Abstract
Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species.A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-γ and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-γ and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset). Pan-reactive mAbs, recognizing cytokines from all NHP species, were further analyzed in capture assays and flow cytometry with NHP peripheral blood mononuclear cells (PBMC).Pan-reactive mAb pairs for IFN-γ well as IL-2 were identified and used in ELISA to measure IFN-γ and IL-2, respectively, in Old and New World NHP PBMC supernatants. The same mAb pairs displayed high functionality in ELISpot and FluoroSpot for the measurement of antigen-specific IFN-γ and IL-2 responses using cynomolgus PBMC. Functionality of pan-reactive mAbs in flow cytometry was also verified with cynomolgus PBMC.The development of well-defined immunoassays functional with a panel of NHP species facilitates improved analyses of cellular immunity and enables inclusion in multiplex cytokine assays intended for a variety of NHP.
Highlights
The similarity of the immune system of humans (HUM) and nonhuman primates (NHP) and the susceptibility of NHP to many human pathogens make NHP valuable animal models for studying immune responses to infectious diseases, preclinical evaluation of new therapeutics as well as for assessment of vaccine-induced immunity
Individual reactivity of monoclonal antibody (mAb) with recombinant HUM and NHP IFN-γ and IL-2 A panel of new mAbs made against HUM IFN-γ (n = 24) and IL-2 (n = 19) were analyzed for reactivity with recombinant HUM IFN-γ and IL-2, respectively
Eight mAbs to IFN-γ and seven mAbs to IL-2 that displayed the strongest reactivity with human cytokines were analyzed by ELISA for their ability to capture human and NHP IFN-γ and IL-2 expressed in transfected HEK cells; in addition, four previously established mAbs to human IFN-γ and two against human IL-2 were included in the analysis (Table 1)
Summary
The similarity of the immune system of humans (HUM) and nonhuman primates (NHP) and the susceptibility of NHP to many human pathogens make NHP valuable animal models for studying immune responses to infectious diseases, preclinical evaluation of new therapeutics as well as for assessment of vaccine-induced immunity. Simian immunodeficiency virus (SIV) infection in non-natural SIV hosts, such as rhesus (RHE), cynomolgus (CYN) and pigtail macaques (PTM), causes AIDS-like symptoms and provide models for assessment of vaccine-induced protective immunity (Sui et al, 2013). Cellular immune responses induced by infection or vaccination are often assessed by analysis of cytokine production and monoclonal antibody (mAb) reagents reactive with cytokines from NHP are essential. Important mAb-based capture assays used to quantify the levels of these cytokines as well as to enumerate the number of cytokine-secreting cells include ELISA and ELISpot, respectively. In addition to the capture assays based on mAb pairs for capture and detection, respectively, single mAbs reagents are used in applications like flow cytometry, immunohistochemistry, neutralization assays and western blot
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