Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella spp - PubMed (original) (raw)

Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella spp

A Al-Mariri et al. Infect Immun. 2001 Oct.

Abstract

In this study, we evaluated the ability of DNA vaccines encoding the bacterioferritin (BFR) or P39 proteins of Brucella spp. to induce cellular and humoral immune responses and to protect BALB/c mice against a challenge with B. abortus 544. We constructed eukaryotic expression vectors called pCIBFR and pCIP39, encoding BFR or P39 antigens, respectively, and we verified that these proteins were produced after transfection of COS-7 cells. PCIBFR or pCIP39 was injected intramuscularly three times, at 3-week intervals. pCIP39 induced higher antibody responses than did the DNA vector encoding BFR. Both vectors elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the specific antigens or Brucella extract. In this report, we also demonstrate that animals immunized with these plasmids elicited a strong and long-lived memory immune response which persisted at least 3 months after the third vaccination. Furthermore, pCIBFR and pCIP39 induced a typical T-helper 1-dominated immune response in mice, as determined by cytokine or immunoglobulin G isotype analysis. The pCIP39 delivered by intramuscular injection (but not the pCIBFR or control vectors) induced a moderate protection in BALB/c mice challenged with B. abortus 544 compared to that observed in positive control mice vaccinated with S19.

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Figures

FIG. 1

FIG. 1

In vitro expression of BFR and P39 was tested by transient transfection of COS-7 cells. The cells were transfected with either pCI (lanes 2 and 4), pCIBFR (lane 1), or pCIP39 (lane 3). After 24 h, the COS-7 cells were washed and lysed. The proteins were separated by SDS-PAGE, and the antigens were then detected by immunoblotting, using rabbit anti-BFR polyserum (lanes 1 and 2), or anti-P39 monoclonal antibody (lanes 3 and 4). Molecular mass standards (lane M.M.) are indicated in kilodaltons.

FIG. 2

FIG. 2

Lymphocyte proliferation assay. BALB/c mice were immunized with pCI, pCIBFR, or pCIP39. T-cell-proliferative responses were measured at week 3 (A) and 12 (B) after the last immunization. Splenocytes from each mouse were prepared and stimulated in vitro with purified recombinant BFR or P39 (1 μg/ml) and Brucella extracts (3 μg/ml) as the antigen. After 72 h of culture, [3H]thymidine was added; 18 h later, cells were harvested and the counts per minute were determined. Each sample was assayed in quadruplicate. Data represent the mean ± standard deviation (error bars) from the four mice.

FIG. 3

FIG. 3

Quantitative ELISA analysis of IFN-γ secretion by murine lymphocytes isolated after DNA immunization (pCI, pCIBFR, or pCIP39) and subsequent stimulation in culture using different antigens. Splenocytes from the immunized mice (four mice/group) were harvested at week 3 (A) and 12 (B) after the last immunization. Assays were performed on supernatants of 2 × 106 lymphocytes per ml. Lymphocytes were restimulated with the indicated antigens. At 96 h after restimulation, supernatants were analyzed for IFN-γ levels. Samples were tested in duplicate.

References

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