Case Study – Human Papilloma Virus
Immunitrack recently undertook a study to identify stably-binding T cell epitopes within the E6 and E7 proteins from human papilloma virus (HPV). E6 and E7 are oncoproteins in high-risk HPV genotypes that mediate the development of cervical cancer when highly expressed. Their role in driving cellular immortalisation and tumour progression has made them prime targets for therapeutic vaccine development for HPV-associated cervical cancer.
The T cell response depends on a patient’s HLA profile since HLA displays epitopes on the surfaces of infected cells. In order to find the best T cell epitopes for vaccine and immune monitoring of patients based on their MHC profile, Immunitrack undertook a large MHC/epitope screening project:
Here, 266 9-mer peptides, overlapping by 8 amino acids and covering the entire sequences of E6 and E7 were designed and applied to our NeoScreen® screening platform (Figure 1).
The NeoScreen® platform may be deployed to assess the affinity of each of these epitopes for 13 MHC I and 10 MHC II molecules (Table 1), as well as the stability of these interactions.
Figure 1. Schematic representation of 266 overlapping 9-mer peptides from HPV E6 and E7.
Table 1. MHC I and MHC II alleles used in the study.
NeoScreen® identifies all 9 IEDB-Confirmed HLA-A*0201 Epitopes
According to the Immune Epitope Database (IEDB), E6 and E7 harbour 9 confirmed T cell epitopes for HLA-A*02:01, and we found all of these among our 13 most stable HLA-A*02:01-binding (MHC I) epitopes (Figure 2 and 3). Remarkably, we also identified the novel HLA-DRB1*03:01 (MHC II)-presented CD4 T-cell epitope that was previously described in a cervical cancer patient vaccinated with HPV 16 or 18 E7-pulsed dendritic cells (Wang et al., 2008). See figure 4.
Figure 2. Stability analysis of 266 overlapping 9-mers from HPV E6 and E7 on A*0201. The red vertical bar immediately beside the Y-axis represents a reference peptide that is a known stably-binding T cell epitope of the tested A*0201 allele. The stability of the other tested peptides, which are indicated as vertical lines were calculated relative to the reference peptide (100 % stability). Peptides represented by black lines are known A*0101-restricted HPV T cell epitopes from the Immune Epitope Database (IEDB).
Figure 3. Tabulated and ranked stability measurements performed in duplicate (referred to as Stab 1 and Stab 2) for the 13 most stably-binding 9-mers. Remarkably, 9 of the top 13 binders are confirmed T cell epitopes (based on IEDB data). NB = non-binding.
Figure 4. Stability analysis of 266 overlapping 9-mers from HPV E6 and E7 on DRB1*01301. Black arrow: T cell epitope identified in Wang et al., (2008).
Superior to In Silico Prediction Methods
In parallel with in vitro binding assays, we used the in silico prediction tool netMHC to predict candidate HLA-A *0201 epitopes among the same 266 9-mers. In our hands, in silico methods trained on MHC-epitope affinity alone predict only 5 of the 9 IEDB-confirmed MHC I T cell epitopes and not the MHC II epitope (Figure 3, see column entitled ‘Pred aff nM’).