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Abstract
One of the shortcomings of vaccinia virus (VACV) as immunization vector is the down-regulation of HLA and costimulatory molecules in antigen presenting cells. To overcome this problem we investigated the use of protein kinase C (PKC) as immune stimulatory agent. Thus several classical and atypical PKCs were inserted into wild-type or attenuated VACV using recombination into the hemagglutinin gene and the expression driven by the VACV 7,5K-IE gene promoter. Recombinant constructs expressing PKC-alpha, -beta, -theta as well as wild-type, constitutive active or dominant negative PKC-zeta constructs were generated. Additional constructs expressing PKB/Akt1 and ICAM-1 were used for comparison. Immature and mature peripheral blood derived-dendritic cells (DC) as well as lymphoid cell lines capable of obtaining a DC-like phenotype upon mitogen stimulation were infected. Disappointingly, VACV-driven PKC overexpression did not significantly enhance expression of various activation markers or costimulatory molecules tested. Neither CD86 nor HLA-DR expression was upregulated and also no influence on the maturation of DC, as measured by DC-SIGN and CD83, was observed. However, VACV did not interfere with LPS induced up-regulation of CD83 and did not lead to substantial apoptosis of infected DC within the first 24 hours.
ACKNOWLEDGMENTS
The authors thank Christine Berling, INSERM, Paris, France for the ICAM expressing vaccinia construct. Constructs containing cDNAs for PKCs have been kindly provided by Gottfried Baier, Med.U. Innsbruck, Austria. Technical assistance of Karolin Lechleitner, and proofreading by Peter Gruber, both Med.U. Innsbruck was appreciated. The work has been supported by FWF grant SFB-F002 (Spezialforschungsbereich Biologische Kommunikationssysteme).
Declaration of Interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
| ABBREVIATIONS | ||
| DC dendritic cells | = | |
| IDC immature dendritic cells | = | |
| DMEM Dulbeco modification of minimal essential medium | = | |
| FACS fluorescence activated cell sorter | = | |
| GM-CSF granulocyte macrophage colony-stimulating factor | = | |
| ICAM intracellular adhesion molecule | = | |
| IL-4 interleukin 4 | = | |
| LPS lipopolysaccharide | = | |
| MEM minimal essential medium | = | |
| MVA modified vaccinia Ankara strain | = | |
| PBMC peripheral blood derived mononuclear cells | = | |
| PBS phosphate-buffered saline solution | = | |
| PKB proteinkinase B | = | |
| PKC proteinkinase C | = | |
| PKC-zeta WT wild-type PKC-zeta | = | |
| PKC-zeta AE constitutive active mutant | = | |
| PKC-zeta KW dominant negative mutant | = | |
| RPMI1640 medium developed at Roswell Park Memorial Institute in the 60s | = | |
| TCID50 tissue culture infective dose | = | |
| TNF-alpha tumor necrosis factor alpha | = | |
| VACV vaccinia virus | = | |
| WR Western Reserve vaccinia strain | = | |
