title: Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.
abstract: Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.
authors.affiliation: a Texas Therapeutics Institute; Brown Foundation Institute of Molecular Medicine; University of Texas Health Science Center at Houston ; Houston , TX , USA.
authors.forename: Weixu
authors.initials: W
authors.lastname: Meng
authors.forename: Leike
authors.initials: L
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Xiong
authors.forename: Xuejun
authors.initials: X
authors.lastname: Fan
authors.forename: Hui
authors.initials: H
authors.lastname: Deng
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bett
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Chen
authors.forename: Aimin
authors.initials: A
authors.lastname: Tang
authors.forename: Kara S
authors.initials: KS
authors.lastname: Cox
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Joyce
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Freed
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Thoryk
authors.forename: Tong-Ming
authors.initials: TM
authors.lastname: Fu
authors.forename: Danilo R
authors.initials: DR
authors.lastname: Casimiro
authors.forename: Ningyan
authors.initials: N
authors.lastname: Zhang
authors.forename: Kalpit
authors.initials: K
authors.lastname: A Vora
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: An
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology; D000921:Antibody-Producing Cells / Q000276:immunology*; D053059:Dengue Vaccines / Q000276:immunology*; D003716:Dengue Virus / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008253:Macaca mulatta; D020539:Sequence Analysis, Protein
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D053059:Dengue Vaccines; D007135:Immunoglobulin Variable Region
keywords: IG; antibody secreting cell; dengue virus; monoclonal antibody; nonhuman primate; single cell PCR; vaccine