DNA vaccine encoding L7/L12-P39 of Brucella abortus induces protective immunity in BALB/c mice
2006; Lippincott Williams & Wilkins; Volume: 119; Issue: 4 Linguagem: Inglês
10.1097/00029330-200602020-00012
ISSN2542-5641
AutoresDeyan Luo, Peng Li, Li Xing, Guangyu Zhao, Wei Shi, Zhang Songle, Xiliang Wang,
Tópico(s)Escherichia coli research studies
ResumoBrucella abortus is a gram-negative, facultative, intracellular bacterium that infects both cattle and humans, causing abortion and infertility in the former and undulant fever, endocarditis, arthritis, and osteomyelitis. Resistance to Brucella depends on acquired cell-mediated immunity (CMI).1 Live attenuated vaccines can stimulate strong CMI response, which are usually very effective against brucellosis and are used to control brucellosis in domestic animals. However, there is no safe and effective vaccine available for human because the vaccine strains used for animals are considered too virulent for humans. A vaccine that will be noninfectious to humans but effective in stimulating a broad protective immune response is needed.2 DNA vaccines seem to offer the best approach to activate cellular immune response.2 A DNA vaccine comprising the Brucella ribosomal L7/L12 gene has been demonstrated to induce significant protective effect in mouse.3 Periplasmic binding protein (P39), existing in all the six species of Brucella, has been proved to be a protective antigen.4 In this study, we constructed the plasmid containing two genes, L7/L12 and P39, to evaluate its protective capacity of immunization. METHODS Animals Sixty-six female BALB/c mice (5 to 6 weeks old, 18 to 22 g, obtained from the Animal Center of the Institute of Microbioiogy and Epidemiology, Academy of Military Medical Sciences, China) were randomly distributed into six experimental groups. The mice were kept in conventional animal facilities and received water and food ad libitum. Plasmids and protein Plasmids pcDNA3.1(+)-L7/L12,5 pcDNA3.1(+)-P39 and fusion protein L7/L12-P39 (rL7/L12-P39), crude Brucella abortus RB51 proteins (CBP, Sigma, USA) were prepared in our laboratory. Bacterial strains Virulent strain A544 and attenuated strain 104M were obtained from our own culture collection. All experiments with live Brucella were performed in biosafety level 3 facilities. E. coli strain DH5α was kept in our laboratory. Construction and preparation of L7/L12-P39 DNA vaccine PCR was performed using four primers which were designed based on conserved amino acids ofL7/L12 and P39 from gene bank. The primer sequences of the L7/L12 were 5′-TAGGTACCATGGCTGATCTCGC-3′ (primer 1) and 5′-TCGGATCCTCCTGAACCCTTGAGTTCAAC-3′ (primer 2). The primer sequences of the P39 were 5′-TATGGATCCCCGGTTGCAGGTG-3′ (primer 3) and 5′-TAGCGGCCGCTTATTTTGCGGCTTC-3′ (primer 4). Theamplified product was purified from the gel, and then the two genes were ligated together into pcDNA3.1(+) vector, and transformed into E. coli DH5α. A colony of E. coli containing pcDNA3.1(+)-L7/L12-P39 was cultured in LuriaBertani broth containing ampicillin 100 μg/ml. Large-scale plasmid DNA isolation was performed and resuspendedin PBS at a concentration of 1000 μg/ml. Immunization In each group, five mice were used for immunogencity test, six for challenge assay. Each mouse was injected with 100 μg plasmid in 100 μl of PBS in tibialis anterior muscle, then was vaccinated at weeks 0, 2 and 4 with pcDNA3.1(+)-L7/L12-P39, pcDNA3.1(+)-L7/L12 and pcDNA3.1(+)-P39, respectively. Mice were injected with 50 μg of rL7/L12-P39 at weeks 0, 2 and 4. Groups of mice immunized with PBS or pcDNA3.1(+) vector were as the negative controls. Mice in the positive control group were vaccinated intraperitoneally with 2 × 108 CFU of 104M in 0.2 ml of PBS. Enzyme-linked immunosorbont Assay (ELISA) Using indirect ELISA determines the IgG, IgG1 and IgG2a