• Vaccine Ontology ID: VO_0000827
• Type: Recombinant vector vaccine
• V antigen gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• Vector: Adenovirus (Ad) gene-transfer vector (Chiuchiolo et al., 2006).
• Preparation: The Y. pestis V antigen gene with mammalian-preferred codons was synthesized by overlap polymerase chain reaction and fused to the human Ig signal sequence for extracellular secretion. The V antigen gene was cloned into a recombinant Ad5–based vector (E1a, partial E1b, and partial E3 deletion), to generate AdsecV. AdNull was used as a control vector with identical backbone but no transgene. The vectors were produced in 293 cells and were purified by double CsCl gradient centrifugation. Dosing was based on particle units (pu), the physical number of Ad particles as measured by spectrophotometry. V antigen was purified as a histidine-tag fusion by use of a Ni-NTA Superflow Column (Qiagen) under native conditions (Chiuchiolo et al., 2006).
• Virulence:
• Description: Ad vectors are excellent candidates for vaccine platforms as they transfer genes effectively to antigen-presenting cells (APCs) in vivo, with consequent activation of APCs, thus conveying immune adjuvant properties and inducing strong, rapid humoral and cellular immune responses against the transgene product (Chiuchiolo et al., 2006).

• Vaccine Ontology ID: VO_0000837
• Type: Subunit vaccine
• Adjuvant:
  • VO 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)

• Vaccine Ontology ID: VO_0000832
• Type: Live, attenuated vaccine
• Preparation: Virulent Y. pestis was modified to produce a potent TLR4-stimulating LPS (Montminy et al., 2006).
• Virulence:

• Tradename: Plague vaccine USP
• Manufacturer: Cutter Biological Inc
• Vaccine Ontology ID: VO_0000847
• Type: Inactivated or "killed" vaccine
• Status: Licensed
• Preparation: USP consists of an aqueous suspension of virulent P.pestis grown on agar medium in Roux bottles. The organisms are harvested in physiological saline and killed by adding formol to an overall concentration of 0.5 %. The standardized suspension is supplied in 20 ml vaccine vials, each ml containing 2*10^9 bacilli (Meyer, 1970).
• Virulence: Side effects, such as malaise, headaches elevated temperature and lymphadenopathy occur in approximately 10% of those immunised with killed whole cells vaccines (Titball et al., 2001).
• Description: The earliest report of a killed whole cell vaccine against plague was in 1897, developed by HalIkine in India. The vaccine caused severe side-effects and was discontinued, although the HalTkine Institute now produces a formaldehyde-killed preparation of strain 195/P (Russell et al., 1995). It was not until 1946 that a killed whole cells vaccine was developed for use in man. Various methods of killing the bacterial cells have been used, including formaldehyde and heat treatment. The vaccine is currently produced by the Commonwealth Serum Laboratories in Australia and is usually given as a course of three doses over a period of two months(Titball et al., 2001). The vaccine currently used in the USA and in the UK is the Plague vaccine, USP (Cutter Biological), a formaldehyde-killed preparation of the highly virulent 195/P strain of Y pestis (Russell et al., 1995).There are no definitive clinical trials which demonstrate the efficacy of killed whole cell vaccines. However, studies in several animal species have demonstrated protection against bubonic plague. Also there is circumstantial evidence for the efficacy of the vaccine in humans derived from data on the incidence of bubonic plague in immunised US servicemen serving in Vietnam during the period 1961–1971. It is possible that differences in the lifestyles of servicemen and Vietnamese civilians were responsible for the reduced incidence of plague in the former group. Evidence for the efficacy of killed whole cells vaccines for the prevention of pneumonic plague is less conclusive. Cases of pneumonic plague have been reported in individuals immunised with this vaccine. More recently it has been shown that mice immunised with this vaccine are protected against subcutaneous challenge, but not against inhalation challenge with Y. pestis. Together, these findings suggest that killed whole cell vaccines do not induce a response which provides protection against pneumonic plague (Titball et al., 2001). Additional problems of the vaccine include the following: production requires special containment; the vaccine is expensive; protection is short-term and annual boosters are required; the incidence of side-effects is high, local reactions occur in 11-24% of vaccinees and systemic effects occur in 4-10% of vaccinees (Russell et al., 1995).

• Vaccine Ontology ID: VO_0004159
• Type: Recombinant vector vaccine
• Status: Research
• Psn gene engineering:
  • Type: Recombinant vector construction
  • Detailed Gene Information: Click Here.
• Vector: Live attenuated Salmonella (Branger et al., 2007).
• Immunization Route: Orally

• Vaccine Ontology ID: VO_0004694
• Type: Recombinant vector vaccine
• Status: Research
• Host Species for Licensed Use: Baboon
• Preparation: Translation enhancer (EMCV-IRES) in combination with a secretory (tPA) signal or secretory (tPA) and membrane anchoring (CHV-gG) signals on in vitro antigen expression of F1 antigen (Osorio et al., 2003).
• Immunization Route: Intramuscular injection (i.m.)

• Vaccine Ontology ID: VO_0000831
• Type: Subunit vaccine
• LcrV from Y. pestis biovar Microtus str. 91001 gene engineering:
  • Type: Protein
  • 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:
  • VO ID: VO_0000142
• 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:

• Vaccine Ontology ID: VO_0000834
• Type: Subunit vaccine
• LcrV from Y. pestis CO92 gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• YopD from Y. pestis CO92 gene engineering:
  • Type: Protein
  • Description: Yop targeting negative regulator; translocon component; important for translocation pore formation.
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0001238
  • Description: R-730 emulsion (Ribi ImmunoChem Research, Inc., Hamilton, Mont.)(Andrews et al., 1999)
• Preparation: The yopD loci from Yersinia pestis was amplified by PCR, cloned, and expressed in Escherichia coli. The purified protein was mixed with an equal volume of the adjuvant to give a final protein concentration of 100 µg/ml (Andrews et al., 1999).
• Virulence:
• Description: Yersinia outer proteins (Yops) are virulence determinants synthesized by the Yersinia species pathogenic for humans, including Y. pestis, the causative agent of plague. The Yop proteins are encoded on a 75-kb plasmid, and in vitro expression from these genes, as well as subsequent secretion and translocation by a Type III secretion system, are regulated by temperature, calcium, and eukaryotic cell contact. There are various functions known for some of the Yops, including translocation and sensor functions in Yop B/D and Yop N. Previous studies showed that antibodies to some Yops are present in convalescent-phase serum from patients infected with Y. pestis, as well as in rodent serum after experimental Y. pestis infection, which suggests that Yops are antigenic. Vaccination with Yop-containing culture supernatants from growth-restricted Yersinia enterocolitica protected mice from a lethal intraperitoneal (i.p.) dose of virulent Y. pestis; however, interpretation is complicated by the likely presence of V antigen in the crude supernatants, as V is known to be a protective antigen (Andrews et al., 1999).

