A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B

Abstract

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-γ 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.

Naturally processed HLA‐DR3‐restricted HHV‐6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 T‐cell responses

“…Six different HHV‐6B proteins, including structural and nonstructural viral proteins, were found to be sources for the eluted peptides. Three of the source proteins, the tegument protein U11 and the viral glycoproteins U39 and U48 (gB and gH, respectively), previously have been shown to be targeted by CD4 Tcell responses , but the epitopes are newly identified here. Three HHV‐6B proteins were newly identified as targets for the CD4 T‐cell response: the capsid protein U56, the hypothetical protein U63, and the glycoprotein U85.…”

“…To increase understanding of how HHV‐6B is recognized and controlled by the cellular immune system, we used an approach not previously applied to this virus, in which naturally processed and presented peptides bound to cellular MHC proteins are identified by immunoaffinity purification, acid elution, and characterization by mass spectrometry. The 25 peptides identified here, corresponding to six distinct immunodominant epitopes, can be added to the set of MHC‐I and MHC‐II epitopes derived from HHV‐6A/B that have already been published . The source of MHC‐II complexes in our studies was the T‐lymphoblastoid cell line SupT1, which is highly susceptible to infection with HHV‐6B.…”

“…All donors tested that had at least one of HLA‐DR alleles present in SupT1.CIITA also had peripheral blood CD4 T cells that recognized that particular MHC‐II bound to an eluted HHV‐6B‐derived peptide epitope. The responses to the eluted peptides were present at low frequency, not surprising for T cells responding to HHV‐6A/B , but could be amplified in vitro using a pool of the eluted peptides to drive a proliferative response. The expanded T‐cell populations responded to HHV‐6B‐infected SupT1.CIITA cells and to single‐MHC APCs pulsed with synthetic peptides, confirming the mass spectrometric identification and the MHC restriction.…”

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

“…Of these, 5 are annotated as having immediate early, 14 are annotated as having early, and 18 are annotated as having late expression kinetics. Eleven of these HHV-6B ORFs have not, to our knowledge, previously been described as HHV-6B CD4 T-cell antigens (30,31,35). Unlike the originally annotated U83 (NCBI accession number NC_000898.1), the reannotated ORF "U83A" was transcribed in the reverse direction and contained one intron, resulting in an entirely new amino acid sequence; interestingly, the full-length U83A protein elicited responses from three donors, supporting its status as a novel HHV-6B ORF.…”

“…These studies have identified HHV-6 U11, U14, U54, and U90, the homologs of CMV UL32, UL25, UL82/83, and IE1, respectively, as targets for CD4 or CD8 T cells (31)(32)(33)(34). Other studies have taken a cross-sectional approach by testing libraries of peptides that were predicted to bind to a particular HLA allele: DRB1*0101 for CD4 T cells (30,35) or HLA-B*0801 for CD8 T cells (36). These studies expanded the number of known T-cell antigens considerably but were limited to single HLA allelic variants.…”