HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplementary Methods Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Mersseman, V. Articles by Reyda, S. Search for Related Content PubMed PubMed Citation Articles by Mersseman, V. Articles by Reyda, S. Agricola Articles by Mersseman, V. Articles by Reyda, S.

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

¹ Institute for Virology, Johannes Gutenberg-Universität, Mainz, Germany
² Institute for Zoology, Department of Cell and Matrix Biology, Johannes Gutenberg-Universität, Mainz, Germany
³ First Department of Internal Medicine, Johannes Gutenberg-Universität, Mainz, Germany

Correspondence
Bodo Plachter
plachter{at}uni-mainz.de

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 IE1_TMY 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 Red-mediated homologous recombination. This method allowed the efficient and seamless insertion of the DNA sequence encoding IE1_TMY 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 IE1_TMY 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.

Supplementary methods describing HCMV BAC recombineering using galK positive/negative selection are available with the online version of this paper.