The present invention relates to novel vaccines for provision of protection against infection with the organism Yersinia pestis and to methods for administering these. Particularly provided are parenterally and orally active vaccines capable of offering protection against bubonic and pneumonic plague particularly by induction of mucosal immunity in both humans and other animals.

Yersinia pestis is the highly virulent causative organism of plague in a wide range of animals, including man. Infection with such organisms results in a high rate of mortality. Studies have shown that the high virulence is due to a complex array of factors encoded by both the chromosome and three plasmids, including the Lcr genes, a fibrinolysin and a capsule.

Man is an occasional host in the natural cycle of the disease, and bubonic plague, characterised by the swelling of local lymph nodes, may occur following the bite of an infected flea. One of the complications of bubonic plague is secondary pneumonia, and in these cases the disease is readily transmitted between humans by airborne droplets. Plague is endemic in regions of North and South America, Africa, China and Asia (see Butler (1983) Plague and Other Yersinia Infections; Plenum Press, New York). Current outbreaks are believed to be part of the fourth world pandemic of the disease, with a clear need to protect individuals living or travelling in endemic areas, and laboratory workers handling the bacterium

The current whole cell vaccines available for prevention of plague are highly heterogeneous, resulting in side effects which make them unsuitable for widespread use (eg Meyer et al (1974) J. Infect Dis 129 supp 13-18 and 85-120; Marshall et al (1974) ibid supp 19-25).

One current vaccine for plague is the Cutter vaccine which comprises formaldehyde killed plague bacilli and is administered to the body by intramuscular injection. However, parenteral immunisation, although effective in inducing systemic immunity, does not effectively induce mucosal immunity (McGhee et al, (1992) Vaccine IQ, 75-88) and cases of pneumonic plague

have been reported in vaccinated individuals (Meyer (1970) Bull WHO 42 p653-666ι So far no vaccine capable of producing a protective immune response at mucosal surfaces has been reported.

The live attenuated EV76 vaccine was tested extensively and useα in the former Soviet Union from 1939, although its efficacy in evoking an immune response in man is questionable (Meyer et al (1974) J. Infect. Dis 129 Supp- 13-18) It has been shown that the virulence of EV76 differs in several animal species, and non-human primates are particularly susceptible to a chronic infection with this strain. In the Western World the vaccine is considered to be unsuitable for mass vaccinations due to the extreme severity of the side effects and the possibility of the strain reverting to full virulence.

Two known Y. pestis antigens are the Y. pestis LcrV (V antigen), and the F1 antigen; both of which have now been found to be capable of evoking protective immune responses in animals. The present inventors have previously provided live orally active vaccine micro¬ organisms capable of expressing V antigen and F1 antigen respectively which each provide good protection against challenge with Y. pestis at up to 10 ³ cfu. These vaccines are the subject of copending patent applications PCT/GB94/02818 and GB 9404577.0.

The present inventors have now suφrisingly found that whereas only the unacceptably hazardous EV live vaccine had been shown to be capable of giving good protection against challenge with 10 ⁹ cfu or more with Y. pestis GB strain, and V and F1 antigens alone only provide full protection against challenge with about 10 ⁵ cfu, by administering a combined vaccine comprising V and F1 antigens they can at least match the protection afforded by EV76 without any of the hazards that have kept the EV vaccine from general use.

Still more advantageously, they have found that the vehicle for administration may be a simple mixture of the two protein components, rather than as a more complex attenuated whole organism. For long term protection and for the purposes of producing mucosal immunity, they have provided preferred forms of vaccine comprising the two components in the form of live attenuated vaccine such as the F1 and V expressing Aro A or C Salmonella

typhi referred to in the aforesaid copending applications, and in more preferred forms a single or double mutant expressing these antigens separately, or a fusion protein comprising both antigens.

Further provided are micro-organisms comprising both of F1 and V types of construct or plasmids of the applicants copending applications referred to above. These contain constructs that are capable of transforming a human or animal gut colonising micro-organism such that it is enabled to express proteins that are equivalent in sequence to F1 and V antigens respectively; these producing a protective immune response against Yersinia pestis in a human or animal body when the micro-organism is administered by oral or parenteral routes, and preferably allow the micro-organism to maintain its ability to colonise the human or animal gut.

A particularly preferred recombinant DNA, plasmid or human or animal gut colonising organism encodes for or expresses all or a protective epitopic part of the mature V protein of Yersinia pestis and all or a protective epitopic part of the mature F1 protein of Yersinia pestis. DNA encoding the whole or part of the F1 antigen and DNA encoding the whole or part of the V antigen could be used directly as a genetic vaccine.

Particularly preferred recombinant DNA encoding for V comprises a DNA sequence as descπbed in SEQ ID No 1 or SEQ ID No 3, more preferably positioned in frame with a promoter such as lacz or nirβ, and preferably in a vector capable of expression and replication in a Salmonella Particularly preferred recombinant DNA encoding for F1 comprises a DNA sequence as described in SEQ ID No 10. SEQ ID No 2 and SEQ ID No 4 show the ammo acid sequences of two preferred V antigen proteins; SEQ ID No 2 being the sequence of the V-antigen itself, and SEQ ID No 4 being that of V-antigen with four extra vector defined N-termmal ammo acids. SEQ ID No 11 is that of an F1 protein as encoded for by SEQ ID No 10.

The preferred DNA constructs used in micro-organisms of the invention allow production of micro-organisms that when orally administered induce local stimulation of the gut-associated

lymphoid tissue (GALT) and, by trafficking of lympnocytes through the common mucosal immune system provide a secondary stimulation of the bronchial associated lymphoid tissue (BALT) In this manner a secretory IgA response is achieved at the respiratory mucosal surface.

The micro-organisms provided by transformation using this DNA in vector or directly inserted format, are preferably attenuated, more preferably attenuated salmonella.

Attenuated micro-organisms such as S. typhimuπum have been well characterised as

/ earners for various heterologous antigens (Curtiss, (1990); Cardenas and Clements, (1992)). Attenuation may be effected in a number of ways, such as by use of the aro A and/or aro C mutation approach (see Hosieth et al (1981 ) Nature 291 , 238-239; Dougan et al (1986) Parasite Immunol 9, 151-160; Chatfield et al (1989) Vaccine 7, 495-498); multiple mutations such as aro A and aro C mutants as described by Hone et al (1991) Vaccine 9, pp 810-816 may also be used. However, any suitably defective organism that is safe for intended use may be employed.

Many other such attenuated deletions and mutations will be known for these and other micro¬ organisms which will render them suitable for transformation with constructs of the present invention for the purposes of expressing vaccine proteins in the gut and/or gut colonisation in animals to be treated for Y. pestis. For human vaccination vectors containing the constructs of the present invention are placed in attenuated S. typhi and that transformed organism used as active agent for a live oral vaccine.

When DNA is used to transform the attenuated micro-organism by direct insertion into micro¬ organism DNA this may be by direct integration into a gene. Alternatively when incoφorated in the form of a plasmid that expresses V or F1 protein or epitopic fragments thereof this may be such that only the V or F1 protein or fragment is expressed or that this is expressed as a fusion peptide with a further protein or peptide fragment, preferably including the other one of the antigemc F1/V components. Such further protein or peptide fragment might be such as

to promote export of mature proteins or peptide through the cell membrane or might be a further Y. pestis antigen.

The lcr gene was cloned from Y. pestis strain KIM by Price et al and its nucleotide sequence published in J Bacteπol (1989) 171 , pp 5646-5653. In the examples below this information was used to design oligonucleotide primers which could amplify the gene from Y. pestis (strain GB) using the polymerase chain reaction (PCR). PCR primers were designed to be complementary to respective sequences flanking the 5' and 3' ends of the IcrV gene but also having 5' end tails containing a restriction enzyme recognition site to enable amplified IcrV gene to be cloned directionally into a plasmid vector (the 5' PCR primer containing an EcorRI site and the 3' primer containing a Sacl site). These restriction enzyme sites are examples only and should not be seen as excluding other restriction enzymes.

In the examples below the constructs of the invention include a lac promoter, but other promoters such as the macrophage promoter (nirβ) may be used.

The production of F1 has been described fully in Oyston et al (1995) Infect. Immun. Vol. 63 No 2 p563 - see page 564 under results: Cloning and Expression of cafl.

The dosage of the antigen components in a vaccine may vary dependent upon an individual animals immune characteristics, but for immunisation in the mouse animal model of the examples below it has been found that 10μg of each of V and F1 per dose were effective in providing full protection when administered in a standard primer and booster schedule.

The antigens may be incoφorated into a conventional pharmaceutically acceptable carrier, no particular limitation being imposed here. Conveniently the antigens have been incoφorated into an oil in water emulsion. Adjuvants may be included in the vaccine composition, and particularly Freund's Incomplete adjuvant IFA has been found to be effective when treating the mouse model.

The carrier may be one suited to parenteral administration, particularly trapeπtoneal administration but optionally oral, in the case of micro-organism based vaccines, or administration in the form of droplets or capsules, such as liposomes or microcapsules as would be effective in deliveπng the composition to the airways of an individual for the puφose of evoking mucosal immune response. T e earner may also compπse a slow-depot release system e.g. Alhydrogel.

Another method of encapsulation includes the use of polymeric structures in particular linear block co-polymers. Biodegradable polymers for example poly-lactic acid with or without glycolic acid or block co-polymer may be used; these may contain the following repeat unit: (POP-POE) _n where POP is polyoxypropylene and POE is polyoxyethylene. Block co- polymers which contain (POP-POE)- are of particular use.

The method constructs micro-organisms and vaccines of the invention wnl πcvv ce exemplified by way of illustration only by reference to the following Sequence -istiπg ^gure and Examples Still further embodiments will be evident to those skilled in the an n the 'ignt of these

Figure 1 illustrates in bar-chart form the survival rates of a number of grcuDS against a challenge of Y pestis

Figure 2 illustrates in graphical form IgG priming responses to intramuscular BSA immunisation in Balb/c mice

SEQUENCE LISTING

SEQ ID No 1 Shows the nucleotide and derived am o acid sequence of a V-encodmg DNA with 6 bases of vector pMAL-p2 or pMAL-c2 into which it is cloned at the 5 ¹ end using the EcoRI site in sequence GAATTC (derived from the 5' end PCR primer) and at the 3' end at the Sail site in sequence GTCGAC (denved from the 3' end PCR primer) The base at position 1006 has been altered by PCR mutagenesis to a T to create a second in frame stop codon The start of the ammo acid sequence is C-termmal to a factor Xa cleavage site

SEQ ID No 2 Shows the ammo acid sequence of the peptide expressed by the insert DNA of the invention, with an additional four am o acids encoded for by the vector (I E + F S) at the N-terminal end

SEQ ID No 3 Shows the nucleotide and derived ammo acid sequence of a second V- encodiπg DNA of the invention with 10 bases of a vector pGEX-5X-2 into which it is cloned shown at the 5' enα using the EcoRI site in sequence GAATTC (GA deπved from the 5' end PCR pπmer) and the Sail site in sequence GTCGAC (GTCGAC denved from the 3' end PCR pπmen The base at position 1006 has been altered by PCR mutagenesis to create a seconα in frame stop codon the base at position 16 has been altered to a C from an A to create the EcoRI site The start of the ammo acid sequence is C-terminal to a factor Xa cleavage site

SEQ ID No 4 Shows the ammo acid sequence of the peptide expressed by the DNA of SEQ ID No 3, with four ammo acids encoded by the vector (G, I, P and G) at the N-terminal end

SEQ ID No 5 Shows the nucleotide sequence of a gene 5' end primer oligonucleotide used to generate V-encodmg DNA used in SEQ ID No 1.

SEQ ID No 6 Shows the nucleotide sequence of a gene 3' end primer oligonucleotide used to generate V-encodmg DNA used in the Examples.

SEQ ID No 7 Shows the nucleotide sequence of a PCR primer oligonucleotide corresponding to the first 21 bases encoding for mature cafl with an additional 5' region encoding for a Sacl site

SEQ ID No 8 Shows the nucleotide sequence of a PCR pπmer oligonucleotide corresponding to the sequence of cafl which encodes a 'stem loop' downstream of the termination codon with an added 5' region encoding Sacl and Accl sites.

