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.

Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

“…This MHC-peptidome study and others in viral infections (9,15,16) or in B cell lines (30,(42)(43)(44)(45) identified clusters of nested peptides. They include optimal epitopes as well as N-and/or Cextended peptides potentially presentable by several HLAs.…”

“…The presentation of vaccinia virus-derived peptides over 12 h (15) and the presentation of both modified vaccinia Ankara (MVA) peptides and HIV peptides from Jurkat cells infected with MVA expressing HIV protein fragments followed during 6 h (16) showed variations in epitope presentation over time. While peptide presentation was linked to protein expression (15,16), defective translation products (defective ribosomal products [DRiPs]) may also contribute to various extents to the pool of MHC-I peptides displayed by cells (35)(36)(37).…”

“…However, considering the relatively low number of MHC-peptide complexes per cell and the potential MS detection limits, the majority of the data on self-, cancer, or pathogen MHC peptidomes come from immortalized cell lines (5)(6)(7)(8) or from models using cell lines engineered to secrete soluble MHC-bound peptide complexes (9)(10)(11), as both systems allow growth of high numbers of cells for peptide isolation. The improvements in peptide isolation and MS-based approaches led to the discovery of numerous MHC-I ligands presented by B cells or by patients' tumors (12)(13)(14) and the identification of virusderived MHC-bound peptides, including vaccinia virus and HIV presented by surface or soluble HLA (5,9,(15)(16)(17). These approaches identified self-and virus-derived noncanonical peptides and demonstrated that direct identification of peptides from infected cells will define the immunopeptidome relevant for the design of HIV immunogens.…”

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry*

“…Along these lines, Ternette et al recently found that 5% of dimethyl sulfoxide in liquid chromatography solvents enhanced the electrospray ionization of HLA class I peptides, improving the total ion count by approximately twofold (personal communication) (72,73). However, the effect of enhancing electrospray ionization and sensitivity might depend on the type of emitter source as well as specific parameters such as voltage, temperature, and gas flow.…”

Minimal Information About an Immuno‐Peptidomics Experiment (MIAIPE)

“…Three accepted methods for the isolation of MHC class I or class II peptides have emerged in the last decades: 1) mild acid elution, where the cell surface MHC class I but not class II complexes are denatured at approximately pH 3.3, therefore releasing the MHC class I peptides while leaving the cells intact18; 2) immunoaffinity purification of the endogenous pMHC from cells, tissues, and body fluids using MHC‐specific monoclonal antibodies bound to solid support19; and 3) immunoaffinity purification of transfected recombinant soluble or membrane‐anchored MHC molecules using anti‐MHC antibodies or antibodies against affinity tags 20. Stable isotope labeling of the cellular proteins has been used in order to follow their synthesis, degradation dynamics, and the MHC presentation of their derived peptides and to assess the degree to which pMHC is derived from defective ribosome products21 and has been applied for the identification of pathogen‐derived pMHC 22. A comprehensive description of such advanced experimental approaches is required, and in this section, we cover aspects related to the extraction of pMHC from the biological samples and their preparation and storage prior to the LC‐MS/MS characterization.…”