CA2179639A1

CA2179639A1 - Vaccine compositions comprising live bacterial vectors for protection against yersinia pestis injection

Info

Publication number: CA2179639A1
Application number: CA002179639A
Authority: CA
Inventors: Richard William Titball; Ethel Diane Williamson; Sophie Emma Clare Leary; Angela Howells; Petra Claire Farquhar Oyston
Original assignee: Individual
Current assignee: UK Secretary of State for Defence
Priority date: 1993-12-24
Filing date: 1994-12-23
Publication date: 1995-07-06
Also published as: WO1995018231A1; JPH09507028A; EP0741786A1; GB9326425D0; AU1322295A

Abstract

Novel DNA constructs are provided that are capable of transforming such that they can be used as live or attenuated vaccines which induce such immune response at mucosal surfaces. Further provided are such transformed microorganisms per se and vaccine compositions containing them. Preferred constructs of the invention are capable of transforming microorganisms such that they express F1 based protein while retaining a capability to establish themselves in human or animal gut environment. Several constructs have been identified that are capable of transforming gut dwelling organisms such as S. typhimurium or S. typhi to enable F1 antigen production, but most of these affect the organism such that it can no longer function effectively in the gut, at least in so far as it cannot express the antigen e.g. being unstable and losing plasmid.

Description

WO 9S/1~231 2 :~ 7 g 6 ~ ~ PCT/GB9 t/02818 VACCINE COMPOSITIONS COMPRISING LIVE 3ACTERIAL VECTORS FOR PRUI~l;llON AGAINST
., YERSINIA PESTIS INJECTION.
The present invention relates to novel vaccines for provision of protection against infection with the organism Yersin~ qt;c (Y
~;i~) and to compositions rr,ntAining theQ. Particularly provided are parentally and orally active vaccines csPable of offering protection against bubonic and I r pla3ue, particularlY by induction of mucosal immunity in both humans and other animals.
y. ~.,c~ic is the highly virulent causative organism of plague in a wide range of animals, including man. Infection with this organism 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 ~1.. and three plasmids, including the Lcr genes (see Straley, (1991) Microb. Pathogen 10: pp87-91), a fibrinolysin (Sodeinde &
Goguen. (1988) Infec. Immun 56: pp2743-2748), and a capsule.
The capsule surrounding Y. r~qtic cells is composed of a protein -polysaccharide complex, the protein component of which is known as Fraction 1 (Fl) (see Baker et al (1952) J. Immunol 68: ppl31-145) which is only fully expressed at 37c. This complex confers resistance to ~ ;u~Lusis, possibly by forming aqueous pores in the membranes of phagocytic cells (Rodrigues et al (1992) J. Med. Biol.
Res. 25: pp75-79). Detection of ~ntihr~ c to F1 is the basis of standard serological tests for the surveillance and diagnosis of pla3ue as infected animals produce a strong humoral response to the antigen (Shepherd et al (1986) J. Clin. Microbiol. 24:
pplO75-1078); Williams et al, (1982) Bull. World Health Organ. 64:
pp745-752) .
The current whole cell vaccines available for prevention of plaG~ue arehighly heL~.u~,G.lGUU~ resulting in side effects which make them unsuitable for widespread use (Reisman (1970) J. Allergy 46:
pp49-55); Meyer et al (1974) J. Infect. Dis. 129: Sl3-S18);