SEQ ID No 9 Shows the nucleotide sequence of a PCR pπmer oligonucleotide corresponding to an internal end region of the cafl gene starting 107 bases downstream from the end of the first oligonucleotide

SEQ ID No 10: Shows the nucleotide sequence of the pFGAL2a construct snowing the fusion of the first few bases of the β-galactosidase sequence in the vector with caf 1 minus :ts signal sequence and having a 5' tail including a Sac I restriction site the sequence is shown up to the cafl AACC 3' end with some vector bases

SEQ ID No 11 : Shows the ammo acid sequence of the protein encoded by oFGALΣa This sequence may be proceeded by Met, Thr, Met, lie, Thr, Asn

SEQ ID No 12: is that of primer FIOU2 used to amplify the F1 operon

SEQ ID No 13: is that of pπmer M4D used to amplify the F1 operon

SEQ ID No 14: is that of primer M3U used to amplify the F1 operon.

SEQ ID No 15: is that of primer FIOD2 used to amplify the F1 operon.

SEQ ID No 16: is the nucleotide and derived amino acid sequence of a DNA fragment encoding an F1-V fusion protein. There is a Sacl cloning site at the 5' end and a Hind III cloning site at the 3' end. Bases 452-472 is a sequence contained in the cloned insert, but derived from PCR primers (not found in Y. pestis DNA).

SEQ ID No 17: is the amino acid sequence of SEQ ID No 16

SEQ ID No 18: is that of primer 5'FAB2 used to amplify the F1 operon including signal sequence.

SEQ ID No 19: is that of primer 3'FBAM used to amplify the F1 operon including signal sequence.

SEQ ID No 20: is the nucleotide and amino acid sequence for F1 antigen as defined by PCR primers detailed in exemplified SEQ ID No 18 and 19 including signal sequence

SEQ ID No 21 . is the ammo acid sequence of SEQ ID No 20.

SEQ ID No 22. is the nucleotide and ammo acid sequence of F1Λ/ fusion protein including a 5 amino acid linker region. The T at position 1522 was modified from G to create a second in frame stop codon.

SEQ ID No 23. is the amino acid sequence of SEQ ID No 22. The linker region referred to in SEQ ID No 22 is at amino acid position 171-176 (bases 523-540 in SEQ. ID No 22).

SEQ ID No 24 shows the nucleotide sequence of a gene 5' end primer oligonucleotide used to generate V-encoding DNA used in SEQ ID No 3.

EXAMPLES

Cloning of SEQ. ID No 3

Materials and Methods: Materials for the preparation of growth media were obtained from Oxoid Ltd. or Difco Laboratories. All enzymes used in the manipulation of DNA were obtained from Boehringer Mannheim UK Ltd. and used according to the manufacturer s instructions. All other chemicals and biochemicals were obtained from Sigma chemical Co unless otherwise indicated. Monospecific rabbit polyclonal anti-V and mouse anti-GST sera were prepared by Dr R Brubaker (Department of Microbiology, Michigan State University) and Dr E D Williamson (Chemical and Biological Defence Establishment), respectively.

Bacterial strains and cultivation: Yersinia pestis GB was cultured aerobically at 28°C in a liquid medium (pH 6.8) containing 15g proteose peptone, 2.5g liver digest, 5g yeast extract. 5g NaC1 per litre supplemented with 80ml of 0.25% haemin dissolved in 0.1 M NaOH (Blood Base Broth). Escherichia coli JM109 was cultured and stored as described by Sambrook et al. Molecular Cloning. A Laboratory Manual.

Production of recombinant V and F1 proteins: Manipulation of DNA, Chromosomal DNA was isolated from Y. pestis by the method of Marmur.

Production of recombinant V-antigen: The gene encoding V-antigen (1crV) was amplified from Y. pestis DNA using the polymerase chain reaction (PCR) with 125pmol of primers homologous to sequences from the 5' and 3' ends of the gene (see Price et al (1989). J. Bacteriol 171 p5646-5653).

The sequences of the 5' primer (V/5'E: GATCGAATTCGAGCCTACGAACAA) and the 3' primer (GGATCGTCGACTTACATAATTACCTCGTGTCA) also included 5' regions encoding the restriction sites EcoRI and Sail, respectively. In addition, two nucleotides were altered from the published sequence of IcrV (Price et al, 1989), so that the EcoRI site was completed and the amplified gene encoded an extra termination codon (TAA). The PCR primers were

prepared with a DNA synthesiser (392 Applied Biosystems) Applied Biosystems. A DNA fragment was obtained after 30 cycles of amplification (95°C, 20secs, 45°C, 20secs, 72°C _, 30secs Perkiπ 9600 GeneAmp PCR System). The fragment was purified, digested with EcoRI and Sail. Iigated with suitably digested plasmid pGEX-5X-2 and transformed into £_ coli J 109 by electroporation (see Sambrook et al 1989). A colony containing the recombinant plasmid (pVG100) was identified by PCR using 30-mer primers (5' nucleotides located at positions 54 and 794: see Price et al 1989) which amplified an internal segment of the IcrV gene.

Expression of rV in ~, cpli. Cultures of E. coli JMl09/pVG100 were grown in LB containing 100μgml ^"1 ampicillin at 37°C until the absorbance (600nm) was 0.3. Isopropyl-β-D- thiogalactopyranoside (IPTG) was then added to the culture to a final concentration of 1mM and growth was continued for a further 5 hours. Whole cell lysates of the bacteria were prepared as described in Sambrook et al and expression of the GSTΛV fusion protein was examined by staining 10% SDS-polyacrylamide gels (Mini-Protean II, BioRad) with Coomassie Brilliant Blue R250 and by Western Blotting (see Sambrook et al). Western Blots were probed with rabbit anti-V serum diluted 1/4000 or mouse anti-GST serum in dilution at 1/1000 and protein bands were visualised with a colloidal gold labelled secondary antibody (Auroprobe BLplus, Cambio).

^Q uantification of GST/V ex ^p ression in vitro Cultures of E. coli JM109 containing pVG100 or pGEX-5X-2 were grown as described above. One ml aliquots were removed from the cultures in logarithmic and stationary phases and the number of viable cells determined by inoculating onto L-agar containing ampicillin. 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 -20°C for 1 hour, thawed and then sonicated on ice at 10°C lower for 3 x 30 sees (model XL2015 sonicator, 3.2mm Microtip probe; Heat Systems Inc.). The sonicates and a standard solution of rV (5μgml ^"1 ) were serially diluted in PBS in a microtitre plate and allowed to bind overnight at 4°C. The quantity of GST/V fusion protein in eacn sonicate was determined in a standard EL1SA using rabbit anti. V serum as the primary antibody Antibodies were incubated in 1% skimmed milk powder in PBS.

Purification of rV. E. coli JM109/pVG100 was grown in 5 x 100 ml volumes of L3 as described above. The cells were harvested by centrifugation ano resuspende _d in 3 mi phosphate buffered saline (PBS). After the addition of 80ul lysozyme (10 mgrm ^' ). the ceil suspension was incubated for 10 min at 22°C. Triton X-100 was addεα to a fins, concentration of 1% and the cells were frozen (-20°C), thawed and sonicated on ice at 70% power for 3 x 30 s (model XL2015 somcator). The lysed cells were centπfuged, and the supernatant was made up to 30ml with PBS and mixed with 5ml of Glutathione Sepaharose 4B (Pharmacia Biotech) which had been washed three times with PBS + 0.1% Triton X-100. The mixture was stirred for 18 hours at 4°C, centrifuged and washed twice in 100 ml PBS, and then packed into a chromatography column (Poly-Prep; Bio-rad) as a 50% slurry The GST/V fusion protein was eluted with 10 ml of 50 mM Tris pH 8.0 containing 5 mM reduced glutathione (Pharmacia Biotech). After dialysis against PBS, the fusion protein was cleaved with factor Xa (Boehringer Mannheim UK Ltd) for 18 hours at 22°C, according to the manufacturer's instructions. Cleaved GST and excess uncleaved GST/V were removed from the solution by affinity adsoφtion, as described above, to leave purified recombinant V (rV).

Immunisation with rV. Six week old female Balb/c mice, raised under specific pathogen-free conditions (Charles. River Laboratoπes, Margate, Kent, UK), were used in this study. A group of 16 mice received a 0.2ml primary immunising dose intraperitoneally (i.p.) of 10.13μg of rV antigen, presented in a 1 :1 water-in-oil emulsion with Incomplete Freund's Adjuvant (IFA). On days 14 and 34, each animal received booster immunising doses, prepared as above. On day 64, 6 animals were sacrificed and their tissues were removed for immunological analyses, as described below. The remaining animals were challenged with Y. pestis. An untreated control group of 16 age-matched mice were divided similarly into groups for tissue sampling and challenge. In a subsequent experiment to determine the degree of protection against higher challenge doses of Y pestis groups of 5 or 6 rV- immunised and control mice were used.

Measurement of serum antibody titre. Blood was sampled by cardiac puncture from mice anaesthetised i.p. with a 0.1ml cocktail containing 6mg of Domitor (Norden Laboratories) and

27μg of Ketalar (Parκe-Davιes) The samples were pooled and the serum was separated The serum antibody titre was measured by a modified ELISA (Willamson and Tiball, (1993) Vaccine 1 1 1253-1258) Briefly, rV (5 μgml ^'1 in PBS) was coated onto a microtitre plate and the test sera were serially diluted in duplicate on the plate Bound antibody was detected using peroxiαase labelled conjugates of anti-mouse polyvalent Ig. The titre of specific antibody was estimated as the maximum dilution of serum giving an absorbance reading greater than 0 1 units after subtraction of the absorbance due to non-specific binding detected in the control sera

Isolation of purified T cells from the spleen A crude suspension of mixed spleen cells was prepared by gently gπnding the spleen on a fine wire mesh. The cells were flushed from the splenic capsule and connective tissue with 2ml of tissue culture medium (DMEM with 20mM L-glutamme, 10 ⁵ U1 ^'1 of penicillin and 100mgl ^"1 of strepomycin). A population of mixed lymphocytes was separated from the spleen cell suspension by density gradient centrifugation of Ficoll-Hypaque (Lymphocyte Separation Medium, ICN Flow). A mixed acπdme orange (0.0003% w/v) and ethidium bromide (0.001% w v) stain was used to determine the percentage of viable cells in the preparation.

The mixed lymphocytes were incubated with sheep anti-mouse IgG-coated Dynabeads (M450), Dynal UK) at a ratio of 1 3 for 30 minutes at 4°C The Dynabead linked B cells were removed by magnetic separation and the remaining T cells were resuspended in DMEM, supplemented with antibiotic and 10% v/v foetal calf serum (FCS) at the desired cell density for seeding to microtitre plates

In vitro proliferation of crude s ^p leen cells and puπfied T cells against rV. Doubling dilutions of rV or Concanavalin A (positive control) in DMEM (range 0-50μgml ^*1 ) were made in the wells of a microtitre plate such that 0 1ml remained in each well. Negative controls consisted of 0 1 ml of DMEM alone An equal volume of the crude spleen cell or puπfied T cell suspension was seeded into each well at a minimum density of 5x10 ⁴ cells and incubated for 72 hours at 37 ^β C (5% C0 ₂ ) One μCi of ³ H thymidme ([methyl[ ³ H]thymidine S.A. 74 GBqmmol ^'1 , Amersham) in 30μl of DMEM supplemented with 10% FCS was aliquoted into each well and

incubation was continued for 24 h The well contents were harvested onto glass fibre filters using a cell harvester (Titertek) and discs representing each well were punched from the filter mats into 1 5ml of scintillation fluid (Cyoscmt, ICN Biomedicais !nc ) to measure the incorporation of H thymidine into cells The cell stimulation inαex was caicuiateα τom - replicates as mean cpm (stιmulated).mean cpm (negative control)

Production of recombinant F1 antigen Cloning of cafl DNA was isolated from Y pestis by the method of Marmur et al (1961) J Mol Biol 3 pp 208-218 A DNA fragment encoding the open reading frame of cafl minus its signal sequence was amplified from this using the polymerase chain reaction (PCR) Oligonucleotides were prepared with a Beckman 200A DNA synthesiser for use in the PCR

pFGAL2a construct Oligonucleotide GATCGAGCTCGGCAGATTTAACTGCAAGCACC (SEQ ID No 7) was synthesised corresponding to the first 21 bases of the cafl gene immediately following the nucleotides encoding the signal sequence with an additional 5' region encoding a Sacl site and the complimentary oligonucleotide CAGGTCGAGCTCGTCGACGGTTAGGCTCAAAGTAG (SEQ ID No 8) corresponding to the sequence which encodes a putative 'stem loop' structure downstream of the cafl termination codon with an added 5' region encoding Sacl and Accl sites A DNA fragment was obtained after 35 cycles of amplification (95°C, 15 sees, 50°C, 15 sees, 72°C, 30 sees using a Perkin Elmer 9600 GeneAmp PCR system) The fragment was purified digested with Sacl and Accl. Iigated into a similarly digested pUC18 plasmid and transformed into E coli JM109 by electroporation. Electroporation was earned out using a Biorad Gene Pulser with 0 2 cm cuvettes at 1 25kV. 25μF, 800 Ohms with a time constant of 20

A pFGAL2a colony containing the cloned cafl gene was identified by PCR using an oligonucleotide TGGTACGCTTACTCTTGGCGGCTAT (SEQ ID No 9) corresponding to an internal region of the gene 128 to 153 nucleotides from the site identified as the signal sequence cleavage site (see Galyov et al (1990)) and the SEQ ID No 2 An F1 expressing E_ coli culture containing the pFGAL2a was grown at 37°C with shaking in Luna Broth with 1mM

isopropyl-β-D-thiogalactopyranoside (IPTG) for 18 hours. Whole cell lysates and periplasmie and cytoptasmic fractions of the bacteria were prepared as described by Sambrook et al

(1989).

