A broadly applicable approach to T cell epitope identification: Application to improving tumor associated epitopes and identifying epitopes in complex pathogens

Abstract

Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens.

Differential Cultivation of Francisella tularensis Induces Changes in the Immune Response to and Protective Efficacy of Whole Cell-Based Inactivated Vaccines

“…We observed that i Ft -MHB induces higher levels of the pro-inflammatory cytokines [IL-1β (Figure 10A), IL-6 (Figure 10C), and TNF-α (Figure 10D)], as compared to i Ft -BHI, while both immunogens induced similar levels of IL-10. Finally, we investigated the presentation of i Ft -MHB versus i Ft -BHI using an Ft- specific T cell hybridoma (FT25 6D10), which is specific to an epitope corresponding to Ft 50 s-ribosomal protein (20) and secretes IL-5 on engagement with cognate major histocompatibility complex (MHC)-peptide complex (13). Co-cultures of FT25 6D10 and APCs (PECs or BMDCs) exposed to i Ft -MHB exhibited higher levels of IL-5 compared to that of i Ft -BHI (Figure 11).…”

Approaches to vaccines against Orientia tsutsugamushi

“…Examples of those techniques include screening of retrovirus-based cDNA libraries (Wang et al, 1998; Aoshi et al, 2005), use of random expression genomic libraries in E. coli to feed macrophages as APCs (Gonçalves et al, 2006), screening of purified proteins (from a genomic library) coupled to beads (Valentino et al, 2011a,b), screening of synthetic minigene libraries (Hondowicz et al, 2012), and screening of plasmid-encoded combinatorial peptide libraries (Siewert et al, 2012). …”

Antigen Discovery for Vaccines Using High‐Throughput Proteomic Screening Technologies

“…For example, Turner and colleagues developed a bead capture system using polyhistidine tag [90,91]. This method has recently been demonstrated to have utility for T-cell antigen discovery using a small (45 ORF) Francisella tularensis expression library [92,93]. HT T-cell screening of proteomes is a new field, and similar studies are in the pipeline.…”