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Infection and Immunity, January 2000, p. 247-256, Vol. 68, No. 1
Cellular and Molecular Microbiology Division,
Institute of Microbiology of the Academy of Sciences of the Czech
Republic, CZ-142 20 Prague 4, Czech Republic,1
and Unité de Biologie des Régulations
Immunitaires, Institut Pasteur, 75724 Paris,
France2
Received 26 July 1999/Returned for modification 2 September
1999/Accepted 19 October 1999
Bordetella pertussis adenylate cyclase (AC)
toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells.
Recombinant AC toxoids have therefore been recently used for delivery
of CD8+ T-cell epitopes into antigen-presenting cells in
vivo and for induction of protective antiviral, as well as therapeutic
antitumor cytotoxic T-cell responses. We have explored the carrier
potential of the ACT molecule by insertional mutagenesis scanning for
new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8+ T-cell epitope of ovalbumin was
incorporated at 10 of these permissive sites along the toxin molecule,
and the capacity of ACT constructs to penetrate into cell cytosol and
deliver the epitope into the major histocompatibility complex (MHC)
class I antigen processing and presentation pathway was examined. While
all six constructs bearing the epitope within the Hly portion of ACT
failed to deliver the epitope to the MHC class I molecules, all four
toxoids with inserts within different permissive sites in the AC domain
efficiently delivered the epitope into this cytosolic pathway, giving
rise to stimulation of a specific CD8+ T-cell hybridoma.
The results suggest that, in contrast to the AC domain, the hemolysin
moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Delivery of CD8+ T-Cell Epitopes into
Major Histocompatibility Complex Class I Antigen Presentation Pathway
by Bordetella pertussis Adenylate Cyclase: Delineation of
Cell Invasive Structures and Permissive Insertion Sites
ka,1
ková,1
ebo1,*
*
Corresponding author. Mailing address: Institute of
Microbiology CAS, Víde
ská 1083, CZ-142 20 Prague
4, Czech Republic. Phone: (4202) 475 2762. Fax: (4202) 472 2257. E-mail: sebo{at}biomed.cas.cz.
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