SDS-PAGE and Western blotting: SDS-polyacrylanπide gel electrophoresis (PAGE) and Western blotting were performed as described by Hunter et al (1993) Infec. Immun. 61. 3958- 3965. Blots were probed with polyclonal antisera raised in sheep (B283) against killed Y. pestis (EV76 strain grown at 37°C) and bound antibody was detected with a horseradish peroxidase-labelled donkey anti-sheep IgG (Sigma).

FXAMPLES 1 AND 2 OF COMBINATION V/F1 VACCINE. COMPARATIVE EXAMPLES EFFECT OF VACCINES ON MORTALITY OF MICE ON CHALLENGE WITH v PESTIS .GB. STRAIN

Animals- Barrier bred female 6-week old Balb/c mice free of mouse oathcgens were obtained from Charles-Reiver Laboratories, Margate. Kent. UK and were used throughout these Examples.

Immunisation: Mice were divided into groups of 5 or 6 and immunised as follows.

Comparative vaccine EV76: A total of 50 mice received a single subcutaneous (s.c.) priming dose of live EV76 vaccine on day 0 of the schedule delivered in a total volume of 100μl.

Comparative vaccine Cutter USP vaccine: A further group of 50 mice were pπmed intramuscularly (i.m.) with 100μl of Cutter vaccine; this representing about 0.2 of the human dose and comprised approximately 2 x 10 ⁸ formaldehyde killed plague bacilli. This dose was administered to each animal again on day 16 of the schedule to effect booster immunisation.

F1 and V vaccines: Groups of 62 mice received a primary immunising dose intra-peπtoneally (i.p.) of either recombinant V-antigen or recombinant F1-antιgen, presented in a 1 :1 water-in- oil emulsion with incomplete Freunds adjuvant (IFA; Sigma). Animals were primed with a 10μg dose of the respective antigen in a total volume of 0.1ml emulsion Animals were boosted with the respective antigen as appropriate on days 14 (V and F1 groups) and 28 (all F1 group and a sub-group of 12 of the V group).

A further group of 12 mice were primed and boosted on days 0 14 and 28 with a combination of 10μg F1 and 10μg V jointly incoφorated into the aqueous phase of a 1 1 water-in-oil emulsion with IFA (final volume 0.1 ml per mouse)

On day 50 of the immunisation schedule, 6 animals were selected at random from each of the treatment grouDS for subsequent analysis of spleen cell responses. The remaining animals in each group were challenged with Y. pestis. An untreated control group of age- matched mice was similarly split.

Multiple LD challenge to determine limits of protection: Mice from each of the immunised groups and untreated controls were divided into groups of 5 or 6 for challenge by the s.c. route with Y. pestis GB strain in the dose range 20 to 2 x 10 ⁹ viable organisms. Challenged mice were closely observed over a 14-day period for the development of symptoms and where appropriate time to death was carefully recorded.

Animals which succumbed to the challenge were autopsied and blood smears, livers and spleens removed for bacteriological analysis.

The results of these challenges on control and test animals is given in Table 1 below; two sets of results being given corresponding two experimental runs with respective controls. From these results it can clearly be seen that while the V antigen is more effective than Cutter, it is inferior to EV76. However, when combined with the less effective F1 the combination is as effective as EV76 without side effects.

TABLE 1

= Example 1 = Example 2

EXAMPLE 3. Production of Attenuated Salmonella for use in oral vaccine-

Expression of recombinant V-antioen from S typhimurium and typhi. Amplified IcrV gene was cloned into three different plasmid vectors:

pMAL-p2: a vector designed to express the cloned gene as a fusion product with a maltose binding protein (MBP). The C-terminus of the MBP is fused to the N-terminus of the V- antigen. The fusion protein so produced on expression is exported to the periplasm. Vector including the V-antigen DNA sequence was designated pVMP100.

pMAL-c2. a vector similar to pMAL-p2 except that MBP-V antigen fusion protein is expressed cytoplasmically. The recombinant plasmid was designated pVMC OO

pGEX-5X-2. a vector designed to express the cloned gene as a fusion protein with glutathione-S-transferase (GST). The C-terminus of GST is fused to the N-terminus of V antigen and the fusion protein is expressed cytoplasmically The recombinant plasmid was designated pVGIOO

All the vectors contain the P _lac promoter and the lacl ^Q gene; the latter encoding the 1ac repressor which turns off transcription from P _tac in Escheπchia coli until IPTG is added. The plasmids contain the origin of replication from pBR322 and as a result replicate to a low copy number in the bacterial cell.

Each of the recombinant plasmids was electroporated into Salmonella typhimuπum strain SL3261, an attenuated strain that has been used extensively as a live vaccine vector for the expression of foreign antigens. It contains a specific deletion mutation in the aroA gene which makes the mutant dependent upon certain aromatics for growth (see Hosieth et al) For producing microorganism suitable for human vaccination use electroporation is into attenuated Salmonella typhi.

The recombinant plasmids all expressed V antigen as shown by Western blotting of £_ typhimurium cultures and probing with a monospecific anti-V antigen polyclonal antiserum supplied by R Brubaker, Dept Microbiology, Michigan State University, East Lansing, Ml 48824-1101 , USA. Recombinant S. typhimurium were mnoeulated intravenously into mice at 5 x 10 ⁷ cfu/dose and shown to colonise the liver and spleen at high levels, between 8 x 10 ⁶ and 5 x 10 ⁸ cfu per organ were recovered. The majority of the bacterial cells recovered were also ampicillin resistant indicating retention of recombinant plasmids

Expression of F1 in S typhimurium The pFGAL2a plasmid was isolated using general techniques described in Sambrook et al (1989) Molecular Cloning; a Laboratory Manual, 2nd Edition Cold Spπng Harbour Laboratory, New York. Purified plasmid was electroporated into S typhimuπum LB5010 (restnction , modification ^* ) and methylated pFGAL2a was suDsequently isolated from the LB5010 for electroporation into S. typhimurium SL3261 (aro A ^' ; Periplasmie and cyptoplasmie fractions were prepared for SDS-PAGE and Western blotting as descπbed above

Stabilit ^y of constmcts Five female Balb/c mice were inoculated intravenously with either 5 x 10 ⁵ or 5 x 10 ⁷ cfu S. tv ^p himuπum containing pFGAl_2a in 200μL phosphate buffered saline Control mice were inoculated similarly with S. tvphimuπum containing pUC18 with no insert After 7 days the mice were killed by cervical dislocation and their livers and spleens removed The organs were homogenised in 10ml phosphate buffered saline using a stomacher on maximum setting for 2 minutes and the homogenate was serially diluted in phosphate buffered saline and placed onto L agar or L agar containing 55μg mr ¹ ampicillin.

F1 operon construct: Attempts to PCR replicate the entire F1 operon as one piece were unsuccessful, so a strategy was developed whereby it was amplified using PCR to produce two discrete fragments using pπmer pairs (A) of SEQ ID No 12 and 13 and (b) of SEQ No 14 and 15 respectively to produce fragments of 3.36kb and 1.89kb from Y. pestis MP6 template DNA Marmur extract of DNA was used without CsC1 ₂ purification. The PCR cycle conditions used were 96°C for 30 seconds, 57°C for 30 seconds and 72 ^β C for 1 minute; total of 30 cycles

These two fragments were digested using Nhe1 and joined together.

This fused fragment, encoding the full length operon (5.25kb), was digested with EcoRI and Sail and then cloned into a number of vectors. When this fragment was cloned into pBR322 and expressed in E. CPU, S. typhimurium LB5010 or SL3261 instability of the recombinant plasmid was noted. To circumvent this problem the operon was cloned into plasmid pLG339, a low copy number plasmid km ^R . The entire F1 operon was also inserted into AroC gene on the chromosome of gene on the chromosome of S. typhimurium using vector pBRD1084.

SEQUENCE LISTING

(1 ) GENERAL INFORMATION:

(i) APPLICANT SECRETARY OF STATE FOR DEFENCE, . .

(ii) TITLE OF INVENTION: VACCINES

(iii) NUMBER OF SEQUENCES: 24

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: SECRETARY OF STATE FOR DEFENCE

(B) STREET: WHITEHALL

(C) CITY: LONDON

(D) STATE: LONDON

(E) COUNTRY: UNITED KINGDOM (GB) (F) ZIP: SW1A 2HB

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE.

(C) CLASSIFICATION:

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1014 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Yersinia pestis

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..987

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :

ATT TCA GAA TTC ATT AGA GCC TAC GAA CAA AAC CCA CAA CAT TTT ATT 48 l ie Ser Glu Phe lie Arg Ala Tyr Glu Gin Asn Pro Gin His Phe He 1 5 10 15

GAG GAT CTA GAA AAA GTT AGG GTG GAA CAA CTT ACT GGT CAT GGT TCT 96 Glu Asp Leu Glu Lys Val Arg Val Glu Gin Leu Thr Gly H s Gl Ser 20 25 3 0

TCA GTT TTA GAA GAA TTG GTT CAG TTA GTC AAA GAT AAA AAT ATA GAT 144 Ser Val Leu Glu Glu Leu Val Gin Leu Val Lys Asp Lys.Asn lie Asp 35 40 45

ATT TCC ATT AAA TAT GAT CCC AGA AAA GAT TCG GAG GTT TTT GCC AAT 192 He Ser He Lys Tyr Asp Pro Arg Lys Asp Ser Glu Val Phe Ala Asn 50 55 60

AGA GTA ATT ACT GAT GAT ATC GAA TTG CTC AAG AAA ATC CTA GCT TAT 240

J Arg Val He Thr Asp Asp He Glu Leu Leu Lys Lys He Leu Ala Tyr

65 70 75 80

TTT CTA CCC GAG GAT GCC ATT CTT AAA GGC GGT CAT TAT GAC AAC CAA 288 Phe Leu Pro Glu Asp Ala He Leu Lys Gly Gly His Tyr Asp Asn Gin

85 90 95

CTG CAA AAT GGC ATC AAG CGA GTA AAA GAG TTC CTT GAA TCA TCG CCG 336 Leu Gin Asn Gly He Lys Arg Val Lys Glu Phe Leu Glu Ser Ser Pro 100 105 110

AAT ACA CAA TGG GAA TTG CGG GCG TTC ATG GCA GTA ATG CAT TTC TCT 384 Asn Thr Gin Trp Glu Leu Arg Ala Phe Met Ala Val Met His Phe Ser 115 120 125

TTA ACC GCC GAT CGT ATC GAT GAT GAT ATT TTG AAA GTG ATT GTT GAT 432 Leu Thr Ala Asp Arg He Asp Asp Asp He Leu Lys Val He Val Asp 130 135 140

TCA ATG AAT CAT CAT GGT GAT GCC CGT AGC AAG TTG CGT GAA GAA TTA 480 Ser Met Asn His His Gly Asp Ala Arg Ser Lys Leu Arg Glu Glu Leu 145 150 155 160

GCT GAG CTT ACC GCC GAA TTA AAG ATT TAT TCA GTT ATT CAA GCC GAA 528 Ala Glu Leu Thr Ala Glu Leu Lys He Tyr Ser Val He Gin Ala Glu 165 170 175

ATT AAT AAG CAT CTG TCT AGT AGT GGC ACC ATA AAT ATC CAT GAT AAA 576 He Asn Lys His Leu Ser Ser Ser Gly Thr He Asn He His Asp Lys 180 185 190

TCC ATT AAT CTC ATG GAT AAA AAT TTA TAT GGT TAT ACA GAT GAA GAG 624 Ser He Asn Leu Met Asp Lys Asn Leu Tyr Gly Tyr Thr Asp Glu Glu 195 200 205

ATT TTT AAA GCC AGC GCA GAG TAC AAA ATT CTC GAG AAA ATG CCT CAA 672 He Phe Lys Ala Ser Ala Glu Tyr Lys He Leu Glu Lys Met Pro Gin 210 215 220

