Individualized Germline IGHV Gene Profiling

Individualized germline IGHV gene databases from NHP MCM01, MCM02, MCM03, MCM04, MCM05, and MCM06 were produced from samples obtained before immunization. In brief, IgM libraries for deep sequencing of antibody repertoires were generated with 5′ multiplex PCR from total PBMC mRNA^{53 (link)}. The mRNA was reverse transcribed with IgM constant region-specific primer containing a unique molecular identifier and a universal outer primer sequence. The cDNA was amplified utilizing the universal 3′ primer and two primer sets covering all gene families and targeting leader and UTR respectively that yield two libraries for each animal. Illumina indices and adapters were introduced by PCR prior to sequencing the library with Illumina’s MiSeq v3 kit. The output library was analyzed with IgDiscover to infer the germline gene IGHV alleles^{31 (link)}. The IGHV database utilized as an input database for the IgDiscover analysis was obtained from genomic DNA sequencing^{54 (link)}, with the addition of non-located (NL) alleles inferred from a larger set of NHPs^{53 (link)}. The heavy chain sequences of the isolated mAbs were assigned to the IGHV individualized database from the respective NHP and a comprehensive IGHJ database obtained from a larger group of NHPs, whereas the light chain was assigned to the IMGT’s light chain database using IgBlast to obtain the SHM and CDR3 sequences.