Effective immune responses rely on T cells. For some vaccines, CD4+ T cells themselves are directly antiviral, and even when antibodies are the final desired immune vaccine response, an effective CD4+ helper T cell response is required. To become activated, CD4+ T cells must first recognize and bind a segment of the pathogen’s surface peptide, known as an epitope, specific for each cell’s unique T cell receptor. Identifying relevant T cell epitopes for a given infection is of high priority for therapeutics and vaccine design, as pieces of the pathogen that contain these binding sites, known as antigens, can be used to induce pathogen-specific T cell responses in vivo.
Identification of CD4+ T cell epitopes is usually performed by determining which T cells become activated when mixed with specific peptides from the microbial proteome. T cell activation is determined by measuring anti-viral cytokine production with a variety of standard methods. However, pathogens with large genomes may contain many sites that T cells recognize. As a result, T cell responses to individual epitopes may fall below the limit of detection for standard readout assays. Furthermore, many readout methods are cell-destructive, prohibiting follow-up studies unless more blood is collected. Standard epitope determination workflows therefore usually require large quantities of blood that increase as the pathogen genome and thus proteome size increases. Large volume blood collection is not always feasible in resource-limited settings, especially in the middle of an outbreak. To mitigate this issue, some researchers use activation-induced markers (AIM) on the T cell surface to detect activated T cells. The reactive T cells are enriched from the larger population, expanded, and used for further studies to determine relevant epitopes. As typically performed, AIM is done using viral peptides for both the enrichment and readout steps. This forms a “circular argument”, and at no point is virus specificity confirmed using the actual pathogen.
The 2016 Zika virus (ZIKV) epidemic continues to contribute significantly to global disease burden, and a vaccine is urgently needed. Previous work has shown that T cells are important to protect from infection, making ZIKV epitope discovery a high priority. Victoria Campbell from the Koelle lab (University of Washington Medicine and Fred Hutch Vaccine and Infectious Disease Division), along with collaborators from Vitalant, La Jolla Institute, and Imperial College London, sought to improve the epitope discovery workflow using T cells from ZIKV-infected people. They recently published this work in ImmunoHorizons.
Using very small blood samples from a previous clinical study, the authors stimulated mixed immune cells from the blood of ZIKV-infected individuals with a whole killed preparation of ZIKV. ZIKV, like some other pathogens, is dangerous in the lab. Collaboration with experts and documentation of complete ZIKV inactivation was essential. The activated T cells were isolated from the bulk population using fluorescence-activated cell sorting and then expanded in vitro. In the subsequent readout step, peptides derived from the ZIKV genome were used to assess which putative epitopes caused T cell activation, using standard T cell activation assays. The readout uses B cells immortalized from less than 1 ml of blood, further economizing. The investigators found that 27 distinct ZIKV peptides elicited IFN-g production in T cells, suggesting their ability to recognize and respond to a ZIKV antigen. The use of whole virus for enrichment followed by ZIKV peptides for readout is a non-circular workflow to discover ZIKV epitopes. Although ZIKV-specific T cells are rare in ZIKV-immune individuals, they can be enriched from quite small blood samples and then expanded for further studies. The AIM system used in this study identifies T cells that respond to both whole virus and to viral peptides and is already being applied to the large SARS-CoV-2 proteome using small patient blood samples.
This work was supported by the National Institutes of Health.
Fred Hutch/UW Cancer Consortium member David Koelle contributed to this research.
Campbell VL, Nguyen L, Snoey E, McClurkan CL, Laing KJ, Dong L, Sette A, Arlehamn CSL, Altmann DM, Boyton RJ, Roby JA, Gale M, Stone M, Busch MP, Norris PJ, Koelle DM. 2020. Proteome-Wide Zika Virus CD4 T Cell Epitope and HLA Restriction Determination. ImmunoHorizons. 2020 Aug 4;4(8):444-453. doi: 10.4049/immunohorizons.2000068.