficient in isolating Env-reactive antibodies (P < 0.0001; χ² = 193).

ADCC activity of selected rMAbs. A subset of 14 antibodies was tested for ADCC activity; the granzyme B activity is shown for each rMAb. The cutoff of reactivity is 5%, shown by a horizontal line. Seven rMAbs mediated ADCC, recapitulating the serum activity (Fig. 1E).