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Vaccine Detail

pCLF4
Vaccine Information
  • Vaccine Name: pCLF4
  • Target Pathogen: Bacillus anthracis
  • Target Disease: Anthrax
  • Vaccine Ontology ID: VO_0000880
  • Type: DNA vaccine
  • Antigen: B. anthracis lethal factor (LF)
  • Lef gene engineering:
    • Type: Protein
    • Description:
    • Detailed Gene Information: Click Here.
  • PagA from B. anthracis str. 'Ames Ancestor' gene engineering:
    • Type: Protein
    • Description:
    • Detailed Gene Information: Click Here.
  • DNA vaccine plasmid: pCI DNA vaccine plasmid
  • Preparation: Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10-254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175-764) of B. anthracis PA cloned into the pCI expression vector. PA, LF, and LFE687C (LF7) were expressed and purified. LFE687C is the full-length enzymatically inactive LF protein containing the indicated aa substitution within the zinc-binding active site (Price et al., 2001).
Host Response

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: Micrometer-diameter gold particles were coated with plasmid pCLF4, pCPA, or a 1:1 mixture of both. Separate groups of female BALB/c mice at 4-5 weeks of age were immunized i.d. in the abdomen via biolistic particle injection on d 0, 14, and 28 with approximately 1 µg of plasmid DNA-coated gold particles for each injection. Immunization groups included mice injected with the same microparticles coated with pCPA, pCLF4, a 1:1 mixture of the pCPA and pCLF4 plasmids, or, as a vector control, the pCI plasmid. For the prime-boost immunization experiments, groups of BALB/c mice were first immunized twice with plasmid DNA as described above and then with a third and final boost of purified antigen emulsified in Freund's incomplete adjuvant. The protein immunizations were administered i.m. Blood samples were obtained 2 weeks following each vaccination, and the sera were pooled and stored at -20°C until analyzed (Price et al., 2001).
  • Immune Response: Titers of anti-LF antibody remain at high levels for much longer periods of time than do titers of anti-PA antibody. The LF antigen appears to be much more immunogenic and produces an immune response which lasts much longer than the response to the PA antigen. Co-administration of the pCPA and pCLF4 plasmids followed by a final protein booster immunization with the recombinant PA and LF7 antigens produced a substantially higher endpoint titer against either PA or LF at the same time-point than the antibody titers resulting from DNA-based immunization alone (Price et al., 2001).
  • Challenge Protocol: Plasmid-immunized BALB/c mice that had received a total of three injections were challenged with purified Letx 2 weeks following the third and final injection. The challenge was conducted by tail vein injection of a previously mixed combination of purified PA and LF proteins (60 μg of PA and 25 to 30 μg of LF per mouse), the equivalent of approximately 5 50% lethal doses (LD50) of Letx (Price et al., 2001).
  • Efficacy: All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. A significant antibody response is generated using DNA-based immunization alone and the levels of antibody produced are sufficient to protect animals against an Letx challenge that is 5 times the LD50. Also, co-administration of the pCPA and pCLF4 plasmids followed by a final protein booster immunization with the recombinant PA and LF7 antigens produced a substantially higher endpoint titer against either PA or LF at the same time point than the antibody titers resulting from DNA-based immunization alone (Price et al., 2001).

Rabbit Response

  • Host Strain: NZW
  • Vaccination Protocol: Groups of rabbits were immunized with various vaccine preparations. The first group was immunized (i.m.) twice using the needleless Biojector device with 500 ug of plasmid DNA (pCPA and/or pCLF4) resuspended in 0.5 ml of sterile phosphate buffered saline (PBS) at 4-week intervals. These animals were boosted by needle (i.m.) 4 weeks later with 200 ug of purified full-length rPA protein or full-length recombinant lethal factor (LF) protein LF7 with a point mutation resuspended in incomplete Freund’s adjuvant. The second group was immunized three times by gene gun with 10 ug plasmid DNA containing the PA63 gene fragment and/or the LF4 gene fragment bound to gold beads, at 4-week intervals. The third group of animals was immunized (s.c.) at 4-week intervals with AVA (lot FAV059), 800 ug rPA protein with Alum, or a mixture of 400 ug rPA and 400 ug rLF7 protein with Alum. Controls consisted of either non-immunized animals or a plasmid vector control not containing the PA and/or LF genes. All rabbits were aerosol challenged with B. anthracis spores, Ames strain, with an average dose of 50 LD50s with a range of 18-169 LD50s. Rabbit sera were collected prior to and following aerosol challenge and titrated for PA antibodies by indirect ELISA (Galloway et al., 2004).
  • Persistence: (Galloway et al., 2004)
  • Side Effects: None were noted (Galloway et al., 2004).
  • Efficacy: The results of this study indicate that DNA-based immunization against PA and LF followed by protein boosting induces significant protective immunity against aerosol challenge in the rabbit model and compares favorably with protein-based immunization (Galloway et al., 2004).
  • Description: None of the rabbits immunized with the DNA vaccines i.d. survived the challenge. Of the 5 vaccinated rabbits that survived, 2 were immunized i.m. with DNA followed with a protein boost and 3 were immunized subcutaneously (s.q.) with recombinant protein. DNA prime-boosted animals mount a protective response against aerosol challenge more than 1 year following the final immunization. Priming immunizations with plasmid DNA appear to set up a substantial memory response which is recalled upon protein boosting. A major factor predicting survival was the ability of the animal to mount a lasting antibody response to PA (Galloway et al., 2004).
References
Galloway et al., 2004: Galloway D, Liner A, Legutki J, Mateczun A, Barnewall R, Estep J. Genetic immunization against anthrax. Vaccine. 2004 Apr 16; 22(13-14); 1604-8. [PubMed: 15068841].
Price et al., 2001: Price BM, Liner AL, Park S, Leppla SH, Mateczun A, Galloway DR. Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein. Infection and immunity. 2001 Jul; 69(7); 4509-15. [PubMed: 11401993].