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Vaccine Comparison

rF1 + rV Yersinia EV76
Vaccine Information Vaccine Information
  • Vaccine Ontology ID: VO_0000830
  • Type: Subunit vaccine
  • Adjuvant: Alhydrogel
    • 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and protective against virulent plague in a number of animal models: mice, guinea pigs, and cynomolgus macaques (unpublished data).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_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 5.8×106 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).
Host Response Host Response

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*109 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*109 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*109 Y. pestis strain EV76. 64% of the vaccinated animals survived a severe subcutaneious challenge infection of 2.3*109 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: 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 4.9×107 CFU and 14 days at 4.9×105 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: 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).

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).
References References
Jones et al., 2000: Jones SM, Day F, Stagg AJ, Williamson ED. Protection conferred by a fully recombinant sub-unit vaccine against Yersinia pestis in male and female mice of four inbred strains. Vaccine. 2000 Sep 15; 19(2-3); 358-66. [PubMed: 10930691].
Jones et al., 2003: Jones SM, Griffin KF, Hodgson I, Williamson ED. Protective efficacy of a fully recombinant plague vaccine in the guinea pig. Vaccine. 2003 Sep 8; 21(25-26); 3912-8. [PubMed: 12922126].
Williamson et al., 2005: Williamson ED, Flick-Smith HC, Lebutt C, Rowland CA, Jones SM, Waters EL, Gwyther RJ, Miller J, Packer PJ, Irving M. Human immune response to a plague vaccine comprising recombinant F1 and V antigens. Infection and immunity. 2005 Jun; 73(6); 3598-608. [PubMed: 15908389].
Meyer, 1970: Meyer KF. Effectiveness of live or killed plague vaccines in man. Bulletin of the World Health Organization. 1970; 42(5); 653-66. [PubMed: 4988692].
Russell et al., 1995: Russell P, Eley SM, Hibbs SE, Manchee RJ, Stagg AJ, Titball RW. A comparison of Plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model. Vaccine. 1995 Nov; 13(16); 1551-6. [PubMed: 8578841].
Titball et al., 2001: Titball RW, Williamson ED. Vaccination against bubonic and pneumonic plague. Vaccine. 2001 Jul 20; 19(30); 4175-84. [PubMed: 11457543].
Williamson et al., 2005: Williamson ED, Flick-Smith HC, Lebutt C, Rowland CA, Jones SM, Waters EL, Gwyther RJ, Miller J, Packer PJ, Irving M. Human immune response to a plague vaccine comprising recombinant F1 and V antigens. Infection and immunity. 2005 Jun; 73(6); 3598-608. [PubMed: 15908389].