ACC ACC ATT CAG GTG GAT GGG AGC GAG AAA AAA ATA GTC TCG ATA AAG 720 Thr Thr He Gin Val Asp Gly Ser Glu Lys Lys He Val Ser He Lys 225 230 235 240

GAC TTT CTT GGA AGT GAG AAT AAA AGA ACC GGG GCG TTG GGT AAT CTG 768 Asp Phe Leu Gly Ser Glu Asn Lys Arg Thr Gly Ala Leu Gly Asn Leu 245 250 255

AAA AAC TCA TAC TCT TAT AAT AAA GAT AAT AAT GAA TTA TCT CAC TTT 816 lys Asn Ser Tyr ≤er Tyr Asn Lys Asp Asn Asn Glu Leu Ser His Phe 260 265 270

GCC ACC ACC TGC TCG GAT AAG TCC AGG CCG CTC AAC GAC TTG GTT AGC 864 Λla Thr Thr Cys Ser Asp Lys Ser Arg Pro Leu Asn Asp Leu Val Ser 275 280 285

CAA AAA ACA ACT CAG CTG TCT GAT ATT ACA TCA CGT TTT AAT TCA GCT 912 Gin Lys Thr Thr Gin Leu Ser Asp He Thr Ser Arg Phe Asn Ser Ala 290 295 300

ATT GAA GCA CTG AAC CGT TTC ATT CAG AAA TAT GAT TCA GTG ATG CAA 960 He Glu Ala Leu Asn Arg Phe He Gin Lys Tyr Asp Ser Val Met Gin 305 310 315 320

CGT CTG CTA GAT GAC ACG TCT GGT AAA TGACACGAGG TAATTATGTA 1007 Arg Leu Leu Asp Asp Thr Ser Gly Lys 325

AGTCGAC 1014

(2) INFORMATION FOR SEQ ID NO:2.

(I) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 329 ammo acids (D) TOPOLOGY: linear

(ii) MOLECULE TYPE, protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

He Ser Glu Phe He Arg Ala Tyr Glu Gin Asn Pro Gin H s Phe He 1 5 10 15

Glu Asp Leu Glu Lys Val Arg Val Glu Gin Leu Thr Gly His Gly Ser 20 25 30

Ser Val Leu Glu Glu Leu Val Gin Leu Val Lys Asp Lys Asn He Asp 35 40 45

He Ser He Lys Tyr Asp Pro Arg Lys Asp Ser Glu Val Phe Ala Asn 50 55 60

Arg Val He Thr Asp Asp He Glu Leu Leu Lys Lys He Leu Ala Tyr 65 70 75 80

Phe Leu Pro Glu Asp Ala He Leu Lys Gly Gly His Tyr Asp Asn Gin

85 90 9 Ξ

Leu Gin Asn Gly He Lys Arg Val Lys Glu Phe Leu Glu Ser Ser Pro 100 105 110

Asn Thr Gin Trp Glu Leu Arg Ala Phe Met Ala Val Met His Phe Ser 115 120 125

Leu Thr Ala Asp Arg He Asp Asp Asp He Leu Lys Val He Val Asp 130 135 140

Ser Met Asn His His Gly Asp Ala Arg Ser Lys Leu Arg Glu Glu Leu 145 150 155 160

Ala Glu Leu Thr Ala Glu Leu Lys He Tyr Ser Val He Gin Ala Glu 165 170 175

He Asn Lys His Leu Ser Ser Ser Gly Thr He Asn He His Asp Lys 180 185 190

Ser He Asn Leu Met Asp Lys Asn Leu Tyr Gly Tyr Thr Asp Glu Glu 195 200 205

He Phe Lys Ala Ser Ala Glu Tyr Lys He Leu Glu Lys Met Pro Gin 210 215 220

Thr Thr He Gin Val Asp Gly Ser Glu Lys Lys He Val Ser He Lys 225 230 235 240

sp Phe Leu Gly Ser Glu Asn Lys Arg Thr Gly Ala Leu Gly Asn Leu 245 250 255

Lys Asn Ser Tyr Ser Tyr Asn Lys Asp Asn Asn Glu Leu Ser H s Phe 260 265 270

Ala Thr Thr Cys Ser Asp Lys Ser Arg Pro Leu Asn Asp Leu Val Ser 275 280 285

Gin Lys Thr Thr Gin Leu Ser Asp He Thr Ser Arg Phe Asn Ser Λla 290 295 300

He Glu Ala Leu Asn Arg Phe He Gin Lys Tyr Asp Ser Val Mec Gin 305 310 315 320

Arg Leu Leu Asp Asp Thr Ser Gly Lys 325

(2) INFORMATION FOR SEQ ID NO.3.

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 1014 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS. double

(D) TOPOLOGY, linear

(ii) MOLECULE TYPE. DNA (genomic)

(iii) HYPOTHETICAL. NO

(iv) ANTI-SENSE. NO

(vi) ORIGINAL SOURCE.

(A) ORGANISM: Yersinia pestis

(ix) FEATURE.

(A) NAME/KEY: CDS

(B) LOCATION: 1..987

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GGG ATC CCC GGA ATT CGA GCC TAC GAA CAA AAC CCA CAA CAT TTT ATT 48 Gly He Pro Gly He Arg Ala Tyr Glu Gin Asn Pro Gin His Phe He

5 10 15

GAG GAT CTA GAA AAA GTT AGG GTG GAA CAA CTT ACT GGT CAT GGT TCT 96 Glu Asp Leu Glu Lys Val Arg Val Glu Gin Leu Thr Gly His Gly Ser 20 25 20

TCA GTT TTA GAA GAA TTG GTT CAG TTA GTC AAA GAT AAA AAT ATΛ GAT _._ _ Ser Val Leu Glu Glu Leu Val Gin Leu Val Lys Asp Lys Asn He Asp 35 40 45

ATT TCC ATT AAA TAT GAT CCC AGA AAA GAT TCG GAG GTT TTT GCC AAT 192 He Ser He Lys Tyr Asp Pro Arg Lys Asp Ser Glu Val Phe Ala Asn 50 55 60

AGA GTA ATT ACT GAT GAT ATC GAA TTG CTC AAG AAA ATC CTA GCT TAT 240 Arg Val He Thr Asp Asp He Glu Leu Leu Lys Lys He Leu Ala Tyr 65 70 75 80

TTT CTA CCC GAG GAT GCC ATT CTT AAA GGC GGT CAT TAT GAC AAC CAA 288 Phe Leu Pro Glu Asp Ala He Leu Lys Gly Gly His Tyr Asp Asn Gin 85 90 95

CTG CAA AAT GGC ATC AAG CGA GTA AAA GAG TTC CTT GAA TCA TCG CCG 336 Leu Gin Asn Gly He Lys Arg Val Lys Glu Phe Leu Glu Ser Ser Pro 100 105 110

AAT ACA CAA TGG GAA TTG CGG GCG TTC ATG GCA GTA ATG CAT TTC TCT 384 Asn Thr Gin Trp Glu Leu Arg Ala Phe Met Ala Val Met His Phe Ser 115 120 125

TTA ACC GCC GAT CGT ATC GAT GAT GAT ATT TTG AAA GTG ATT GTT GAT 432 Leu Thr Ala Asp Arg He Asp Asp Asp He Leu Lys Val He Val Asp 130 135 140

TCΛ ΛTG ΛAT CAT CAT GGT GAT GCC CGT AGC AAG TTG CGT GAA GAA TTA 480 Ser Met Asn His His Gly Asp Ala Arg Ser Lys Leu Arg Glu Glu Leu 145 150 155 160

GCT GAG CTT ACC GCC GAA TTA AAG ATT TAT TCA GTT ATT CAA GCC GAA 528 Ala Glu Leu Thr Ala Glu Leu Lys He Tyr Ser Val He Gin Ala Glu 165 170 175

ATT AAT AAG CAT CTG TCT AGT AGT GGC ACC ATA AAT ATC CAT GAT AAA 576 He Asn Lys His Leu Ser Ser Ser Gly Thr He Asn He His Asp Lys 180 185 190

TCC ATT AAT CTC ATG GAT AAA AAT TTA TAT GGT TAT ACA GAT GAA GAG 624 Ser He Asn Leu Met Asp Lys Asn Leu Tyr Gly Tyr Thr Asp Glu Glu 195 200 205

ATT TTT AAA GCC AGC GCA GAG TAC AAA ATT CTC GAG AAA ATG CCT CAA 672 He Phe Lys Ala Ser Ala Glu Tyr Lys He Leu Glu Lys Met Pro Gin 210 215 220

ACC ACC ATT CAG GTG GAT GGG AGC GAG AAA AAA ATA GTC TCG ATA AAG 720 Thr Thr He Gin Val Asp Gly Ser Glu Lys Lys He Val Ser He Lys 225 230 235 240

GAC TTT CTT GGA AGT GAG AAT AAA AGA ACC GGG GCG TTG GGT AAT CTG 768 Asp Phe Leu Gly Ser Glu Asn Lys Arg Thr Gly Ala Leu Gly Asn Leu 245 250 255

AAA AAC TCA TAC TCT TAT AAT AAA GAT AAT AAT GAA TTA TCT CAC TTT 816 Lys Asn Ser Tyr Ser Tyr Asn Lys Asp Asn Asn Glu Leu Ser His Phe 260 265 270

GCC ACC ACC TGC TCG GAT AAG TCC AGG CCG CTC AAC GAC TTG GTT AGC 864 Ala Thr Thr Cys Ser Asp Lys Ser Arg Pro Leu Asn Asp Leu Val Ser 275 280 285

CAA AAA ACA ACT CAG CTG TCT GAT ATT ACA TCA CGT TTT AAT TCA GCT 912 Gin Lys Thr Thr Gin Leu Ser Asp He Thr Ser Arg Phe Asn Ser Ala 290 295 300

ATT GAA GCA CTG AAC CGT TTC ATT CAG AAA TAT GAT TCA GTG ATG CAA 960 He Glu Ala Leu Asn Arg Phe He Gin Lys Tyr Asp Ser Val Met Gin 305 310 315 320

CGT CTG CTA GAT GAC ACG TCT GGT AAA TGACACGAGG TAATTATGTA 1007 Arg Leu Leu Asp Asp Thr Ser Gly Lys 325

AGTCGAC 1014

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 329 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Gly He Pro Gly He Arg Ala Tyr Glu Gin Asn Pro Gin His Phe He 1 5 10 15

Glu Asp Leu Glu Lys Val Arg Val Glu Gin Leu Thr Gly His Gly Ser 20 25 30

Ser Val Leu Glu Glu Leu Val Gin Leu Val Lys Asp Lys Asn He Asp 35 40 45

He Ser He Lys Tyr Asp Pro Arg Lys Asp Ser Glu Val Phe Ala Asn 50 55 60

Arg Val He Thr Asp Asp He Glu Leu Leu Lys Lys He Leu Ala Tyr 65 70 75 80

Phe Leu Pro Glu Asp Ala He Leu Lys Gly Gly His Tyr Asp Asn Gin

85 90 95

Leu Gin Asn Gly He Lys Arg Val Lys Glu Phe Leu Glu Ser Ser Pro 100 105 110

Asn Thr Gin Trp Glu Leu Arg Ala Phe Met Ala Val Met His Phe Ser 115 120 125

Leu Thr Ala Asp Arg He Asp Asp Asp He Leu Lys Val He Val Asp 130 135 140

Ser Met Asn His His Gly Asp Ala Arg Ser Lys Leu Arg Glu Glu Leu 145 150 155 160

Ala Glu Leu Thr Ala Glu Leu Lys He Tyr Ser Val He Gin Ala Glu

165 170 175

He Asn Lys His Leu Ser Ser Ser Gly Thr He Asn He His Asp Lys 180 185 190

Ser He Asn Leu Met Asp Lys Asn Leu Tyr Gly Tyr Thr Asp Glu Glu 195 200 205

He Phe Lys Ala Ser Ala Glu Tyr Lys He Leu Glu Lys Met Pro Gin 210 215 220

Thr Thr He Gin Val Asp Gly Ser Glu Lys Lys He Val Ser He Lys 225 230 235 240

Asp Phe Leu Gly Ser Glu Asn Lys Arg Thr Gly Ala Leu Gly Asn Leu 245 250 255

Lys Asn Ser Tyr Ser Tyr Asn Lys Asp Asn Asn Glu Leu Ser His Phe 260 265 270

Ala Thr Thr Cys Ser Asp Lys Ser Arg Pro Leu Asn Asp Leu Val Ser 275 280 285

Gin Lys Thr Thr Gin Leu Ser Asp He Thr Ser Arg Phe Asn Ser Ala 290 295 300

He Glu Ala Leu Asn Arg Phe He Gin Lys Tyr Asp Ser Val Met Gin 305 310 315 320

Arg Leu Leu Asp Asp Thr Ser Gly Lys

325

(2) INFORMATION FOR SEQ ID NO 5 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH. 28 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY linear

(n) MOLECULE TYPE. DNA (genomic)

(iii) HYPOTHETICAL. NO

(iv) ANTI-SENSE. NO

(vi) ORIGINAL SOURCE.

