A primer set for comprehensive amplification of V-genes from rhesus macaque origin based on repertoire sequencing

摘要

Recombinant antibodies serve as therapeutic molecules for a broad range of applications. High affinity antibodies are typically isolated following an active and effective immunization. Human-like antibodies may be obtained from immunized nonhuman primates (NHP), such as rhesus macaque, when immunized human origin is not available. For the isolation of such antibodies, strategies like phage and yeast display, are employed. These strategies are primarily based on the amplification of the rearranged variable (V) regions coded by mRNA, obtained from lymphatic source of immunized animals. To amplify these genomic sequences, designated set of primers are required, ideally covering the immune animal V-gene repertoire. Such primer sets are commonly designed based on the germline repertoire of specific animals according to immunoglobulin databases. However, In case of rhesus macaque, however, the known immunoglobulin germline V-gene database is still limited. The emergence and continuous improvements in high-throughput sequencing (HTS) technologies now enable the profiling of an immune repertoire for both basic and applicative studies, among which is the identification and expression of novel alleles. We report here on the profiling of non-immunized rhesus macaque (Macaca mulatta) expressed antibody repertoire, using HTS and advanced tailored bioinformatics tools. This analysis resulted in 32,480 and 73,354 complete heavy and light variable gene (V-H and V-L) sequences, respectively. Further analysis of these sequences, using the IgDiscover tool, resulted in the identification of 102, 214 and 48 inferred V-H, V-kappa and V-lambda germline sequences, respectively, of which over 50% are novel alleles. This dataset, together with other recently published datasets, enabled the design of a comprehensive primer set (v2018), which demonstrated the broadest coverage of rhesus macaque germline genes identified up to date. The newly designed primer set was confirmed for its extent of coverage of the V-genes in various datasets of rhesus macaque germlines as well as the expressed repertoire mapped in this study. Among other things, an improvement of 28% and 50% in the coverage of the VH and VL expressed repertoire was demonstrated in comparison to a primer set we have previously designed. This primer set can be further used for various applications that require the complete coverage of the NHP V-gene repertoire.