Abstract
Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we generated one to three peptide pools for each of the 82 KSHV ORFs. IFN-γ ELISpot analysis of PBMCs from 19 patients with a history of KSHV-associated disease and 24 healthy donors (11 KSHV seropositive) detected widely varied responses. Fifty six of the 82 ORFs were recognized by at least one individual but there was little overlap between participants. Responses to at least one ORF pool were observed in all 19 patients and in 7 seropositive donors. Four seropositive donors and 10 seronegative donors had no detectable responses while 3 seronegative donors had weak responses to one ORF. Patients recognised more ORFs than the donors (p=0.04) but the response intensity (spot forming units: SFU per million cells) was similar in the two groups. In four of the responding donors, individual peptides eliciting the predominant responses were identified: three donors responded to only one peptide per ORF, while one recognized five. Using intracellular cytokine staining in four participant samples, we detected peptide-induced IFN-γ, MIP1-β, and TNF-α as well as CD107a degranulation, consistent with multifunctional effector responses in CD8+ and CD4+ T cells. Sequence analysis of TCRs present in peptide specific T-cell clones generated from two participants showed both mono- and multi-clonotypic responses. Finally, we molecularly cloned the KSHV specific TCRs and incorporated the sequences into retroviral vectors to transfer the specificities to fresh donor cells for additional studies. This study suggests that KSHV infected individuals respond to diverse KSHV antigens, consistent with a lack of shared immunodominance and establishes useful tools to facilitate KSHV immunology studies.
Highlights
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that establishes a prevalently latent and, in most individuals, asymptomatic lifelong infection [1]
Four hundred and thirty-two Research Donor Program (RDP) participants were tested for antibodies to KSHV using ELISA assays detecting anti-KSHV IgG against the lytic antigen K81 and the latent antigen LANA, encoded by ORF73
ELISpot analysis using a library of peptides covering the entire KSHV proteome revealed no immunodominant INF-γ responses to KSHV antigens
Summary
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that establishes a prevalently latent and, in most individuals, asymptomatic lifelong infection [1]. KSHV causes malignancies, including Kaposi’s sarcoma (KS)[2], and primary effusion lymphoma (PEL)[3]; as well as multicentric Castleman’s disease (MCD) a lymphoproliferative disorder [4]. The most common KSHV-associated disease is KS, which can occur in HIV uninfected people, especially older men in the Mediterranean (Classic KS); sub-Saharan Africans (endemic KS); and transplant recipients (iatrogenic KS) but most frequently develops in those with HIV infection (AIDS KS). The elevated risk of KS in immunocompromised hosts and the risk-reduction following recovery of T-cell function indicate that a loss of cell-mediated responses plays an important role in KS development. The critical role of T cells in maintaining control of chronic herpesviruses is demonstrated in the natural history of Epstein-Barr virus (EBV), human cytomegalovirus (HCMV) and herpes simplex virus (HSV)[8,9,10]
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