Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells.
Bottom Line: We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with the class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 were derived from the HIVconsv immunogen.MVA.HIVconsv infection generally altered the composition of HLA class I-associated human (self) peptides, but these changes corresponded only partially to changes in the whole cell host protein abundance.Identification and quantitation of HLA class I-associated peptides by Q-MS will not only find broad application in T-cell epitope discovery but also inform vaccine design and allow evaluation of efficient epitope presentation using different delivery strategies.
Affiliation: The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom email@example.com.Show MeSH
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Mentions: The HIVconsv gene is inserted into the thymidine kinase locus of the MVA genome under the control of the early/late 7.5 promoter. Jurkat cells were chosen as a model cell line for analysis of the HLA-associated peptidome, as they are easily expandable in cell culture, which includes the possibility of transformation with labeled amino acids, and they exhibit good levels of HLA class I expression. Before analysis of the immunopeptidome of MVA.HIVconsv-infected Jurkat cells, the levels of the whole-protein HIVconsv expression was assessed in a time course employing a C-terminal Pk tag recognized by monoclonal antibody (MAb) (33) in a Western blot analysis. This indicated that the HIVconsv protein reached a peak level by 5 h, maintained this level for at least 8 h, and almost disappeared by 24 hpi (Fig. 1A). To quantify the expression of the HIVconsv protein more accurately, protein levels were monitored following MVA.HIVconsv infection of Jurkat cells by using Q-MS analyses of trypsin-digested whole-cell protein extracts. The amounts of HIVconsv protein peaked at 12 hpi (Fig. 1B). Both the Western blot and quantitative LC-MS/MS techniques yielded similar results, and notably, both methods detected the HIVconsv protein with comparable sensitivities. The cytoplasmic localization of the HIVconsv protein was confirmed by immunofluorescence at 24 hpi (Fig. 1C). The half-life of the HIVconsv protein was previously determined by pulse-chase experiments using DNA expression vectors and Semliki Forest virus replicons to be 1 to 2 h (21; our unpublished observations). It is notable that the HIVconsv protein was detected at maintained levels at 24 hpi by LC-MS/MS, whereas the signal faded substantially in the Western blot analysis. This may be due to a loss of the C-terminal Pk tag due to processing, which is essential for detection by immunoblotting but not for LC-MS/MS. Since the HIVconsv protein could be identified as early as 2 hpi, and it has been reported that MVA infection interferes with MHC peptide presentation and induces apoptosis (34), we concluded that the first 3.5 h after MVA.HIVconsv infection would be the most suitable window for studying the early kinetics of the HIVconsv-derived immunopeptidome.
Affiliation: The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom firstname.lastname@example.org.