Long-Term Immunity against Actual Poxviral HLA Ligands as Identified by Differential Stable Isotope Labeling

Abstract

Viral peptides are presented by HLA class I on infected cells to activate CD8+ T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8+ T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.

Discovering naturally processed antigenic determinants that confer protective T cell immunity

“…We limited the analysis to peptides that contain 9-11 residues, because the large majority conforms to this length while a small fraction contains longer peptides. the protective potential of individual antigenic determinants, which were mostly focused on a few rapidly detectable specificities (31)(32)(33)(34). Therefore, the actual array of critical antigenic determinants driving protective TCD8 responses during a natural infection or after vaccination remains unknown.…”

“…As alternatives to algorithm-based epitope prediction, mass spectrometry-based proteomics approaches to sequence peptides eluted from HLA class I molecules have been used to identify VACV epitopes (32,36,37). A significant advantage to this approach is that it directly informs whether an antigenic determinant is natu-…”

“…Hence, only limited preclinical mouse studies have addressed rally processed, thereby narrowing the search for potential vaccine targets to those determinants that are efficiently presented during an infection (38). While only a few such epitopes have been evaluated for immune recognition (32,37), none were ever interrogated for their protective potential. Therefore, it remains unclear whether the proteomics approach is superior for TCD8 epitope discovery over currently existing computational approaches and whether eluted peptides inform relevant vaccine targets.…”

Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation

“…By intracellular cytokine staining (ICS) assays, we found that 32% of IFN-␥ secreting cells had a specific response versus 1% without virus. Later, this CD8 ϩ cell line was used to identify TAP-independent epitopes with target cells previously pulsed with the VACV synthetic peptides previously reported as HLA-B*0702 epitopes identified from either HLA-B*0702 transgenic mice (A34R(82-90), D1R(808 -817), and J2R(116 -124)) (20) or human vaccines (AC1L(97-106), D1R(686 -694), F4L (6 -14), and J6R(303-311)) (21,22). In addition, 14 HLA-B7 potential ligands from a VACV proteome-based in silico prediction of high binding were also included in the study to identify new epitopes (supplemental Table 2).…”

The Viral Transcription Group Determines the HLA Class I Cellular Immune Response Against Human Respiratory Syncytial Virus*

“…Although multiple factors determine both the nature and the immunodominance of the T-cell responses of individual epitopes, some studies have shown the importance of some viral elements such as temporal virus protein expression. Thus, specific T-cell recognition of early but not late viral antigens was found both for herpesvirus (46) and some vaccinia virus strains (47,48). However, although a tight correlation between onset of protein expression and vaccinia virus epitope display has been found, no connection between immunodominance and epitope abundance was described for this poxvirus (49).…”