isotypes in mouse serum. Briefly, polystyrene plates (Nunc, Denmark) were coated with rL7/L12-P39. After the plates were washed, a sheep anti-mouse immunoglobulin-horseradish peroxidase conjugate (Zhongshan, China) was added and the reaction was developed with orthopheny-lenediamine-H202. The cutoff value for the assay was calculated as the mean specific optical density plus or subtract 3 standard deviations (SD) for 20 sera from nonimmunized mice assayed at dilutions of 1:100. The titer of each serum was calculated as the last serum dilution while a specific optical density higher than the cutoff value. Splenocyte cultures and lymphocyte proliferation Mice were sacrificed, and their spleens were removed under aseptic conditions. Spleen cell suspensions from immunized and control mice were prepared in RPMI 1640 (Gibco, USA) supplemented with 10% fetal calf serum. Splenocytes were cultured at 37°C with 5% CO2 in a 96-well flat-bottom plate at a concentration of 4 × 105 viable cells/well in the presence of CBP 4 μg/ml or concanavalin A (ConA) 2.5 μg/ml (Sigma, USA). The cells were cultured for 3 days and pulsed with MTS (Boda, China) 5 μg/ml per well for 4 hours, and the stimulation index (SI, the ratio of mean absorption value at 450 nm in the stimulated group to the unstimulated group) was measured. Cytokine ELISPOT For detection of cytokines, the supernatant of cultured spleen cells was collected after 48 hours of antigen stimulation and tested for the cytokines by antigen-capture ELISAPOT using MultiScreenTM-IPSterilePlateγ (DIACLONE Biosciences, USA). All assays were performed in triplicate. The spot number of interferon-γ (IFN-γ) in the supernatants was calculated. Protective experiment Five weeks after vaccination (day 60), six mice from each group were challenged by intraperitoneal injection of 1.25×104 CFU of A544. Two weeks later, their spleens were macerated, and dilutions were plated to determine the number of Brucella CFU per spleen. This experiment was repeated three times. Statistical analysis All results were expressed as mean±SD. One-way analysis of variance was used to test the statistical significance by SPSS12.0. P value of less than 0.05 was considered statistically significant. RESULTS Immune response Mice immunized with pcDNA3.1(+)-L7/L12-P39 had a higher IgG titers than those in other DNA vaccine groups, but lower than those in rL7/L12-P39 and 104M groups after the third immunization. (Table 1) None of the animals inoculated with PBS showed specific anti-L7/L12-P39 antibodies. Subisotype analysis of these antibodies (IgG1 and IgG2a) indicated that the predominant anti-L7/L12-P39 antibodies were IgG2a at day 60 post-vaccination. The specific IgG2a titers were higher than the IgG1 titers (IgG2a/IgG1 > 1, Fig. 1). All the titers were higher than those in pcDNA3.1(+)-L7/L12 and pcDNA3.1(+)-P39 groups. T lymphocytes from mice immunized with pcDNA3.1(+)-L7/L12-P39 showed a significant proliferation response to ConA or CBP [P<0.01 compared with pcDNA3.1(+)-L7/L12 or pcDNA3.1(+)-P39 groups] with a SI of 1.39. (Fig. 2) Supernatant of spleen cell from pcDNA3.1(+)-L7/L12-P39 immunized mice contained high levels of IFN-γ compared to the other groups (P<0.01), except for 104M group.Table 1: Antibody titers of special IgG in mouse serum after three rounds of immunization.Fig. 1.: Comparision of titers of different subclass antibodies in sera (1:100) of immunized mice. Group 1: PBS, Group 2: pcDNA3.1(+)-P39, Group 3: pcDNA3.1(+)-L7/L12, Group 4: pcDNA3.1(+)-L7/L12-P39, Group 5: rL7/L12-P39, and Group 6: 104M. * P<0.01 compared with pcDNA3.1(+)-P39 and pcDNA3.1(+)-L7/L12 groups.Fig. 2.: Proliferation of T lymphocyte in sera of immunized mice. Group 1: PBS, Group 2: pcDNA3.1(+)-P39, Group 3: pcDNA3.1(+)-L7/L12, Group 4: pcDNA3.1(+)- L7/L12-P39, Group 5: rL7/L12-P39, and Group 