Rhesus macaque and human immunoglobulin sequence analysis. (a–c) Ancestral clonal sequences identify HCDR3 indels and VDJ gene contributions and confirm the acquisition of a five-residue EDDYG motif for rhesus lineages with significant somatic mutation within D gene segments. HCDR3 alignments of the rhesus antibodies (a) 42056-a.01, (b) 6561-a.01, and (c) V031-a.01 to their respective VDJ germline genes and ancestral lineage intermediate sequences identified from peripheral memory B cells at the indicated time points. Mismatches to the germline V, D, and J genes are highlighted. (d) Human V2 apex broadly neutralizing lineage D genes are aligned with their rhesus D gene homologs. Nucleotide differences in rhesus homologs are colored in blue. Non-homolog rhesus D genes encoding the “YYD” motif in human DH3-3*01 are included below that alignment. Rhesus DH3-15*01 is included for reference with the unique three-residue anionic motif (EDD) underlined. All rhesus sequences are labeled in red. (e) Left, proportion of reading frame usage among naïve rhesus B cells derived from DH4-25*01, the rhesus homolog of the human PGT145 lineage DH4-17*01 gene, in the peripheral Indian rhesus macaque repertoire. Right, box plots showing in order: the frequency of DH4-17*01 usage in reading frame two among all naïve B cells in the peripheral rhesus repertoire; HCDR3 length distribution of naïve rhesus B cells in the peripheral rhesus repertoire derived from DH4-17*01 in reading frame two; HCDR3 net charge distribution of naïve rhesus B cells in the peripheral repertoire derived from DH4-17*01 in reading frame two. Charge calculations only consider amino acid residues and not predicted sites of tyrosine sulfation.