T cell epitope mapping of the e-protein of West Nile virus in BALB/c mice

Abstract

West Nile virus (WNV) is a zoonotic virus, which is transmitted by mosquitoes. It is the causative agent of the disease syndrome called West Nile fever. In some human cases, a WNV infection can be associated with severe neurological symptoms. The immune response to WNV is multifactorial and includes both humoral and cellular immunity. T-cell epitope mapping of the WNV envelope (E) protein has been performed in C57BL/6 mice, but not in BALB/c mice. Therefore, we performed in BALB/c mice a T-cell epitope mapping using a series of peptides spanning the WNV envelope (E) protein. To this end, the WNV-E specific T cell repertoire was first expanded by vaccinating BALB/c mice with a DNA vaccine that generates subviral particles that resemble West Nile virus. Furthermore, the WNV structural protein was expressed in Escherichia coli as a series of overlapping 20-mer peptides fused to a carrier-protein. Cytokine-based ELISPOT assays using these purified peptides revealed positive WNV-specific T cell responses to peptides within the different domains of the E-protein.

Figure 1. Expression and purification of recombinant GST tagged peptides.

SDS-PAGE showing crude lysates of protein-expressing bacteria and purification steps of peptide E471 showing a moderate expression (a) and peptide E131 showing very low expression (b). Lane 1, crude lysate; lane 2, lysate supernatant after centrifugation; lane 3, size marker; lanes 4–5, respectively elution fraction 1 and 2 of recombinant peptide after glutathione affinity purification.

Figure 2. Detection of cellular and humoral immune response following pDNA-based vaccination.

IFN-γ production by (a) CD4-depleted and (c) CD8-depleted splenocytes after stimulation with purified recombinant GST tagged E-protein derived peptides. The WNV E-protein specific T-cell repertoire in BALB/c mice was expanded by two DNA vaccinations. Splenocytes obtained two weeks after the boost were stimulated with different recombinant GST tagged E-protein derived peptides and the numbers of cells producing IFN-γ were determined via ELISPOT. (b) Detection of serum IgG1 and IgG2a titers to the WNV E-protein two weeks after the boost via ELISA.

Figure 3. Location of the peptide sequences in the E protein that, based on our in vivo experiments, contain strong CD4+ (underlined) and CD8+ (bold) T cell epitopes.

The shown amino acid sequence is that of the E protein of lineage 1 WNV strain Ita09. Sequences that are in bold and underlined contain strong CD4+ as well as CD8+ T cell epitopes.