Fase 4. Examinar la solución obtenida.
1.2.11 Niveles de aprendizajes
3.1 Introduction
The aim of the work presented in this chapter was to identify immunogenic peptide epitopes derived from the amino acid sequence of the CMV pp65 protein which were able to bind to the HLA-A2 class I molecule, and which could potentially serve in the induction of a CMV-specific T cell response.
T cell epitope determination has been greatly influenced by the understanding of the rules for peptide selection by MHC molecules. One of those rules is the presence of allele-specific motifs, that is particular amino acids that are preferred in specific sequence
positions. The amino acids which determine these motifs have been defined as “anchor residues” (or dominant anchor residues), because specific pockets inside the peptide binding groove of the MHC molecule accommodate the side-chain residues of these amino acids on the peptide, and this interaction seems to “anchor” the peptide to the MHC molecule (Silver gr a/., 1992; Young, 1995). For example, the dominant anchor residues described for peptides which bind to HLA-A2 are Leucine, Valine, Isoleucine or Methionine at position 2 and Valine, Leucine, Isoleucine or Alanine at position 9 or 10 (Falk et al., 1991; Hunt et a l , 1992) (Figure 3.1). These residues bind in the pockets B
and F respectively on the peptide binding groove of the HLA-A2 molecule (Saper et al., 1991). Some other positions in the peptide sequence have also been identified as being important in the determination of peptide binding to an HLA molecule. These later appear to interact with particular pockets in the peptide binding site, and are enriched for specific
amino acids defined as “secondary anchor residues” (Ruppert et al., 1993). Thus,
depending on the presence or absence of particular dominant anchor or secondary anchor
residues in set positions of the peptide sequences, shown in Figure 3.1 for HLA-A2, the binding affinity of the peptide for a given HLA molecule may either be favoured or reduced (Drijfhout et al., 1995; Ruppert et al., 1993). In several instances these peptide binding motifs, in combination with peptide binding assays, have proven useful in the identification of cytotoxic T cell peptide epitopes from a given protein (Hill et a l , 1992; Pamer et a l ,
1991).
Bearing this in mind, in the present study, the identification of potential CTL peptide epitopes from the CMV pp65 protein was performed using the following strategy: Firstly the pp65 amino acid sequence was screened for peptides containing the anchor binding motifs for HLA-A2. The HLA-A2 molecule was chosen since this molecule is the most common HLA class I allele in the Caucasoid population, and its peptide binding motif is known (Falk et al., 1991; Rammensee et a l, 1995; Ruppert et al., 1993). Thus a list of candidate peptides with the potential to bind to HLA-A2 molecules was obtained. The peptides were then synthesised, and peptide binding assays were performed to exclude weak binding peptides. Two peptide binding assays were employed, one of them the T2 stabilisation assay, takes advantage of the HLA-A2 positive mutant cell line T2, which has a reduced capacity to present endogenously processed peptides. The processing deficiency of the T2 cell line is due to a deletion in the MHC class I region, which includes the TAP genes (Cerundulo et al., 1990). Thus, endogenous peptides cannot be translocated from the cytosol into the endoplasmic reticulum, where they could bind and stabilise MHC class I molecules. Consequently T2 cells mainly express unstable, empty HLA-A2 molecules on
their surface. Such molecules can be stabilised by the addition of HLA-A2 binding peptides, leading to a higher density of class I molecules on their surface, which can be
monitored by immunofluorescence analysis (Hobohm and Meyerhans, 1993; Stuber et ah,
1992). Thus it represents a simple read-out system to study the binding of the pp65 derived peptides to the HLA-A2 molecule (Cerundulo, 1991).
Another method used in this study to evaluate the relative affinity of selected peptides for HLA-A2 molecules was a peptide binding competition assay, which takes advantage of a fluoresceinated reference peptide known to bind with high affinity to HLA-A2 molecules. Peptides are tested for their abihty to compete for the binding of the reference peptide to HLA-A2, and thus a measurement of their affinity for this molecule can be made. Thus, while the T2 stabilisation assay gives a qualitative assessment of the affinity of peptides for the HLA-A2 molecule, the peptide-binding competition assay allows the semiquantitative determination of the binding capacity of peptides under investigation (Burg et al., 1995).
a) 9-mer peptides "C
.e
.e
L I D V M E V L Y V V S F V I F A F T W P A W T W C I V A R M IZ D E R K H R K H D E R K H b) 10-mer peptidesI
B 1 F I W V V M L L L F V V V 1 W I I A A Ï M G G M M 1 2 3 4 5 6 7 8 9 10 D D R P R D R E E K K E K P H H R H K HFigure 3 .1 . R e sid u e s which conform to the p e p tid e binding m o t i f f o r H L A -A 2 m olecules.
Dominant anchor residues for HLA-A2 molecules in positions 2 and 9 (in nonamers) or 10 (in decamers) are shown in red, whilst amino acids identified as secondary anchor residues are shown in black. The association of the latter with no binding (or poor) or good binding to HLA-A2 is indicated (Adapted from Ruppert et al (Ruppert et al., 1993)).