Yersinia enterocolitica as a vehicle for a naked DNA vaccine encoding Brucella abortus bacterioferritin or P39 antigen

Abstract

Brucella is a facultative intracellular parasite that causes brucellosis in animals and humans. The protective immune response against Brucella involves both humoral and cell-mediated immunity. In previous studies, we demonstrated that the T-dominant Brucella antigens bacterioferritin (BFR) and P39 administered either as CpG adjuvant recombinant proteins or as naked-DNA plasmids induced a specific Th1-biased immune response in mice. In order to improve the protection conferred by the BFR and P39 vaccines and to evaluate the additive role of antilipopolysaccharide (anti-LPS) antibodies, we used live attenuated Yersinia enterocolitica serotypes O:3 and O:9 as delivery vectors for naked-DNA plasmids encoding these BFR and P39 antigens. Following two intragastric immunizations in BALB/c mice, the Yersinia vectors harboring a DNA vaccine encoding BFR or P39 induced antigen-specific serum immunoglobulin and Th1-type responses (both lymphocyte proliferation and gamma interferon production) among splenocytes. Moreover, as expected, antibodies recognizing Brucella abortus 544 lipopolysaccharide were detected in O:9-immunized mice but not in O:3-treated animals. Animals immunized with O:9 organisms carrying pCI or with O:9 organisms alone were found to be significantly resistant to infection by B. abortus 544. Our data demonstrated that pCI plasmids encoding BFR or P39 and delivered with live attenuated strains of Yersinia O:3 or O:9 can trigger Th1-type responses. The fact than only O:9 vectors induced a highly significant protective immunity against B. abortus 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.

FIG. 1.

Time course of persistence of bacteria in spleen and PP. Groups of nine mice were immunized i.g. with 1 × 10⁸ to 3 × 10⁸ CFU of Y. enterocolitica O:9(pCIP39) (A) or O:9 (B) and sacrificed after 2, 7, or 21 days (three mice at each time). The spleen and PP were recovered, homogenized, and plated on agar plates. Data are means ± standard deviations (n = 3).

FIG. 2.

Western blot of sera from recombinant-Yersinia-immunized mice. Recombinant BFR (A) and P39 (B) were subjected to SDS-PAGE and then transferred to nitrocellulose, which was subsequently probed with sera from immunized mice (four mice per group). (A) Lanes 2, 3, 4, and 5 were probed with sera from mice immunized with O:3(pCIP39), O:3 alone, O:9(pCIP39), and O:9 alone, respectively. Lane 6 was probed with anti-P39 monoclonal antibody as a control. (B) Lanes 2, 3, 4, and 5 were probed with sera from mice immunized with O:3(pCIBFR), O:3 alone, O:9(pCIBFR), and O:9 alone, respectively. Lane 6 was probed with rabbit anti-BFR polyclonal antibody as a control. Lanes 1 contain prestained molecular markers. Sizes (in kilodaltons) are indicated on the left.

FIG. 3.

Proliferative T-cell responses among splenocytes recovered from BALB/c mice. The assays were performed 14 days after two i.g. inoculations with attenuated Y. enterocolitica serotype O:3 or O:9 harboring pCI, pCIBFR, or pCIP39 or with Yersinia strains alone. Splenic cells were stimulated in quadruplicate with 10 μg of attenuated O:3 or O:9 lysate or the recombinant proteins BFR or P39 per ml or were not stimulated (RPMI). Data are means ± standard deviations of quadruplicate measurements from four mice.