(A) ORGANISM: Yersinia pestis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5.

GATCGAATTC ATTAGAGCCT ACGAACAA 28

(2) INFORMATION FOR SEQ ID NO:6.

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 32 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY, linear

(n) MOLECULE TYPE. DNA (genomic)

(mi HYPOTHETICAL. NO

(ivi ANTI-SENSE. NO

(vi) ORIGINAL SOURCE.

(A) ORGANISM- Yersinia pestis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GGATCGTCGA CTTACATAAT TACCTCGTGT CA 32

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE. DNA (genomic)

(iii) HYPOTHETICAL. NO

(ιv) ANTI-SENSE. NO

(vi) ORIGINAL SOURCE.

(A) ORGANISM. Yersinia pestis

(xi) SEQUENCE DESCRIPTION SEQ ID NO 7

GGCAGATTTA ACTGCAAGCA CC

.2) INFORMATION FOR SEQ ID NO 8

(I) SEQUENCE CHARACTERISTICS

(A) LENGTH 35 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS double

(D) TOPOLOGY linear

(n) MOLECULE TYPE DNA (genomic)

(in) HYPOTHETICAL NO

(iv) ANTI-SENSE NO

(vi) ORIGINAL SOURCE

(A) ORGANISM Yersinia pestis

(xi) SEQUENCE DESCRIPTION SEQ ID NO 8

GAGGTCGAGC TCGTCGACGG TTAGGCTCAA AGTAG

(2) INFORMATION FOR SEQ ID NO 9

(i) SEQUENCE CHARACTERISTICS (A) LENGTH 25 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS. double

(D) TOPOLOGY: linear

Oil MOLECULE TYPE. DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE. NO

(vi) ORIGINAL SOURCE.

(A) ORGANISM: Yersinia pestis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

TGGTACGCTT ACTCTTGGCG GCTAT 25

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 541 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Yersinia pestis

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 2..454

(ix) FEATURE:

(A) NAME/KEY: misc_recomb

(B) LOCATION: 1..6

(ix) FEATURE:

(A) NAME/KEY: misc_recomb

(B) LOCATION: 536..541

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

G AGC TCG GCA GAT TTA ACT GCA AGC ACC ACT GCA ACG GCA ΛCT CTT 46

Ser Ser Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu 1 5 10 15

GTT GAA CCA GCC CGC ATC ACT ATT ACA TAT AAG GAA GGC GCT CCΛ ΛTT 94 Val Glu Pro Ala Arg He Thr He Thr Tyr Lys Glu Gly Al a Pro He 20 25 2 0

BAD ORIGINAL ft

ACA ATT ATG GAC AAT GGA AAC ATC GAT ACA GAA TTA CTT GTT GGT ACG 142 Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr 35 40 45

CTT ACT CTT GGC GGC TAT AAA ACA GGA ACC ACT AGC ACA TCT GTT AAC 190 Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn 50 55 60

TTT ACA GAT GCC GCG GGT GAT CCC ATG TAC TTA ACA TTT ACT TCT CAG 238

J Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin

65 70 75

GAT GGA AAT AAC CAC CAA TTC ACT ACA AAA GTG ATT GGC AAG GAT TCT 286 Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser 80 85 90 95

AGA GAT TTT GAT ATC TCT CCT AAG GTA AAC GGT GAG AAC CTT GTG GGG 334 Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly 100 105 110

GAT GAC GTC GTC TTG GCT ACG GGC AGC CAG GAT TTC TTT GTT CGC TCA 382 Asp Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser 115 120 125

ATT GGT TCC AAA GGC GGT AAA CTT GCA GCA GGT AAA TAC ACT GAT GCT 430 He Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala 130 135 140

GTA ACC GTA ACC GTA TCT AAC CAA TAATCCATAT AGATAATAGA TAAAGGAGGG 484 Val Thr Val Thr Val Ser Asn Gin 145 150

CTATTATGCC CTCCTTTAAT ATTTATGAAT TATCCTACTT TGAGCCTAAC CGTCGAC 541

(.2) INFORMATION FOR SEQ ID NO:11 :

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 151 ammo acids

(B) TYPE, ammo acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11 :

Ser Ser Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val 1 5 10 15

Glu Pro Ala Arg He Thr He Thr Tyr Lys Glu Gly Ala Pro He Thr 20 25 30

He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr Leu 35 40 45

Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe 50 55 60

Thr Asp Ala Λla Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin Asp 65 70 75 80

Gly Asn Asn His G n Phe Thr Thr Lys Val He Gly Lys Asp Ser Arg

35 90 95

Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp 100 105 110

Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser He 115 120 125

Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val 130 135 140

Thr Val Thr Val Ser Asn Gin 145 150

(2) INFORMATION FORSEQ IDNO:12:

(i) SEQUENCECHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Yersinia pestis

(xi) SEQUENCE DESCRIPTION SEQ ID NO 12

TCGCCCGGGA ATTCCGAACA TAAAT GGTT CAGTGGCC 38

(2) INFORMATION FOR SEQ ID NO 13

(i) SEQUENCE CHARACTERISTICS (A) LENGTH 29 base pairs (B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE DNA (genomic)

(in) HYPOTHETICAL NO

(iv) ANTI-SENSE NO

(vi) ORIGINAL SOURCE

(A) ORGANISM Yersinia pestis

(xi) SEQUENCE DESCRIPTION SEQ ID NO 13

GGCGTATTCC TCGCTAGCAA TGTTTAACG 29

(2) INFORMATION FOR SEQ ID NO 14

(i) SEQUENCE CHARACTERISTICS (A) LENGTH 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE. DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Yersinia pestis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

ATCGTTAAAC ATTGCTAGCG AGGAATACGC C 31

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

.iv) ANTI-SENSE NO

■ i ORIGINAL SOURCE.

(A) ORGANISM- Yersinia pestis

(xi) SEQUENCE DESCRIPTION' SEQ ID NO: 15.

GΛTAGATCTG TCGACTGAAC CTATTATATT GCTTCGCGC 39

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1462 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE. DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE. NO (vi) ORIGINAL SOURCE.

(A) ORGANISM. Yersinia pestis

(ix) FEATURE

(A) NAME/KEY: CDS

(B) LOCATION. 8..1447

(xi)SEQUENCEDESCRIPTION:SEQIDNO:16:

GAGCTCG GCA GAT TTA ACT GCA AGC ACC ACT GCA ACG GCA ACT CTT GTT 49 Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val 1 5 10

GAA CCA GCC CGC ATC ACT CTT ACA TAT AAG GAA GGC GCT CCA ATT ACA 97 Glu Pro Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala Pro He Thr 15 20 25 30

ATT ATG GAC AAT GGA AAC ATC GAT ACA GAA TTA CTT GTT GGT ACG CTT 145 He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr Leu 35 40 45

ACT CTT GGC GGC TAT AAA ACA GGA ACC ACT AGC ACA TCT GTT AAC TTT 193 Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe 50 55 60

ACA GAT GCC GCG GGT GAT CCC ATG TAC TTA ACA TTT ACT TCT CAG GAT 241 Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin Asp 65 70 75

GGA AAT AAC CAC CAA TTC ACT ACA AAA GTG ATT GGC AAG GAT TCT AGA 289 Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser Arg 80 85 90

GAT TTT GAT ATC TCT CCT AAG GTA AAC GGT GAG AAC CTT GTG GGG GAT 337 Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp 95 100 105 110

GAC GTC GTC TTG GCT ACG GGC AGC CAG GAT TTC TTT GTT CGC TCA ATT 385 Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser He 115 120 125

GGT TCC AAA GGC GGT AAA CTT GCA GCA GGT AAA TAC ACT GAT GCT GTA 433

J Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val

130 135 140

ACC GTA ACC GTA TCT AAC CAA GGA TCC ATC GAA GGT CGT ATT AGA GCC 481 Thr Val Thr Val Ser Asn Gin Gly Ser He Glu Gly Arg He Arg Ala 145 150 155

TAC GAA CAA AAC CCA CAA CAT TTT ATT GAG GAT CTA GAA AAA GTT AGG 529 Tyr Glu Gin Asn Pro Gin His Phe He Glu Asp Leu Glu Lys Val Arg 160 165 170

GTG GAA CAA CTT ACT GGT CAT GGT TCT TCA GTT TTA GAA GAA TTG GTT 577 Val Glu Gin Leu Thr Gly His Gly Ser Ser Val Leu Glu Glu Leu Val 175 180 185 190

CAG TTA GTC AAA GAT AAA AAT ATA GAT ATT TCC ATT AAA TAT GAT CCC 625 Gin Leu Val Lys Asp Lys Asn He Asp He Ser He Lys Tyr Asp Pro 195 200 205

AGA AAA GAT TCG GAG GTT TTT GCC AAT AGA GTA ATT ACT GAT GAT ATC 673 Arg Lys Asp Ser Glu Val Phe Ala Asn Arg Val He Thr Asp Asp He 210 215 220

GAA TTG CTC AAG AAA ATC CTA GCT TAT TTT CTA CCC GAG GAT GCC ATT 721 Glu Leu Leu Lys Lys He Leu Ala Tyr Phe Leu Pro Glu Asp Ala He 225 230 235

CTT AAA GGC GGT CAT TAT GAC AAC CAA CTG CAA AAT GGC ATC AAG CGA 769 Leu Lys Gly Gly His Tyr Asp Asn Gin Leu Gin Asn Gly He Lys Arg 240 245 250

GTA AAA GAG TTC CTT GAA TCA TCG CCG AAT ACA CAA TGG GAA TTG CGG 817 Val Lys Glu Phe Leu Glu Ser Ser Pro Asn Thr Gin Trp Glu Leu Arg 255 260 265 270

GCG TTC ATG GCA GTA ATG CAT TTC TCT TTA ACC GCC GAT CGT ATC GAT 865 Ala Phe Met Ala Val Met His Phe Ser Leu Thr Ala Asp Arg He Asp

275 280 285

GAT GAT ATT TTG AAA GTG ATT GTT GAT TCA ATG AAT CAT CAT GGT GAT 913 Asp Asp He Leu Lys Val He Val Asp Ser Met Asn His His Gly Asp 290 295 300

GCC CGT AGC AAG TTG CGT GAA GAA TTA GCT GAG CTT ACC GCC GAA TTA 961 Ala Arg Ser Lys Leu Arg Glu Glu Leu Ala Glu Leu Thr Ala Glu Leu 305 310 315

ΛΛG ATT TAT TCΛ GTT ATT CAA GCC GAA ATT AAT AAG CAT CTG TCT AGT 1009 Lys He Tyr Ser Val He Gin Ala Glu He Asn Lys His Leu Ser Ser 220 325 330

ΛGT GGC ΛCC ΛTA AAT ΛTC CAT GAT AAA TCC ATT AAT CTC ATG GAT AAA 105"? Ser Gly Thr He Asn He His Asp Lys Ser He Asn Leu Met Asp Lys 225 340 345 350

AAT TTA TAT GGT TAT ACA GAT GAA GAG ATT TTT AAA GCC AGC GCA GAG 1105 Asn Leu Tyr Gly Tyr Thr Asp Glu Glu He Phe Lys Ala Ser Ala Glu 355 360 365

TAC AAA ATT CTC GAG AAA ATG CCT CAA ACC ACC ATT CAG GTG GAT GGG 1153 Tyr Lys He Leu Glu L s Met Pro Gin Thr Thr He Gin Val Asp Gly 370 375 380

AGC GAG AAA AAA ATA GTC TCG ATA AAG GAC TTT CTT GGA AGT GAG AAT 1201 Ser Glu Lys Lys He Val Ser He Lys Asp Phe Leu Gly Ser Glu Asn 385 390 395

AAA AGA ACC GGG GCG TTG GGT AAT CTG AAA AAC TCA TAC TCT TAT AAT 1249 Lys Arg Thr Gly Ala Leu Gly Asn Leu Lys Asn Ser Tyr Ser Tyr Asn 400 405 410

AAA GAT AAT AAT GAA TTA TCT CAC TTT GCC ACC ACC TGC TCG GAT AAG 1297 Lys Asp Asn Asn Glu Leu Ser His Phe Ala Thr Thr Cys Ser Asp Lys 415 420 425 430