6: 104M. SI: stimulation index. * P<0.01 compared with pcDNA3.1(+)-P39 and pcDNA3.1(+)-L7/L12 groups.Protective efficacy Immunization with pcDNA3.1(+)-L7/L12-P39 resulted in a significantly higher degree of protective efficacy than that of pcDNA3.1(+)-L7/L12 and pcDNA3.1(+)-P39 groups (P<0.05, respectively). Vaccination with live 104M induced 2.21log protection [P<0.01 compared with pcDNA3.1(+)-L7/L12-P39 group]. No significant difference in the number of CFU was seen between groups injected with PBS (Table 2).Table 2: Protective efficacy challenged with A544 after immunization with DNA vaccine (mean ± SD)DISCUSSION The development of new-generation vaccine systems to prevent brucellosis is needed to overcome the disadvantages of the live vaccines currently used. At the same time, for an intracellular pathogen, including Brucella, requires the generation of a Th1-type immune response. The DNA vaccine can express an antigen, then successfully present antigen to a naive T cell resulting in the activation of T cell and the interaction between other molecules on the surfaces of the T cell. The Th1 subset CD4+ lymphocytes secrete IFN-γ, a crucial cytokine upregulating the macrophage activity and reflecting the Th1-type immune response. The CD8+ T lymphocytes are able to lyse infected cells.5 DNA vaccine shows many advantages, such as no risk of infection, induction of a long-lived immune response, better stability than live attenuated vaccines, easy preparation, and low cost.6 Immunization with plasmid DNA coding the interesting antigen represents a novel and promising method in vaccine research and development. A number of studies have demonstrated that after naked DNA immunization, the antigen is naturally processed and mainly presented on major histocompatibility complex class I, including CD8+ cytotoxic T cells (CTLs), and Th1 subset CD4+ lymphocytes.2,6,7 Injection of plasmid DNA containing the L7/L12-P39 gene sequence elicited specific humoral and cellular immune responses in BALB/c mice. Two weeks after the first immunization, we found a weak titer of specific IgG in mice immunized with the plasmid pcDNA3.1(+)-L7/L12-P39. By the end of the experiment, this response was higher than that at the begin and in the pcDNA3.1(+)-L7/L12 and pcDNA3.1(+)-P39 only groups. The result depend on several factors, including the amount of L7/L12-P39 protein expressed, epitope, and structure.2,8 The induction of a T-cell immune response after DNA immunization was evaluated by measuring lymphocyte proliferation and cytokine production after in vitro stimulation of splenic cells with ConA or CBP. Both ConA and CBP induced a high T-cell-proliferative response and synthetic peptides induced a high level of IFN-γ. These indicate that immunization with the pcDNA3.1(+)-L7/L12-P39 plasmid induces Th1 cellular response. The predominance of IgG2a over IgG1 also supports this conclusion. The pcDNA3.1(+)-L7/L12-P39 vaccine induces higher protective level than protein vaccine, which may reflect the different processing and presenting mechanism for endogenous antigen (e.g., DNA vaccine) and exogenous antigen (e.g., protein). Among the three DNA vaccines, the pcDNA3.1 (+)-L7/L12-P39 DNA vaccine could provide the highest protective level against Brucella infection, which may reflect that there are more T cell epitopes in the divalent DNA vaccine than in univalent. In conclusion, inoculation of plasmid DNA containing the L7/L12-P39 gene leads to both antibody and CMI responses of Th1 type, and confers protection against A544 challenge. It is better than the pcDNA3.1(+)-L7/L12 and pcDNA3.1(+)-P39 only we studied before. This indicates we can conjunct two genes to improve immune response and might find another way to develop the new-generation vaccine systems.9
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