Exogenous introduction of an immunodominant peptide from the non-structural IE1 protein of human cytomegalovirus into the MHC class I presentation pathway by recombinant dense bodies.
Exogenous introduction of particle-associated proteins of human cytomegalovirus (HCMV) into the major histocompatibility complex (MHC) class I presentation pathway by subviral dense bodies (DB) is an effective way to sensitize cells against CD8 T-cell (CTL) recognition and killing. Consequently, these particles have been proposed as a platform for vaccine development. We have developed a strategy to refine the antigenic composition of DB. For proof of principle, an HCMV recombinant (RV-VM3) was generated that encoded the immunodominant CTL determinant IE1TMY from the IE1 protein in fusion with the major constituent of DB, the tegument protein pp65. To generate RV-VM3, a bacterial artificial chromosome containing the HCMV genome was modified by applying positive/negative selection based on the expression of the bacterial galactokinase in conjunction with lambda Red-mediated homologous recombination. This method allowed the efficient and seamless insertion of the DNA sequence encoding IE1TMY in frame into the pp65 open reading frame (UL83) of the viral genome. RV-VM3 expressed its fusion protein to high levels. The fusion protein was packaged into DB and into virions. Its delivery into fibroblasts by these viral particles led to the loading of the MHC class I presentation pathway with IE1TMY and to efficient killing by specific CTLs. This demonstrated that a heterologous peptide, not naturally present in HCMV particles, can be processed from a recombinant, DB-derived protein to be subsequently presented by MHC class I. The results presented here provide a rationale for the optimization of a vaccine based on recombinant DB.
Mersseman
Véronique
V
Institute for Virology, Johannes Gutenberg-Universität, Mainz, Germany.
Besold
Katrin
K
Institute for Virology, Johannes Gutenberg-Universität, Mainz, Germany.
Reddehase
Matthias J
MJ
Institute for Virology, Johannes Gutenberg-Universität, Mainz, Germany.
Wolfrum
Uwe
U
Institute for Zoology, Department of Cell and Matrix Biology, Johannes Gutenberg-Universität, Mainz, Germany.
Strand
Dennis
D
First Department of Internal Medicine, Johannes Gutenberg-Universität, Mainz, Germany.
Plachter
Bodo
B
Institute for Virology, Johannes Gutenberg-Universität, Mainz, Germany.
Reyda
Sabine
S
Institute for Virology, Johannes Gutenberg-Universität, Mainz, Germany.
D017951:Antigen Presentation / Q000276:immunology; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000276:immunology; D015316:Genetic Therapy / Q000379:methods; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D017874:Immediate-Early Proteins / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology / Q000378:metabolism*; D014765:Viral Vaccines; D014771:Virion / Q000378:metabolism
D015395:Histocompatibility Antigens Class I; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D014765:Viral Vaccines