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Vaccine Detail

Recombinant Y. pestis V antigen vaccine
Vaccine Information
  • Vaccine Name: Recombinant Y. pestis V antigen vaccine
  • Target Pathogen: Yersinia pestis
  • Target Disease: Plague
  • Vaccine Ontology ID: VO_0000831
  • Type: Subunit vaccine
  • LcrV from Y. pestis biovar Microtus str. 91001 gene engineering:
    • Type: Protein
    • Description:
    • Detailed Gene Information: Click Here.
  • LcrV from Y. pestis KIM 10 gene engineering:
    • Type: Protein
    • Description: Protein coding
    • Detailed Gene Information: Click Here.
  • LcrV from y. pestis CO92 gene engineering:
    • Type: Protein
    • Description: Protein coding; V antigen; low calcium response protein V; functions in needle complex protein export; Yop secretion and targeting control protein; important for translocation pore formation; induces IL-10 production by macrophages; interacts with Toll-like receptor 2.
    • Detailed Gene Information: Click Here.
  • Adjuvant: incomplete Freunds adjuvant
  • Preparation: The gene encoding V antigen (lcrV) was amplified from Y. pestis DNA by PCR. The fragment was purified, digested with EcoRI and SalI, ligated with suitably digested plasmid pGEX-5X-2, and transformed into E. coli JM109 by electroporation. One-milliliter aliquots were removed from the cultures in the logarithmic and stationary phases, and the number of viable cells was determined by inoculating the aliquots onto L agar containing 100 mg of ampicillin/ml. The cells were harvested from a second 1-ml aliquot by centrifugation and resuspended in 1 ml of phosphate-buffered saline (PBS). The cell suspension was frozen at 2208C for 1 h, thawed, and then sonicated on ice at 10% power three times for 30 s each. The sonicates and a standard solution of rV (5 mg/ml) were serially diluted in PBS in a microtiter plate and allowed to bind overnight. The GST-V fusion protein was eluted with 10 ml of 50 mM Tris containing 5 mM reduced glutathione. After dialysis against PBS, the fusion protein was cleaved with factor Xa at an enzyme/fusion protein ratio of approximately 1:200 by weight. Cleaved GST and excess uncleaved GST-V (but not factor Xa) were removed from the solution by affinity adsorption to leave purified rV (Leary et al., 1995). Other recombinant V antigen (rV) expression systems, besides the N-terminal GST fusion pGEX-5X-2, include the pGEX-6P-2 systems from Pharmacia Biotech and the C-terminal CBD fusion (IMPACT I) system from New England Biolabs (Carr et al., 1999).
  • Virulence:
Host Response

Mouse Response

  • Host Strain: Six-week-old female BALB/c mice (Charles River Laboratories, Margate, Kent, United Kingdom)
  • Vaccination Protocol: A group of 16 mice received a 0.2-ml primary immunizing dose of 10 mg of rV, presented in a 1:1 water-in-oil emulsion with incomplete Freund’s adjuvant. On days 14 and 34, each animal received booster doses. On day 64, six animals were sacrificed, and their tissues were removed for
    immunological analyses. The remaining animals were challenged with Y. pestis. An untreated control group of 16 age-matched mice was
    divided similarly into groups for tissue sampling and challenge. Groups of 5 to 10 mice from the immunized and control groups were challenged subcutaneously with 0.1-ml aliquots of Y. pestis GB at various cell densities. The mice were observed for 14 days (Leary et al., 1995).
  • Persistence: All of the rV-immunized animals survived the challenge, although the controls succumbed with a mean time to death of 119 +/- 4.9 h (Leary et al., 1995).
  • Side Effects: No side effects noted.
  • Efficacy: When used to inoculate mice, purified rV elicited solid protective immunity against a subcutaneous challenge with up to 3.74 3 106 CFU of Y. pestis GB (Leary et al., 1995).
  • Description: The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase–V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Immunogenicity of recombinant V antigen was then tested in vivo. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague (Leary et al., 1995).
References
Carr et al., 1999: Carr S, Miller J, Leary SE, Bennett AM, Ho A, Williamson ED. Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems. Vaccine. 1999 Aug 20; 18(1-2); 153-9. [PubMed: 10501245].
Leary et al., 1995: Leary SE, Williamson ED, Griffin KF, Russell P, Eley SM, Titball RW. Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague. Infection and immunity. 1995 Aug; 63(8); 2854-8. [PubMed: 7622205].