Abstract
The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design.
Highlights
Human respiratory syncytial virus (HRSV)1 [1], a member of the Paramyxoviridae family of the Mononegavirales order, is the single most important cause of serious lower respiratory tract illnesses, such as pneumonia and bronchiolitis in infants and young children [2,3,4]
Despite the immune mechanisms involved in HRSV disease and protection are not completely understood, it is known that the cytotoxic T lymphocytes (CTLs) are required to clear virus-infected cells [9]
Translated viral mRNA yields proteins that can be further degraded by proteasomes [12], and in some cases, by other cytosolic proteases [13], which generate an extremely diverse pool of peptides both in sequence and length that can be translocated to the endoplasmic reticulum (ER) lumen by transporters associated with antigen processing
Summary
Mice—HLA-A*0201 [19] -B*0702 [20], and -B*2705 [21] transgenic mice were bred in our animal facilities in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Spanish “Comision Nacional de Bioseguridad” of the “Ministerio de Medio Ambiente y Medio Rural y Marino” (accreditation number 28079 –34A). The search was not limited by enzymatic specificity; the peptide tolerance was set to 0.005 Da, and the fragment ion tolerance was set to 0.5 Da [17, 31] This search was not limited by any methodological bias (e.g. individual protein selection or HLA consensus scoring algorithm use). The different RMA-S transfectant cells were incubated at 26 °C for 16 h This allowed for empty HLA class I molecule expression (without antigenic peptide) at the cell membrane that was stable at 26 °C but not at 37 °C. The cells were maintained at 37 °C for an additional 2 h and collected for flow cytometry This method allowed for the empty HLA class I molecules to become internalized, and we were able to discriminate between bound or unbound peptides.
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