• Vaccine Ontology ID: VO_0000840
• Type: Subunit vaccine
• Adjuvant:
  • VO ID: VO_0001241
  • Description: Immunization with purified recombinant LcrV (rLcrV) is sufficient to generate protective immunity to both bubonic plague and pneumonic plague in mice, guinea pigs, and non-human primates. LcrV injection of animals triggered release of interleukin-10, a cytokine that suppresses innate immune functions. LcrV also prevents the release of proinflammatory cytokines (gamma interferon and tumor necrosis factor ) in murine and human macrophages. Considering the immune modulatory properties of rLcrV, there are concerns regarding the safety of LcrV vaccines in humans. Thus, there is an emphasis upon searching for variants with reduced immune modulatory properties. rV10, a variant lacking amino acids 271 to 300 of LcrV, displayed a significant decrease in its ability to induce interleukin-10 and to suppress tumor necrosis factor or gamma interferon release (DeBord et al., 2006).
• Preparation: Escherichia coli BL21(DE3) carrying prV10 was grown overnight at 37 °C in Luria-Bertani medium (Difco) with 100 µg/ml ampicillin. Bacteria were diluted in fresh medium and grown to an optical density at 600 nm of 0.8 to 1.0. T7 polymerase expression was induced with 1 mM isopropyl-ß-D-thiogalactopyranoside, and bacterial growth was continued for 3 hours at 37°C. Cells were harvested by centrifugation at 10,000 x g for 10 min. Bacterial sediment was suspended in 20 ml of Tris-HCl (pH 7.5)-150 mM NaCl (column buffer) containing 100 µM phenylmethylsulfonyl fluoride, and cells were disrupted by two passages through a French pressure cell at 14,000 lb/in2. The lysate was subjected to ultracentrifugation at 40,000 x g for 30 min, and the soluble fraction was applied to a nickel nitrilotriacetic acid column (1-ml bed volume) preequilibrated with 10 ml of column buffer. The column was washed with 10 ml of the same buffer, followed by a second (10 ml of column buffer with 10% glycerol) and a third (10 ml of column buffer with 10% glycerol and 20 mM imidazole) washing. Bound protein was eluted in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 10% glycerol containing 250 mM imidazole. Purified proteins were subjected to three sequential Triton X-114 (Sigma) phase separations to remove endotoxins. Purified proteins were applied to a G-25 (Amersham) gel filtration column to remove residual Triton X-114 and then retrieved by phosphate-buffered saline elution. Lipopolysaccharide contamination of purified proteins was assayed with Limulus amebocyte lysate (QCL-1000; Cambrex, New Jersey) and determined to be less than 1 ng/100 µg of purified protein. Protein concentrations were determined by the bicinchoninic acid assay (Pierce Technology, Rockford, IL). Proteins were aliquoted at 1 mg/ml and stored at –80 °C for further use. Purified recombinant rV10 vaccine antigens were emulsified with Alhydrogel (DeBord et al., 2006).
• Virulence:
• Description: Immunization with purified recombinant LcrV (rLcrV) is sufficient to generate protective immunity to both bubonic plague and pneumonic plague in mice, guinea pigs, and non-human primates. LcrV injection of animals triggered release of interleukin-10, a cytokine that suppresses innate immune functions. LcrV also prevents the release of proinflammatory cytokines (gamma interferon and tumor necrosis factor ) in murine and human macrophages. Considering the immune modulatory properties of rLcrV, there are concerns regarding the safety of LcrV vaccines in humans. Thus, there is an emphasis upon searching for variants with reduced immune modulatory properties. rV10, a variant lacking amino acids 271 to 300 of LcrV, displayed a significant decrease in its ability to induce interleukin-10 and to suppress tumor necrosis factor or gamma interferon release (DeBord et al., 2006).

• Vaccine Ontology ID: VO_0000830
• Type: Subunit vaccine
• Adjuvant:
  • VO ID: VO_0001241
  • Description: This vaccine utilizes a combination of the Fraction 1 antigen (F1) and the V antigen of Y. pestis in an optimum molar ratio. The F1 and V antigens, in recombinant form in this vaccine, are natural virulence factors of Y. pestis. Fraction 1 antigen, the major protein component of the capsule surrounding Y. pestis cells, is expressed only at 37 °C and it is believed to have anti-phagocytic activity. High anti-F1 titres have been correlated with survival following plague infection. Recombinant F1 antigen (rF1) has been produced by cloning the caf operon from Y. pestis into Escherichia coli and the protection provided by highly purified native F1 and recombinant F1 against Y. pestis has been demonstrated not to differ. The V antigen of Y. pestis is a secreted protein that is thought to act both as a regulatory protein and as a virulence factor. The V antigen has a key role in the Type III secretion process utilised by Y. pestis to translocate cytotoxic and anti-phagocytic Yersinia outer proteins (Yops) into the host cell. Supporting evidence for this role has been gained and the V antigen has been visualised on the bacterial cell surface (Jones et al., 2003).
    Both the rF1 and rV proteins, administered in alhydrogel, have been demonstrated to be highly immunogenic and protective against virulent plague in a number of animal models: mice, guinea pigs, and cynomolgus macaques (unpublished data). Further, the combination of rF1 plus rV is additive in the protection conferred on the vaccinee. In the mouse, the combined immunoglobulin G1 (IgG1) titer to rF1 plus rV has been shown to correlate with protection against challenge. Further, protection against plague in the mouse has been demonstrated by the passive transfer of antiserum specific for rF1 plus rV from immunized BALB/c mice into naïve SCID/beige mice (Williamson et al., 2005).
• Preparation: The rF1 and rV antigens were produced in Escherichia coli from the expression systems previously described, under Good Manufacturing Practice conditions. The vaccine was formulated by adsorption to 20% (vol/vol) adjuvant at the required concentrations of each protein such that concentrations in the range 10 µg rF1 + 10 µg rV per ml up to 80 µg rF1 + 80 µg rV per ml were achieved in a final concentration of 0.26% (wt/vol) alhydrogel to achieve a molar ratio for rF1 to rV of 2:1. The formulated vaccine was designated rYP002 (Williamson et al., 2005).
• Virulence:
• Description: This vaccine utilizes a combination of the Fraction 1 antigen (F1) and the V antigen of Y. pestis in an optimum molar ratio. The F1 and V antigens, in recombinant form in this vaccine, are natural virulence factors of Y. pestis. Fraction 1 antigen, the major protein component of the capsule surrounding Y. pestis cells, is expressed only at 37 °C and it is believed to have anti-phagocytic activity. High anti-F1 titres have been correlated with survival following plague infection. Recombinant F1 antigen (rF1) has been produced by cloning the caf operon from Y. pestis into Escherichia coli and the protection provided by highly purified native F1 and recombinant F1 against Y. pestis has been demonstrated not to differ. The V antigen of Y. pestis is a secreted protein that is thought to act both as a regulatory protein and as a virulence factor. The V antigen has a key role in the Type III secretion process utilised by Y. pestis to translocate cytotoxic and anti-phagocytic Yersinia outer proteins (Yops) into the host cell. Supporting evidence for this role has been gained and the V antigen has been visualised on the bacterial cell surface (Jones et al., 2003).
  Both the rF1 and rV proteins, administered in alhydrogel, have been demonstrated to be highly immunogenic and protective against virulent plague in a number of animal models: mice, guinea pigs, and cynomolgus macaques (unpublished data). Further, the combination of rF1 plus rV is additive in the protection conferred on the vaccinee. In the mouse, the combined immunoglobulin G1 (IgG1) titer to rF1 plus rV has been shown to correlate with protection against challenge. Further, protection against plague in the mouse has been demonstrated by the passive transfer of antiserum specific for rF1 plus rV from immunized BALB/c mice into naïve SCID/beige mice (Williamson et al., 2005).

• Vaccine Ontology ID: VO_0000845
• Type: Recombinant vector vaccine
• LcrV from Y. pestis CO92 gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• Preparation: Recombinant plasmids for recovery of VSV recombinants expressing the low calcium response protein V (LcrV) of Y. pestis were using PCR amplification of the lcrV gene. The PCR product was digested, purified and ligated to XhoI–NheI digested pVSV1XN or pVSVXN2 to generate pVSV-LcrV1 and pVSV-LcrV5, respectively. The lcrV gene was also cloned at the fifth position of the glycoprotein exchange vector pVSV(GNJ)XN-1, where the ORF of the New Jersey serotype G replaced the G of the parent Indiana serotype genome, to generate pVSV(GNJ)-LcrV5. Recombinant VSVs expressing LcrV were recovered from these plasmids (Palin et al., 2007).
• Virulence:
• Description: Recombinant vesicular stomatitis virus (VSV)-based vector is potentially a candidate plague vaccine. VSV is a negative-strand RNA virus encoding five structural proteins: nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G) and RNA dependent RNA polymerase (L). It is a natural pathogen of livestock; human infection is rare and usually asymptomatic. VSV recombinants expressing foreign genes can be generated from plasmid DNA. VSV can accommodate insertion of large foreign genes whose expression can be controlled based on the site of gene insertion in the VSV genome. VSV induces potent humoral and cellular immune responses in a variety of animal models. VSV naturally infects mucosal surfaces to elicit strong systemic immunity and possible local mucosal immunity. The extremely low VSV seropositivity in the general population is also an added advantage. Previous studies have shown that recombinant VSV-based vectors expressing appropriate foreign antigens are highly effective vaccines that protect against challenges with numerous viral pathogens (Palin et al., 2007).

