Abstract
Rhesus macaques (Macaca mulatta) are used as a human-relevant animal species for the evaluation of vaccines and as a source for cloning monoclonal antibodies (mAbs) that are highly similar to human-derived antibodies. Although antibody-secreting plasmablasts in humans are well-defined and can be easily isolated for mAb cloning, it remains unclear whether the same phenotypic markers could be applied for isolating antibody-secreting plasmablasts from Chinese rhesus macaques. In this study, we evaluated a series of cell surface and intracellular markers and identified the phenotypic markers of plasmablasts in Chinese rhesus macaques as CD3−CD14−CD56−CD19−CD27−CD20−/lowCD80+HLA-DR+CD95+. After influenza virus vaccination, the plasmablasts in peripheral blood mononuclear cells (PBMCs) increased transiently, peaked at day 4–7 after booster vaccination and returned to nearly undetectable levels by day 14. Antigen-specific enzyme-linked immunosorbent spot (ELISPOT) assays confirmed that the majority of the plasmablasts could produce influenza virus-specific antibodies. These plasmablasts showed transcriptional characteristics similar to those of human plasmablasts. Using single-cell PCR for immunoglobulin heavy and light chains, most mAbs cloned from the CD3−CD14−CD56−CD19−CD27−CD20−/lowCD80+HLA-DR+CD95+ plasmablasts after vaccination exhibited specific binding to influenza virus. This study defined the phenotypic markers for isolating antibody-secreting plasmablasts from Chinese rhesus macaques, which has implications for efficient cloning of mAbs and for the evaluation of plasmablast response after vaccination or infection in Chinese rhesus macaques.
Highlights
Non-human primates (NHPs) are widely used as a model for the evaluation of human vaccines and for cloning monoclonal antibodies against influenza virus, HIV, Ebola virus, and other pathogens [1,2,3,4,5,6]
The cell surface markers for identifying plasmablasts have been established as CD3−CD19+CD20−/lowCD27hiCD38hi [7, 11]
We first assessed the cross-reactivity of a variety of anti-human antibodies to ensure that they recognize the same proteins from Chinese rhesus macaques (Supplementary Table 1)
Summary
Non-human primates (NHPs) are widely used as a model for the evaluation of human vaccines and for cloning monoclonal antibodies (mAbs) against influenza virus, HIV, Ebola virus, and other pathogens [1,2,3,4,5,6]. Antibody-secreting plasmablasts are a good source for cloning antigen-specific mAbs after vaccination or infection with a pathogen [7,8,9,10]. Plasmablasts peak around 7 days after influenza virus vaccination [7, 12], and influenza virus-specific antibody-secreting cells account for up to 6% of all B cells [7]. The phenotypic markers used to characterize antibody-secreting cells in rhesus macaques have been inconsistent among different laboratories. The research on the phenotypic markers of plasmablasts from Chinese rhesus macaques is even more lacking. One study focused on antibody-secreting plasma cells in bone marrow from Chinese rhesus macaques [13], but there have been no further studies on plasmablasts in the peripheral blood. Chinese rhesus macaques have been increasingly used in biomedical research, possibly due to the restricted supply of Indian rhesus macaques, as India banned the exportation of rhesus macaques, while the exportation of Chinese rhesus macaques has increased in recent decades [14, 15]
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