Abstract
Dengue envelope (E) protein is a dominant antigen for vaccine development and E-based vaccines have shown partial or full protection against live-virus challenge in non-human primates. Generally, T cell responses can be investigated with peptides. However, hundreds of over-lapping peptides need to be synthesized to cover the whole sequence of a protein, which brings the cost up to a much higher level than purchasing a protein. We have developed an enzyme-linked immunospot (ELISpot) assay that uses intact E proteins instead of peptides for assessing IFN-gamma (IFN-γ) responses. The assay relies on professional antigen presenting cells, dendritic cells, to process and present the E proteins to stimulate T cells.
Peripheral blood mononuclear cells (PBMCs) from dengue-exposed and naïve subjects were selected for the assay development. IFN-γ production ranged from 53 to 513 spot forming units (SFUs) and 0–45 SFUs per million PBMCs in dengue-exposed and naive subject groups, respectively. The assay allowed quantification of E-specific IFN-γ secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Our results suggest that the dendritic cell-based IFN-γ assay is a useful tool for assessing immunological memory for clinical research.
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The views expressed in this chapter are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, the U.S. Government or the Henry Jackson Foundation (HJF). MS is an employee of the U.S. Government and PS is a HJF government contract employee. Title 17 U.S.C. article 105 provides that “Copyright protection under this title is not available for any work of the United States Government.” Title 17 U. S.C. article 101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties. The study protocol was approved by the Naval Medical Research Center Institutional Review Board in compliance with all applicable federal regulations governing the protection of human subjects.
Financial SupportThis work was funded by a grant from the Military Infectious Disease Research Program (MIDRP), U.S. Army Medical Research and Materiel Command, Fort Detrick, MD, work unit number A0311.
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Sun, P., Simmons, M. (2018). Dendritic Cell-Based ELISpot Assay for Assessing T-Cell IFN-γ Responses in Human Peripheral Blood Mononuclear Cells to Dengue Envelope Proteins. In: Kalyuzhny, A. (eds) Handbook of ELISPOT . Methods in Molecular Biology, vol 1808. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8567-8_17
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DOI: https://doi.org/10.1007/978-1-4939-8567-8_17
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