Journal of Visualized Experiments www.jove.com Copyright © 2019 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License January 2019 | 143 | e58595 | Page 1 of 7 Video Article Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination Katherine Schultheis 1 , Hubert Schaefer 2 , Holly M. Pugh 1 , Bryan S. Yung 1 , Janet Oh 1 , Jacklyn Nguyen 1 , Laurent Humeau 1 , Kate E. Broderick 1 , Trevor R.F. Smith 1 1 Inovio Pharmaceuticals, Inc 2 Mycotic and Parasitic Agents and Mycobacteria, Robert Koch Institute Correspondence to: Katherine Schultheis at kschultheis@inovio.com URL: https://www.jove.com/video/58595 DOI: doi:10.3791/58595 Keywords: Immunology and Infection, Issue 143, Enzyme-linked immune spot assay, guinea pigs, T-lymphocytes, vaccination, interferon-gamma, administration, cutaneous Date Published: 1/20/2019 Citation: Schultheis, K., Schaefer, H., Pugh, H.M., Yung, B.S., Oh, J., Nguyen, J., Humeau, L., Broderick, K.E., Smith, T.R. Optimized Interferon- gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination. J. Vis. Exp. (143), e58595, doi:10.3791/58595 (2019). Abstract The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals. Video Link The video component of this article can be found at https://www.jove.com/video/58595/ Introduction The protocol described here permits the detection of interferon-gamma (IFN-γ) secreting cells after antigen recall in a peripheral blood mononuclear cell (PBMC) population harvested from Hartley guinea pigs. We have applied the assay to characterize the kinetics and magnitudes of antigen-specific T cell responses to an Influenza vaccination-regimen in the guinea pig. We believe this protocol will significantly propel the pre-clinical development of vaccination programs in this highly relevant animal model. T cells elicited by vaccines play an essential role in the protection against infectious agents and immunotherapeutic pathways associated with other diseases. The importance of T cells has been highlighted in multiple vaccine studies. Immunization of ferrets and mice with a plasmid DNA (pDNA) encoding for H5 hemagglutinin and N1 neuraminidase provided protection from morbidity and mortality in an influenza virus challenge in the absence of neutralizing antibodies, indicating the importance of T cell immunity 1 . In addition to strong neutralizing humoral response, T-cells play a crucial role for not only viral clearance 2 but also protection from infection with respiratory syncytial virus (RSV) in mice 3 . In humans, pre- existing CD8+ T cells were associated with decreased disease severity during the H1N1 pandemic in 2009 4 . CD4+ T cell counts together with certain cytokine plasma concentration are correlated with disease severity in RSV-infected children 5 . The guinea pig has gained prominence as a laboratory model for research and development in various areas of medicine such as skin sensitization, nutritional research, studies of the auditory system and, most relevant for this work, infectious diseases. It was crucial for the discovery of vaccines against tuberculosis and diphtheria. More recently the guinea pig is used as a model for Influenza 6 and Ebola 7 . Furthermore, possessing physiological similarities to human skin 8 , the guinea pig offers an accessible small animal model for dermal drug delivery methods. In contrast to its importance as a laboratory model, the availability of guinea pig specific assays and probes to characterize immune responses remains limited 9 . Basic cellular assays such as the IFN-γ enzyme-linked immunospot (ELISpot-assay) that is routinely used in pre-clinical and clinical research to enumerate T cell responses have not been available. The first solid-phase enzyme-linked immunospot assays were used to determine the number of specific antibody-secreting cells in a diverse B cell-population 10,11 . The format has advanced to detect cells secreting cytokines including IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF, TNF-alpha, TNF-beta, granzyme B, and IFN-γ. The ELISpot assay possesses high sensitivity; potentially each cytokine-producing cell can be detected. The limit of detection for ELISpot assays has been reported to be lower than 10 spots per 100,000 PBMCs 12 . Recently a guinea-pig IFN-γ specific antibody pair was made available 13 , and this has opened up the opportunity to address this deficiency in the field.