Immunopeptidomic analysis of influenza A virus infected human tissues identifies internal proteins as a rich source of HLA ligands

Abstract

CD8+ and CD4+ T cells provide cell-mediated cross-protection against multiple influenza strains by recognising epitopes bound as peptides to human leukocyte antigen (HLA) class I and -II molecules respectively. Two challenges in identifying the immunodominant epitopes needed to generate a universal T cell influenza vaccine are: A lack of cell models susceptible to influenza infection which present population-prevalent HLA allotypes, and an absence of a reliable in-vitro method of identifying class II HLA peptides. Here we present a mass spectrometry-based proteomics strategy for identifying viral peptides derived from the A/H3N2/X31 and A/H3N2/Wisconsin/67/2005 strains of influenza. We compared the HLA-I and -II immunopeptidomes presented by ex-vivo influenza challenged human lung tissues. We then compared these with directly infected immortalised macrophage-like cell line (THP1) and primary dendritic cells fed apoptotic influenza-infected respiratory epithelial cells. In each of the three experimental conditions we identified novel influenza class I and II HLA peptides with motifs specific for the host allotype. Ex-vivo infected lung tissues yielded few class-II HLA peptides despite significant numbers of alveolar macrophages, including directly infected ones, present within the tissues. THP1 cells presented HLA-I viral peptides derived predominantly from internal proteins. Primary dendritic cells presented predominantly viral envelope-derived HLA class II peptides following phagocytosis of apoptotic infected cells. The most frequent viral source protein for HLA-I and -II was matrix 1 protein (M1). This work confirms that internal influenza proteins, particularly M1, are a rich source of CD4+ and CD8+ T cell epitopes. Moreover, we demonstrate the utility of two ex-vivo fully human infection models which enable direct HLA-I and -II immunopeptide identification without significant viral tropism limitations. Application of this epitope discovery strategy in a clinical setting will provide more certainty in rational vaccine design against influenza and other emergent viruses.

Immunopeptidome profiling of human coronavirus OC43-infected cells identifies CD4 T cell epitopes specific to seasonal coronaviruses or cross-reactive with SARS-CoV-2

“…Of fourteen peptides tested, we observed robust T cell responses to eleven. In other systems, characterization of naturally-processed, MHC-bound peptides by mass spectrometry of infected cells has proven to be an efficient route for T cell epitope discovery [31,32,[32][33][34][35][36][37][38][39]65,66]. We observed a correlation between the observed T cell response and epitope abundance in the overall immunopeptidome, whereas a significant correlation was not observed for the predicted or even observed peptide binding affinity.…”

“…T cell epitope identification can be approached in different ways, including screening of overlapping peptide libraries, predicting potential epitopes using MHC-binding prediction algorithms, or identifying naturally processed and presented peptides eluted from purified MHC molecules isolated from infected cells. In this work, we used the latter method, which has proven to be very efficient in identifying immunogenic peptides in human T cell responses to vaccinia virus [31–33], HHV-6B [34], influenza [35], measles [36], EBV [37], and SARS-CoV-2 [38] and in mouse responses to vaccinia virus where this was validated extensively [39]. Here, we identified and characterized naturally-processed viral epitopes presented by HEK293 cells transfected with master transcriptional regulator CIITA and infected with OC43.…”

The HLA class-II immunopeptidomes of AAV capsids proteins

“…MHC-associated peptide proteomics (MAPPs) is a recent technology that gives access to the HLA class II immunopeptidome, defined as the naturally displayed peptides by HLA class II (DR, DQ and DP) molecules on the surface of human dendritic cells. Recent studies have leveraged MAPPs to identify immunopeptidomes of different viruses, including SARS-CoV-2, influenza, and HIV ( 16 – 21 ) as well as immunogenicity risk assessment of preclinical molecules in the industry ( 22 ). However, despite the importance of AAVs in gene therapies, no previous MAPPs studies have been performed on AAV capsids.…”

T Cell Epitope Discovery in the Context of Distinct and Unique Indigenous HLA Profiles

“…This may change for different viruses, strains and infection models. Thus, the development of models that more closely mimic the in vivo setting is of great interest (170) and a consideration for the success of this strategy. For viruses with low infection efficiency of cell lines, or for which high physical containment is required, analysis of presentation of specific viral proteins can also be performed by transfection with expression constructs for viral antigens (171).…”