The Viral Transcription Group Determines the HLA Class I Cellular Immune Response Against Human Respiratory Syncytial Virus*

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.

Structural and Nonstructural Viral Proteins Are Targets of T-Helper Immune Response against Human Respiratory Syncytial Virus

“…HRSV Infection of the Human JY Epstein-Barr-Transformed Cell Line-JY cells (HLA-DRB1*0404, -DRB4*0101, -DRB1*1301, and -DRB3*0101 or -DRB3*0201) were incubated with the HRSV Long strain and assayed at different time points for the presence of HRSV antigens using flow cytometry, as previously described, with either Epstein-Barr-transformed human B-cell lines (11) or other cell lines (12), to obtain a persistently infected JY-cell line that synthesized HRSV viral proteins and secreted infectious virus several months after infection (13).…”

“…Electrospray-Ion Trap Mass Spectrometry Analysis-Peptide mixtures recovered after the ultrafiltration step were concentrated using Micro-Tip reversed-phase columns (C 18 , 200 l, Harvard Apparatus, Holliston, MA) (13). Each C 18 tip was equilibrated with 80% acetonitrile in 0.1% TFA, washed with 0.1% TFA, and then loaded with the peptide mixture.…”

“…The HLA-DR/ peptide complexes were isolated via affinity chromatography from the soluble cell extract fraction with the L243 (HB55) mAb, which is specific for a monomorphic pan-HLA-DR class II determinant (14). The HLA-bound peptides were eluted at 4°C with 0.1% aqueous trifluoroacetic acid (TFA), separated from the large subunits, and concentrated with a Centricon 3 column (Amicon, Beverly, MA), exactly as previously described (13).…”

“…The tip was then washed with an additional volume of 0.1% TFA, and the peptides were eluted with 80% acetonitrile in 0.1% TFA. The peptide samples were then concentrated to ϳ20 l using vacuum centrifugation (13,15).…”

“…The HLA class II peptides recovered from immunoprecipitated HLA-DR-specific mAb, were analyzed by LC-MS/MS using an Orbitrap XL mass spectrometer (Thermo Scientific, San Jose, CA) that was fitted with a capillary HPLC (Eksigent, Dublin, CA) (13,15). The peptides were resolved on homemade Reprosil C18-Aqua capillary columns (75 micron inner diameter) (16) with a 7-40% acetonitrile gradient for 2 h in the presence of 0.1% formic acid.…”