TCC AGG CCG CTC AAC GAC TTG GTT AGC CAA AAΛ ACA ΛCT GΛG 2~~ TCT 12 4 Ξ Ser Arg Pro Leu Asn Asp Leu Val Ser Gin Lys Thr Thr Gin Leu Ser 435 440 445

GAT ATT ACA TCA CGT TTT AAT TCA GCT ATT GAA GCΛ GTG ΛAC GGT TTC 12 92 Asp He Thr Ser Arg Phe Asn Ser Ala He Glu Ala Leu ΛΞΓ. Λrg Phe 450 455 460

ATT CAG AAA TAT GAT TCA GTG ATG CAA CGT CTG CTA GAT GAC ACG TCT 1441 He Gin Lys Tyr Asp Ser Val Met Gin Arg Leu Leu Asp Asp Thr Ser 465 470 475

GGT AAA TGACACTAGA AGCTT 1462

Gly Lys 480

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 480 amino acids

(B) TYPE: ammo acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17

Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Λla Thr Leu Val Glu Pro 1 5 10 15

Ala Arg _le Thr Leu Thr Tyr Lys Glu Gly Ala Pro He Thr He Met 20 25 30

Λsp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu 35 40 45

Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp 50 55 60

Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin Asp Gly Asn 65 70 75 80

Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser Arg Asp Phe

85 90 95

Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val 100 105 110

Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser He Gly Ser

115 120 125

Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val 130 135 140

Thr Val Ser Asn Gin Gly Ser He Glu Gly Arg He Arg Ala Tyr Glu 145 150 155 160

Gin Asn Pro Gin His Phe He Glu Asp Leu Glu Lys Val Arg Val Glu 165 170 175

Gin Leu Thr Gly His Gly Ser Ser Val Leu Glu Glu Leu Val Gin Leu 180 185 190

Val Lys Asp Lys Asn He Asp He Ser lie Lys Tyr Asp Pro Arg Lys 195 200 205

Asp Ser Glu Val Phe Ala Asn Arg Val He Thr Asp Asp He Glu Leu 210 215 220

Leu Lys Lys He Leu Ala Tyr Phe Leu Pro Glu Asp Ala He Leu Lys 225 230 235 240

Gly Gly His Tyr Asp Asn Gin Leu Gin Asn Gly He Lys Arg Val Lys 245 250 255

Glu Phe Leu Glu Ser Ser Pro Asn Thr Gin Trp Glu Leu Arg Ala Phe 260 265 270

Met Ala Val Met His Phe Ser Leu Thr Ala Asp Arg He Asp Asp Asp 275 280 285

He Leu Lys Val He Val Asp Ser Met Asn His His Gly Asp Ala Arg 290 295 300

Ser Lys Leu Arg Glu Glu Leu Ala Glu Leu Thr Ala Glu Leu Lys He 305 310 315 320

Tyr Ser Val He Gin Ala Glu He Asn Lys His Leu Ser Ser Ser Gly 325 330 335

7hr He Asn He His Asp Lys Ser He Asn Leu Met Asp Lys Asn Leu 340 345 350

:yr Gly Tyr :hr Asp Glu Glu He Phe Lys Ala Ser Ala Glu Tyr Lys 355 360 365

He Leu Glu Lys Met Pro Gin Thr Thr He Gin Val Asp Gly Ser Glu 370 375 380

Lys Lys He Val Ser He Lys Asp Phe Leu Gly Ser Glu Asn Lys Arg 385 390 395 400

Thr Gly Ala Leu Gly Asn Leu Lys Asn Ser Tyr Ser Tyr Asn Lys Asp 405 410 415

Asn Asn Glu Leu Ser His Phe Ala Thr Thr Cys Ser Asp Lys Ser Arg 420 425 430

Pro Leu Asn Asp Leu Val Ser Gin Lys Thr Thr Gin Leu Ser Asp He 435 440 445

Thr Ser Arg Phe Asn Ser Ala He Glu Ala Leu Asn Arg Phe He Gin 450 455 460

Lys Tyr Asp Ser Val Met Gin Arg Leu Leu Asp Asp Thr Ser Gly Lys 465 470 475 480

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Yersinia pestis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

ATAAGACTGT GCTAGCTAGA GGTAATATAT G 31

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE DNA (genomic)

(in) HYPOTHETICAL NO

(vi) ORIGINAL SOURCE

(A) ORGANISM Yersinia pestis

(xi) SEQUENCE DESCRIPTION SEQ ID NO 19

GATGGATCCT TGGTTAGATA CGGTTACG 28

(2) INFORMATION FOR SEQ ID NO.20

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 547 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS. double

(D) TOPOLOGY- linear

(II) MOLECULE TYPE. DNA (genomic)

(in) HYPOTHETICAL NO

(iv) ANTI-SENSE NO

(vi) ORIGINAL SOURCE

(A) ORGANISM Yersinia pestis

(ix) FEATURE.

(A) NAME/KEY CDS

(B) LOCATION 29 538

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

ATAAGACTGT GCTAGCTAGA GGTAATAT ATG AAA AAA ATC AGT TCC GTT ATC 52

Met Lys Lys He Ser Ser Val He 1 5

GCC ATT GCA TTA TTT GGA ACT ATT GCA ACT GCT AAT GCG GCA GAT TTA 100 Ala He Ala Leu Phe Gly Thr He Ala Thr Ala Asn Ala Ala Asp Leu 10 15 20

ACT GCA AGC ACC ACT GCA ACG GCA ACT CTT GTT GAA CCA GCC CGC ATC 148 Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg He 25 30 35 40

ACT CTT ACA TAT AAG GAA GGC GCT CCA ATT ACA ATT ATG GAC AAT GGA 196 Thr Leu Thr Tyr Lys Glu Gly Ala Pro He Thr He Met Asp Asn Gly 45 50 55

AAC ATC GAT ACA GAA TTA CTT GTT GGT ACG CTT ACT CTT GGC GGC TAT 244 Asn He Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr 60 65 70

AAA ACA GGA ACC ACT AGC ACA TCT GTT AAC TTT ACA GAT GCC GCG GGT 292 Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ala Gly 75 80 85

GAT CCC ATG TAC TTA ACA TTT ACT TCT CAG GAT GGA AAT AAC CAC CAA 340 Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin Asp Gly Asn Asn His Gin 90 95 100

TTC ACT ACA AAA GTG ATT GGC AAG GAT TCT AGA GAT TTT GAT ATC TCT 388 Phe Thr Thr Lys Val He Gly Lys Asp Ser Arg Asp Phe Asp He Ser 105 110 115 120

CCT AAG GTA AAC GGT GAG AAC CTT GTG GGG GAT GAC GTC GTC TTG GCT 436 Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu Ala 125 130 135

ACG GGC AGC CAG GAT TTC TTT GTT CGC TCA ATT GGT TCC AAA GGC GGT 484 Thr Gly Ser Gin Asp Phe Phe Val Arg Ser He Gly Ser Lys Gly Gly 140 145 150

AAA CTT GCA GCA GGT AAA TAC ACT GAT GCT GTA ACC GTA ACC GTA TCT 532 Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser 155 160 165

AAC CAA GGATCCATC 547

Asn Gin 170

(2) INFORMATION FOR SEQ ID NO:21 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 170 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Met: Lys Lys He Ser Ser Val He Ala He Ala Leu Phe Gly Thr He 1 5 10 15

Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala 20 25 30

Thr Leu Val Glu Pro Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala 35 40 45

Pro He Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val 50 55 60

Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser 65 70 75 80

Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr

85 90 95

Ser Gin Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys 100 105 110

Asp Ser Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu 115 120 125

Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val 130 135 140

Arg Ser He Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr 145 150 155 160

Asp Ala Val Thr Val Thr Val Ser Asn Gin 165 170

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1530 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Yersinia pestis

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 13. 1515

(xi) SEQUENCE DESCRIPTION: SEQ IDNO:22:

TAGAGGTAAT AT ATG AAA AAA ATC AGT TCC GTT ATC GCC ATT GCA TTA 48 Met Lys Lys He Ser Ser Val He Ala He Ala Leu 1 5 10

TTT GGA ACT ATT GCA ACT GCT AAT GCG GCA GAT TTA ACT GCA AGC ACC 96 Phe Gly Thr He Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr 15 20 25

ACT GCA ACG GCA ACT CTT GTT GAA CCA GCC CGC ATC ACT CTT ACA TAT 144 Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg He Thr Leu Thr Tyr 30 35 40

AAG GAA GGC GCT CCA ATT ACA ATT ATG GAC AAT GGA AAC ATC GAT ACA 192 Lys Glu Gly Ala Pro He Thr He Met Asp Asn Gly Asn He Asp Thr 45 50 55 60

GAA TTA CTT GTT GGT ACG CTT ACT CTT GGC GGC TAT AAA ACA GGA ACC 240 Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr

65 70 75

ACT AGC ACA TCT GTT AAC TTT ACA GAT GCC GCG GGT GAT CCC ATG TAC 288 Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr 80 85 90

TTA ACA TTT ACT TCT CAG GAT GGA AAT AAC CAC CAA TTC ACT ACA AAA 336 Leu Thr Phe Thr Ser Gin Asp Gly Asn Asn His Gin Phe Thr Thr Lys 95 100 105

GTG ATT GGC AAG GAT TCT AGA GAT TTT GAT ATC TCT CCT AAG GTA AAC 3S4 Val He Gly Lys Asp Ser Arg Asp Phe Asp He Ser Pro Lys Val Asn 110 115 120

GGT GAG AAC CTT GTG GGG GAT GAC GTC GTC TTG GCT ACG GGC AGC CAG 432 Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gin 125 130 135 140

GAT TTC TTT GTT CGC TCA ATT GGT TCC AAA GGC GGT AAA CTT GCA GCA 480 Asp Phe Phe Val Arg Ser He Gly Ser Lys Gly Gly Lys Leu Ala Ala 145 150 155

GGT AAA TAC ACT GAT GCT GTA ACC GTA ACC GTA TCT AAC CAA GGA TCC 528 Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gin Gly Ser 160 165 170

ATC GAA GGT CGT ATT AGA GCC TAC GAA CAA AAC CCA CAA CAT TTT ATT 576 He Glu Gly Arg He Arg Ala Tyr Glu Gin Asn Pro Gin His Phe He 175 180 185

GAG GAT CTA GAA AAA GTT AGG GTG GAA CAA CTT ACT GGT CAT GGT TCT 624 Glu Asp Leu Glu Lys Val Arg Val Glu Gin Leu Thr Gly His Gly Ser 190 195 200

TCA GTT TTA GAA GAA TTG GTT CAG TTA GTC AAA GAT AAA AAT ATA GAT 672 Ser Val Leu Glu Glu Leu Val Gin Leu Val Lys Asp Lys Asn He Asp 205 210 215 220

ATT TCC ATT AAA TAT GAT CCC AGA AAA GAT TCG GAG GTT TTT GCC AAT 720 He Ser He Lys Tyr Asp Pro Arg Lys Asp Ser Glu Val Phe Ala Asn 225 230 235

AGA GTA ATT ACT GAT GAT ATC GAA TTG CTC AAG AAA ATC CTA GCT TAT 768 Arg Val He Thr Asp Asp He Glu Leu Leu Lys Lys He Leu Ala Tyr 240 245 250

TTT CTA CCC GAG GAT GCC ATT CTT AAA GGC GGT CAT TAT GAC AAC CAA 816 Phe Leu Pro Glu Asp Ala He Leu Lys Gly Gly His Tyr Asp Asn Gin 255 260 265

CTG CAA AAT GGC ATC AAG CGA GTA AAA GAG TTC CTT GAA TCA TCG CCG 864 Leu Gin Asn Gly He Lys Arg Val Lys Glu Phe Leu Glu Ser Ser Pro 270 275 280

AAT ACA CAA TGG GAA TTG CGG GCG TTC ATG GCA GTA ATG CAT TTC TCT 912 Asn Thr Gin Trp Glu Leu Arg Ala Phe Met Ala Val Met His Phe Ser 285 290 295 300

TTA ACC GCC GAT CGT ATC GAT GAT GAT ATT TTG AAA GTG ATT GTT GAT 960 Leu Thr Ala Asp Arg He Asp Asp Asp He Leu Lys Val He Val Asp 305 310 315

TCA ATG AAT CAT CAT GGT GAT GCC CGT AGC AAG TTG CGT GAA GAA TTA 1008 Ser Met Asn His His Gly Asp Ala Arg Ser Lys Leu Arg Glu Glu Leu 320 325 330

