• Vaccine Name: Microencapsulated Caf1 and LcrV vaccine
• Target Pathogen: Yersinia pestis
• Target Disease: Plague
• Vaccine Ontology ID: VO_0000837
• Type: Subunit vaccine
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0001241
  • Description: Current killed whole cell vaccines have been shown to cause a number of transient side effects, require frequent boosting to maintain immunity, and their efficacy against pneumonic infection is questionable. Thus, a new vaccine for plague based on the protective protein sub-units capsular Fraction 1 (Caf1) and LcrV is under development. The Caf1 molecule is a temperature-regulated capsular protein of Y. pestis which is maximally expressed at 37 °C and which has a role in resistance of phagocytosis. The LcrV antigen of Y. pestis is a 37 kDa secreted protein that is known to have a role in modulation of host defence mechanisms by down-regulating production of IFN-γ and TNF-α and and up-regulation of the anti-inflammatory cytokine IL-10. It is also believed to act as a virulence factor with a key role in the Type III secretion system of Y. pestis. Immunisation with recombinant LcrV antigen adsorbed to the adjuvant alhydrogel has been shown to confer protection against virulent challenge by both the sub-cutaneous and airborne routes. The use of the rCaf1 and rLcrV subunits in combination has been shown to have an additive effect on protection in murine models of infection and the two sub-units when formulated with alhydrogel are being taken forward into clinical trials as a vaccine for human use (Elvin et al., 2006)
• Preparation: Recombinant Caf1 antigen was produced in Escherichia coli strain JM101 containing plasmid pAH34L, encoding the caf operon of Y. pestis strain GB. The rCaf1 was purified by ammonium sulfate fractionation followed by gel filtration chromatography. The LcrV antigen was expressed as a fusion protein with glutathione-S-transferase (GST) in E. coli using the plasmid pVG 110 for expression. The recombinant LcrV antigen was cleaved from the fusion protein with Factor Xa (Boehringer Mannheim UK Ltd.) and then purified by affinity chromatography. Microspheres were prepared using a modified solvent evaporation process. Freeze-dried rCaf1 or rLcrV (2 mg) was resuspended to form a PVA internal phase, which was then added to a polymer solution and sonicated on ice to form a water-in-oil primary emulsion. This was then added to PVA and homogenised to form a water-in-oil-in-water double emulsion. The microspheres were stirred overnight at room temperature to remove the solvent by the process of evaporation. Residual PVA and solvent were then removed by washing the microspheres. Briefly, the microspheres were centrifuged to form a pellet. The supernatant was removed and the pellet re-suspended in water twice. The final pellet was re-suspended in 2 ml water and freeze-dried. Microencapsulated rCaf1 was mixed with microencapsulated rLcrV and free antigen in solution was added to give a range of doses from 25 to 100 μg of each sub-unit (Elvin et al., 2006).
• Virulence:
• Description: Current killed whole cell vaccines have been shown to cause a number of transient side effects, require frequent boosting to maintain immunity, and their efficacy against pneumonic infection is questionable. Thus, a new vaccine for plague based on the protective protein sub-units capsular Fraction 1 (Caf1) and LcrV is under development. The Caf1 molecule is a temperature-regulated capsular protein of Y. pestis which is maximally expressed at 37 °C and which has a role in resistance of phagocytosis. The LcrV antigen of Y. pestis is a 37 kDa secreted protein that is known to have a role in modulation of host defence mechanisms by down-regulating production of IFN-γ and TNF-α and and up-regulation of the anti-inflammatory cytokine IL-10. It is also believed to act as a virulence factor with a key role in the Type III secretion system of Y. pestis. Immunisation with recombinant LcrV antigen adsorbed to the adjuvant alhydrogel has been shown to confer protection against virulent challenge by both the sub-cutaneous and airborne routes. The use of the rCaf1 and rLcrV subunits in combination has been shown to have an additive effect on protection in murine models of infection and the two sub-units when formulated with alhydrogel are being taken forward into clinical trials as a vaccine for human use (Elvin et al., 2006)

Mouse Response

• Host Strain: Female 5-6-week-old BALB/c mice (Charles River UK).
• Vaccination Protocol: Microencapsulated rCaf1 (0.5 mg spheres, 7 μg) was mixed with microencapsulated rLcrV (0.5 mg spheres, 14 μg) and free antigen in solution was added to give a range of doses from 25 to 100 μg of each sub-unit. A group of mice was immunised with microencapsulated rCaf1 + rLcrV only (7 and 14 μg, repectively). For intra-nasal immunisation, animals were lightly anaesthetised with Halothane mixed with oxygen. The microencapsulated and free antigens were applied to the nostrils in a volume of 50 μl PBS by pipette (Elvin et al., 2006).
• Persistence: There was the presence of antibody to both vaccine antigens at days 45 and 60 p.i. in animals dosed by both the i.n. and i.m. routes (Elvin et al., 2006).
• Immune Response: High serum IgG levels as well as transudated IgG in the lungs of vaccinated animals were observed. Cytokine and proliferative T-cell responses were also found in the spleen and draining lymph nodes following either i.m. or i.n. immunisation, indicating that either immunisation route can induce strong immune responses (Elvin et al., 2006).
• Side Effects: No side effects noted.
• Challenge Protocol: Animals were challenged by the sub-cutaneous route with either 10^5 or 10^7 MLD of Y. pestis GB. For aerosol challenge, animals received a dose of 10^4 MLD Y. pestis (Elvin et al., 2006).
• Efficacy: Protection against both bubonic and pneumonic forms of the disease following a single i.m. or i.n. administration of microencapsulated rCaf1 + rLcrV was demonstrated. The best level of protection was provided by 100 μg of each sub-unit and so this dose level was taken forward to studies culminating in aerosol challenge. In animals immunised by the intra-muscular route, there were no deaths when challenged at day 45 or day 60 p.i. In the intra-nasally immunised animals, however, there was 50% mortality when challenged at day 45 p.i. Yet, when intranasally immunised animals were challenged by the airborne route at day 60, there was 100% protection against challenge. None of these animals showed any signs of illness at any point during the 14-day observation period following challenge (Elvin et al., 2006).