Plague vaccine research and development
Williamson CBD Sector, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
1. Summary, 606
2. Introduction, 606
3. A recombinant subunit vaccine for plague, 606
4. Production of the recombinant F1 + V vaccine, 606
5. Vaccine efficacy, 607
6. Surrogate marker of protective efficacy, 607
7. Conclusions, 607
8. References, 607
A fully recombinant subunit vaccine is described which provides enhanced protection against bubonic plague and a new prophylactic against the pneumonic form of the disease. The vaccine comprises two subunit antigens termed F1 and V which are individually immunogenic and protective and have additive protective effect in combination. The vaccine has been demonstrated efficacious in small animal models, including a strain of mice with a targeted gene deletion for interleukin 4 (IL4T mice). The mechanism of protection conferred by the vaccine is principally antibody-mediated and antibody titre to F1 + V in the mouse correlates with protection. Surrogate markers of efficacy in man have been identified and will be applied to support clinical trialling of the vaccine. This vaccine could be used to counter bubonic and pneumonic plague in areas of the world where the disease is endemic.
Plague is an ancient disease which has not been eradicated from the modern world. Each year there are several thousand reported cases of the disease world-wide and plague has been classified as a re-emerging disease by the World Health Organization. Endemic areas for the disease exist in China, Africa, Asia and South America (Perry and Fetherston 1997).
Killed whole cell vaccines for plague have been used in man since the late 1890s and these have been shown to protect against the bubonic form of the disease. There is, however, experimental evidence that protection against the pneumonic form of the disease is limited (Meyer 1970; Williamson et al. 1997). A live attenuated vaccine, EV76, which had questionable efficacy in evoking an immune response in humans (Meyer 1970; Meyer et al. 1974a, b) and some severe side-effects, was used in the past. The virulence of EV76 also differs from species to species with chronic infections occurring in nonhuman primates. At CBS Porton Down, work has been ongoing for the last nine years on a fully recombinant subunit vaccine for plague which in animal models provides enhanced protection against both bubonic and pneumonic infections and which is a new candidate human vaccine.
3. A RECOMBINANT SUBUNIT VACCINE FOR PLAGUE
The vaccine comprises recombinant proteins termed the F1 (Fraction 1) and V (Virulence) antigens. In the native form, these proteins are natural virulence factors of the causative organism of plague, Yersinia pestis. F1 antigen is a capsular protein which is assembled by the organism on its surface and which therefore has structural properties (Titball et al. 1997; Miller et al. 1998). The V antigen is an intracellular antigen which is thought to be secreted by the bacterium onto the cell surface during and following contact with a eukaryotic cell (Petterson et al. 1999). As virulence factors, F1 has antiphagocytic properties and the V antigen has a key role in the Type III secretion machinery employed by Y. pestis to deliver a range of Yersinia outer proteins (YOPs) into the host cell.
Individually, the F1 and V recombinant proteins are immunogenic and protective against live organism challenge. In combination, they are additive in the protection they provide against very high challenge levels with virulent plague in a small animal model (Williamson et al. 1997, 2001).
4. PRODUCTION OF THE RECOMBINANT F1 + V VACCINE
Recombinant F1 antigen was produced by cloning the entire caf operon which encodes the expression and assembly of the F1 antigen in Y. pestis into E. coli JM101 using plasmid pAH 34 L for expression (Titball et al. 1997). In this vector the F1 antigen is exported and assembled on the surface of the cell in a manner identical to the Y. pestis F1 antigen, so that the E. coli cells were encapsulated as for Y. pestis. Recombinant F1 antigen is sloughed off from the cell surface and collected from the supernatant fluid.
A number of methods have been used to characterize the rF1 product to ensure its homology with the native protein. Western blotting identified the protein as F1 when compared to the ATCC F138G-1 material. Purity was assessed as greater than 90% by SDS-PAGE, revealing the monomeric form of F1 with a molecular weight of 15·5 Kda. However, FPLC analysis indicated that multimerization of F1 can occur, with molecular weights exceeding 200 MDa in some cases. The multimeric forms of the protein can be reduced to the monomeric form by boiling in SDS, which indicated that the aggregation was due to hydrogen bonds and hydrophobic interactions (Miller et al. 1998). The reduced structure had some loss of protective efficacy which was restored when the protein re-aggregated (Miller et al. 1998), indicating that the protective efficacy of this protein was highly dependent on its conformation.
Recombinant V antigen was also produced in E. coli JM101. The plasmid pVG 110 containing the V antigen gene sequence (lcr V) was cloned into E. coli resulting in the V antigen being produced intracellularly as a fusion protein with glutathione-s-transferase (Carr et al. 2000). The fusion protein was cleaved off with a recombinant enzyme during the downstream purification process. The rV antigen was characterized, identified by SDS-PAGE and by Western blotting. In the monomeric form the protein had a molecular weight of 37 KDa, but there was evidence that it also formed multimers, and this propensity to aggregate was important for its protective efficacy.
5. VACCINE EFFICACY
The small animal model used to investigate the protective efficacy of the vaccine against plague was the mouse. In both male and female BALB/c mice, protection was demonstrated against challenge with 106 median lethal doses (MLD) by the subcutaneous route and 104 MLD by the inhalational route (Jones et al. 2001). Protection against challenge was also demonstrated by the passive transfer of serum from immune BALB/c mice into naïve SCID/Bge mice (Green et al. 1999). Other strains of mice, including C57 BL/6, CBA and CB6F1, with different genetic backgrounds were used in efficacy studies, with challenge by the subcutaneous and inhalational routes and the vaccine was highly efficacious in these strains also (Jones et al. 2001). The F1 + V vaccine was used to immunize mice with a targeted deletion in the gene encoding IL4 (IL4T mice) and these mice were fully protected by the vaccine, indicating that the F1 + V vaccine induced protection against plague even in the absence of IL-4 driven immune responses, previously thought to be necessary for the development of a protective immune response (Elvin and Williamson 2000). Thus the protection afforded by this vaccine is robust, at least in a range of mouse strains of varied genetic background.
Vaccine efficacy has been clearly demonstrated in the small animal model, against both the pneumonic and bubonic forms of plague. Licensing of the vaccine for human use entails a demonstration of its safety and immunogenicity and a prediction of its efficacy. The demonstration of a correlation between an F1 + V-specific IgG titre, specifically of the IgG1 subclass, with protection in the BALB/c mouse (Williamson et al. 1999), will facilitate the identification of surrogate markers of vaccine efficacy when the vaccine is trialled in man.
6. SURROGATE MARKER OF PROTECTIVE EFFICACY
Since there was a correlation between antibody titre induced by the vaccine and protective efficacy in the mouse (Williamson et al. 1999), and it was possible to transfer hyperimmune serum into naïve mice and protect the latter against plague challenge (Williamson et al. 1997), the passive transfer of serum from a human vaccine into a naïve mouse, with challenge of the latter, provides a surrogate marker of the vaccine’s efficacy in man.
A highly efficacious recombinant subunit vaccine for plague has been identified. The efficacy observed in the small animal model was attributable to its effect in antagonizing two of the natural virulence mechanisms of this pathogen. There is evidence that the protective efficacy of the two subunits which comprise the vaccine are additive in effect. The recombinant F1 + V vaccine is now in development as a candidate human vaccine to protect against plague.