Extended Data Fig. 9: THX mice vaccinated with Pfizer-BioNTech 162b2 COVID-19 mRNA mount a class-switched and somatically hypermutated antibody response to SARS-CoV-2 Spike S1 RBD.
a, Spleen huB cell huVHDJH-Cμ, huVHDJH-Cγ and huVHDJH-Cα1 transcripts in COVID-19 mRNA-vaccinated THX mice (n=3, same mice as in Fig. 7e, f) were analyzed for CDR3 length, R:S mutations and huB cell clonal size. Left, CDR3 length distribution in recombined huVHDJH-CH transcripts. Colors denote different antibody isotypes; color gradients denote different THX mice. Middle, huVHDJH-Cμ, huVHDJH-Cγ and huVHDJH-Cα1 huB cell clonal size depicted as scatter plots. Bars depict the mean with s.e.m. Right, means of total, S and R VH mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 depicted as histograms. R:S data are from one THX mouse representative of 3 THX mice. b–c, Spleen RBD-specific huB cell huVHDJH-CH, huVκJκ and huVλJλ transcripts in 3 additional COVID-19 mRNA-vaccinated THX mice were reverse transcribed and amplified by RT-PCR. Paired huVHDJH and huVκJκ or huVλJλ gene segments from 100 single cells were used to make recombinant human monoclonal antibodies. (b) Left, huVH, huVκ or huVλ gene family member expression in 100 recombinant human monoclonal antibodies, as depicted by pie charts. Colors depict different huVH, huVκ or huVλ gene families; color gradients denote individual gene family members. The 100 human monoclonal antibodies showed predominant utilization of V3, V4 and V1, together with Vκ3, Vκ1 and Vκ2 as well as Vλ1 and Vλ2 genes. Middle, mutation frequency (change/base) of recombined huIgH VHDJH and huIg VκJκ or VλJλ regions in recombinant human monoclonal antibodies, as depicted by scatter plots. Each dot represents a single sequence and the bar depicts the mean with s.e.m. Right, CDR3 length distribution in paired VHDJH and human immunoglobulin VκJκ or VλJλ human monoclonal antibodies. huIgH CDR3 lengths varied between 5 and 25 amino acids, peaking at 12, 13 and 15 amino acids; those of huVκ and huVλ varied between 5 and 13 amino acids, peaking at 9 amino acids. c, Forty-five expressed recombinant human monoclonal antibodies were selected for analysis of paired huIgH and huIgL genes based on their highest RBD-binding activity (> 1.0 OD by specific ELISA). Shown are 27 huIgM (blue), 5 huIgG (red) and 13 huIgA (green) monoclonal antibodies. Consistent with the higher haploid representation of V3, V4 and V1 gene families, V3 genes were the most frequently utilized (35 human monoclonal antibodies), particularly V3-23 (10 human monoclonal antibodies), V3-30 (8), V3-9 (4) and V3-7 (3), followed by V4 (7) and V1 (3) genes. Twenty-two human monoclonal antibodies utilized JH4, 9 JH6 and 6 JH3, with JH1, JH5 and JH2 accounting for the remaining 8 human monoclonal antibodies. Thirty-five human monoclonal antibodies utilized Vκ genes, with Vκ3-11 as the most frequently utilized (8 human monoclonal antibodies) followed by Vκ1-44 (4) and Vκ4-1 (4). Ten human monoclonal antibodies utilized Vλ genes, with Vλ1-44 and Vλ2-14 (2 and 2 human monoclonal antibodies) as the most frequently utilized.
