• Vaccine Ontology ID: VO_0004417
• Type: DNA vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Rabbit
• Lef gene engineering:
  • Type: DNA vaccine construction
  • Description: This DNA vaccine expressed lethal factor (LF) (Hermanson et al., 2004).
  • Detailed Gene Information: Click Here.
• Vector: VR1012 (Hermanson et al., 2004)
• Immunization Route: Intramuscular injection (i.m.)

• Vaccine Ontology ID: VO_0004386
• Type: DNA vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Rabbit, guinea pig, mouse
• pagA gene engineering:
  • Type: DNA vaccine construction
  • Description: Vector pWRG7079 expressed protective (PA) gene of B. anthracis (Riemenschneider et al., 2003).
  • Detailed Gene Information: Click Here.
• Vector: pWRG7079 (Riemenschneider et al., 2003)
• Immunization Route: Gene gun

• Vaccine Ontology ID: VO_0004481
• Type: DNA vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Rabbit
• PA83 gene engineering:
  • Type: DNA vaccine construction
  • Description: Vector VR1012 expressed the PA construct that was chemically synthesized (Retrogen, San Diego) to include an amino terminal human tissue plasminogen activator (hTPA) leader peptide (replacing the Bacillus leader peptide) fused to a PA83 sequence (Hermanson et al., 2004).
  • Detailed Gene Information: Click Here.
• Vector: VR1012 (Hermanson et al., 2004)
• Immunization Route: Intramuscular injection (i.m.)

• Vaccine Ontology ID: VO_0004416
• Type: DNA vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Rabbit
• PA83 gene engineering:
  • Type: DNA vaccine construction
  • Description: This DNA vaccine expressed the protective antigen (PA) (Hermanson et al., 2004).
  • Detailed Gene Information: Click Here.
• Vector: VR1012 (Hermanson et al., 2004)
• Immunization Route: Intramuscular injection (i.m.)

• Vaccine Ontology ID: VO_0004479
• Type: DNA vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Rabbit
• pagA gene engineering:
  • Type: DNA vaccine construction
  • Description: Vector pVAX1 expressed the mature 83 kDa full-length PA protein (without the 29 aminoacid prokaryotic secretory signal sequence) (Luxembourg et al., 2008).
  • Detailed Gene Information: Click Here.
• Vector: pVAX1 (Luxembourg et al., 2008)
• Immunization Route: Intramuscular injection (i.m.)

• Vaccine Ontology ID: VO_0000521
• Type: DNA vaccine
• Antigen: B. anthracis PA and LF (Hermanson et al., 2004)
• PagA from B. anthracis str. 'Ames Ancestor' gene engineering:
  • Type: DNA vaccine construction
  • Description: The PA construct is chemically synthesized to include an amino terminal human tissue plasminogen activator (hTPA) leader peptide fused to a PA83 sequence (amino acids 30–764) with the furin cleavage site deleted (SRKKRS, amino acids 192–197). This construct, designated PA83 furin, is cloned into the mammalian expression vector VR1012 (Hermanson et al., 2004).
  • Detailed Gene Information: Click Here.
• Lef from B. anthracis str. A2012 gene engineering:
  • Type: Protein
  • Description: contains Bacillus anthracis lethal factor x-ray crystal structure derived domain I; LF; vaccine construct
  • Detailed Gene Information: Click Here.
• Vector: VR1012 (Hermanson et al., 2004)
• Preparation: The PA construct is chemically synthesized to include an amino terminal human tissue plasminogen activator (hTPA) leader peptide fused to a PA83 sequence (amino acids 30–764) with the furin cleavage site deleted (SRKKRS, amino acids 192–197). This construct, designated PA83 furin, is cloned into the mammalian expression vector VR1012. The LF coding sequences are derived from the B. anthracis LF93 protein sequence, codon-optimized, and chemically synthesized as above to include the hTPA leader peptide. The LF domain I–III is PCR amplified from this clone by using a forward and reverse primer pair to amplify the 1,740-bp fragment encoding the hTPA leader peptide fused to LF amino acids 34–583. The LF domain I is also derived from the LF93 plasmid by PCR amplification using forward and reverse primer pairs to amplify an 876-bp fragment encoding an hTPA leader peptide fused to LF amino acids 34–295. Both LF genes are cloned into the VR1012 vector (Hermanson et al., 2004).
• Virulence: The virulence of B. anthracis in rabbits, non-human primates, and humans is primarily the result of a multicomponent toxin secreted by the organism. The toxin consists of three separate gene products, designated protective antigen (PA), lethal factor (LF), and edema factor (EF), that are encoded on a 184-kb plasmid designated pXO1 (Hermanson et al., 2004).
• Description: DNA vaccines provide an attractive technology platform against bioterrorism agents due to their safety record in humans and ease of construction, testing, and manufacture. Monovalent and bivalent anthrax plasmid DNA (pDNA) vaccines encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins have been designed and tested for their immunogenicity and ability to protect rabbits from an aerosolized inhalation spore challenge. Immune responses after two or three injections of cationic lipid-formulated PA, PA + LF, or LF pDNAs were at least equivalent to two doses of anthrax vaccine adsorbed (AVA) (Hermanson et al., 2004).

• Vaccine Ontology ID: VO_0000880
• Type: DNA vaccine
• Antigen: B. anthracis lethal factor (LF)
• Lef gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• PagA from B. anthracis str. 'Ames Ancestor' gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• Vector: pCl (Price et al., 2001)
• 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).

