Vaccine 25 (2007) 5071–5085 A fusogenic peptide expressed on the surface of Salmonella enterica elicits CTL responses to a dengue virus epitope R. Luria-Perez a,b,c , L. Cedillo-Barron b,∗∗ , L. Santos-Argumedo b , V.F. Ortiz-Navarrete b , A. Oca ˜ na-Mondragon a , C.R. Gonzalez-Bonilla a,∗ a Medical Research Unit on Immunology and Infectious Diseases, Infectology Hospital, National Medical Center “La Raza”, IMSS, M´ exico City, Mexico b Department of Biomolecular Medicine, CINVESTAV, M´ exico City, Mexico c Flow Cytometry Unit, Hospital Infantil de M´ exico “Dr. Federico G´ omez”, Secretar´ ıa de Salud, M´ exico City, Mexico Received 16 June 2006; received in revised form 23 March 2007; accepted 30 March 2007 Available online 19 April 2007 Abstract Attenuated Salmonella strains are used widely as live carriers of antigens because they elicit both mucosal and systemic immunity against passenger antigens. However, they generally evoke poor cytotoxic T cell (CTL) responses because Salmonella resides within vacuolar compartments and the passenger antigens must travel to the cytosol and be processed through the MHC class I-dependent pathway to simulate CTLs. To address this problem, we designed a fusion protein to destabilize the phagosome membrane and allow a dengue epitope to reach the cytosol. The fusion protein was displayed on the bacterial surface of Salmonella enterica serovar Typhimurium SL3261 through the  domain of the autotransporter MisL. The passenger  domain contained, from the N-terminus, a fusogenic sequence, the NS3 protein 298–306-amino acid CTL epitope from the dengue virus type 2, a molecular tag, and a recognition site for the protease OmpT to release it to the milieu. Display of the fusion protein on the bacterial surface was demonstrated by IFA and flow cytometry using antibodies against the molecular tag. Cleavage of the fusogenic protein–dengue peptide was demonstrated by flow cytometry using OmpT+ Escherichia coli strains. The recombinant Salmonella strains displaying the fusogenic–dengue peptide were able to lyse erythrocytes, induced specific proliferative responses, and elicited CTL responses. These results suggest that the recombinant fusion proteins containing fusogenic sequences provide a promising system to induce CTLs by live vector vaccines. © 2007 Elsevier Ltd. All rights reserved. Keywords: Vaccines; Autotransporters; Fusogenic peptides 1. Introduction Attenuated Salmonella vaccine vectors generally elicit adequate antibody responses, especially when the passenger ∗ Corresponding author at: Unidad de Investigaci´ on M´ edica en Inmunolog´ ıa e Infectolog´ ıa, Hospital de Infectolog´ ıa CMN “La Raza”, IMSS, Avenida Vallejo esq. Jacarandas sn, Colonia “La Raza”, M´ exico DF 0200, P.O. Box 15-095, Mexico. Tel.: +55 5724 5900x24321; fax: +52 55 5583 0626. ∗∗ Corresponding author at: Department of Biomolecular Medicine, CIN- VESTAV, Avenida Instituto Polit´ ecnico Nacional No. 2506, San Pedro Zacatenco, Mexico DF 07360, Mexico. E-mail addresses: lcedillo@cinvestav.mx (L. Cedillo-Barron), crgb@prodigy.net.mx (C.R. Gonzalez-Bonilla). antigen is exposed on the bacterial surface [1,2]. Neverthe- less, the induction of cell-mediated immunity tends to be more variable [3]. Although the reasons for such variability are not clearly understood, Salmonella has several mecha- nisms to interfere with dendritic cell function and antigen processing by the class I-dependent pathway [2–5]. Once taken by professional phagocytic cells, Salmonella remains in the phagosome and its antigens do not reach the cytosolic processing pathway efficiently. Because cytotoxic T lympho- cytes (CTLs) play a key role in the clearance of intracellular pathogens and neoplastic cells, it would be advantageous to find methods to enhance antigen presentation through the cytosolic pathway. 0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.03.047