• Vaccine Ontology ID: VO_0004158
• Type: DNA vaccine
• Status: Research
• Antigen: F1 devoid of its putative signal peptide (deF1) (Grosfeld et al., 2003).
• caf1 gene engineering:
  • Type: DNA vaccine construction
  • Detailed Gene Information: Click Here.
• Vector: pCI
• Immunization Route: Gene Gun

• Vaccine Ontology ID: VO_0004314
• Type: DNA vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• Caf1 from Y. pestis biovar Microtus str. 91001 gene engineering:
  • Type: DNA vaccine construction
  • Description: Vector pGT146-mIL-12 expressed the proplague epitope capsular antigen (F1-Ag) (Yamanaka et al., 2008).
  • Detailed Gene Information: Click Here.
• LcrV from Y. pestis biovar Microtus str. 91001 gene engineering:
  • Type: DNA vaccine construction
  • Description: Vector pGT146-mIL-12 expressed the proplague epitope virulence antigen (V-Ag) (Yamanaka et al., 2008).
  • Detailed Gene Information: Click Here.
• Vector: pBudCE4.1 (Yamanaka et al., 2008)
• Immunization Route: intranasal immunization

• Vaccine Ontology ID: VO_0004573
• Type: DNA vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• YscF from Y. pestis KIM 10 gene engineering:
  • Type: DNA vaccine construction
  • Description: Vector pJW4303 had two copies of yscF gene in tandem (Wang et al., 2008).
  • Detailed Gene Information: Click Here.
• Vector: pJW4303 (Wang et al., 2008)
• Immunization Route: Gene gun

• Vaccine Ontology ID: VO_0004252
• Type: Subunit vaccine
• Status: Research
• Antigen: Purified F1 antigen (Honko et al., 2006).
• YPMT1.84 (F1 capsule antigen) gene engineering:
  • Type: Recombinant protein preparation
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0001331
• Immunization Route: intranasal immunization

• Vaccine Ontology ID: VO_0000829
• Type: Subunit vaccine
• Caf1 from Y. pestis biovar Microtus str. 91001 gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0001241
  • Description: Yersinia pestis fraction 1 capsular antigen (F1) is a plasmid (pFra)-encoded, proteinaceous capsule, synthesized in large quantities by the pathogen, and reported to confer antiphagocytic properties on Y. pestis by interfering with complementmediated opsonization. The protein is highly immunogenic and has been indirectly associated with eliciting a protective immune response in humans, as evidenced by the detection of high levels of anti-F1 antibody in F1-immunized volunteers (Andrews et al., 1996).
• Preparation: Partially pure cell-associated (capsular) F1 was extracted and isolated. HIB containing xylose was inoculated with a loop of Y. pestis CO92 Pgm2 Lcr2 from a plate stock and grown. The bacteria were then harvested by centrifugation, and the supernatant was retained for isolation of cell-free F1. The cell pellets were next resuspended and recentrifuged before the supernatants were pooled. Crude cell-associated capsular F1 from the salt extract supernatants was precipitated with ammonium sulfate. Protein that precipitated was collected by centrifugation. Purified, detoxified Y. pestis and E. coli supernatant F1 and cell-extracted F1 were adsorbed to the adjuvant (Andrews et al., 1996).
  
• Virulence:
• Description: Yersinia pestis fraction 1 capsular antigen (F1) is a plasmid (pFra)-encoded, proteinaceous capsule, synthesized in large quantities by the pathogen, and reported to confer antiphagocytic properties on Y. pestis by interfering with complementmediated opsonization. The protein is highly immunogenic and has been indirectly associated with eliciting a protective immune response in humans, as evidenced by the detection of high levels of anti-F1 antibody in F1-immunized volunteers (Andrews et al., 1996).
  

• Vaccine Ontology ID: VO_0004157
• Type: Subunit vaccine
• Status: Research
• YscF from Y. pestis CO92 gene engineering:
  • Type: Recombinant protein preparation
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0001238
  • Description: Ribi adjuvant system (RAS) R-730 monophosphoryl lipid-A (Corixa, Hamilton, MT) (Swietnicki et al., 2005)
• Immunization Route: Subcutaneous Injection

• Vaccine Ontology ID: VO_0000836
• Type: Subunit vaccine
• YscF from Y. pestis CO92 gene engineering:
  • Type: Protein
  • Description: YscF expressed and purified from E. coli was highly alpha-helical and formed relatively stable aggregates under physiological conditions.
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000139
• Preparation: To facilitate its purification, YscF was cloned into the overexpression plasmid pET24b (Novagen) to yield a hexahistidine-tag on the C-terminus of YscF (HT-YscF). Purified HT-YscF with the adjuvant was then used for innoculation (Matson et al., 2005).
• Description: Due to the severity of plague and its potential for use as a bioweapon, better preventatives and therapeutics for plague are desirable. Subunit vaccines directed against the F1 capsular antigen and the V antigen (also known as LcrV) of Y. pestis are under development. However, these new vaccine formulations have some possible limitations: the F1 antigen is not required for full virulence of Y. pestis and LcrV has a demonstrated immunosuppressive effect. These limitations could damper the ability of F1/LcrV based vaccines to protect against F1-minus Y. pestis strains and could lead to a high rate of undesired side effects in vaccinated populations. Thus, the use of other antigens in a plague vaccine formulation may be advantageous. Desired features in vaccine candidates would be antigens that are conserved, essential for virulence and accessible to circulating antibody. Several of the proteins required for the construction or function of the type III secretion system (TTSS) complex could be ideal contenders to meet the desired features of a vaccine candidate. Accordingly, the TTSS needle complex protein, YscF, was selected to investigate its potential as a protective antigen (Matson et al., 2005).

• Vaccine Ontology ID: VO_0000828
• Type: Live attenuated
• Preparation: The EV76 strain is a pigmentation negative mutant of Y. pestis which was derived from a fully virulent strain. The vaccine has been in use since 1908 and is given as a single dose of 5.8×10^6 cfu. Findings suggest that immunisation with the EV76 vaccine will provide protection against both bubonic and pneumonic plague in man. However, the safety of this vaccine in man is questionable, because the EV76 strain is not avirulent (Titball et al., 2001).
• Virulence: A febrile response was reported in 20% of vaccinees, accompanied by headache, weakness, and malaise. Erythema surrounding the site of vaccination, reaching dimensions as large as 15 cm2, was frequently reported. Some severe systemic reactions even required hospitalization. Numerous unsuccessful attempts were made to reduce the incidence of side effects by administering the vaccine by different routes, such as scarification, inhalation, and even intraocularly (Williamson et al., 2005).
• Description: A live, attenuated vaccine has also been used since 1908 using the avirulent strain of Y pestis, EV76. This strain is not capable of assimilating chromatophores such as Congo Red or haemin from artificial media, and is described as Pigmentation (Pgm) -. Virulent strains of Y. pestis are Pgm+. Although the organisms in the vaccine could multiply following administration, and could therefore theoretically enhance protection, there appeared to be wide variation in the immunogenicity and virulence between different EV76 vaccine preparations. The vaccine caused severe side-effects when used and there was also the danger of reversion to a fully virulent form. A large part of the Vietnamese population was vaccinated with EV76 between 1967 and 1969 (Russell et al., 1995).

• Vaccine Ontology ID: VO_0000833
• Type: Subunit vaccine
• Adjuvant:
  • VO ID: VO_0001260
  • Description: TiterMax (Hunter’s TiterMax R-1; CytRx Corp., Norcross, Ga.) (Nakajima et al., 1995).
• Preparation: Fusion between the structural gene of staphylococcal protein A (PA) present on the vector plasmid pRIT5 and that of V antigen (LcrV) obtained from the lcr plasmid of Y. pseudotuberculosis resulted in the PA-V antigen peptide (PAV), encoded on pPAV13 carried by protease-deficient Escherichia coli BL21, which contained 305 N-terminal amino acids from PA plus 259 C-terminal amino acids from V antigen and could be purified to homogeneity in one step by immunoglobulin G affinity chromatography. Rabbit antibodies raised against one or more epitopes present within an internal portion of the V antigen domain of PAV have accounted for protection against experimental plague. PAV was diluted in phosphate buffer to 2 mg/ml and emulsified separately with an equal volume of the adjuvant (Nakajima et al., 1995).
• Virulence:

• Type: Live, attenuated vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• guaA gene engineering:
  • Type: Gene mutation
  • Description: This guaBA operon is from Yersinia pestis (Oyston et al., 2010).
  • Detailed Gene Information: Click Here.
• guaB gene engineering:
  • Type: Gene mutation
  • Description: This guaBA operon is from Yersinia pestis (Oyston et al., 2010).
  • Detailed Gene Information: Click Here.
• Immunization Route: Intravenous injection (i.v.)

