ProImmune’s REVEAL® & ProVE® Rapid Epitope Discovery System validates novel epitopes important for CD8+ T cell control of Human Herpesvirus 8

Lepone L., et al. (2010). Monofunctional and polyfunctional CD8+ T cell responses to human herpesvirus 8 lytic and latency proteins. Clin Vaccine Immunol. 17(10): 1507-16. [PubMed ID 20719985]

Human herpesvirus 8 (HHV-8) is the causative agent of Kaposi’s sarcoma, the most common cancer affecting HIV-infected individuals. Preventing or mitigating the effects of HHV-8 infection would therefore be a major health benefit. CD8+ T cell responses are known to be important for the control of this family of viruses, so Lauren Lepone and her colleagues at the University of Pittsburgh investigated these responses to two HHV-8 lytic proteins (gB and K8.1) and two latency proteins (LANA-1 and K12).

Cytotoxic T Lymphocytes (CTL) responding to these proteins are known to be rare, so the team used their own enhanced ELISpot technique to find reactive CTL. They stimulated PBMC with peptide-loaded autologous monocyte derived dendritic cells for a week before ELISpot assay, then used this approach to map ‘hotspots’ of CTL reactivity in the four HHV-8 proteins. This enabled them to narrow down the candidate regions to define 10 potential novel viral epitopes.

Lepone et al turned to ProImmune’s REVEAL® MHC-Peptide Binding Assays to quantitate the binding of their 10 candidate epitopes to HLA-A*02:01, comparing them with five known epitopes and with positive and intermediate binding controls. The data supported their conclusions that these peptides may act as T cell epitopes, showing binding levels comparable to established epitopes (figure 1).

MHC class I REVEAL binding assay results

Figure 1. MHC-peptide binding assay results. The binding score for each peptide is shown relative to a positive control of a known T cell epitope with very strong HLA A*02:01 binding properties. Binding scores are shown for ten novel epitopes (black bars), five known epitopes (grey bars) and the positive and intermediate control peptides (striped bars). Starred epitopes were made as ProVE® Pentamers.

To further characterize the CTL responses to their epitopes, the team commissioned ProVE® MHC Pentamers for the two epitopes with the highest binding scores, and also three characterized HHV-8 epitopes for comparison. Their staining analysis showed the presence of Pentamer-reactive CTL in eight HLA-A*02:01–positive and HHV-8 seropositive donors tested, for all of their epitopes including the two previously unidentified sequences (figure 2).

HLA A*02:01 Pentamer staining in peripheral blood samples from 8 HHV-8-seropositive donors

Figure 2: HLA A*02:01 Pentamer staining in peripheral blood samples from 8 HHV-8-seropositive donors. PBMC from HLA A*02:01-positive HHV-8-seropositive donors were stained with ProVE® MHC Class I- Pentamer complexes specific for LANA-1281-289, LANA-11116-1124 K1217-25, gB492-500, and K8.1135-143. Newly-discovered epitopes are starred. Average response is indicated by the black bar and response levels in seronegative donors indicated by the dotted line.

In new, as yet unpublished experiments, the team tell us that they have found that the presence of CD4+CD25+ regulatory T cells is detrimental to a CTL response to HHV-8. Viewed together, their work demonstrates that through characterization of HHV-8 epitopes, using technologies such as those provided by ProImmune, we are gathering information that aids our understanding of HHV-8 pathogenesis and will lead us to a strategy for vaccine development.

This work was carried out at the University of Pittsburgh

Images reproduced with permission from permission from the American Society for Microbiology