Extended Data Fig. 10: RBD-KLH-vaccinated THX mice mount a class-switched and somatically hypermutated antibody response to SARS-CoV-2 Spike S1 RBD.
a, b, THX mice were injected i.p. with RBD-KLH (100 μg in 100 μl alum) or nil (100 μl alum) on day 0, boosted (100 μg in 100 µl PBS or 100 μl PBS alone) on day 21 and euthanized on day 28. Total serum human immunoglobulin and RBD-specific huIgM, huIgG and huIgA antibodies in RBD-KLH-immunized (n=6) and non-immunized (n=6) THX mice measured by specific ELISAs (total human immunoglobulin concentrations expressed as μg eq ml−1 and RBD-specific human antibody titers as OD readings at different dilutions or RUs). In the histograms, each dot represents an individual mouse and the bar depicts the mean with s.e.m. Statistical significance was assessed by two-sided Student’s unpaired t-test (NS, not significant; **P<0.01, ***P<0.001). c–f, Spleen huB cell huVHDJH-CH transcripts in RBD-KLH-immunized THX mice (n=3 of the 6 as in a–b, THX 488, 490, 492) were analyzed for CDR3 length, clonal expansion and intraclonal diversification. (c) CDR3 length distribution in huVHDJH-CH transcripts. Colors denote different antibody isotypes; color gradients denote different THX mice. (d) huVH mutation frequency (change/base) in huVHDJH-Cγ (2.7+0.08x10-2, mean+s.e.m.) transcripts depicted as scatter plots (left) and pie charts (middle). Each dot represents a single sequence and the bar depicts the mean with s.e.m. Right, means of total, S and R huVH mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of huVHDJH-Cμ and huVHDJH-Cγ transcripts depicted as histograms. R:S data are from one THX mouse representative of 3 THX mice. (e) huVHDJH-Cμ and huVHDJH-Cγ huB cell clonal size and diversity in RBD-KLH-immunized THX mice (THX 488, 490, 492) depicted as scatter plots. Bars depict the mean with s.e.m. (f) huVHDJH-Cμ and huVHDJH-Cγ huB cell clones and intraclonal diversification, as depicted by TreeMaps and phylogenetic trees. Individual rectangle or square (unique color) area reflects huB cell clone size. In THX mice 488, 490 and 492, the 20 largest huVHDJH-Cμ and huVHDJH-Cγ huB cell clones accounted for about one-tenth of huVHDJH-Cμ huB cells and one-fourth of huVHDJH-Cγ huB cells.
