• Vaccine Name: Ag85B and ESAT-6 fusion protein
• Target Pathogen: Mycobacterium tuberculosis
• Target Disease: Tuberculosis
• Vaccine Ontology ID: VO_0000536
• Type: Conjugate vaccine
• Antigen: Two immunodominant antigens, Ag85B and ESAT-6, were used in the analysis. ESAT-6 is a 6-kDA early secretory antigenic target. The two antigens are combined as a fusion protein in the vaccine (Olsen et al., 2001).
• EsxA (ESAT-6) gene engineering:
  • Type: Fusion protein ESAT6-Ag85B
  • Description: Fusion protein created in E. coli using His-tag cloning system pMCT6. Proteins were affinity purified, separated via ion exchange chromatography, and analyzed with SDS-PAGE and Western blots (Olsen et al., 2001).
  • Detailed Gene Information: Click Here.
• Ag85B from M. tuberculosis H37Rv gene engineering:
  • Type: Fusion protein Ag85B-ESAT6
  • Description:
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0001250
  • Description: 1 25 μg amount of monoposphoryl lipid A (MPL) adjuvant was added to 250μg dimethyl dioctadecylammonium bromide (DDA) emulsifier per 0.01-50μg of vaccine.
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0001261
• Preparation: M tuberculosis Erdamn and H37Rv were grown in media, while BCG Danish 1331 was acquired as a freeze dried-vaccine and replenished (Olsen et al., 2001). Recombinant ESAT-6 was obtained along with short-term culture filtrate. Esat6 and ag85B coding regions were obtained by PCR amplification from M. tuberculosis H37Rv chromosomal DNA using selected primers. PCR products were digested by two restriction endonucleases and cloned into pMCT6 prior to sequencing. The E. coli XL-1 Blue was used to express the His-tagged protein prior to purification, protein anion-exchange chromatography, and dialysis. PCR fragments per gene were joined at a HindIII site, cloned into pMCT6, expressed, and later purified to create a fusion protein and a chimeric plasmid.
  
  

Mouse Response

• Host Strain: C57BL/6J (H-2^b), specific pathogen-free female; B6CBAF1
• Vaccination Protocol: Female mice 6-12 weeks old were housed within a BSL-3 enclosure. Mice were immunized with 0.01-50 μg vaccine emulsed in 250 μg dimethyl dioctadecylammonium bromide (DDA) and 25 μg of monophosphoryl lipid A (MPL) aduvant. Vaccines were injected 3 times subcutaneously on back at 2-week intervals (Olsen et al., 2001). BCG Danish 1331 dose (5x10^4 baccilli/mouse) was administered via subcutaneous injection at base of tail simultaneously with first subunit vaccination. No booster injections were included (Olsen et al., 2001).
• Persistence: Mice were sacrificed at either 2 or 6 weeks post-innoculation via i.v. or aerosol routes, respectively prior to organ removal and bacterial enumeration(Olsen et al., 2001).
• Challenge Protocol: Prechallenge immunity was assessed 5 weeks post-first vaccination (Olsen et al., 2001).
  Mice were challenged at either 10 or 30 weeks post-immunization via aerosol route (100 CFU of M. tuberculosis Erdman per lung) or intravenous route (0.2 ml volume containing 5x10^4 CFU M. tuberculosis H37Rv in PBS) (Olsen et al., 2001).
• Efficacy: A statistical reduction in the number of bacteria was observed for vaccine doses as low as .01μg. The 0.1-10μg dose range showed a higher level of protection versus the .01μg dose. 50 μg doses resulted in reduced protection in lung and spleen, whereas, 10 μg showed significant levels of protection in spleen. The fusion protein Ag85B-ESAT-6 showed a statistically significant improvement in reduction of bacteria versus the individual Ag85B and ESAT-6 components (Olsen et al., 2001). Both recombinant proteins induced long-lived memory immunity when challenged 10 or 30 weeks post-first vaccination, while BCG showed significantly lower protection levels at the later challenge points(Olsen et al., 2001).
• Description: Blood lymphocytes were purified and pooled from eight mice per group and cultured in triplicate as 2x10^5 cells in 200μl of RPMI 1640 with 2-mercaptoethanol, glutamine, penicillin-streptomycin, and 5% v/v fetal calf serum. Mycobacterial antigens administered as 5-1.3μg/ml. Interferon gamma amounts were determined using ELISA (Olsen et al., 2001).
• Information about this animal model: Mouse Model for TB research