Coupling of Single Molecule, Long Read Sequencing with IMGT/HighV-QUEST Analysis Expedites Identification of SIV gp140-Specific Antibodies from scFv Phage Display Libraries.

Seung Yub Han,Ismael Ben F Fofana,Alesia Antoine,Gintaras Deikus,Matthew Slein,Marie-Paule Lefranc,Robert P Sebra,Patrice Duroux,Sofia Kossida,David Howard,Bryant Chang,Melissa L Smith,Woo Sung Chang

doi:10.3389/fimmu.2018.00329

Abstract

The simian immunodeficiency virus (SIV)/macaque model of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome pathogenesis is critical for furthering our understanding of the role of antibody responses in the prevention of HIV infection, and will only increase in importance as macaque immunoglobulin (IG) gene databases are expanded. We have previously reported the construction of a phage display library from a SIV-infected rhesus macaque (Macaca mulatta) using oligonucleotide primers based on human IG gene sequences. Our previous screening relied on Sanger sequencing, which was inefficient and generated only a few dozen sequences. Here, we re-analyzed this library using single molecule, real-time (SMRT) sequencing on the Pacific Biosciences (PacBio) platform to generate thousands of highly accurate circular consensus sequencing (CCS) reads corresponding to full length single chain fragment variable. CCS data were then analyzed through the international ImMunoGeneTics information system® (IMGT®)/HighV-QUEST (www.imgt.org) to identify variable genes and perform statistical analyses. Overall the library was very diverse, with 2,569 different IMGT clonotypes called for the 5,238 IGHV sequences assigned to an IMGT clonotype. Within the library, SIV-specific antibodies represented a relatively limited number of clones, with only 135 different IMGT clonotypes called from 4,594 IGHV-assigned sequences. Our data did confirm that the IGHV4 and IGHV3 gene usage was the most abundant within the rhesus antibodies screened, and that these genes were even more enriched among SIV gp140-specific antibodies. Although a broad range of VH CDR3 amino acid (AA) lengths was observed in the unpanned library, the vast majority of SIV gp140-specific antibodies demonstrated a more uniform VH CDR3 length (20 AA). This uniformity was far less apparent when VH CDR3 were classified according to their clonotype (range: 9–25 AA), which we believe is more relevant for specific antibody identification. Only 174 IGKV and 588 IGLV clonotypes were identified within the VL sequences associated with SIV gp140-specific VH. Together, these data strongly suggest that the combination of SMRT sequencing with the IMGT/HighV-QUEST querying tool will facilitate and expedite our understanding of polyclonal antibody responses during SIV infection and may serve to rapidly expand the known scope of macaque V genes utilized during these responses.

Highlights

Nonhuman primates are an important animal model for numerous human diseases, as there is great similarity between the human and macaque genomes [1,2,3,4,5,6,7]
In order to focus our analyses on highly accurate, full-length single chain fragment variable (scFv) fragments, SMRT sequencing data were processed using the latest CCS2 algorithm and filtered by size (700–1,200 bp) and accuracy (99.9%, QV30) (Figure 1C)
Techniques that rely on antigen-based selection of single B cells or screening of single colonies following phage display generally result in a low number of sequences and are not always suited to examine the expected level of complexity in such a system

Summary

Introduction

Nonhuman primates are an important animal model for numerous human diseases, as there is great similarity between the human and macaque genomes [1,2,3,4,5,6,7]. The variable heavy (VH) and variable light (VL) domains of macaque antibody heavy (H) and light (L) chains can be generated using polymerase chain reaction (PCR) conditions and oligonucleotide primers based on human IG nucleotide sequences This has been shown far for VH rather than VL genes, the use of rhesus specific primers for amplification may function for both variable domains [9, 11, 12]. Virus-specific antibodies are abundantly produced during the course of HIV/SIV infection in humans or macaques [5, 17,18,19,20] These antibodies, which primarily target the envelope glycoprotein (Env) on the surface of HIV/SIV virions, generally do not provide protection due in part to their inability to neutralize the virus. The presence of hypervariable loops and extensive glycosylation are all components of a complex escape mechanism that limits the potential potency of antibody-mediated neutralization [5, 21]

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