• Vaccine Ontology ID: VO_0002941
• Type: Live, attenuated vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse, guinea pig
• IpxM gene engineering:
  • Type: Gene mutation
  • Description: This IpxM mutant is from Yersinia pestis CO92 (Feodorova et al., 2007).
  • Detailed Gene Information: Click Here.
• Immunization Route: subcutaneous injection

• Vaccine Ontology ID: VO_0002942
• Type: Live, attenuated vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• nlpD gene engineering:
  • Type: Gene mutation
  • Description: This nlpD mutant is from Yersinia pestis (Tidhar et al., 2009).
  • Detailed Gene Information: Click Here.
• Immunization Route: subcutaneous injection

• Vaccine Ontology ID: VO_0002943
• Type: Live, attenuated vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• pcm gene engineering:
  • Type: Gene mutation
  • Description: This pcm mutant is from Yersinia pestis (Flashner et al., 2004).
  • Detailed Gene Information: Click Here.
• Immunization Route: subcutaneous injection

• Type: Live, attenuated vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• smpB gene engineering:
  • Type: Gene mutation
  • Description: This smpB/ssrA mutant is from Yersinia pestis (Okan et al., 2010).
  • Detailed Gene Information: Click Here.
• ssrA gene engineering:
  • Type: Gene mutation
  • Description: This smpB/ssrA mutant is from Yersinia pestis (Okan et al., 2010).
  • Detailed Gene Information: Click Here.
• Immunization Route: intranasal immunization

• Vaccine Ontology ID: VO_0002946
• Type: Live, attenuated vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• YopH from Y. pestis strain: CO92, biovar: Orientalis gene engineering:
  • Type: Gene mutation
  • Description: This yopH mutant is from Yersinia pestis CO92 (Bubeck and Dube, 2007).
  • Detailed Gene Information: Click Here.
• Immunization Route: intranasal immunization

• Vaccine Ontology ID: VO_0004273
• Type: Subunit vaccine
• Status: Research
• Antigen: YopE(69-77) peptide (Lin et al., 2011).
• YopE gene engineering:
  • Type: Recombinant protein preparation
  • Description: YopE(69-77) protein was sythezised by New England Peptide (Gardner, MA) (Lin et al., 2011).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000143
  • Description: 1 μg cholera toxin (CT; List Biological Laboratory, Campbell, CA) (Lin et al., 2011).
• Immunization Route: intranasal immunization

Human Response

• Host Strain: Healthy adult males.
• Vaccination Protocol: Twenty-four healthy adult males were studied in a double-blind, ascending-dose design, such that groups of six individuals received the vaccine at dose levels of 5 µg,10 µg, 20 µg, and 40 µg of each subunit in a dose volume of 0.5 ml; however, data was reported for only 20 of the 24. Attached to each dose group were two individuals who were administered placebo (alhydrogel in PBS). The vaccine or placebo was administered to individuals in a two-dose intramuscular regimen, with the priming dose on day 1 and the booster dose on day 21. For any one individual, the dose level given for priming and boosting was identical. Equal aliquots of individual serum samples, collected at day 35 from volunteers in all the vaccine dose groups, were pooled by dose group prior to dilution in PBS. The pooled samples were used to passively immunize groups of five BALB/c female mice (Charles River United Kingdom) intraperitoneally (Williamson et al., 2005).
• Persistence: Antibodies to rV were produced within two weeks of the first dose at day 1, with greater levels observed after the booster dose at day 21. Across the dose range studied, at least 50% of the maximum mean anti-rV titer was retained for up to 3 months following the first dose. Antibodies to rF1 were also generated within two weeks of the first dose at day 1, with greater levels observed after the booster dose at day 21. Across the dose range studied, at least 50% of the maximum mean anti-rF1 titer was retained for up to 3 months following the first dose (Williamson et al., 2005).
• Immune Response: The number of individuals determined to have a titer of competing antibody increased to a maximum of 18/20 at day 28, seven days after administration of the second dose. The data gained indicate that the vaccine was immunogenic in recipients at all dose levels tested, although data from only three subjects were available at the 20-µg dose level and the lowest dose level (5 µg) was suboptimal.The titer of specific IgG developed by individuals at 21 and 28 days post-initial vaccination and 7 days postboost correlated with the development of a titer of antibody competing for binding to the rV antigen and with transferable protective immunity (Williamson et al., 2005).
• Side Effects: No side effects noted.
• Description: This is a Phase I safety and immunogenicity trial in healthy volunteers.

Human Response

• Host Strain: Male subjects varying in age (21 to 59 yrs, avg. 39).
• Vaccination Protocol: 12 male human subjects were inoculated with 1 ml of 1*10^9 Y. pestis strain EV76.
• Persistence: On the 28th day, the average MPI was 8.6 and 3 sera yielded an MPI below 5 (Meyer, 1970).
• Immune Response: An antibody response compatible with some degree of immunity was recorded in 33% of the subjects. The MPI (average 9.2) was within the range charactersitc for human subjects given a single inoculation of killed vaccine suspension containing 3.6*10^9 Y. pestis. Booster inoculations produced no local or systemic reactions (Meyer, 1970).
• Side Effects: Systematic reactions included general malaise, aching, and anorexia. At least half of the group was incapacitated for as long as 72 hours; 2 were hospitalized for 48 hours. Local reactions were confined to hot, erythematous, moderately indurated, painful areas. The unpleasant side effects thus discourages mass vaccinations (Meyer, 1970).
• Description: 28 macaques were inoculated with 1*10^9 Y. pestis strain EV76. 64% of the vaccinated animals survived a severe subcutaneious challenge infection of 2.3*10^9 virulent P.pestis strain 195/P. Inoculations of 1 ml of the same suspension used for macaques were made for the human subjects (Meyer, 1970).

Mouse Response

• Host Strain: Female, seven-week old BALB/c mice were obtained from Taconic.
• Vaccination Protocol: Mice were immunized in a single vaccination by 2 intramuscular injections, with 50 microliter of the vaccine preparation divided evenly between the quadriceps on each side. AdsecV doses ranged from 10^8 to 10^11 pu. Ad vectors were diluted with saline to the specified dose (Chiuchiolo et al., 2006).
• Persistence: AdsecV induces high IgG titers within 2 weeks after a single immunization (Chiuchiolo et al., 2006).
• Side Effects: No side effects noted.
• Challenge Protocol: Four weeks after vaccination, the mice were infected intranasally with 3 × 10^3 cfu of Y. pestis strain CO92. Assessment of the data at 15 days after challenge showed that the mortality of mice was dependent on the vaccine dose.
• Efficacy: All of the mice in the group vaccinated with 10^11 pu of AdsecV survived the Y. pestis challenge. The 10^9- and 10^8-pu doses were not protective; the mice in those groups died according to the same time frame as did the mice in the control groups that received either saline or 10^11 pu of AdNull. Thus mice immunized with a single dose of AdsecV are shown to be protected from a lethal intranasal challenge of Y. pestis (Chiuchiolo 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).

Mouse Response

• Host Strain: Wild-type C57BL/6 or Rag1-/- mice (Jackson Laboratories)
• Vaccination Protocol: Mice were vaccinated with a single dose (1 x 10^3 or 1 x 10^5 CFU) of Y. pestis KIM1001-pLpxL (Montminy et al., 2006).
• Side Effects: None noted.
• Challenge Protocol: At 30–40 d post vaccination, both vaccinated and naive mice were challenged subcutaneously with virulent Y. pestis KIM1001 at doses between 1 x 10^3 CFU and 1 x 10^6 CFU or by intranasal administration, mimicking pneumonic disease, of 5 x 10^3 CFU or 5 x 10^4 CFU. Survival was monitored every 12 h during acute infection up to 21 or 28 d. For collection of organs, mice were killed by pentobarbital overdose 48 h after intravenous infection or 72 h after subcutaneous infection and spleens were homogenized in PBS to obtain bacterial titers and for cytokine analysis.
• Efficacy: All mice vaccinated with Y. pestis KIM1001-pLpxL survived, whereas all naive mice died, demonstrating that Y. pestis producing potent LPS is an effective vaccine against both bubonic and pneumonic plague (Montminy et al., 2006).

