Immunoproteomic Lessons for Human Respiratory Syncytial Virus Vaccine Design

Abstract

Accurate antiviral humoral and cellular immune responses require prior recognition of antigenic peptides presented by human leukocyte antigen (HLA) class I and II molecules on the surface of antigen-presenting cells. Both the helper and the cytotoxic immune responses are critical for the control and the clearance of human respiratory syncytial virus (HRSV) infection, which is a significant cause of morbidity and mortality in infected pediatric, immunocompromised and elderly populations. In this article we review the immunoproteomics studies which have defined the general antigen processing and presentation rules that determine both the immunoprevalence and the immunodominance of the cellular immune response to HRSV. Mass spectrometry and functional analyses have shown that the HLA class I and II cellular immune responses against HRSV are mainly focused on three viral proteins: fusion, matrix, and nucleoprotein. Thus, these studies have important implications for vaccine development against this virus, since a vaccine construct including these three relevant HRSV proteins could efficiently stimulate the major components of the adaptive immune system: humoral, helper, and cytotoxic effector immune responses.

Keywords: Antigen processing; HLA; T cells; immune response; immunoproteomics; mass spectrometry; respiratory infectious disease; vaccine.

Figure 1

Overview of the HLA immunoproteomic approach. Both large amounts of pathogen-modified and healthy cells expressing the HLA class I or II molecules of interest are lysed in the presence of protease inhibitors. The HLA class I complexes are immunoprecipitated by affinity chromatography with specific anti-HLA mAbs and the HLA/peptide complexes are denatured. The resulting peptide mixtures recovered after an ultra-filtration step are separated by high performance liquid chromatography and analyzed by mass spectrometry. Bioinformatics tools are then used to obtain the sequences of the pathogen ligands. Finally, synthetic peptides, which can be used in functional analysis such as HLA/peptide stability assays, ELISPOT, intracellular cytokine staining assays or cytotoxicity assays, are generated.

Figure 2

HLA class I ligands from HRSV identified by mass spectrometry and their relation to the viral mRNA, proteome, and T cell cytotoxic immune response. A panel: Pie chart representation of the HRSV genome indicating the relative abundance of mRNAs from the viral transcription, which was measured as the mRNA molar ratio percentage [21,41,42]. The abbreviations used for viral proteins were NS1 (non-structural protein 1), NS2 (non-structural protein 2], N (nucleoprotein), P (phosphoprotein), M (matrix protein), SH (small hydrophobic protein), G (glycoprotein), F (fusion protein), M2-2 (matrix protein 2], and L (polymerase), which were grouped into three different transcription groups: 3′ (white), intermediate (IM) (gray), and 5′(black) separated by arrows as in previously publications [21,41,42]. For each protein included into its respective transcription group, the proteome percentage is shown in B panel; the HLA class I ligand percentage identified by mass spectrometry in studies [35,36,37,38] is shown in C panel; the CTL epitope percentage from [37] is shown in D panel, and the total IFN-γ⁺ immune response percentage detected in the HLA class I-transgenic mice from [37] is shown in E panel.

Figure 3

HLA class II ligands from HRSV identified by mass spectrometry and their relation to the viral mRNA, proteome, and T cell helper immune response. Pie chart representation of the HRSV genome indicating the relative abundance of mRNAs due to viral transcription (A panel) and the percentage of the proteome (B panel) for each protein, as described in Figure 1. For each protein included in its respective transcription group, the HLA class II ligand percentage identified by mass spectrometry in study [48] is shown in C panel, and the CD4⁺ epitope percentage from [48] is shown in D panel. The total of either IL-4⁺ or IFN-γ⁺ immune helper response percentages that were detected in the HLA class II-transgenic mice in study [48] is shown in the E and F panels, respectively.