Purification, immunogenicity and protective potency of the F1 antigen from Yersinia pestis

Abstract

Yersinia pestis, an organism endemic in much of the world, is the causative agent of pneumonic and bubonic plague. The disease is severe with a high mortality rate. Most plague vaccines are formalised whole cells, which give limited and short-lived immunity. An acellular vaccine could prove to be more effective than using killed whole cells, without the associated side effects. The 17.5kDa F1 antigen of Y. pestis has been shown to be immunogenic and protective in mice (Simpson et at., 1990) and therefore it might be a suitable component of an acellular vaccine. In this study, the conditions for the production and purification of Fl from culture of Y. pestis were examined with a view to increasing the yield of F1 antigen. The optimal conditions were to grow Y. pestis MRE 1447 in a chemically defined medium at pH7.4 for 48h at 37°C and these conditions were transferred to a 25-litre fermenter for the larger-scale production of F1 antigen. Typically, 40mg of F1 antigen of ≥90% purity was obtained from 1L of culture supernatant. The purified F1 was characterised using biochemical, structural and immunological methods to confirm the integrity of the purified antigen. Immunological methods using anti-F1 monoclonal and polyclonal antibody identified the purified antigen as F1. Structural studies on the glycosylation of the protein showed that no post-translational glycosylation of the protein could be detected by the methods used in this study. The caf1 sequence was successfully cloned into Escherichia coli to produce a recombinant F1 antigen. This recombinant antigen proved to be both immunogenic and protective but yields of recombinant protein were consistently low. Purified culture-derived native F1 and a recombinant V antigen (produced at CBD, DERA) were used to assess controlled-release vaccine delivery systems in poly(lactideco-glycolide) PLG micro spheres and liposomes. Recombinant V was used in addition to F1 to assess a combination subunit vaccine with F1 given alone as an acellular vaccine. F1 antigen encapsulated in PLG microspheres induced high serum antibody titres when injected i.p. in mice; mucosal IgA was also detected. Mice immunised with Fl in Alhydrogel or PLG micro spheres were protected against sub-cutaneous challenge with Y. pestis. F1 antigen surface-labeled onto liposome vesicles stimulated high serum titres in Balb/c mice; a mucosal response was also induced and mice were protected against subcutaneous challenge with up to 1 x 105 organisms. This strongly indicated that immunising with F1 formulated in PLG micro spheres and liposomes induced a potent immune response and that protection was achieved after only one dose. However, increased protection was observed when a combination vaccine of F1 and rV was administered in PLG microspheres, liposomes or Alhydrogel given in multiple doses. The efficacy of different delivery routes against sub-cutaneous Y. pestis challenge was also investigated. Administration by the i.p. route gave consistent protection against subcutaneous plague challenge. The i.n. route also proved effective for both PLG microspheres and liposomes when multiple doses were used. Oral delivery failed to give adequate protection against sub-cutaneous infection, even after multiple doses.