Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells

Abstract

Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). Here, we evaluated the immunopeptidome of Jurkat cells infected with the vaccine candidate MVA.HIVconsv, which delivers HIV-1 conserved antigenic regions by using modified vaccinia virus Ankara (MVA). 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. Human vaccine recipients responded to the peptide sequences identified by LC-MS/MS. Peptides derived from the conserved HIV-1 regions were readily detected as early as 1.5 h after MVA.HIVconsv infection. Four of the seven conserved peptides were monitored between 0 and 3.5 h of infection by using quantitative mass spectrometry (Q-MS), and their abundance in HLA class I associations reflected levels of the whole HIVconsv protein in the cell. While immunopeptides delivered by the incoming MVA vector proteins could be detected, all early HIVconsv-derived immunopeptides were likely synthesized de novo. 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.

Importance: The vast changes in cellular antigen presentation after infection of cells with a vectored vaccine, as shown here for MVA.HIVconsv, highlight the complexity of factors that need to be considered for efficient antigen delivery and presentation. 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.

FIG 1

HIVconsv protein expression dynamics using MVA as a delivery vector. HIVconsv expression was monitored in cells infected with MVA.HIVconsv for the indicated durations by Western blotting using a V5 antibody (A), label-free quantitative LC-MS/MS (average abundance of 4 tryptic peptides) (B), and immunofluorescence at 24 hpi (C).

FIG 2

Characteristics of peptides eluted from Jurkat cells infected with MVA.HIVconsv. (A and B) Numbers of unique identified peptide sequences in the time course experiment (A) and their size distribution for each experiment (B). (C) Amino acid distribution of all eluted 8- to 12-mer peptides from all four samples in comparison to the known major anchor residues for the HLA allele of Jurkat cells, as listed above the graphs. The represented single-letter abbreviation for each amino acid is scaled by size according to the relative frequency of the amino acid at the indicated position in the eluted peptide (WebLogo 3.4). (D) Frequency of predicted binding of the identified peptide sequences to the HLA alleles of Jurkat cells, as determined with the NetMHC 3.4 server.

FIG 3

Correlation analysis of HIVconsv epitope and HIVconsv protein abundances. (A) Representative extracted ion chromatograms of the indicated peptide sequences plotted according to their retention time. (B) Average calculated abundances from duplicate analyses as determined by PEAKS. (C) R² values calculated for each immunopeptide by using Pearson's correlation to total HIVconsv protein levels (average abundance of 63 tryptic peptides).

FIG 4

Spectral matches for HIVconsv-derived peptides. MS fragment spectra show the experimentally acquired spectrum of the indicated peptide in the indicated sample in comparison to a spectrum of the synthetic peptide counterpart acquired under the same conditions. Fragment ions are indicated for the peptide sequence above each spectrum, and the most intense ion mass peaks are labeled in the experimental spectrum as follows: b, singly charged N-terminal fragment ion; y, singly charged C-terminal fragment ion; 0, loss of H₂O; *, loss of NH₂; ++, doubly charged fragment ion. The detected mass of the intact peptide parent ion is stated for each spectrum.

FIG 5

Responses in humans vaccinated with HIVconsv recognizing eluted, MS-identified peptides. (A) Schematic view of the HIVconsv immunogen, with the 14 conserved regions of HIV-1 that were combined in the immunogen represented as colored boxes and with the original HIV-1 region stated (Gag, Pol, Vif, and Env). Letters above the boxes (A, B, C, and D) indicate the clade of origin. The positions of eluted peptides identified in the HIVconsv immunogen are indicated by red bars. Genomic regions of the HXB2 strain are shown as gray rectangles. TAGs, epitope tag sequences; LTR, long terminal repeat; nt, nucleotides. (B) The magnitude of the response (in SFU/10⁶ PMBCs) is plotted for each individual, summed for all 15-mer peptides containing MS-identified sequences (left y axis), in addition to the percent magnitude compared to the total magnitude of responses to all 15-mer peptides spanning the full HIVconsv immunogen (right y axis). The breadth of the response is indicated by the number of peptides generating a response in each individual subject (MS-identified/total number of peptides, indicated above each bar). Individual responses to the indicated peptide sequences are shown below each graph column, and HLA genotypes for each volunteer are indicated. Matching HLA types are highlighted in green, and matching alleles from an identical HLA supertype are highlighted in violet. P, placebo recipient.

FIG 6

Determination of the origin of MVA.HIVconsv-specific protein using SILAC. Shown are the results from two independent experiments (top row, experiment 1 [Exp.1]; bottom row, experiment 2 [Exp.2]). Labeled peptide (newly made) abundances are plotted as red curves, and unlabeled (incoming virus particle-derived) peptide abundances are depicted in green. Single tryptic peptide abundances are plotted for the MVA proteins VLTF-4, TBP-I1, and PAP-S, as measured in both experiments. For the HIVconsv protein, average abundances of 5 (experiment 1) or 23 (experiment 2) tryptic peptides are plotted, reflecting the abundance of the HIVconsv protein at the indicated time points after infection with MVA.HIVconsv.

FIG 7

The immunopeptidome reflects changes of the cellular proteome in MVA.HIVconsv-infected cells. (A) The most relevant pathways that are affected by MVA.HIVconsv infection at 3.5 hpi, as identified by Ingenuity Pathway Analysis, for all proteins according to the abundances of the corresponding HLA-associated peptides (left) and the intracellular proteins (right), as determined by label-free quantitative LC-MS/MS. All changes in abundance were determined between the 3.5-h time point and the negative control. (B) Correlation factors between the trend of protein abundance and the trend of the HLA-associated peptide abundance at 0, 1.5, 2.5, and 3.5 hpi. A proportional correlation was assigned a value of +1, and an inverse correlation was assigned a value of −1. Factors are plotted for all proteins along the x axis, for which both protein and immunopeptide abundances were determined.