GCT GAG CTT ACC GCC GAA TTA AAG ATT TAT TCA GTT ATT CAA GCC GAA 1056 Ala Glu Leu Thr Ala Glu Leu Lys He Tyr Ser Val He Gin Ala Glu 335 340 345

ATT AAT AAG CAT CTG TCT AGT AGT GGC ACC ATA AAT ATC CAT GAT AAA 1104 He Asn Lys His Leu Ser Ser Ser Gly Thr He Asn He His Asp Lys 350 355 360

TCC ATT AAT CTC ATG GAT AAA AAT TTA TAT GGT TAT ACA GAT GAA GAG 1152 Ser He Asn Leu Met Asp Lys Asn Leu Tyr Gly Tyr Thr Asp Glu Glu 365 370 375 380

ATT TTT AAA GCC AGC GCA GAG TAC AAA ATT CTC GAG AAA ATG CCT CAA 1200 He Phe Lys Ala Ser Ala Glu Tyr Lys He Leu Glu Lys Met Pro Gin 385 390 395

ACC ACC ATT CAG GTG GAT GGG AGC GAG AAA AAA ATA GTC TCG ATA AAG 1248 Thr Thr He Gin Val Asp Gly Ser Glu Lys Lys He Val Ser He Lys 400 405 410

GAC TTT CTT GGA AGT GAG AAT AAA AGA ACC GGG GCG TTG GGT AAT CTG 1296 Asp Phe Leu Gly Ser Glu Asn Lys Arg Thr Gly Ala Leu Gly Asn Leu 415 420 425

AAA AAC TCA TAC TCT TAT AAT AAA GAT AAT AAT GAA TTA TCT CAC TTT 1344 Lys Asn Ser Tyr Ser Tyr Asn Lys Asp Asn Asn Glu Leu Ser His Phe 430 435 440

GCC ACC ACC TGC TCG GAT AAG TCC AGG CCG CTC AAC GAC TTG GTT AGC 1392 Ala Thr Thr Cys Ser Asp Lys Ser Arg Pro Leu Asn Asp Leu Val Ser 445 450 455 460

CAA AAA ACA ACT CAG CTG TCT GAT ATT ACA TCA CGT TTT AAT TCA GCT 1440

Gin Lys Thr Thr Gin Leu Ser Asp He Thr Ser Arg Phe Asn Ser Ala 465 470 475

ATT GAA GCA CTG AAC CGT TTC ATT CAG AAA TAT GAT TCA GTG ATG CAA 1488 He Glu Ala Leu Asn Arg Phe He Gin Lys Tyr Asp Ser Val Met Gin 480 485 490

CGT CTG CTA GAT GAC ACG TCT GGT AAA TGACACTAGA AGCTT 1530

Arg Leu Leu Asp Asp Thr Ser Gly Lys 495 500

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 501 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Met Lys Lys He Ser Ser Val He Ala He Ala Leu Phe Gly Thr He 1 5 10 15

Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala 20 25 30

Thr Leu Val Glu Pro Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala 35 40 45

Pro He Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val 50 55 60

Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser 65 70 75 80

Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr

85 90 95

Ser Gin Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys 100 105 110

Asp Ser Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu 115 120 125

Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val 130 135 140

Arg Ser He Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr 145 150 155 160

Asp Ala Val Thr Val Thr Val Ser Asn Gin Gly Ser He Glu Gly Arg 165 170 175

He Arg Ala Tyr Glu Gin Asn Pro Gin His Phe He Glu Asp Leu Glu 180 185 190

Lys Val Arg Val Glu Gin Leu Thr Gly His Gly Ser Ser Val Leu Glu 195 200 205

Glu Leu Val Gin Leu Val Lys Asp Lys Asn He Asp He Ser He Lys 210 215 220

Tyr Asp Pro Arg Lys Asp Ser Glu Val Phe Ala Asn Arg Val He Thr 225 230 235 240

Asp Asp He Glu Leu Leu Lys Lys He Leu Ala Tyr Phe Leu Pro Glu 245 250 255

Asp Ala He Leu Lys Gly Gly His Tyr Asp Asn Gin Leu Gin Asn Gly 260 265 270

He Lys Arg Val Lys Glu Phe Leu Glu Ser Ser Pro Asn Thr Gin Trp 275 280 285

Glu Leu Arg Ala Phe Met Ala Val Met His Phe Ser Leu Thr Ala Asp 290 295 300

Arg He Asp Asp Asp He Leu Lys Val He Val Asp Ser Met Asn His 305 310 315 320

His Gly Asp Ala Arg Ser Lys Leu Arg Glu Glu Leu Ala Glu Leu Thr 325 330 335

Ala Glu Leu Lys He Tyr Ser Val He Gin Ala Glu He Asn Lys His 340 345 350

Leu Ser Ser Ser Gly Thr He Asn He His Asp Lys Ser He Asn Leu 355 360 365

Met Asp Lys Asn Leu Tyr Gly Tyr Thr Asp Glu Glu He Phe Lys Ala 370 375 380

Ser Ala Glu Tyr Lys He Leu Glu Lys Met Pro Gin Thr Thr He Gin 385 390 395 400

Val Asp Gly Ser Glu Lys Lys He Val Ser He Lys Asp Phe Leu Gly 405 410 415

Ser Glu Asn Lys Arg Thr Gly Ala Leu Gly Asn Leu Lys Asn Ser Tyr 420 425 430

Ser Tyr Asn Lys Asp Asn Asn Glu Leu Ser His Phe Ala Thr Thr Cys 435 440 445

Ser Asp Lys Ser Arg Pro Leu Asn Asp Leu Val Ser Gin Lys Thr Thr 450 455 460

Gin Leu Ser Asp He Thr Ser Arg Phe Asn Ser Ala He Glu Ala Leu 465 470 475 480

Asn Arg Phe He Gin Lys Tyr Asp Ser Val Met Gin Arg Leu Leu Asp 485 490 495

sp Thr Ser Gly Lys 500

2) INFORMATION FOR SEQ ID NO 24

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 24 base pairs

(B) TYPE, nucieic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY linear

(n) MOLECULE TYPE. DNA (genomic)

(iii) HYPOTHETICAL. NO

(iv) ANTI-SENSE. NO

(vi) ORIGINAL SOURCE.

(A) ORGANISM: Yersinia pestis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

24

As discussed previously in this application, the V and FI antigen may DΘ microenca _D sulateo The V and F1 antigens may be separately microencaDsulateα The comDineα microencapsulated sub-units may be used for immunisation The present inventors _D eneve thai the protection affordeα by the combined microencaDSulated suo-units is superior to that provided by existing plague vaccines and that there is an additive effect in com _D ining the suD-units The protective efficacy of the combined microencapsulated sub-units may further be enhanced by co-adm istering an adjuvant for example cholera toxin B sub-unit (CTB) Micr oencapsulation of the sub-unit vaccine prolongs release of the vaccine in vivo permits oral intra-nasal or inhalational delivery and gives scope for targett g

The microencapsulation of the combined V and F1 sub-unit vaccine is described below Also demonstrated is that this formulation is able to induce both mucosal and systemic immunity against plague

The microencapsulation of sub-units was effected in PLA 2000 using a solvent evaporation technique

Immunisation with the microencapsulated sub-units was carried out as follows

Groups of 21 mice received a primary immunising dose of 25ug of either V antigen or F1 antigen presented in mierospheres resuspended in PBS for intra-peπtoneal (i p ) injection Further groups of 21 mice received a combination of 25μg of each of the F1 and V antigens presented in mierospheres A dose of 25μg of F1 was delivered in a total mass of 5 42mg of spheres whilst 25ug of V was contained in 2.08mg of spheres The required mass of mierospheres was re-suspended in 100ul PBS per animal for injection Animals were boosted with the respective aπtιgen(s). as appropriate, on days 14 and 28

Two further groups of 21 mice were primed and then boosted i p on days 14 and 28 with a combination of 10μg F1 and 10ug V jointly incorporated in the aqueous phase of 25% (v/vj susDension of alhydrogel (Alhydrogel 1 3%, Superfos. Denmark) in PBS

Selected grouos of animals received in addition a dose of 10μg CTB (Sigma. Poole) mcorporateα into the delivery vehicle at each dosing point.

Control groups ^, eacn of 21 mice, received either alhydrogel only (100μl of 25% solution) cr CTB cniy ⁽ 10ug in 100ul PBS) or remained untreated.

in order to compare the protective efficacy of immunisation with combined sub-units, free or microencapsulated. against that provided by the Greer vaccine, (purchased from Greer laboratories) animals were challenged s.c. with virulent Y.pestis.

There was a 60% survival rate in Greer vaccmees against a challenge of 2 x 10 ⁵ cfu Y.pestis (Fig. 1 ). By comparison, 80 % of the combined microencapsulated F1 + V (group 1) survived this challenge and there was 100% survival in group 2 (μV + μF1 + 10μg CTB). Thus the combined microencapsulated formulation was protective against virulent Y. pestis with no evidence of side effects.

In summary treatment groups were:

Group Treatment

¹ 25μg microencapsulated F1 (μ F1) + 25μg microencapsulated V (μV) i.p.

2. 25μg u F1 + 25μg μV + 10μg CTB i.p.

3. 25μg u F1 i.p.

4 25μV ι.p.

5 25μg F1 + 25μg V in alhydrogel i.p. 6. Greer vaccine 0.1ml i.m.

Micro-encapsulation may also be carried out with block co-polymers in the following experiments model protein antigen BSA was used The Dreparation and characterisation of mierospheres is as follows Protein-loaded mierospheres were prepared bv an oιl/wate ^r solvent evaporation method procedure previously described see R L Hunter and B Bennet The J Immunol , 133(6), 3167-3175 (1984), with some modifications Polymer (pciv-D-lactic acid) Resomer 206, Boehringer Ingelheim, Germany, 125 or 250mg) solution ,n acetone (22 5ml), containing model protein (antigen) BSA (at 15-25% theoretical loaαing iβvel) and 0 11 %w/v Pluronic L101 (or 0 09% w/v L121 ) available from Zeneca probe sonicated for 10 seconds and then added to the aqueous phase (22 5ml), mixing at 100 rom for 5 minutes and rotary evaporated until the organic solvent had been removed The resulting colloid was washed and freeze-dned Mierospheres with an average diameter of - 1 μm (as determined by Malvern Mastersizer) and protein loadings -0 5-1 0% produced in this fabrication condition External morphology of the resulting mierospheres were analysed by scanning electron microscopy (SEM) Surface characteπstics were defined in terms of zeta potential and hydrophobicity

Hydrophobicity measurements hydrophobicity of mierospheres was quantified using hydrophobic interaction chromatography (HIC) as previously reported see H O Alpar and A J Almeida, Eur J Pharm Biopharm 40, (4), 198-202 (1994) Mierospheres were eluted from a series of agaroses which were modified with hydrophobic residues The retention of mierospheres in octyl agarose was used as an index of hydrophobicity

Immunisation A study was designed to establish the effects of differences in the type of microsphere and surface properties of the immune response Female Balb/c mice (five per group) were injected i m with a single dose of BSA either encapsulateα in Pluronic formulated mierospheres or free in 100μl alone or suspended in the presence of surfactant The control group of mice received the same amount of antigen encapsulateα into mierospheres containing PVA as emulsion stabiliser Tail tip blood samples were removed periodically for 2 months The serum from each sample was analysed for anti- BSA antibody using an enzyme-linked immuπosorbent assay (ELISA) Results are represented graphically in Figure 2

The presence of Pluronic L101 and L121 endows the surface with a much higher degree of hyαrcDnobiαty comoared to PVA formulations (70% retained on octyl agarose column as opposed to 30% iatex control 95% retained). A hydrophobic surface would facilitate macropnage interactions and subsequent uptake and would therefore be much more likely to mediate increased immune response. Figure 2 shows the effect of different BSA formulations in eliciting an immunoresponse. Batches formed by Pluronic L101 had a more enhanced effect on the plasma anti-BSA antibody titre than those formed by PVA. Batches formed by Pluronic L121 were slightly inferior to those of PVA mierospheres in inducing good primary antibody response after delivery of only a small dose of antigen (1 μg). The higher serum IgG level obtained with Pluronic L101 preparations as compared to other preparations is noted and may partly be due to the higher surface hydrophobicity.

