Fig. 2: Expansion of public IGHV1-2 BCR lineages with both affinity matured and non-detectable binding underscore the elicitation of D368R sensitive antibodies.

Single D368R-sensitive memory IgG B cells (CD19⁺/IgD^-/IgM^-/IgG⁺/ GL7^-/CD38⁺/122E-PE⁺/122E-APC-Cy7⁺/122E D368R-APC^-) from animals receiving the heterologous immunization regimen (see also Fig. 1F, Supplementary Figs. 3–5) were sorted from n = 3 biologically independent IGHV1-2 HC2 animals. A Sequencing the memory B cells yielded 68 BCRs in Mouse A, 70 BCRs in Mouse B, and 68 BCRs in Mouse C (see also Supplementary Data 1). Public B cell pathways were first assigned based on clonotype (usage of a shared CDRH3 sequence) using Cloanalyst software^{52,70,71,72,73,74,75,76,77}. These public B cell pathways were then further subdivided into public clonal lineages (public CDRH3 + shared CDRL3) revealing four clonal lineages that were reproducibly expanded in all the mice (see also Supplementary Data 1). B Fabs from each public clonal lineage recombinantly and then tested for affinity to original sorting probes (122E Env or 122E Env-D368R). Binding was evaluated using bilayer interferometry (BLI). The equilibrium dissociation constant (KD) values were calculated by applying a 1:1 binding isotherm using vendor-supplied software. KD values above 100 µM are beyond the limit of detection for this instrument¹⁷. The low affinity Lin1 and Lin2 BCR sequences chosen for this analysis contain a conserved pattern of SHM that was independently reproduced within each public clonal lineage within each mouse (Supplementary Figs. 7, 8). Data are from one vaccination experiment that yielded antigen specific IgG memory BCRs in the three mice; details of each individual B cell clone are provided in Supplementary Data 1.