Mouse Response

• Host Strain: Balb/C (Charles River), NIH/S (Harlan Olac Inc.), outbred Porton strain (bred in-house).
• Vaccination Protocol: Mice, immunized with the Plague vaccine, USP, were given 0.1 ml of neat vaccine (1.8-2.2 X 10’ formaldehyde-killed bacilli) on day 0 and a booster of identical volume on day 8 (Russell et al., 1995).
• Persistence: In all cases, animals showed signs of infection within 24-48 h of challenge with the high doses of Y pestis. The first deaths occurred within 60 h, irrespective of the route and murine strain. The average time-to-death, however, varied according to the administration route. For the intraperitoneal and intranasal routes these were similar with little variation between strains. The average times to death by the intraperitoneal route were 3 days, 2-3 days and 3 days for Porton, Balb/C and NIH/S, respectively. When administered by the intranasal route, the average times were 3 days, 2 days and 2-3 days. After subcutaneous challenge the times to death were 5-6 days, 4 days and 4 days, respectively (Russell et al., 1995).
• Side Effects: Mice immunized with the Plague vaccine, USP, developed local lesions at the site of the injection of the challenge including grey/white caseation of the focal lymphatic glands. This lesion would either track to other sites between the dermis and the muscle fascia or penetrate the muscle fascia forming a lesion between bundles of muscles. In some cases the lesions ulcerated and began healing (Russell et al., 1995).
• Challenge Protocol: The mice were challenged either s.c. or i.n. with Y. pestis GB strain and observed for 14 days.
• Efficacy: Plague vaccine, USP, induced protection against a subcutaneous challenge with Y.pestis strain GB. Two series of trials were conducted, both with the challenge strain growing exponentially. In the first trial mice were challenged with multiples of the MLD up to 50 000. There was 100% mortality in the control groups, treated with 5 and 50 MLDs, l/5 deaths in the vaccinated group challenged with 50 MLD, and 2/5 deaths when challenged with the highest dose. In the second trial, in which challenge doses were extended from 4200 to 4 200 000 MLD, between 40 and 60% of the animals died at each dose. Thus USP conferred protection against the s.c. challenge with 5 x l0^3 MLD of Y. pestis strain GB. However, this protection does not extend to challenge with high multiplicities of the infectious dose (Russell et al., 1995).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Female BALB/c mice, 6-8 weeks of age, were purchased from Harlan. Mice were deprived of food and water for 4 h prior to immunization and resupplied 30 min after. RASVs (recombinant attenuated Salmonella vaccine) were grown in LB broth to an OD600 of 0.9 and concentrated to a final concentration of 5 x 10^10 CFU/ml in phosphate-buffered saline containing 0.01% gelatin (BSG). Mice were orally immunized with 20 μl of RASV suspensions two times with a one week interval between immunizations. Blood samples were collected on days 0, 7, 35, and 49 (Branger et al., 2007).
• Challenge Protocol: A subcutaneous challenge with virulent Y. pestis strain, CO92, (biovar orientalis) was performed 28 days after the second immunization. Each animal received by subcutaneous injection 3,000 CFU or 600 CFU in the first experiment and 1300 or 60 CFU in a second experiment. Mice were observed daily, and mortality was recorded for 14 days after the challenge. The surviving animals were euthanized after 14 days to obtain blood samples for serological analysis (Branger et al., 2007).
• Efficacy: For mice immunized with RASV expressing rPsn, 2/8 (1,300 CFU) and 4/8 (60 CFU) vaccinated animals died versus 6/8 and 5/8 respectively of the RASV controls, with a delay in time to death in the mice immunized with rPsn. The survival rate of mice vaccinated with RASV-rPsn was significantly higher than that of the RASV controls at the two challenge doses (p<0.045). A combined analysis of the results from groups immunized by RASV-Psn showed significant protection 14 days after challenge (p<0.004). In terms of the onset of death, there was a significant difference between the RASV-rPsn group and the RASV control over all the experiments (Branger et al., 2007).

Mouse Response

• Vaccination Protocol: Mice were injected with RCN-IRES-YpF1, RCN-IRES-tPA-YpF1, or RCN-IRES-tPA-YpF1-gG (10^7 pfu/0.1 ml, footpad) (Osorio et al., 2003).
• Vaccine Immune Response Type: VO_0003057
• Challenge Protocol: On day 84 PI, mice were injected subcutaneously with 200 μl of a suspension containing 562 cfu of Y. pestis, approximately 28 LD50 (Osorio et al., 2003).
• Efficacy: These recombinant viruses generated protective immune responses that resulted in survival of 80% of vaccinated mice upon challenge with Y. pestis. Of the RCN-based vaccines we tested, the RCN-IRES-tPA-YpF1 recombinant construct was the most efficacious. Mice vaccinated with this construct withstood challenge with as many as 1.5 million colony forming units of Y. pestis (7.7 x 10(4)LD(50)). Interestingly, vaccination with F1 fused to the anchoring signal (RCN-IRES-tPA-YpF1-gG) elicited significant anti-F1 antibody titers, but failed to protect mice from plague challenge (Osorio et al., 2003).

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 10^6 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).

Mouse Response

• Host Strain: Female, 8-week-old, Hsd:ND4 Swiss Webster outbred mice (Harlan Sprague Dawley, Indianapolis, Ind.).
• Vaccination Protocol: Thirty micrograms of each Yop-adjuvant mixture was administered to two groups each of 8 to 14 mice, followed by one boost of 30 µg at 30 days post-primary vaccination. Mice in all groups were subsequently boosted with 30 µg of each antigen (15 µg s.c. and 15 µg i.p.) on day 60. A control group was vaccinated with the adjuvant R-730 emulsion alone (Anderson et al., 1996).
• Side Effects: None noted.
• Efficacy: YopD offered protection against challenge with the virulent, nonencapsulated C12, with a statistically significant increase in mean survival time (26.2+/-1.7 days, P < 0.001) in one experiment. In a second experiment, the mean survival time was again highly significant (22.4+/-3.1 days, P = 0.006). The overall average mean survival time was 28 days (Andrews et al., 1999). The ability of YopD to protect mice against nonencapsulated Y. pestis C12 strongly suggests that at least one of the Yops may be important in eliciting a protective immune response against lethal Y. pestis s.c. challenge. The failure of YopD to protect against encapsulated organisms as it protects against the nonencapsulated strain may result from a masking effect of the F1 capsule on secreted YopD, which blocks the antibody-antigen interaction at the surface of the bacterium. Experiments are currently being conducted to examine these hypotheses (Andrews et al., 1999).
• Description: Significant protection of mice against challenge with encapsulated CO92 was not observed in any of the Yops except YopD (Andrews et al., 1999).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Groups of 10 BALB/c mice were immunized with adjuvant alone or with 50 µg of rV10 on day 0, followed by a booster with an equal dose on day 21. Blood from 5 mice in each immunization set was taken on days 0, 14, 28, and 42 after primary immunization to measure the generation of specific antibodies (DeBord et al., 2006).
• Side Effects: No side effects mentioned.
• Challenge Protocol: On day 43, mice were challenged with 100,000 MLD of Y. pestis CO92 via subcutaneous injection (DeBord et al., 2006). An intranasal infection model of Y. pestis CO92 was also developed using groups of 10 BALB/c mice following the aforementioned 2-dose immunization regimen. Mice were infected on day 43 with 2570 MLD of Y. pestis CO92 delivered by the intranasal route. Animals were monitored for 14 days for signs of lethal disease or death and time-to-death was recorded (DeBord et al., 2006).
• Efficacy: Mice were protected against lethal challenge in all cases, whereas mice receiving adjuvant alone succumbed to disease within 4 days after infection with an average time-to-death of 2.5 days (DeBord et al., 2006).
• Description: In contrast to Yersinia pestis LcrV, the recombinant V10 variant does not suppress the release of proinflammatory cytokines by immune cells. Immunization with rV10 generates robust antibody responses that protect mice against bubonic plague and pneumonic plague, suggesting that rV10 may serve as an improved plague vaccine (DeBord et al., 2006).

