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 D 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.

A Novel Neutralizing Antibody Specific to the DE Loop of VP1 Can Inhibit EV-D68 Infection in Mice

“…The sequences of the monkey Ig H chain variable gene (V H ) and L chain variable gene (V L ) chains were amplified by RT-PCR and nested PCR using the methods and primers in Table I ( 24). In brief, Ig V H and V L were amplified in 96-well PCR plates containing sorted single B cells.…”

“…To determine the Ab properties of the isolated memory B cells, we amplified the Ab gene by cDNA synthesis from single memory B cells followed by nested PCR (24,31,32). Two rounds of specific PCR were performed with two sets of optimal primers for monkey IgG genes (Table I).…”

Disrupting LILRB4/APOE Interaction by an Efficacious Humanized Antibody Reverses T-cell Suppression and Blocks AML Development

“…Heavy-and light-chain constructs were cotransfected into human embryonic kidney freestyle 293 (HEK293F) cells using transfection reagent PEI (Sigma; ref. 18). After 7 days of expression, supernatants were harvested and antibodies were purified by affinity chromatography using protein A resin as we reported before (Repligen; ref.…”

“…After 7 days of expression, supernatants were harvested and antibodies were purified by affinity chromatography using protein A resin as we reported before (Repligen; ref. 18). A panel of 26 purified rabbit monoclonal antibodies (mAb) was generated and used for this study.…”

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning

“…In such cases, the secondary screening takes place following the expression and production of recombinant mAbs. Importantly, single B cell isolation by FACS is translatable across species, and has been described for humans, rhesus macaques, rabbits, and mice (12, 19–22). …”