Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity. © 2013 Kaabinejadian et al

Peptide specific CD8⁺ responses from PBMC isolated from 12 WNV infected subjects stimulated with peptides.

<p>Each bar represents the mean percentage of the CD8⁺ T cells from all 12 donors that express 1 (purple), 2 (blue), 3 (green), 4 (yellow) or 5 (red )markers [4 cytokines (IL-2, IFN-γ, TNF-α, and MIP-1β) + degranulation factor (CD107 a)] when exposed to each peptide.</p

CTL reactivity of identified WNV epitopes.

<p>PBMC isolated from naturally infected individuals were stimulated with a pool of 6 WNV peptides in the presence of IL-2 for 9 days. On day 10, cultures were restimulated with individual peptides for 6 h and T cell responses were tested by Intracellular Cytokine Staining. Y-axis  =  %IFN-γ positive CD3⁺/CD8⁺ T cells. Each dot represents the response from a single WNV subject. The average response to GTL9 was significantly higher than the response to the negative control (DMSO). SEB: Staph Enterotoxin B *: significant increase from negative control. Significance was determined by one-way ANOVA followed by Tukey’s test; P<0.05.</p

Identified WNV-derived peptides presented by HLA-A*11:01.

<p>Identified WNV-derived peptides presented by HLA-A*11:01.</p

Polyfunctional profiles of WNV-specific CD8⁺ T cells.

<p>All possible combinations of five functions (CD107a, IL-2, MIP-1β, IFN- γ, and TNF-α) produced by WNV epitope-specific CD8⁺ T cells are shown on the X-axis. Functional profiles are grouped and color-coded according to number of functions and summarized in the pie charts. Each slice of the pie corresponds to the mean percentage of CD8⁺ T cells producing five (red), four (dark blue), three (green), two (light blue), or one (yellow) function.</p

Heterogeneity of CTL responses to WNV.

<p><b>A.</b> PBMC isolated from subjects were stimulated with WNV peptides as described in materials and methods and T cell responses were tested by Intracellular Cytokine Staining. Y-axis  =  %IFN-γ positive CD3⁺/CD8⁺ T cells. Each bar represents responses from CD3⁺/CD8⁺ T cells from each subject to the identified WNV peptides. GTL9 (red), KSY9 (blue), AII11 (green), AVV9 (yellow), RVL9 (purple) and KNM9 (brown). Histograms represent patient-specific responses to WNV epitopes minus the negative control (DMSO) plus 2× STD. <b>B.</b> Number of patients that recognized each epitope.</p

Direct epitope discovery of WNV epitope GTL9.

<p>(A) RP-HPLC profile of HLA-A*11:01 peptides from uninfected (blue) and WNV-infected (red) cells. (B) MS ion spectra of HPLC fraction 50 from uninfected (black) and infected (red) cells. MS data were recorded for 300 scans. (C) Overlay of the MS/MS fragmentation pattern of ion 466.7 in the infected HPLC fraction 50 (identified as the GTL9 peptide; red) with the MS/MS fragmentation pattern for the GTL9 synthetic peptide (black). (D) Florescence polarization competitive binding assay with the GTL9 synthetic peptide and HLA-A*11:01.</p

Location of A*11:01 (red) and A*02:01 (black) WNV peptide epitopes on the WNV polyprotein.

<p>C, nucleocapsid; M, membrane; E, envelope glycoprotein; NS, nonstructural.</p