Mouse Response

• Host Strain: Six to eight week-old male and female BALB/c, CBA, CB6F1 and C57BL mice, raised under specific-pathogen-free conditions (Charles River Laboratories, Margate, Kent, UK).
• Vaccination Protocol: Mice were divided into groups of 10 for immunisation that consisted of a total of 0.1 cm^3 primary immunising dose of 10 μg of rF1 and 10 μg of rV adsorbed to 25% v/v Alhydrogel in phosphate-buffered saline. The immunising dose was equally divided between intramuscular sites in each hind leg. Individuals were randomly selected from each strain and used as untreated controls for the challenge and immune response analysis. At day 21 or 28 mice were boosted receiving doses as described above (Jones et al., 2000).
• Persistence: All strains of mice were protected against challenge with Y. pestis GB following immunisation. Male CBA and CB6F1 mice were less well protected against s.c. challenge than either the BALB/c or C57BL6. Nevertheless, the mean time to death for CBA following s.c. challenge was 13.5 days at 4.9×10^7 CFU and 14 days at 4.9×10^5 CFU; significantly delayed compared with the 3.1 days mean time to death seen in unimmunised control mice. A similar difference was apparent in CB6F1 mice and for both strains following aerosol challenge. Mortalities in both the CBA and CB6F1 mice after aerosol challenge were dose-independent. All survivors tested for Y. pestis after 15 days were found to have no bacteria in the spleen, blood, lung or liver (Jones et al., 2000).
• Side Effects: No side effects noted.
• Efficacy: The recombinant vaccine is capable of inducing protective immunity in all four strains of mice representing H-2b, H-2d, H-2k, haplotypes and a H-2b/H-2d F1 hybrid. Although breakthrough in protection was observed in male mice, both sexes raised good antibody responses to the vaccine and these were maintained for greater than 1 year in the female mice.
  The challenge data from the first trial suggested that CBA and CB6F1 males were less well protected than female mice but the BALB/c and C57BL6 data showed males of those strains were as well protected as females (Jones et al., 2000).
  
• Description: Significant differences in the ability to develop an antibody response to the foreign antigen were observed between various strains of mouse depending on their H-2 genes and were related to the ability of each strain of mouse to raise antibodies after immunisation with low doses of antigen. To investigate the possibility that potent immune responses seen in BALB/c female mice were a function of their H-2 haplotype, this study immunised males and female of four strains of mice each with different haplotypes; BALB/c (H-2d), CBA (H-2k). C57BL6 (H-2b) and CB6F1 (the F1 hybrid from a cross between BALB/c and C57BL6). CB6F1 mice co-express both the H-2d and H-2b forms of the MHC molecule (Jones et al., 2000) .

Mouse Response

• Host Strain: Six to 8 week-old Balb/C female mice (Charles River laboratories).
• Vaccination Protocol: Animals were anesthetized with methoxyfluorane (Metafane; Medical Developments Australia, Pty. Ltd.) and then inoculated with 1 × 10^6 pfu of virus in a total volume of 25 μl. Some groups of mice also received 1 × 10^6 pfu of the boosting virus VSV(GNJ)-LcrV5 at about 1-month post-prime (Palin et al., 2007).
• Persistence: The prime+boost protection was durable, lasting at least 5 months after the boost (Palin et al., 2007).
• Side Effects: None noted.
• Efficacy: Highly significant protection was obtained in the VSV-LcrV1-prime + boost group where 9 out of 10 animals survived challenge. Protection in the VSV-LcrV5-prime + boost group was also significant, in which four out of nine mice survived challenge (Palin et al., 2007).

Mouse Response

• Host Strain: ICR, BALB/c and DBA/2
• Vaccination Protocol: For gene gun immunization, plasmid DNA was precipitated onto 1-μm-diameter gold particles at a ratio of 2 μg per milligram of gold and loaded onto Gold-Coat tubing as suggested by the manufacturer (Bio-Rad). Polyvinylpyrrolidone (MW, 360,000) was used as an adhesive at a concentration of 0.05 mg/ml. Agarose gel electrophoresis was used to determine the amount of DNA. Vaccination was carried out with three immunizations of 0.5 μg of DNA at 2-week intervals. Gene gun shots were directed into exposed abdominal dermis (Grosfeld et al., 2003).
• Challenge Protocol: Immunized mice were challenged by s.c. injection of the indicated amounts of Y. pestis Kimberley53 strain suspension (0.1 ml). Animals were monitored daily for survival for a period of 3 weeks (Grosfeld et al., 2003).
• Efficacy: Immunized mice all survived lethal challenge with Y. pestis (Grosfeld et al., 2003).

Mouse Response

• Vaccine Immune Response Type: VO_0000286
• Efficacy: Following pneumonic challenge, the best efficacy was obtained in mice primed with IL-12(Low)/F1-V vaccine with 80% survival compared to mice immunized with IL-12(Low)/F1, IL-12(Low)/V, or IL-12(Low) vector DNA vaccines (Yamanaka et al., 2008).

Mouse Response

• Vaccine Immune Response Type: VO_0003057
• Efficacy: In the YscF-2 immunized group, 60% of mice survived the intranasal challenge, which was significant as compared to either wt.YscF or tPA.YscF DNA vaccines according to Fisher's exact test (p < 0.05) (Wang et al., 2008).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: For intratracheal immunization, mice were anesthetized with Avertin (2,2,2-tribromoethanol [Sigma]; tert-amyl alcohol [Fisher]) by intraperitoneal injection and suspended from a length of wire by their front incisors. With the use of a sterile gel-loading tip inserted gently into the trachea, 10 μg F1 antigen and the indicated amount of flagellin or the flagellin mutant 229 were administered in a total of 50 μl pyrogen-free phosphate-buffered saline (PBS). For intranasal immunization, small volumes (9 to 12 μl total) containing antigen and adjuvant in PBS were administered to the nostrils of anesthetized mice (Honko et al., 2006).
• Challenge Protocol: Mice were challenged intranasally with 10 μl of culture diluted in PBS to ∼1.8 × 10^7 CFU/ml, a dose equivalent to 150 50% lethal doses (LD50s) (Honko et al., 2006).
• Efficacy: BALB/c mice immunized and boosted i.n. with flagellin and F1 had a 93% survival rate, versus only 7% in the control group, following challenge with a dose equivalent to 100× the LD50 (Honko et al., 2006).

Mouse Response

• Host Strain: female, 8- to.10-week-old outbred (Hsd:ND4) Swiss Webster mice (Harlan Sprague Dawley,Indianapolis, Ind.).
• Vaccination Protocol: Mice were immunized with two hundred microliters of each antigen-adjuvant mixture, containing 10 mg of F1. After 30 days, the animals were boosted with the identical dose at the same inoculation site. Four negative control groups, consisting of 10 mice each, were immunized with the adjuvants only. F1 antibody titers of all animal groups were measured 26 days after the second F1-immunizing dose (Andrews et al., 1996).
• Side Effects: No side effects mentioned.
• Challenge Protocol: The 11 immunized and control animal groups to receive the s.c. challenge were administered 100 50% lethal doses (LD50) of wild-type Y. pestis CO92 (LD50 5 1.9 CFU) 28 days after the F1 booster dose. The second set of 11 animal groups was next exposed in a nose-only exposure chamber to a dynamic aerosol containing the virulent organisms diluted to give an inhaled dose of about 100 LD50 (LD50 5 2.3 3 10^4 CFU). All infected animals were monitored daily for disease symptoms and/or death until 28 days postchallenge. At day 28, surviving animals were bled for anti-F1 titer and euthanized, their spleens were cultured on blood agar base, and viable organisms were counted.
• Efficacy: F1 antigen evoked a high degree of protection in animals challenged s.c. with 100 LD50 of wild-type Y. pestis (70-100% survival). Protection also appeared to be independent of the source of the antigen, whether cell derived or cell free, from either Y. pestis or E. coli, and the adjuvant used. However, results do suggest that F1 combined with Alhydrogel, the only adjuvant approved for human use, elicits good protective immunity at the challenge dose administered. Although not achieving the level of protection seen against s.c. challenge, F1 also protected the majority of immunized mice by aerosol (65-84% survival) (Andrews et al., 1996).

