Synthesis and ex vivo profiling of chemically modified cytomegalovirus CMVpp65 epitopes.
The effect of substituting unnatural hydrophobic amino acids into the critical MHC binding residues of an HLA-A*0201-restricted cytomegalovirus CMVpp65 epitope, NLVPMVATV, has been investigated. A new set of peptides containing the amino acids tert-butyl glycine (Tgl), cyclohexyl glycine (Chg), neo-pentyl glycine (Npg), cyclohexyl alanine (Cha) and cyclo leucine (Cyl), at either position 2, to mimic Leu, or position 9, to mimic Val, have been synthesised. Immunological profiling using class I MHC stabilisation assays to assess MHC binding affinity, and enzyme-linked immunospot (ELISPOT) assays to assess the ability of the modified peptides to re-stimulate a specific cytotoxic T-lymphocyte (CTL) response, compared to the native epitope, have been performed. It was found that the majority of the unnatural substitutions resulted in a decrease in either HLA-A*0201 binding affinity or cytotoxic T-cell activity. However, the HLA-A*0201 binding affinity was unrelated to the ability to re-stimulate a T-cell response. Minimisation and molecular dynamics studies proved helpful in dissecting the ELISPOT responses. Two principal peptide binding modes were found by minimisation, designated kinked and straight. Peptides that bound in a kinked conformation were poor at re-stimulating a T-cell response. Of the peptides that bound in a straight conformation, molecular dynamics (MD) simulations revealed that those capable of re-stimulating the strongest responses had the greatest degree of flexibility (as determined by RMSD values across the MD simulation) around the P6 residue, one of the residues important for T-cell receptor recognition.
Jones
Matthew A
MA
School of Chemistry, University of Birmingham, Birmingham, B15 2TT, UK.
Notta
Jatinder K
JK
Cobbold
Mark
M
Palendira
Mainthan
M
Hislop
Andrew D
AD
Wilkie
John
J
Snaith
John S
JS
D000956:Antigens, Viral / Q000737:chemistry; D003587:Cytomegalovirus / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry; D010750:Phosphoproteins / Q000138:chemical synthesis* / Q000737:chemistry; D014763:Viral Matrix Proteins / Q000138:chemical synthesis* / Q000737:chemistry
D000956:Antigens, Viral; D000939:Epitopes; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa