(SV5) epitope [T. Hanke, P. Szawlowski, R. E. Randall, J. Gen. Virol. 73, 653 (1992)] in pCDNA3 (Invitrogen). In vitro–translated, radiolabeled proteins were mixed with 2 g of CaN (Sigma), 2 g of CypA (Sigma) and 10 M CsA (Sandoz) where appropriate, absorbed with protein A–Sepharose (Sigma), and immunopre- cipitated with 2 g of anti-HA (Boehringer) or 5 g of anti-SV5 (Serotec). Complexes were analyzed by autoradiography or protein immunoblot analysis with polyclonal anti-CaN (Chemicon) or monoclonal anti- CaN(B) (Sigma). 10. J. E. Miskin, C. C. Abrams, L. K. Dixon, unpublished observations. 11. Vero cells were infected with wild-type BA71V ASFV or SV5-A238L recombinant ASFV (12) for 4 to 10 hours. BSC1 cells were infected with modified vac- cinia ankara (MVA) expressing T7 RNA polymerase [G. Sutter, M. Ohlmann, V. Erfle, FEBS Lett. 371,9 (1995)] and transfected with pT7–SV5-A238L or pT7–SV5-IB. Proteins were radiolabeled, and the lysed cell extracts were immunoprecipitated and an- alyzed as before (9). 12. Regions flanking the A238L ORF were cloned into KS vector (Stratagene), and the -Gal gene downstream from the ASFV vp72 promoter [ J. M. Rodriguez, F. Almazan, E. Vinuela, J. F. Rodriguez, Virology 188, 67 (1992)] was cloned between them. In addition, SV5- tagged A238L was cloned downstream from the A238L promoter. Recombinant ASF viruses A238L and SV5-A238L were isolated by using X-Gal (5- bromo-4-chloro-3-indoxyl--D-galactopyranoside). Recombinant baculovirus expressing A238L (A238L– Bac) was constructed with use of the BAC TO BAC baculovirus expression system (Life Technologies). Anti-A238L was raised in rabbits by using bacterially expressed A238L purified by SDS-polyacrylamide gel–electrophoresis. 13. J. G. Neilan et al., Virology 235, 377 (1997). 14. D. A. Fruman, S.-Y. Pai, C. B. Klee, S. J. Burakoff, B. E. Bierer, in Methods: Companion Methods Enzymol. 9, 146 (1996). 15. C. Loh et al., J. Biol. Chem. 271, 10884 (1996). 16. K. T.-Y. Shaw et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11205 (1995); A. Rao, C. Luo, P. G. Hogan, Annu. Rev. Immunol. 15, 707 (1997). 17. Messenger RNA from RS-2 cells was converted to cDNA by using anchored oligo dT 25 . PCR-amplified fragments corresponding to nucleotides 1309 to 1669 of the NFATc ORF (GenBank U08015) were cloned into pT7-Blue2 (Novagen), and the nucleotide sequence was determined. 18. The A238L ORF was cloned in pCDNA3. The reporter plasmids used were NFAT-luc, mutant NFAT-luc (mNFAT-luc), and AP-1-luc. Transfected RS-2 cells were treated after 16 hours with (where appropriate) 40 nM PMA (Sigma), 4 M ionomycin (Sigma), and 1 M CsA (Sandoz), and luciferase activity was assayed 24 hours later. 19. B. Franz et al., EMBO J. 13, 861 (1994); T. Kanno and U. Siebenlist, J. Immunol. 157, 5277 (1996). 20. We thank S. J. Elledge for two-hybrid reagents; R. Hay for pT7-SV5-IB; G. R. Crabtree for the NFAT-luc reporter plasmid; E. McKenzie, I. O’Beirne, and A. Lennard for mNFAT-luc and AP-1-luc; and Sandoz Pharmaceuticals for CsA. Supported by the Biotech- nology and Biological Sciences Research Council and Ministry of Agriculture Fisheries and Food. 10 April 1998; accepted 8 June 1998 Delivery of Epitopes by the Salmonella Type III Secretion System for Vaccine Development Holger Ru ¨ssmann,* Homayoun Shams, Fernando Poblete, Yixin Fu, Jorge E. Gala ´n,† Ruben O. Donis Avirulent strains of Salmonella typhimurium are being considered as antigen delivery vectors. During its intracellular stage in the host, S. typhimurium resides within a membrane-bound compartment and is not an efficient inducer of class I–restricted immune responses. Viral epitopes were successfully delivered to the host-cell cytosol by using the type III protein secretion system of S. typhi- murium. This resulted in class I–restricted immune responses that protected vaccinated animals against lethal infection. This approach may allow the ef- ficient use of S. typhimurium as an antigen delivery system to control infections by pathogens that require this type of immune response for protection. The success of global vaccination programs requires efficacious vaccines that are stable and easy to administer (1). Viable carrier systems offer the greatest potential for inno- vative approaches to develop polyvalent vac- cines. Efficient protection against infectious agents often requires the action of both hu- moral and cellular immune mechanisms. Therefore, an ideal polyvalent antigen deliv- ery system should be capable of stimulating all desired effector cell populations of the immune system. Live replicating bacteria and viruses that stimulate complex immune re- sponses have been rendered avirulent and endowed with the ability to express foreign proteins derived from pathogenic microor- ganisms (2). Avirulent strains of Salmonella typhimurium are being widely considered as delivery systems for heterologous antigens because of their ability to induce complex mucosal and systemic immune responses af- ter oral administration (3). A characteristic feature of these bacteria is their ability to invade nonphagocytic cells such as those of the intestinal epithelium (4). After internal- ization, S. typhimurium remains confined to a membrane-bound compartment insulated from the cytosolic environment of the host cell (5). Localization within the “internaliza- tion” vacuole prevents delivery of expressed foreign antigens to the major class I antigen presentation pathway, thereby hampering the use of Salmonella vaccine carriers when this type of response is crucial for protection (for example, viral infections) (6). An attempt to circumvent this problem has been the use of Salmonella to deliver plasmid DNA to ex- press antigens within the host-cell cytosol (7). Contact of S. typhimurium with host cells results in activation of a specialized protein secretion system (type III) that is encoded in a pathogenicity island at centisome 63 of its chromosome (4). This protein secretion sys- tem delivers a set of bacterial effector pro- teins into the host-cell cytosol, which leads to stimulation of signal transduction pathways that result in a variety of responses such as actin cytoskeleton reorganization and activa- tion of transcription factors (4). In an effort to improve the ability of Salmonella to elicit class I–restricted immune responses to those epitopes, we investigated the potential of this system to deliver heterologous epitopes into the host-cell cytosol. To this end, we chose SptP, a S. typhimurium effector protein that is delivered into the host cell through the cen- tisome 63 type III secretion system but is not required for efficient bacterial entry into nonphagocytic cells (8). We constructed a chimeric form of SptP that carries a class–I restricted epitope consisting of residues 366 to 374 from the influenza virus nucleoprotein (IVNP 366 –374 ) found to be immunodominant in mice of the H-2 b haplotype (9). The epi- tope was introduced at a permissive site of SptP (10) located between the two predicted independent domains of this protein (Fig. 1) (8). The chimeric SptP-IVNP 366 –374 protein was secreted into the culture supernatant of both wild-type S. typhimurium and the isogenic avirulent aroA sptP mutant strain SB824 at concentrations indistinguishable from those of wild-type SptP (Fig. 1). Both strains efficiently delivered SptP-IVNP 366 –374 into the cytosol of infected cultured epithelial cells (Fig. 1). In contrast, and as expected, the isogenic S. typhimurium sipD mutant strain SB221 did H. Ru ¨ssmann, Y. Fu, J. E. Gala ´n, Department of Mo- lecular Genetics and Microbiology, School of Medi- cine, State University of New York at Stony Brook, Stony Brook, NY 11794 –5222, USA. H. Shams, F. Poblete, R. O. Donis, Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583– 0905, USA. *Present address: Max von Pettenkofer-Institut, Pet- tenkofer Strasse 9a, 80336 Munich, Germany. †To whom correspondence should be addressed. E- mail: galan@asterix.bio.sunysb.edu R EPORTS www.sciencemag.org SCIENCE VOL 281 24 JULY 1998 565