• Vaccine Name: Mtb72f fusion protein
• Target Pathogen: Mycobacterium tuberculosis
• Target Disease: Tuberculosis
• Vaccine Ontology ID: VO_0004108
• Type: Fusion protein with adjuvant
• PepA gene engineering:
  • Type: Fusion protein from co-expressed ORFs
  • Description: Two open reading frames for M. tuberculosis antigenic proteins Mtb32 and Mtb39 (Rv0125 and Rv1196, respectively) were combined and expressed as a single recombinant polyprotein of size 72kDa. Each ORF was PCR amplified and subcloned into plasmids prior to transformation into E. coli. Correct inserts and orientation were identified by restriction digests and DNA sequencing. The final Mtb72f fusion protein acts as an activator of IFN-gamma in CD4 and CD8 T cells (Skeiky et al., 2004).
  • Detailed Gene Information: Click Here.
• PPE18 gene engineering:
  • Type: Fusion protein from co-expressed ORFs
  • Description: Two open reading frames for M. tuberculosis antigenic proteins Mtb32 and Mtb39 (Rv0125 and Rv1196, respectively) were combined and expressed as a single recombinant polyprotein of size 72kDa. Each ORF was PCR amplified and subcloned into plasmids prior to transformation into E. coli. Correct inserts and orientation were identified by restriction digests and DNA sequencing. The final Mtb72f fusion protein acts as an activator of IFN-gamma in CD4 and CD8 T cells (Skeiky et al., 2004).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0001264
  • Description: Adjuvants included AS02A or AS01B (Skeiky et al., 2004).
• Preparation: See genetic engineering section for details regarding construction of rMtb72F. Vaccines included rMtb72F combined with either of two adjuvants, or Mtb72F alone (Skeiky et al., 2004). Immunization dose was either 50 or 43 μl with 1x PBS (pH 6.8) mixed with 50 μl of AS01B or 57 μl of AS02A, respectively (Skeiky et al., 2004).
  
• Description: Mice were immunized three times (at 3 week intervals) with 8μg of recombinant Mtb72F (rMtb72F) (Skeiky et al., 2004).

Mouse Response

• Host Strain: C57/BL6 female mice
• Vaccination Protocol: Mice were age-matched (4-6 weeks) per experiment, and immunized 3 times (2 wk apart) with 8μg of rMtb72f and selected adjuvants. Immunization dose was increased from 8μg using 50 or 43 μl of PBS with 50 or 57 μl of AS01B or AS02A adjuvants. In total, 100 μl of vaccine was injected via i.m. (tibialis) route with 50 μl/leg (Skeiky et al., 2004). Additional culturing protocols were included for mononuclear and splenic cells (see (Skeiky et al., 2004)). Mice immunized with 5x10^4BCG in base of tail served as positive controls. Negative controls were injected with saline, adjuvant alone, or DNA vector.
  
• Persistence: The extent of protection with Mtb72F represents the longest documented survival end point reported for any defined subunit vaccine (~40% survival after 80 weeks post-challenge) (Skeiky et al., 2004).
• Challenge Protocol: Thirty days after the last immunization, mice were challenged by low-dose aerosol exposure (50-100 bacteria in lungs) with M. tuberculosis H37Rv strain. Mice were euthanized four weeks later prior to lung and spleen homogenate preparation.
• Efficacy: Mice immunized with DNA developed an Mtb72f Ab response of the IgG2a but not IgG1 subclasses which was directed against N-terminal (Mtb32c) molecules. Strong IFN-gamma responses were elicited in response to rMtb72F, rMtb32c, Mtb39, and PPD treatments. Mice immunized with rMtb72f and either adjuvant mounted strong IgG1 and IgG2a responses against Mtb72F. The AS01B-Mtb72F formulation elicited robust CD8+ T cell responses (directed against Mtb32c), whereas the AS02A-Mtb72F formulation were weaker than the AS01B formulation. However, both adjuvants when combined with Mtb72f could elicit ~0.6 log reduction in bacteria burden of mouse lungs. The Mtb72F-DNA provided 0.7-1.0 log reduction in bacterial load, which is comparable to BCG results (Skeiky et al., 2004).
• Information about this animal model: Mouse Model for TB research

Guinea pig Response

• Vaccination Protocol: Similar to mouse protocol, the guinea pigs were immunized three times (3 weeks apart) with 200μg Mtb72F-DNA with 250μl of 1xPBS @ pH 7.0, 20-μg dose of rMtb72f, or a mixture of these components in a total 250uL dose containing AS02A. Guinea pigs were immunized with 125μl of final formula in the leg (Skeiky et al., 2004). BCG administered to some animals via intradermal route. ***
• Persistence: Evidence suggests that the protection can last for > 1 year (Skeiky et al., 2004).
• Challenge Protocol: Animals were challenged with virulent H37Rv strain via aerosol route after 13 weeks, with nebulizer providing 20-50 bacteria into the lungs (Skeiky et al., 2004).
• Efficacy: After 30 wk postchallenge, four of five guinea pigs immunized with rMtb72F and three of five immunized with Mtb72F-DNA were alive, whereas after 70 weeks, two of five in DNA-immunized groups were still alive.