• Vaccine Name: B. melitensis 16M DNA vaccine expressing Omp31
• Target Pathogen: Brucella spp.
• Target Disease: Brucellosis
• Vaccine Ontology ID: VO_0000325
• Type: DNA vaccine
• Antigen: The antigen for this vaccine were found in eukaryotic expression vectors called pTargeTomp31, which encoded the outer membrane protein (omp31) of B. melitensis 16M (Gupta et al., 2007).
• Omp31 gene engineering:
  • Type: DNA vaccine construction
  • Description: The nucleotide sequence of the gene coding for the outer membrane protein omp31 reported for B. melitensis were synthesized by Qiagen. The primers were used to amplify a target sequence of 720 bp within a gene code for the production of a 28–31 kDa outer membrane protein (omp31) specific to the B. melitensis. The omp31 gene of B. melitensis was amplified with primers 5′-TGACAGACTTTTTCGCCGAA-3′ and 5′-TATGGATTGCAGCACCGC-3′. This 720 bp B. melitensis DNA fragment encoding omp31 was cloned in pTargeT mammalian expression system vector. The resultant plasmid (pTargeTomp31) contained the omp31 gene. Competent Escherichia coli JM109 was transformed with pTargeTomp31. Ampicillin-resistant colonies were grown in Luria-Bertani medium containing 100 μg/ml of ampicillin at 37 °C with agitation at 225 rpm. Protein expression was induced by adding 1 mM isopropyl-β-d-thiogalactopyranoside and incubating transformed cells for 4 h. Bacteria were pelleted by centrifugation and frozen at −20 °C. Bacterial cells were suspended in a solution consisting of 50 mM Tris, 5 mM EDTA, and 1% Triton X-100 at a pH of 8.0 and sonicated for three 1-min cycles at 4 °C. Inclusion bodies were pelleted at 20,000 × g for 30 min at 4 °C and washed twice with suspension solution without Triton X-100. Inclusion bodies were solubilized in a solution containing 50 mM Tris, 5 mM EDTA, and 8 M urea at a pH of 8.0 at room temperature overnight with agitation. After centrifugation, soluble protein was purified by chromatography through Ni-agarose. rOmp31 was adsorbed with Sepharose-polymyxin B to eliminate LPS contamination. Plasmid DNA for in vitro transfection or mouse immunization was extracted from a 16-h culture and purified using the Endo-Free Plasmid purification kit. Plasmid DNA was adjusted to a final concentration of 1 mg/ml in PBS and stored at −80 °C (Gupta et al., 2007) .
  • Detailed Gene Information: Click Here.
• DNA vaccine plasmid:
  • DNA vaccine plasmid name:
  • DNA vaccine plasmid VO ID: VO_0000318
• Preparation: B. melitensis 16M, the virulent strain, was isolated from the stomach content of the aborted fetus of the goat. B. melitensis DNA fragment encoding omp31 was cloned in pTargeT mammalian expression system vector. The plasmid was then purified and made into a vaccine consisting of 100 μg of pTargeTomp31 in 50 μl sterile saline (Gupta et al., 2007).
• Virulence: The strain isolated from the goat was virulent, bu the plasmid created was quite attenuated (Gupta et al., 2007).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Specific-pathogen-free 4-week-old BALB/c female mice were obtained. 25 mice were randomly divided in to five groups of 5 mice each and received intramuscular injections in the tibialis anterior muscles with 100 μg of pTargeTomp31 in 50 μl sterile saline by using a 1-ml insulin syringe with a 28-gauge needle. Similarly, another set of 6 weeks old 25 female mice (divided in five groups of 5 mice each) were sham immunized with saline only, the control group. From both the DNA immunized and sham immunized mice, one group of 5 mice each was exclusively used in order to obtain sera to test antibody and spleen cells for cytokine production in response to omp31 or B. melitensis extract. Three vaccinations at 3-week intervals were performed (Gupta et al., 2007).
• Immune Response: Mice injected with pTargeTomp31 have good IgG2a and IgG1 titers to anti-omp31 antibodies. Antibodies against omp31 could already be detected 1 week after the first pTargeTomp31 DNA injection. Immunization with pTargeT did not induce any production of anti-omp31 antibodies. Humoral response measured 18 weeks postvaccination indicates that pTargeTomp31 DNA vaccine leads to the generation of long-lived IgG1 and IgG2a responses. pTargeTomp31 DNA vaccination resulted in specific T-cell proliferation in response to omp31 or to Brucella extracts. This specific induced proliferative response was also found at 8 weeks postvaccination, although to a lesser extent. In contrast, immunization with pTargeT appeared to have no effect on the level of T-cell proliferative response. The ConA mitogen was able to induce T-cell proliferation in all cases (Gupta et al., 2007).
• Challenge Protocol: Mice immunized three times with pTargeTomp31 or pTargeT were infected 9 weeks later the last DNA immunization with B. melietensis 16M. Seven, 14, 21 and 28 days after challenge, mice were killed and the number of bacteria in the spleens was quantitated (Gupta et al., 2007).
• Efficacy: No significant difference in the number of the bacteria isolated from the spleens of the saline vaccinated and pTargeT-vaccinated animals was observed. The mice, which were immunized by pTargeTomp31, showed a significant level of protection at 28 days after challenge. Mice immunized with DNA vaccine made anti-Brucella serum antibody. This vaccine provided the moderate degree of protection to the mice (Gupta et al., 2007).