It was also mentioned earlier in the present application that DNA encoding the wnoie or oa-r of the F1 antigen and DNA encoding the whole or part of the V antigen could be used αirect.y as a genetic vaccine By way of example this may be carried out as follows

1/ DNA encoding Y.pestis F1 and V is obtained by Polymerase Chain Reaction (PCR, amplification of specific regions of the Y.pestis genome or by isolation of these genes from previously constructed plasmid clones e.g. for V exemplified sequence I D no 3

2/ The F1 and V genes are cloned into mammalian expression vector plasmids such that the genes are situated downstream of a eukaryotic promoter Suitable plasmids include pCMVβ (purchased from Clontech), in which the cytomegalovirus Immediate Early promoter is used F1 and V may be cloned individually or in combination and may be cloned as fusions with such genes as glutathione S-transferase, or eukaryotic signal sequences which may stabilise the expressed protein and may facilitate export from mammalian ceils

3/ The recombinant plasmids are propagated in Escheπchia coli and stocks are purified for transfection into a mammalian animal model and for immunisation of expeπmental animals by the intra-muscular or intra-dermal or mhalational routes

Example 1 To construct a DNA vaccine expressing V antigen, the plasmid vector pCMV was digested with restriction enzyme Not 1 to remove the lac Z gene coding sequence The digested plas lα was treated with Klenow enzyme to create blunt-ended vector DNA An ssp 1 restriction fragment containing the coding sequence for a fusion protein of V antigen ana glutathione-S transferase was isolated from recombinant plasmid pVGIOO and ligated to the vector DNA The V sequence used in this case is that described in exemplified Seq ID No 3 The recombinant plasmid was transformed into E coli strain Nova Blue Punfieα plasmid was inoculated into Balb/c mice by intra-muscular injection Immunoglobuliπ responses to V antigen were detected in the serum of inoculated animals

/

Example 2 To construct a DNA vaccine expressing F1 antigen, PCR pnmers were designed to amplify the complete cafl open reading frame This encodes F1 and its signal peptide which directs export of the protein from the bacterial cell The PCR pnmers had "tails" at their 5' ends which contained restriction enzyme recognition sites to allow directional insertion into a plasmid vector The sequences of the PCR pnmers, 5'FAB2 and 3'FBAM, are given in exemplified Seq. ID No 18 and Seq. ID No. 19, respectively

5'FAB2 and 3'FBAM were used to amplify a PCR fragment, the sequence of which is given in exemplified Seq ID no 20 The PCR fragment was digested with restriction enzymes Nhe 1 and Bam HI and cloned into the plasmid pBKCMV which had been digested with the same enzymes The resulting plasmid, pF1AB was transformed into E. coli Nova Blue and purified plasmid was used to inoculate Balb/c mice by intra-muscular injection Immunoglobulin responses to F1 were found in inoculated animals.

Example 3- To construct a DNA vaccine expressing both F1 and V the ccαing seαuence for V was inserted into the DNA vaccine expressing F1 . detailed in exarri _D le 2. A linker region coding for 6 ammo acids was positioned between the F1 and V ccαing seαuences tc allow each of the proteins to attain their conformational shape indepenαantly The linker- coding sequence was obtained by digesting the recombinant plasmid p/acP/6 with Bam HI and Hind III. The linker-V DNA was ligated with the plasmid pFIAB wnicn nad also _D een digested with Bam HI and Hind III. The resulting plasmid. pFVAB. was transformed into cells of E. coli Nova Blue and stocks of plasmid were purified for further use. The nucleotide and derived am o acid sequence of the F1 V fusion are given in exemplified Seq. ID No. 22.

An example of a fusion protein comprising both F1 and V antigens is described below.

Enzymes and reagents.

Materials for the preparation of growth media were obtained from Oxoid Ltd or Difco Laboratories. All enzymes used in the manipulation of DNA were obtained from Boehringer Mannheim UK Ltd and used according to the manufacturer's instructions. All other chemicals and biochemicals were obtained from Sigma Chemical Co unless otherwise indicated. Monospecific rabbit polyclonal anti-V serum was supplied by Dr R Brubaker (Department of Microbiology, Michigan State University) and mouse anti-F1 IgA monoclonal antibody (Mab) F13G8-1 was obtained from the American Type Culture Collection.

Bacterial strains and cultivation.

Yersinia pestis GB was cultured aerobically at 28°C in a liquid medium (pH 6.8) containing 15 g proteose peptone, 2.5 g liver digest, 5 g yeast extract, 5 g NaCl per litre, supplemented with 80 ml of 0.25% haemin dissolved in 0.1M NaOH (Blood Base broth) Eschenchia coli JM109 was cultured and stored as described by Sambrook et al (Sambrook J et al. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, New York).

Manipulation of DNA.

Chromosomal DNA was isolated from Y. pestis by the method of Marmur (Marmur. J 1961 J Mol Biol 3: 208-218). The genes encoding F1 antigen (cafl) and V antigen (IcrV) were amplified from Y. pestis DNA using PCR with 125 pmol of primers homologous to sequences from the 5' and 3' ends of the gene (Galyov, E E et al. 1990. FEBS Lett

277:230-232 Price S B et al 1989 J Bacteπol 171 : 5646-5653) althougn for cafl only the region encoding the mature F1 antigen was amplified The seαuences of the - " 5 primer (F/5'B GATCGAGCTCGGCAGATTTAACTGCAAG

CACC), the F1 3' primer (Flιnk/3'A GCATGGATCCTTGGTTAGATACGGTTACGGT the V 5' primer (Vlιnk/5'A ATGGATCCATCGAAGGTCGTATTAGAGCCTACGAACAA) aπα the V 3' primer (VG/3 ^' A GCATAAGCTTCTA ^* GTGTCATTTACCAGACGT) also inciuαeα 5 tails encoding the restriction sites Sacl, BamHI BamHI and Hindlll respectively In addition the nucleotide A ^* was altered from the published sequence of IcrV (Price S B et al 1989 J Bacteπol 171 : 5646-5653) to include an extra termination codon (TAA) in the amplified DNA The tail of primer Vlink 5'A also included nucleotides encoding the factor Xa cleavage sequence lle-Glu-Gly-Arg The PCR primers were prepared with a DNA synthesiser (model 392. Applied Biosystems) DNA fragments were obtained after 30 cycles of amplification (95°C, 20 s, 45°C, 20 s, 72'C, 30 s, model 9600 GeneAmp PCR System, Perkm Elmer) and the fragments were purified The cafl PCR product was digested with Sacl and BamHI, ligated with suitably digested plasmid pUC18 and transformed into E. coli JM109 by electroporation Subsequently, the .c/V-lmker PCR product was digested with BamHI and Hindlll, and ligated into the intermediate plasmid to form the recombinant plasmid placFV6 A colony containing placFV6 was identified by PCR using 30-mer primers (5' nucleotides located at positions 54 and 794 (Price S B et al 1989 J Bacteπol 171 : 5646-5653) which amplified an internal segment of the IcrV gene To confirm the nucleotide sequence of the cloned insert, sequencing reactions containing placFVδ and primers designed from the cat ^* 7 and IcrV genes were performed using an automated Taq polymerase cycle sequencing protocol with fluorescently labelled dideoxy nucleotides (CATALYST Molecular Biology Labstation Applied Biosystems) The reaction products were analysed using an automated DNA sequencer (model 373A Applied Biosystems)

The DNA sequence and derived ammo acid seαuence of the cloneα fusion protein is shown in Example 1 The fusion protein consists of F1 and V antigens separated by a six- amino acid linker Gly-Ser-lle-Glu-Gly-Arg It is cloned downstream of the lac promoter and

in-frame with the sector-encoded LacZ' fragment. Thus, the complete fusion protein encoαes nine aαditional ammo acids at the N-terminus (Met-Thr-Met-lle-Thr-Asn-Ser-Ser-

Seπ and it accumulates in the cytoplasm.

Expression of the F1/V fusion protein in E. coli.

Cultures of E. coli JM109/placFV6 were grown in LB containing 100 μgml ^_1 ampicillin at 37°C until the absorσance (600nm) was 0.3. Isopropyl- -D-thiogalactopyranoside (IPTG) was then added to the culture to a final concentration of 1 mM and growth was continued for a further 5 h. Whole cell lysates of the bacteria were prepared as described by Sambrook et al (Sambrook J et al. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press. New York) and expression of the F1Λ fusion protein was examined by SDS-PAGE on 10-15% gradient gels (Phastsystem, Pharmacia Biotech) and Western blotting (Sambrook J et al. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, New York). Western blots were probed with rabbit anti-V serum at a dilution of 1/4000 or Mab F13G8-1 at a dilution of 1/250, and protein bands were visualised with a colloidal gold labelled secondary antibody (Auroprobe BLplus, Cambio) or an anti-mouse IgA secondary antibody conjugated to horse radish peroxidase (Sigma).

A fusion protein with an approximate molecular weight of 54.2 kDa was detected in lysates of JM109/placFV6 by Western blotting with anti-V and anti-F1 sera. This product was not detected in control lysates of JM109/pUC18.

Electroporation into Salmonella typhimurium SL3261.

Plasmid DNA was extracted and purified from JM109/placFV6 using a Qiaprep kit (Qiagen) and electroporated into S. typhimurium strain LB5010 (r- m ⁺ ). Subsequently, modified placFVΘ was isolated and electroporated in S. typhimurium strain SL3261 (aroA his). For inoculation into mice, bacteria were grown in LB containing 100 μgml ^"1 ampicillin for 18 h

without shaking After washing, the cells were resuspended in 10% glycεrol ,n phosphate buffered saline (PBS) and stored at -70°C The cell suspensions were αe ^τ' rosteα ana dilutea in PBS as required prior to injection

Immunisation with SL3261/placFV6.

Six week old female Balb/c mice raised under specific pathogen-free conαitions (Charles- River Laboratories, Margate Kent, UK), were used in this study A group of 19 mice received 0 1 ml immunising doses of approximately 5x10 ⁶ cfu of SL3261/pFV6 on days 0 and 14 by the intravenous (iv) route To retain placFV6 in vivo, mice were also injected subcutaneously (sc) with 50μl ampicillin tπhydrate suspension (150 mgml" Penbπtin injectable suspension POM, SmithKhne Beecham Animal Health) for 5 days after each immunisation In addition, groups of 15 mice were immunised iv on day 0 with a single 0 1 ml dose of approximately 5x10^ cfu of SL3261 or intrapeπtoneally (ip) on days 0 and 14 with 0 1 ml of a mixture of 10 μg V and 10 μg F1 adsorbed to Alhydrogel An untreated group of 10 age-matched mice were used as controls

On day 7, five mice from the groups receiving SL3261/placFV6 or SL3261 were sacrificed and their spleens were removed The organs were homogenised in 5 ml of PBS with a stomacher (Seward Medical Ltd) for 30 sec. The homogenates were serially diluted in PBS and inoculated on to L-agar or L-agar containing 100 μgml ^"1 ampicillin to determine the number of bacteria per spleen

S typhimur ⁱ um Actual ose Average cfu per spleen ± se _m a % recombinant

L-agar L-amp

SL3261/placFV6 3.3χ10 ⁶ cfu 1030 ± 294 380 ± 135 37%

SL3261 1 6x10 cfu 1.85x10 ⁷ ±

3.57x10 ^s

^a standard error of the mean

Measurement of serum antibody titre.

On day 42, blood was sampled from the tail vein of mice immunised with SL3261/placFV6 and pooled. The serum anti-V and anti-F1 titres were measured by a modified ELISA (Williamson, E D and R W Titball. 1993. Vaccine 11 :1253-1258). Briefly, V (5 μgml ^"1 in

PBS) or F1 (2 μgml ^"1 ) were coated on to a microtitre plate and the test sera were serially diluted in duplicate on the plate. Bound antibody was detected using peroxidase labelled conjugates of anti-mouse polyvalent Ig. The titre of specific antibody was estimated as the maximum dilution of serum giving an absorbance reading greater than 0.1 units, after subtraction of the absorbance due to non-specific binding detected in the control sera. The serum antibody titre was also determined for all groups of mice prior to challenge with Y. pestis

On day 42, the anti-V and antι-Fl titres of mice receiving SL3261/placFV6 were 1 :5120 and 1 2560, respectively.

86

Challenge with Y. pestis.

On day 57, groups of 5 or 7 mice from the immunised and control groups were challenged subcutaneously with 0.1 ml aliquots of Y. pestis strain GB containing 7 _. 36x10 ² or 7.36x10 ⁴ cfu. Strain GB was isolated from a fatal human case of plague and has a median lethal dose (MLD) of < 1 cfu in Balb/c mice by the s.c. route (Russell, P et al. 1995. Vaccine 13: 1551-1556). The mice were observed for 14 days and, where appropriate _, the time to death was recorded. A post-mortem was carried out on all animals where possible. To test for the presence of Y. pestis, samples of blood, liver and spleen were smeared on to Congo Red agar and incubated at 28°C for 48 h.

^• standard error of the mean