Fig 2: Frequency of B Cell Subsets in Peripheral Blood of HIV-1 Env-Immunized Neonatal and Adult MacaquesFrequency of eight B cell subsets, including naive, IgM+ memory, IgG+ memory, IgM− IgG− memory, IgM+ plasmablasts, IgM− plasmablasts, marginal zone-like (in log scale), and a rare population of undefined B cells (Query 1; Ki67+CD1c+CD27+ IgM2+) among CD20+CD3− CD14− CD16− lymphocytes from CH505-SOSIP-immunized neonatal (blue) and adult (red) macaques in studies 2 and 3 measured by flow cytometry 2 weeks after the first, third, and sixth immunizations. Horizontal bars represent the group mean. nparLD was performed on SOSIP-immunized animals combined in studies 2 and 3 (p = 0.007 for naive, p = 0.69 for IgM+ memory, p = 0.0005 for IgG+ memory, p = 0.0002 for IgM− IgG− memory, p = 0.027 for IgM+ plasmablasts, p = 0.04 for IgM plasmablasts, p = 0.00009 for marginal zone-like, and p = 0.0007 for Query 1). The scales of the y axis are different on each graph.

Fig 3: S1-RBD-specific MBCs in SARS-CoV-2-infected individuals make a poor tertiary response(A–E) Total log10 number of S1-RBD tetramer-binding total MBCs (A), plasmablasts (B), CD21+ cl-MBCs (C), CD21‒ CD27+ pb-MBCs (D), or CD21‒ CD21‒ CD11c+ at-MBCs (E) per million total B cells in the peripheral blood of SARS-CoV-2-naive (black circles) or SARS-CoV-2-infected (red circles) subjects (n = 5–12) at the indicated times after the second vaccination. Box and whisker plots are formatted as in Figure 1C. Values were compared with one-way ANOVA. ∗p < 0.05, ∗∗∗∗p < 0.0001, ns, not significant.

Fig 4: Identification of S1-RBD–specific B cells by flow cytometry.B cells were identified in PBMC samples by flow cytometry as cells with the (A) light scatter properties of lymphocytes and (B) a side scatter area versus width profile of singlets (C) that lacked B lineage–negative markers and did not bind a viability dye and (D) expressed CD19. (E and F) S1-RBD–binding B cells were identified as B cells that expressed low amounts of decoy and bound the S1-RBD tetramer. The frequencies of these cells are shown in the gates. (E) Examples of a sample before vaccination or (F) samples from a participant 5 weeks after the first vaccination and 1 week after the second or (G) 28 weeks after the first vaccination and 24.5 weeks after second are shown. S1-RBD–specific naive cells were identified as (H) CD19+CD20+ and (M) IgM+IgD+ cells. S1-RBD–specific plasmablasts were identified as (I) CD19loCD20‒ and (J) CD27hiCD38hi cells and (N) further divided by expression of IgA and lack of expression of IgM. S1-RBD–specific memory B cells were identified as (K) CD19+CD20+ and (L) CD27+CD38lo cells and further divided by lack of expression of (O) IgM and IgD and (P) expression of IgA or IgG.

Fig 5: Secondary response by mRNA-vaccine- and SARS-CoV-2-induced primary MBCs(A) Flow cytometry plots of the indicated cell types and molecules, pre-gated as described in Figure S2, for samples from SARS-CoV-2-naive subjects on day 28 after the first vaccination or on days 7, 14, or 33 after the second vaccination. Percentages of cells in each gate are indicated. CD19hi CD11c+ at-MBCs are shown in the gate in the fourth plot in each row.(B–F) Total log10 number of S1-RBD tetramer-binding non-plasmablasts (nPB) (B), plasmablasts (C), CD21+ cl-MBCs (D), CD21‒ CD27+ pb-MBCs, (E), or CD21‒ CD27‒ CD11c+ at-MBCs (F) per million total B cells in the peripheral blood of SARS-CoV-2-naive subjects (n = 8–29) at indicated times after the first (primary) or second (secondary) vaccination (black circles) or SARS-CoV-2-infected individuals at the time of (primary) or the indicated times after (secondary) the first vaccination (red circles). CD21‒ CD27‒ CD11c‒ MBCs were not included in the analysis because the nature of these cells is unknown. Box and whisker plots are formatted as in Figure 1C. Values were compared with one-way ANOVA. ∗p < 0.05, ∗∗p < 0. 01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns = not significant. Any cell per million value that was ≤1 was set to 0 on the log10 cell per million plots. Such values were at or below the limit of detection.(G) Flow cytometry plots of the indicated cell types and molecules as in (A) for samples from SARS-CoV-2-infected subjects before vaccination or on days 7, 14, or 28 after the first vaccination.