• Vaccine Ontology ID: VO_0000412
• Type: Subunit vaccine
• Antigen: Brucella melitensis 16M P39, a putative periplasmic binding protein (Al-Mariri et al., 2001).
• B. melitensis M5 P39 gene engineering:
  • Type: recombinant protein
  • Description: The bfr gene of Brucella melitensis 16M was subcloned into a pET-15b expression vector that contains a polyhistidine tag, and the resulting plasmid pET-15b-bfr was introduced in E. coli BL21(DE3). After 2 to 4 of induction with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), bacterial cells from a 100-ml culture were washed once and then sonicated. The lysate was centrifuged for 10 min at 9,000 × g at 4°C. The pellet was kept frozen at −70°C. After it had thawed, the pellet was resuspended in a lysis buffer. The resulting lysate was centrifuged at 9,000 × g for 20 min at 4°C (Al-Mariri et al., 2001). P39 was then purified based on metal chelate affinity chromatography as described previously (Letesson et al., 1997).
  • Detailed Gene Information: Click Here.
• Adjuvant: A synthetic phosphorothioate oligodeoxynucleotide containing an unmethylated, consensus immunostimulatory CpG motif (5′-purine-purine-CpG-pyrimidine-pyrimidine-3′ oligodeoxynucleotide [CpG ODN]). Specifically, the immunostimulatory CpG 1826 (5′-TCCATGACGTTCCTGACGTT-3′) was used (Al-Mariri et al., 2001).
• Preparation: The bfr gene of Brucella melitensis 16M was subcloned and expressed in an expression vector. The protein was purified and mixed with the CpG ODN as the vaccine (Al-Mariri et al., 2001).
• Virulence: Not reported.
• Description: P39 has been found to be a Brucella protective antigen (Al-Mariri et al., 2001).

Mouse Response

• Host Strain: Balb/c mice
• Vaccination Protocol: Female BALB/c mice at 4 weeks of age were separated into nine groups of 12 mice. Groups 1, 2, and 3 received PBS, CpG ODN, and non-CpG ODN, respectively, and served as negative controls. Groups 4 and 5 were injected with the purified P39 and bacterioferrin (BFR) alone, respectively. Groups 6 and 7 were injected with the recombinant protein with CpG ODN adjuvant. Finally, groups 8 and 9 received the recombinant antigens with the non-CpG ODN. Vaccines were prepared in PBS and contained combinations of the following: 20 μg of recombinant protein and/or 20 μg of oligonucleotides when needed. Vaccines were given intramuscularly (i.m.) into the left tibial anterior muscles in a total volume of 50 μl three times at 3-week intervals (Al-Mariri et al., 2001).
• Persistence: Not reported.
• Side Effects: None reported.
• Challenge Protocol: Three weeks after the last injection, the remaining mice of each group were challenged by the intraperitoneal route (i.p.) with 5 × 10⁴ CFU of B. abortus strain 544 in 100 μl of PBS. An additional group of eight mice vaccinated i.p. with B19 (1x10⁵ CFU) was challenged 4 weeks later in the same way and served as a vaccinated control. Spleen colonization with the challenge strain was determined at 4 and 8 weeks postinfection (Al-Mariri et al., 2001).