Dataset Information

Genome-Wide Approach to the CD4 T-Cell Response to Human Herpesvirus 6B.

ABSTRACT: Human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) are population-prevalent betaherpesviruses with intermittent lytic replication that can be pathogenic in immunocompromised hosts. Elucidation of the adaptive immune response is valuable for understanding pathogenesis and designing novel treatments. Knowledge of T-cell antigens has reached the genome-wide level for CMV and other human herpesviruses, but study of HHV-6 is at an earlier stage. Using rare-cell enrichment combined with an HLA-agnostic, proteome-wide approach, we queried HHV-6B-specific CD4 T cells from 18 healthy donors with each known HHV-6B protein. We detected a low abundance of HHV-6-specific CD4 T cells in blood; however, the within-person CD4 T-cell response is quite broad: the median number of open reading frame (ORF) products recognized was nine per person. Overall, the data expand the number of documented HHV-6B CD4 T-cell antigens from approximately 11 to 60. Epitopes in the proteins encoded by U14, U90, and U95 were mapped with synthetic peptides, and HLA restriction was defined for some responses. Intriguingly, CD4 T-cell antigens newly described in this report are among the most population prevalent, including U73, U72, U95, and U30. Our results indicate that selection of HHV-6B ORFs for immunotherapy should consider this expanded panel of HHV-6B antigens.IMPORTANCE Human herpesvirus 6 is highly prevalent and maintains chronic infection in immunocompetent individuals, with the potential to replicate widely in settings of immunosuppression, leading to clinical disease. Antiviral compounds may be ineffective and/or pose dose-limiting toxicity, and therefore, immune-based therapies have garnered increased interest in recent years. Attempts at addressing this unmet medical need begin with understanding the cellular response to HHV-6 at the individual and population levels. The present study provides a comprehensive assessment of HHV-6-specific T-cell responses that may inform the development of cell-based therapies directed at this virus.

SUBMITTER: Hanson DJ

PROVIDER: S-EPMC6600184 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Genome-Wide Approach to the CD4 T-Cell Response to Human Herpesvirus 6B.

Hanson Derek J DJ Tsvetkova Olga O Rerolle Guilhem F GF Greninger Alexander L AL Sette Allesandro A Jing Lichen L Campbell Victoria L VL Koelle David M DM

Journal of virology 20190628 14

Human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) are population-prevalent betaherpesviruses with intermittent lytic replication that can be pathogenic in immunocompromised hosts. Elucidation of the adaptive immune response is valuable for understanding pathogenesis and designing novel treatments. Knowledge of T-cell antigens has reached the genome-wide level for CMV and other human herpesviruses, but study of HHV-6 is at an earlier stage. Using rare-cell enrichment combined with an HLA-agno ...[more]

PMID: 31043533

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Project description:Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-? production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6B.

Project description:The "omics" revolution of recent years has simplified the study of RNA transcripts produced during viral infection and under specific defined conditions. In the quest to find new and differentially expressed transcripts during the course of human Herpesvirus 6B (HHV-6B) infection, we made use of large-scale RNA sequencing to analyze the HHV-6B transcriptome during productive infection of human Molt-3 T-cells. Analyses were performed at different time points following infection and specific inhibitors were used to classify the kinetic class of each open reading frame (ORF) reported in the annotated genome of HHV-6B Z29 strain. The initial search focussed on HHV-6B-specific reads matching new HHV-6B transcripts. Differential expression of new HHV-6B transcripts were observed in all samples analyzed. The presence of many of these new HHV-6B transcripts were confirmed by RT-PCR and Sanger sequencing. Many of these transcripts represented new splice variants of previously reported ORFs, including some transcripts that have yet to be defined. Overall, our work demonstrates the diversity and the complexity of the HHV-6B transcriptome.IMPORTANCERNA sequencing (RNA-seq) is an important tool for studying RNA transcripts, particularly during active viral infection. We made use of RNA-seq to study human Herpesvirus 6B (HHV-6B) infection. Using six different time points, we were able to identify the presence of differentially spliced genes at 6, 9, 12, 24, 48 and 72 hours post-infection. Determination of the RNA profiles in the presence of cycloheximide (CHX) or phosphonoacetic acid (PAA) also permitted identification of the kinetic class of each ORF described in the annotated GenBank file. We also identified new spliced transcripts for certain genes and evaluated their relative expression over time. These data and next-generation sequencing (NGS) of the viral DNA have led us to propose a new version of the HHV-6B Z29 GenBank annotated file, without changing ORF names in order to facilitate trace back and correlate our work with previous studies on HHV-6B.

Dataset Information

Genome-Wide Approach to the CD4 T-Cell Response to Human Herpesvirus 6B.

Publications

Genome-Wide Approach to the CD4 T-Cell Response to Human Herpesvirus 6B.

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