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Vaccine Detail

Recombinant PA domain 4
Vaccine Information
  • Vaccine Name: Recombinant PA domain 4
  • Target Pathogen: Bacillus anthracis
  • Target Disease: Anthrax
  • Vaccine Ontology ID: VO_0000629
  • Type: Subunit vaccine
  • Antigen: PA domain 4 from B. anthracis strain Sterne
  • PagA from B. anthracis str. 'Ames Ancestor' gene engineering:
    • Type: Protein
    • Description:
    • Detailed Gene Information: Click Here.
  • Adjuvant: Alhydrogel
    • Adjuvant name: Alhydrogel
    • VO adjuvant ID: VO_0001241
    • Description: Domain 4 contains the dominant protective epitopes of PA and comprises amino acids 596-735 of the carboxy terminus of the PA polypeptide. Cell intoxication is thought to occur when full-length PA (PA83) binds to the cell surface receptor via domain 4, which contains the host cell receptor binding site. After binding to the host cell receptor, the N-terminal amino acids (1-167, i.e. domain 1a) of domain 1, which contains a furin protease cleavage site, are cleaved off, exposing the LF or EF binding site located in domain 1b and the adjacent domain 3. Domains 2 and 3 then form part of a heptameric pore on the cell surface, the LF or EF binds to its receptor, and the whole toxin complex undergoes receptor-mediated endocytosis into the cell. After acidification of the endosome, the toxin is translocates into the cell cytosol, where it exerts its cytotoxic effect. Therefore, inhibition of the binding and entry of the toxin complex, particularly lethal toxin, into the host cell is clearly important for the prevention of infection. The crystal structure of PA shows domain 4 to be more exposed than the other three domains, which are closely associated with each other. This structural arrangement may make the epitopes in domain 4 the most prominent for recognition by immune effector cells (Flick-Smith et al., 2002).
  • Preparation: DNA encoding the PA domains, which comprise amino acids 1-258, 168-487, 1-487, 168-595, 1-595, 259-735, 488-735, 596-735, and 1-735 (fusion proteins GST1, GST1b-2, GST1-2, GST1b-3, GST1-3, GST2-4, GST3-4, GST4, and GST1-4, respectively), was PCR amplified from B. anthracis strain Sterne DNA and cloned into the XhoI and BamHI sites of the expression vector pGEX-6-P3. Proteins produced by this system were expressed as fusion proteins with an N-terminal glutathione S-transferase (GST) protein. Immunization was done with rPA, with recombinant GST control protein, or with fusion proteins comprising domains 1, 4, and 1 to 4, which had the GST tag removed by incubation with PreScission Protease and removal of the GST on a glutathione Sepharose column (Flick-Smith et al., 2002).
  • Virulence: (Flick-Smith et al., 2002)
  • Description: Domain 4 contains the dominant protective epitopes of PA and comprises amino acids 596-735 of the carboxy terminus of the PA polypeptide. Cell intoxication is thought to occur when full-length PA (PA83) binds to the cell surface receptor via domain 4, which contains the host cell receptor binding site. After binding to the host cell receptor, the N-terminal amino acids (1-167, i.e. domain 1a) of domain 1, which contains a furin protease cleavage site, are cleaved off, exposing the LF or EF binding site located in domain 1b and the adjacent domain 3. Domains 2 and 3 then form part of a heptameric pore on the cell surface, the LF or EF binds to its receptor, and the whole toxin complex undergoes receptor-mediated endocytosis into the cell. After acidification of the endosome, the toxin is translocates into the cell cytosol, where it exerts its cytotoxic effect. Therefore, inhibition of the binding and entry of the toxin complex, particularly lethal toxin, into the host cell is clearly important for the prevention of infection. The crystal structure of PA shows domain 4 to be more exposed than the other three domains, which are closely associated with each other. This structural arrangement may make the epitopes in domain 4 the most prominent for recognition by immune effector cells (Flick-Smith et al., 2002).
Host Response

Mouse Response

  • Host Strain: Female A/J mice
  • Vaccination Protocol: Mice were vaccinated with 10 µg of protein adsorbed to a 20% (vol/vol) solution of 1.3% Alhydrogel on days 1 and 28 of the study. Also included were groups of mice that were immunized with rPA (expressed and purified from B. subtilis), with recombinant GST control protein, or with fusion proteins comprising domains 1, 4, and 1-4, which had the GST tag removed by incubation with PreScission Protease and removal of the GST on a glutathione Sepharose column. Blood samples from mice were collected 37 days after primary immunization for serum antibody analysis by enzyme-linked immunosorbent assay. Mice were challenged i.p. with either 105 or 106 spores of the B. anthracis STI strain (equivalent to 102 or 103 minimum lethal doses [MLDs], respectively) on day 70 of the immunization regimen and were monitored for 14 days postchallenge to determine their protected status (Flick-Smith et al., 2002).
  • Persistence: (Flick-Smith et al., 2002)
  • Side Effects: No side effects noted (Flick-Smith et al., 2002).
  • Efficacy: At the lower challenge level of 102 MLDs, mice in the GST1-2-, GST4-, and cleaved 4-immunized groups were all fully protected. All mice in the groups immunized with fusion proteins containing domain 4 were fully protected against challenge with 103 MLDs of STI spores (Brey, 2005).
References
Brey, 2005: Brey RN. Molecular basis for improved anthrax vaccines. Advanced drug delivery reviews. 2005 Jun 17; 57(9); 1266-92. [PubMed: 15935874].
Flick-Smith et al., 2002: Flick-Smith HC, Walker NJ, Gibson P, Bullifent H, Hayward S, Miller J, Titball RW, Williamson ED. A recombinant carboxy-terminal domain of the protective antigen of Bacillus anthracis protects mice against anthrax infection. Infection and immunity. 2002 Mar; 70(3); 1653-6. [PubMed: 11854261].