Mouse Response

• Host Strain: Swiss
• Vaccination Protocol: Groups of 10 female, 8- to 10-week-old outbred (Hsd:ND4) Swiss Webster mice were inoculated with purified, recombinant YscF protein. To improve immunogenicity, each protein antigen was adsorbed with Ribi adjuvant system (RAS) R-730 monophosphoryl lipid-A (Corixa, Hamilton, MT). Two hundred microliters of each antigen–adjuvant mixture containing 20 μg of recombinant protein was administered at a single subcutaneous (s.c.) site on the backs of the animals. After 30 days, the animals were boosted with an identical dose at the same injection site. The F1–V vaccine candidate was included in the experiment to serve as a positive control (Swietnicki et al., 2005).
• Challenge Protocol: Each of the vaccinated and control animals designated to receive s.c. challenges was administered 130 50% lethal doses (LD50) of wild type Y. pestis CO92 30 days after the booster dose. The s.c. LD50 for adult mice challenged with CO92 is 1.9 CFU. The mice were observed daily for 28 days, at which time the survivors were killed (Swietnicki et al., 2005).
• Efficacy: Immunization with recombinant YscF protein confers significant protection against challenge with live wild type Yersinia pestis CO92 strain. Six out of ten YscF vaccinated mice survived a lethal challenge with Y. pestis while 100% of the mice that were inoculated with R-730 adjuvant alone succumbed to infection (Swietnicki et al., 2005).

Mouse Response

• Host Strain: 6-to 8-week-old female Swiss-Webster mice.
• Vaccination Protocol: Mice immunized 40 μg/mouse HT-YscF in PBS or PBS alone (control mice) emulsified 1:1 with CFA. Experimental mice were boosted with 40 μg/mouse HT-YscF in IFA after two weeks and with 20 μg/mouse HT-YscF in IFA at 4 weeks post-immunization. Negative control mice were boosted with PBS emulsified with IFA according to the same schedule. Two weeks following the final booster immunization, sera were collected from the HT-YscF-immunized and the PBS-immunized mice to assay for HT-YscF reactivity. Sera from 22 mice from the HT-YscF-immunized and the PBS-immunized groups were tested for total IgG reactivity (Matson et al., 2005).
• Side Effects: None noted.
• Challenge Protocol: After establishing that the HT-YscF immunized mice had developed a strong antibody response to HT-YscF, the mice were challenged with Y. pestis. Two weeks after the final immunization, groups of 10 mice were challenged i.v. via the retro-orbital sinus with 10^1 to 10^6 CFU Y. pestis KIM5 (pgm-) in PBS. The mice were observed for 19 days after challenge, and the average doses required to kill 50% of the mice (LD50) for the treatment groups were calculated.
• Efficacy: Mice immunized with HT-YscF demonstrated a strong antibody response to YscF and provided protection to the vaccinated mice from lethal Y. pestis challenge (Matson et al., 2005).

Mouse Response

• Host Strain: Balb/C, Porton outbred, and NIH/S
• Vaccination Protocol: The median lethal dose (MLD) of a pathogenic strain of Yersinia pestis was established by, three routes of administration (s.c., i.p., i.n.) in three strains of mouse. There was no significant difference in the MLDs in the different strains of mouse. The MLD by the subcutaneous route in Balb/C and an outbred line was approximately I c.fu.; the MLD following the intraperitoneal administration was tenfold higher.
  Y pestis EV76 strain was retrieved from the storage beads. Fresh BAB broth was seeded with an overnight culture and incubated. Neat seeded broth (0.1 ml) was administered as a single injection on day 0. One group received the 28°C cultured bacteria and the other the 37°C culture. The
  mice were then challenged subcutaneously (Russell et al., 1995).
• Persistence: In all cases, animals showed signs of infection within 24-48 h of challenge with the high doses of Y pestis. The first deaths occurred within 60 h, irrespective of the route and murine strain. The average time-to-death, however, varied according to the administration route.
  Where deaths did occur the mean time to death was ten days in EV76 (37”C-grown)-vaccinated mice and four days in mice vaccinated with EV76 grown at 28°C (Russell et al., 1995).
• Side Effects: EV76 caused side-effects in nearly all of the mice - some severe - and even one death attributable to the vaccine. Autopsy revealed damage to the spleen, consisting of purulent abscesses over the surface of the organ and apical necrosis. In other animals, the vaccine caused paralysis in the injected limb which did not improve over the total study period. There appeared to be both sensory and motor dysfunction (Russell et al., 1995).
• Efficacy: EV76 induced protection against a subcutaneous challenge with at least 5 x l0^3 MLD of Y. pestis strain GB. The fatality rate has been reported to be approximately 1% of vaccinees (Russell et al., 1995).

Mouse Response

• Host Strain: 5 to 6 weeks of age Female Swiss-Webster mice (Charles River Laboratories, Wilmington, MA).
• Vaccination Protocol: Mice received a primary immunization on day 0 consisting of 25 ml of emulsion (containing 25 mg of homogenous PA or PAV) and then identical booster immunizations were given on days 21 and 35. Adjuvant emulsified with an equal volume of phosphate buffer alone was also used as a negative control (Nakajima et al., 1995).
• Persistence: During immunization of mice with PAV, serum antibodies directed against highly purified recombinant V antigen became evident by week 4 and achieved a maximum titer (optical density of ~0.6) by week 6 (Nakajima et al., 1995).
• Immune Response: Injected PAV but not PA markedly suppressed TNF-a and IFN-g normally induced upon infection of control mice with avirulent lcrV or Lcr2 mutants of Y. pestis and promoted in vivo survival of these isolates as well as salmonellae and Listeria monocytogenes (Nakajima et al., 1995).
• Side Effects: No side effects noted.
• Challenge Protocol: It is established that an ~70-kb Lcr plasmid enables Yersinia pestis, the causative agent of bubonic plague, to multiply in focal necrotic lesions within visceral organs of mice by preventing net synthesis of the cytokines tumor necrosis factor alpha (TNF-a) and gamma interferon (IFN-g), thereby minimizing inflammation (Lcr1). Rabbit antiserum raised against cloned staphylococcal protein A-V antigen fusion peptide (PAV) is known to passively immunize mice against 10 minimum lethal doses of intravenously injected Lcr1 cells of Y. pestis. In this study, injected PAV suppressed TNF-a and IFN-g in mice challenged with avirulent V antigen deficient Y. pestis (lcrV or Lcr2) and promoted survival in vivo of these isolates. Active immunization of mice with PAV protected against 1,000 minimum lethal doses of intravenously injected Lcr1 cells of Y. pestis. The progressive necrosis provoked by Lcr1 cells of Y. pestis in visceral organs of nonimmunized mice was replaced after active immunization with PAV by massive infiltration of neutrophils and mononuclear cells (which generated protective granulomas indistinguishable from those formed against avirulent Lcr2 mutants in nonimmunized mice). Significant synthesis of TNF-a and IFN-g occurred in spleens of mice actively immunized with PAV after challenge with Lcr1 cells of Y. pestis. These findings suggest that V antigen contributes to disease by suppressing the normal inflammatory response (Nakajima et al., 1995).
• Efficacy: PAV but not PA provided absolute protection against 10 MLD of Y. pestis (Nakajima et al., 1995).

Mouse Response

• Persistence: A guaBA mutant is attenuated in mice (Oyston et al., 2010).
• Efficacy: A guaBA mutant induced significant protection from challenge with wild type Yersinia pestis in mice (Oyston et al., 2010).

Mouse Response

• Persistence: An IpxM mutant is attenuated in both inbred and outbred mice (Feodorova et al., 2007).
• Efficacy: An IpxM mutant conferred modest protection from challenge with wild type Y. pestis in Balb/c mice and significant protection from challenge in outbred mice (Feodorova et al., 2007).