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

• Vaccine Immune Response Type: VO_0000286
• Immune Response: High titers of anti-LF and neutralizing antibody to lethal toxin (Letx) were achieved in all rabbits (Hermanson et al., 2004).
• Efficacy: Eight or nine animals in each group were challenged with 100x LD(50) of aerosolized anthrax spores 5 or 9 weeks after vaccination and 5 of 9 animals receiving LF pDNA survived. In addition, the time to death was significantly delayed in the others (Hermanson et al., 2004).

Rabbit Response

• Vaccine Immune Response Type: VO_0000286
• Immune Response: All rabbits vaccinated with the DNA vaccine or with AVA vaccine developed antibody responses predictive of protective immunity to anthrax challenge (Riemenschneider et al., 2003).
• Efficacy: Evaluating antibody responses by ELISA and TNA revealed that all rabbits vaccinated with the DNA vaccine developed antibody responses predictive of protective immunity to anthrax challenge. After a fourth vaccination, titers rebounded and the rabbits were challenged by subcutaneous injection of 100 LD50 of B. anthracis Ames strain heat-shocked spores - 9/10 rabbits given the PA DNA vaccine survived (Riemenschneider et al., 2003).

Rabbit Response

• Vaccine Immune Response Type: VO_0000286
• Efficacy: All animals receiving PA or PA plus LF pDNA vaccines were protected (Hermanson et al., 2004).

Rabbit Response

• Vaccine Immune Response Type: VO_0000286
• Immune Response: High titers of anti-PA and neutralizing antibody to lethal toxin (Letx) were achieved in all rabbits (Hermanson et al., 2004).
• Efficacy: Eight or nine animals in each group were challenged with 100x LD(50) of aerosolized anthrax spores 5 or 9 weeks after vaccination. An additional 10 animals vaccinated with PA pDNA were challenged >7 months postvaccination. All animals receiving PA pDNA vaccines were protected (Hermanson et al., 2004).

Rabbit Response

• Vaccine Immune Response Type: VO_0000286
• Efficacy: Anthrax toxin neutralizing antibodies were also induced in rabbits immunized with electroporation with ED50 values comparable to those previously found to be protective in rabbits immunized with rPA (Luxembourg et al., 2008).

Rabbit Response

• Host Strain: New Zealand White
• Vaccination Protocol: Plasmid DNA was prepared from overnight cultures of transformed XL-2 Blue bacteria in Terrific Broth plus 50 µg/ml kanamycin sulfate and processed by using Endo-free Giga kits. One milliliter of sterile water for irrigation (SWFI) was added to a vial containing a dried film of 3.75 µmol each of a 1:1 mixture of cationic lipid and colipid and vortex mixed for 5 minutes. The liposome suspension was diluted to 1.5 mM with SWFI and added to an equal volume of pDNA and vortex mixed briefly. The final molar ratio of all formulations was 4:1, DNA/cationic lipid (Hermanson et al., 2004). Two- to five-kilogram female New Zealand White rabbits were injected bilaterally in the quadriceps muscles with 1 ml of pDNA formulated with Vaxfectin or DMRIE/DOPE (0.5 ml per leg). Rabbits vaccinated with PA, LF, or vector received 1 mg of that pDNA whereas rabbits co-injected with PA + LF pDNAs received a mixture of 0.5 mg of each plasmid. Groups of rabbits receiving three doses were injected on days 0, 28, and 56; rabbits receiving only two doses were injected on study days 0 and 28. Rabbits immunized with AVA were injected unilaterally with 50 µl of AVA diluted to 0.5 ml in PBS on days 28 and 56. Prebleeds and biweekly postvaccination bleeds were taken for all groups for analysis of serum antibodies (Hermanson et al., 2004).
• Persistence: Spore challenge induced a significant increase in the Letx neutralization titer in group 4 rabbits, suggesting that there was limited spore germination after challenge in these animals. This post-challenge increase in Letx neutralization titer, however, was smaller than the increase seen in AVA- or LF pDNA-vaccinated rabbits challenged at week 12 (Hermanson et al., 2004).
• Immune Response: Both the PA and the LF pDNAs generate anti-PA and anti-LF antibody responses, respectively, when injected alone or co-injected. Furthermore, co-injection of PA and LF pDNAs does not cause detectable interference in the immunogenicity of either of the pDNAs (Hermanson et al., 2004). Immune responses after two or three injections of cationic lipid-formulated PA, PA + LF, or LF pDNAs were at least equivalent to two doses of anthrax vaccine adsorbed (AVA). High titers of anti-PA, anti-LF, and neutralizing antibody to lethal toxin were achieved in all rabbits.
• Side Effects: none reported (Hermanson et al., 2004)
• Challenge Protocol: Eight or nine animals in each group were challenged with 100x LD50 of aerosolized anthrax spores 5 or 9 weeks after vaccination. An additional 10 animals vaccinated with PA pDNA were challenged over 7 months post-vaccination.
• Efficacy: All animals receiving PA or PA + LF pDNA vaccines were protected. In addition, 5/9 animals receiving LF pDNA survived, and the time to death was significantly delayed in the others. Groups receiving three immunizations with PA or PA + LF pDNA showed no increase in anti-PA, anti-LF, or Letx neutralizing antibody titers post-challenge, suggesting little or no spore germination (Hermanson et al., 2004).

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).