Row H of the 96-well plate was used as blank control, which contains only lysis buffer without sorted cells. 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 EFNA1 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 Purpureaside C 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. Keywords:nonhuman primate, antibody secreting cell, single cell PCR, monoclonal antibody, IG, vaccine, dengue virus == Introduction == Nonhuman primates (NHPs) are widely used in preclinical studies for development of human vaccines. They can also be used to gain confidence in defining vaccine images and their ability to elicit the desired immune response, derived from reverse vaccinology approaches. Vaccine efficacy evaluation has historically relied on the immune response to experimental vaccine in NHPs including rhesus macaque.1-4Antibody elicitation is one of the key attributes of immune response to vaccines. However, due to technical limitations, antibody response to vaccination is measured as overall serum binding and/or as the development of a functional titer such as Purpureaside C neutralization to the vaccines or targeted viruses. While serum antibody titers provide a general humoral response to experimental vaccines, polyclonal antibodies are of little value for defining the critical components of the host humoral response and this is particularly true for pathogens with sero-type variants and complex antigens. Profiling monoclonal antibodies (mAbs) generated from vaccinated NHPs can reveal crucial aspects of the immunologic response to a vaccine such as antigen epitopes for generating high affinity, neutralizing, cross-reactive antibodies, and inter-relationship of binding and neutralizing antibodies to analyze in vivo maturation of responses post vaccination. Cloning of immunoglobulins (IG) or antibodies from infected or immunized rhesus macaques has mostly been accomplished using methods which include phage display, immortalization of B cells, or single cell cloning of memory B cells.5-8Antibody secreting cells originating from activated memory B cells possess unique properties including their ability to produce large amounts of IG in response to ongoing infection or immunization, and are enriched in specificity for the antigens of interest. Therefore, antigen-specific IG can be isolated from single antibody secreting cells Purpureaside C efficiently without antigen pre-screening. This is a useful approach in evaluation of candidate vaccines for which the antigens are not well defined. Methods for cloning human IG from human antibody secreting cells have been developed,9-15and several protocols for cloning monoclonal antibodies from NHPs plasma/memory B cells have also been reported.7,9,16Here we report a robust and efficient process for cloning IG from single rhesus macaques antibody secreting cells that achieves higher cloning efficiency than that previously reported,7and validate the Purpureaside C method with an experimental dengue vaccine. This methodology is expected to have general application for studies of IG in response to experimental vaccines using preclinical NHPs models. == Results == == Immunization of rhesus macaques and single cell sorting of antibody secreting cells == A tetravalent live attenuated dengue virus followed by a boost with recombinant dengue virus envelope glycoprotein was used for rhesus macaque (Macaca mulatta) immunization, described in details in the methods. In order to determine the peak of the humoral response for optimal peripheral blood mononuclear cells (PMBCs) sampling, we carried out a time course fluorescence activated cell sorter (FACS) analysis of macaque immunized with theMMR IIvaccine of live attenuated measles, mumps and rubella virus vaccine. The PBMCs were collected from days 4, 5, 6, and 7 postMMR IIvaccination and analyzed by FACS for cell surface markers (CD19, surface IgG (sIgG), CD138) as well as intracellular IgG (IC IgG) and proliferation Ki67 markers. At day 7, a unique population was clearly observed, which was IC IgG and Ki67 positive (Fig. 1A). Based on this observation, we subsequently focused on the PBMCs isolation on day 7 post vaccination. Blood samples (10 ml per subject) were.