Interleukin-7 (IL-7), the principal cytokine implicated in thymopoiesis and peripheral T-cell homeostasis, is presently under evaluation in human diseases characterized by persistent lymphopenia. Unexpectedly, before the eventual IL-7-driven T-cell expansion, all treated patients showed a profound T-cell depletion 24 hours after injection. The current study uses the rhesus macaque model to investigate the mechanisms involved in this IL-7-induced T-cell depletion. We identify a new critical function of IL-7 that induces massive and rapid T-cell migration from the blood into various organs, including lymph nodes, parts of the intestine, and the skin. This homing process was initiated after the induction of chemokine receptor expression by circulating T cells and the production of corresponding chemokines in target organs. Finally, we demonstrate that the IL-7-induced cell cycling is initiated within these organs before T cells migrate back into the bloodstream, indicating that T-cell homing is required for in vivo IL-7 function. (Blood. 2009;114:816-825)

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 l 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. INTRODUCTIONInfection with human cytomegalovirus (HCMV; family Herpesviridae, subfamily Betaherpesvirinae) may cause significant morbidity and mortality in individuals with immature or compromised immune-defence functions, but proceeds asymptomatically in most healthy adults (Pass, 2001). Prevention of HCMV infection or disease by a vaccine has been ranked as a high-priority goal (Stratton et al., 2001). Although several vaccine approaches have been developed, none have yet entered routine clinical practice (Pepperl-Klindworth & Plachter, 2006;Plotkin, 2004;Schleiss & Heineman, 2005;Zhong & Khanna, 2007). We have demonstrated that HCMV dense bodies (DB) provide a promising basis for vaccine development (Pepperl et al., 2000;Pepperl-Klindworth et al., 2002). DB are enveloped, subviral particles devoid of capsids and viral DNA. They are released from infected fibroblast cultures and enter cells presumably via the normal HCMV entry processes. The major constituent of DB is pp65 (ppUL83) (Varnum et al., 2004), which is a prominent target of the CD4 and CD8 T-cell (CTL) responses following natural infection (Beninga et al., 1995;McLaughlin-Taylor et al., 1994;Wills et al., 1996). Although pp65 by itself appears to be an attractive antigen for the design of a vaccine, other HCMV proteins may also be important and should thus be considered (Reddehase, 2002). One of these, as far as the CTL response is co...

Development of a vaccine against human cytomegalovirus (HCMV) infection has been identiWed as a high priority goal in biomedical research, yet no vaccine has been licensed until now. Recombinant subviral dense bodies (recDB) are a promising basis for the establishment of such a vaccine. In this article, strategies for the generation of recDB, based on recombination-mediated genetic engineering of the 230 kb HCMV DNA genome in E. coli are outlined. Analysis of viral mutants that were constructed in this process provided the proof-of-principle that heterologous antigens can be packaged into recDB and that these particles prime CD8 T cell responses against the recombinant antigen upon their application to HLA-A2 transgenic mice.

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