Mouse Response

• Persistence: An nlpD mutant is highly attenuated in mice (Tidhar et al., 2009).
• Efficacy: An nlpD mutant induced significant protection from challenge with wild type Y. pestis in mice (Tidhar et al., 2009).
• Host TNF-alpha response
  • Description: The nlpD-null mutant preserves the ability to translocate Yop effectors into host cells as evidenced by suppression of TNF-α secretion from infected RAW264.7 macrophages as compared to other mutant strains. TNF-alpha levels were similar to the levels found in wild type infection, which was significantly less than Yop mutants (Tidhar et al., 2009).
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Mouse Response

• Persistence: A pcm mutant is highly attenuated in mice (Flashner et al., 2004).
• Efficacy: A pcm mutant induced significant protection from challenge with wild type Y. pestis in mice (Flashner et al., 2004).

Mouse Response

• Persistence: An smpB/ssrA mutant is attenuated in mice (Okan et al., 2010).
• Efficacy: An smpB/ssrA mutant induces significant protection from challenge with wild type Y. pestis in mice (Okan et al., 2010).

Mouse Response

• Persistence: A yopH mutant is highly attenuated in mice (Bubeck and Dube, 2007).
• Efficacy: A yopH mutant induced significant protection from challenge with wild type Y. pestis in mice (Bubeck and Dube, 2007).

Mouse Response

• Host Strain: C57BL/6
• Vaccination Protocol: Mice were lightly anesthetized by isoflurane and immunized intranasally with a 15 &mu;l saline solution containing 1 or 10 &mu;g peptide and 1 &mu;g cholera toxin (CT; List Biological Laboratory, Campbell, CA). Mice were immunized on days 0, 7, and 21, and challenged with Y. pestis strain D27 on day 37 or day 56 (Lin et al., 2011).
• Challenge Protocol: Mice were lightly anesthetized by isoflurane and infected intranasally with 20 or 200 median lethal doses (MLD) Y. pestis strain D27 in 30 &mu;l saline. The intranasal MLD of Y. pestis strain D27 is ∼1 x 10^4 CFU when the bacteria are grown and administered, as described above (Lin et al., 2011).
• Efficacy: 83% of mice immunized with YopE(69-77) peptide survived lethal challenge with Y. pestis D27 (Lin et al., 2011).

Rat Response

• Host Strain: Rattus norvegicus
• Vaccination Protocol: Rats were vaccinated with F1 antigen in Freund's complete adjuvant. The rats received an injection of 500 ug of F1 followed by booster injections of 200 ug of F1 at 7 and 14 days. These rats were challenged 6 weeks later with 3.5 * 10^3 Y. pestis 195/P(Williams et al., 1979) .
• Persistence: Survival among vaccinated rats was in direct proportion to the titre of F1 antibody titre present at the time of challenge. In vaccinated rats that died, death was directly correlated with the F1 antibody titre at the time of challenge (Williams et al., 1979).
• Side Effects: None noted.
• Efficacy: Inoculation of 1*10^3 to 5*10^5 Y. pestis survived at rates of 6% at titres less than 1:16, 46% at titres of 1:32-1:64, 90% at titres of 1:128-1:256, and 96% at titres of 1:512-1:1024. Rats vaccinated with F1 antigen and rats that had been infected previously were challenged i.n. with 8.9 *10^4 Y. pestis and subsequently demonstrated similar rates of survival that was 0 at titres less than 1:128, 86% at titres of 1:128-1:256, and 100% at titres of 1:512-1:1024 (Williams et al., 1979).
• Description: Exposure to F1, either through vaccination or infection, stimulates the production of antibodies that can be measured quantitatively. At present, the passive haemagglutination (PHA) and haemagglutination-inhibition techniques are most widely employed for this purpose because the procedures are convenient and exhibit great sensitivity and specificity. Although the occurrence of antibody to F1 in man or animals suggests that some degree of protection against reinfection has been acquired, the relationship between the serological titre of F1 antibody and immunity to plague has not been clearly defined. The work reported here investigated the correlation between titre and protection with reference to the PHA procedure for measuring F1 antibody (Williams et al., 1979).

Monkey Response

• Host Strain: Vervets of three different races or subspecies (Ethiopian race, Kenya race, and Cercopithecus aethiops pygerythrus).
• Vaccination Protocol: Ethiopian race: 16 vervets were vaccinated with 160 million EV (51f) organisms, 16 million organisms, and 1.6 million organisms, and then challenged on the 41st day after vaccination.
  Kenya race: 20 vervets were vaccinated with 218 million organisms, and then challenged on the 52nd day after vaccination.
  Cercopithecus aethiops pygerythrus: 50 vervets were vaccinated with 100 million and 100,000 organisms, and then challenged with 8,770 organisms of highly virulent strain of P. pestis 166 days after vaccination (Meyer, 1970).
• Persistence: Deaths occurred within 6-28 days following vaccination (Meyer, 1970).
• Side Effects: Symptoms included anatomical lesions and bacteriological findings of bubonic-septicaemic plague (Meyer, 1970).
• Efficacy: Survivors that were challenged showed complete immunity except for 3 of 10 Cercopithecus aethiops pygerythrus (Meyer, 1970).

Guinea pig Response

• Host Strain: Six- to eight-week-old Dunkin Hartley female guinea pigs (Charles River Laboratories, Margate, Kent, UK).
• Vaccination Protocol: Guinea pigs (18, divided into three groups of six) were immunised with 50 μg rF1+50 μg rV adsorbed to 25% (v/v) Alhydrogel in 0.3 ml PBS. Individual guinea pigs were randomly selected and used as untreated controls for the challenge and immune response analysis. Guinea pigs were boosted at day 21, exactly as for the priming immunisation.
  In order to raise antiserum for the passive immunisation of mice, a further group of 6 guinea pigs was immunised with 50 μg rF1+50 μg rV in 0.3 ml of 25% (v/v) Alhydrogel in PBS on days 1, 21 and 115. Additional groups of six guinea pigs were immunised with either 0.3 ml USP KWCV or the same volume of USP KWCV supplemented with 50 μg of rV antigen. These animals also received boosting immunisations on days 21 and 115.
  Three groups of six guinea pigs immunised with rF1+rV vaccine were challenged subcutaneously on day 90 with 0.1 ml aliquots of Y. pestis strain GB at various cell densities between 105 and 108 colony-forming units (CFU)/ml. A naïve control group of six guinea pigs was also challenged. The passively immunised Balb/c mice were challenged subcutaneously with either 10 or 1000 MLD Y. pestis strain GB and were observed for 8 days post-challenge (Jones et al., 2003).
• Persistence: Immunised guinea pigs were challenged at day 90 and were fully protected at the lowest challenge dose (105 CFU), surviving for 26 days post-challenge. However, there was loss of protection as the challenge dose was increased so that five of six animals were protected against 106 CFU and only three of six against 107 CFU. All of the naïve control animals had succumbed to a challenge of 104 CFU (Jones et al., 2003).
• Side Effects: Buboe development occurred although protection against challenge was achieved in rF1+rV-immunised guinea pigs (Jones et al., 2003).
• Efficacy: 50% of the guinea pigs had responded strongly to rF1 after two doses of vaccine and the remainder had responded less well. While at the lowest challenge level, the IgG response to F1 was adequate to protect; it may be that it was insufficient against the highest challenge level, so that 50% of the animals succumbed to infection. Too high and too frequent dosing with F1 antigen or whole killed plague bacilli in the guinea pig is not efficacious (Jones et al., 2003).
• Description: The efficacy of the rF1+rV vaccine in protecting guinea pigs against subcutaneous challenge with a virulent Y. pestis strain was assessed and compared with that of the USP KWCV. Previously, deficiencies in efficacy of KWCV formulations have been attributed to the lack of V antigen in these formulations and so the effect on the protective efficacy of the KWCV of supplementing it with rV antigen has been determined by passive transfer of immune guinea pig sera into naive mice with subsequent challenge of the mice with a virulent plague strain (Jones et al., 2003).

Guinea pig Response

• Persistence: An IpxM mutant is attenuated in guinea pigs (Feodorova et al., 2007).
• Efficacy: An IpxM mutant conferred significant protection from challenge with wild type Y. pestis in guinea pigs (Feodorova et al., 2007).