with 5 104 CFU of S19 4 or 8 weeks before the challenge, and another group was injected i.g. of O:3 or O:9 can trigger Th1-type responses. The fact than only O:9 vectors MYH9 induced a highly significant protective immunity against 544 infection pointed out the crucial role of anti-LPS antibodies in protection. The best protection was conferred by a serotype O:9 strain carrying pCIP39, confirming the importance of the P39 T-cell antigen in this mechanism. strain 19 (S19) has been used successfully for eradication of brucellosis in cattle (47). However, as this vaccine is virulent for humans and is responsible for abortion in pregnant animals (5, 48), alternative vaccinal approaches are needed. The bacterioferritin (BFR) and P39 antigens of were previously identified as T-dominant antigens (16, 17). Recently, we demonstrated that both BFR and P39 antigens elicited a strong Th1-dominated immune response in mice, using either the purified recombinant proteins with CpG oligodeoxynucleotide adjuvant (3) or naked-DNA vectors (pCIP39 or pCIBFR) encoding these antigens (3a). Whatever the candidate vaccine, only the P39 antigen was able to protect against 544 challenge. Nevertheless, the protection achieved was not comparable in quality or in Lurbinectedin duration to the protection conferred by the S19 live vaccine. A probable explanation for this difference could be that the live smooth strain S19 elicits not only protective cell-mediated immunity (CMI) against a panel of T-cell epitopes but also humoral responses mostly against the lipopolysaccharide (LPS) O chain. These antibodies were demonstrated to be partially protective (4, 45, 70). So, in order to improve protection, we tested an approach by which a live bacterial vector delivers the selected antigens. In such delivery vectors, the foreign genes may be present under the control of procaryotic promoters, enabling bacteria to express the heterologous protein and to deliver it to the immune system of the host (26, 27, 30). Alternatively, the live vector may deliver a naked-DNA vaccine plasmid in which the gene encoding the foreign antigen is under the Lurbinectedin control of a eucaryotic promoter (11, 18, 19, 55). We preferred the latter approach, assuming that intracellular antigen expression and plasmid-borne CpG motifs would drive a Th1-oriented immune response, including major histocompatibility complex class I-specific CD8+ cells, which are the keystones of the protective immune response against serovar Typhimurium, as a delivery vector for several reasons. First, organisms are able to persist in tissues for several days after immunization, and they replicate the naked-DNA vaccine during their division, thereby increasing the amount of plasmidic DNA. Finally and more importantly, structural and immunological studies revealed that and serotype O:9 share common epitopes on their LPS O chains (A and CY epitopes) (52). This is not the case for O:3, which has an antigenically unrelated LPS (2). Animals infected with serotype O:9 organisms but not with O:3 organisms produced antibodies cross-reacting with smooth LPS (S-LPS) in diagnostic tests (25, 67). In addition, it has been demonstrated that monoclonal antibodies specific for these epitopes were protective against infection in passive transfer experiments (12, 45, 41). This will allow us to test the relative role of anti-LPS antibodies by using serotype O:3 and O:9 strains as delivery vectors for the naked-DNA vaccines. We report here the use of attenuated strains of serotypes O:3 and O:9 to deliver eucaryotic expression vectors (pCIBFR and pCIP39) in BALB/c mice. We analyzed the immune response (humoral and cellular) against BFR and P39 and against extracts and compared the elicited protection against a 544 challenge to that elicited by the live vaccinal S19 strain. MATERIALS AND METHODS Bacterial strains and growth conditions. 544 was obtained from J.-M. Verger (Institut National de la Recherche Agronomique, Pathologie Infectieuse et Immunologie, Nouzilly, France), and S19 and the serotype O:3 and O:9 strains harboring virulence plasmid pYV+ (wild types) Lurbinectedin were obtained from J. Godfroid (Centre d’Etude et de Recherche Vtrinaire et Agrochimique, Brussels, Belgium). organisms were grown.
with 5 104 CFU of S19 4 or 8 weeks before the challenge, and another group was injected i