WO 95118231 , r~ '7#18 ~179639 Marshall et nl (1974) J. Infect. Dis. 129: S19-S25). Fl has been proposed as being the primary immunogen in whole cell vaccines (Williams et al (1980) Bull. World. Health Organ. 58: pp753-756;
Chen et al, (1976) J. Infect. Dis. 133: pp302-309) and nay therefore be a suitable candidate on which to base an improved vaccine against plague.
The cafl gene encoding Fl antigen has been cloned and sequenced (Galyov et al (l990) FEBS Letters 277: pp230-232) and ., 'nAnt F1 expressed and purified from 13~ induced a protective response in BALB/c mice sufficient to protect against challenge with 105 virulent plague bacilli (Simpson et al (l990) Am. J. Trop. Med. Hyg.
43(4): pp389-396). Such resistance to infection by Y. T)F~Rtic is correlated with high titres of F1 antibody.
It is known to orally administer gcn~tlr~lly engineered organisms which express antigenic proteins for the purpose of inducing antigen production (EP 0474891) and it is further known that use of such route may result in mucosal immunity (Cardenas & Clements (1992) Clin.
Microbiol Rev 5 (3) pp328-342 and (1992) Vaccine lO (4) pp263).
FUL L1I~ULe~ it has been reported that dLLe..~lc.Led bacteria have been prepared thst are protective against enteric di8eases, including genus Yersinia (US 6865709).
The current vaccine for plague is the Cutter vaccine which comprises formaldehyde killed plague bacilli and is administered to the body by illL. lnr injection. However, parenteral i c~tinn, although effective in inducing systemic immunity, does not effectively induce mucosal immunity (McGhee et al, (1992) Vaccine ~, 75-88). sO far no Y . rC~cti R vaccine capable of producing a protective immune response st mucosal surfaces has been developed.
The present inventors have now provided . 'n~nt DNA .u..~L.
that when incu-~ u.c.Led into DNA of a microorganism, particularly ~ WO 95/18231 217 9 ~ 3 ~ PCT/GB9~/Oz818 , of a human or animal gut cnlnnic~ne micrnnr~Anl~, are cspable of transforming it such that it i9 able to express e protein which produces a protective immune response against yprsin~A DPct~c in the human or animal body when the gut rr,lnnlc~ng microorgani8m is administered by oral routes.
Preferred forms of the present invention provide such DNA CU--~LLU-,L,that transform such a microorganism while allowing it to maintain its ability to colonise the human or animal gut and thus provide sustained administration, ie. by exposure of antigen to the human or animal body immune system, preferably with systemic invasion of the human or animal body.
Further provided are vectors eg.plasmids, rnntp~n;nF the DNA
cu-l~LLu~L~ of the invention, that are capable of transforming a human or animal gut cnlnnicing microorganism such that it is capable of expressing a protein which produces a protective immune response against YerqlnlP DPqtic in a human or animal body when the microorganism is administered by oral or parenteral routes, preferably allowing the microorganism to maintain ability to colonise the human or animal gut, and preferably s-lhcP~ ntly systemically to invade the body.
Still further provided are microorganisms, preferably hum_n or animal gut rnlnnic~ne microorganisms, transformed with a vector cnntAinln~
L'~ ~ nAnt DNA, eg. a plasmid rnntAinine nAnt DNA, according to the invention such that it is enabled to express a protein which produces a protective immune response against YerciniP D~octiq in a human or animal body when the microorganism is administered by oral or parenteral routes, and preferably allows the microorganism to maintain its ability to colonise the human or animal gut, and preferably invade systemically. The protective response provided preferably includes protection at mucosal surfaces.
ûne preferred 1~ ' npnt DNA, and plasmids comprising it, are wo ssllsnl 2 ~ 7 ~ ~ 3 ~ F~ QI8 ~hnL~,~Le.lsed in that they comprise a lacZ promoter in frame with a sequence encoding for all or part of the cafl antigen. Still further preferred, 1 nPnt DNA utilises a caflR positive regulator derived from the F1 operon itself. It is further preferred that the caflM
fragment is included for the purposes of assisting export of mature protein through the cell wall of the host organism, ie. the transformed cell and/or the caflA fragment that encodes proteins thought to be important in anchoring the F1 subunit to the cell wall.
Where the .. ' nPnt DNA includes the lacZ promoter it i3 preferably incuL~ ed into a vector such as pUC18 that has the all or part of the cafl encoding sequence inserted in frame with the lacZ promoter bviously the microorganisms of the invention sre preferably l microorganisms not capable of causing disease in humans or animals, eg. such as the SP1_~ P aro A or aro C mutants, prefer~bly of species ~P1_nn~11n tv~hi-~-ri or a ~Plmnn~1lP tv~hi.
Preferred vaccine compositions are provided as such microorganisms together with a 1' rAlly acceptable carrier, eg. saline or buf fer .
Att~nllPte~l microorganisms such as S. tvmhim~1ri have been well .ised as carriers for various heterologous antigens (Curtiss, ( 1990 ); New Generation Vaccines , Woodrow & Levine ( eds ~ Marcel Dekker Inc. New York; Cardenas and Clements, (1992) ibid).
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). Many other such Ptti nll~t~ng deletions and mutations will be known for these and other microorganisms which will render them suitable for transformation with c~ .u~s of the present invention for the purposes of expressing vaccine proteins in the gut and/or gut rnlnnicPtinn in animals to be treated for ~.2~, with systemic invasion and ~nlnnlcPtinn WO 9~/18231 ~17 g G 3 9 PCT/GB94/02818 following. For human v~rrinAtinn ~LLG,-u~Led 5. tv~h~ is the preferred microorganism.
A particularly preferred 1 ~ nPnt DNA, and plagmid or human or animal gut rr,lnni~ing microorganism incuL~u,~Llng it, encodes for or expresses all or part af the mature cafl (Fl~ protein of ~:S~
~;i~. A particularly preferred L~ ' 'nAnt DNA comprises a DNA
sequence as described in SEQ ID No 4.
The inventors have determined the sequence of a still further preferred L'l ' n~nt DNA which when included within suitable vectors within, or integrated directly into the LIILI 1 DNA of, gut dwelling microorganisms results in still stronger expression of protective Fl, Fl fusion or Fl truncate proteins. This L~ ' 'n~mt DNA i8 particul~rly ch~racterised in that it it comprises the complete Fl operon including caflR, a positive regulator of Fl expression;
caflM, encoding for the proposed chaperone sequence which assists in export of the Fl sub-units across the cell wall; caflA, thought to encode a protein which anchors the Fl into the cell wall; all in addition to the cafl gene encoding the Fl subunit or a truncate or fusion product thereof.
The method, cu..~LLu~Ls, microorganisms and vaccines of the invention will now be exemplified by way of illustration only by reference to the following Sequence listing, Figure and Examples. Still further will oe evident to those skilled in the art in the light of these.
SEQUENCE LISTING:
SEQ ID No l: is the sequence of a PCR primer nl~gnn~rlPntide CC~ "U .ding to the first 21 bases encoding for mature cafl with an additionPl 5' region encoding for a SacI site.

WO 95/18231 ~ , PCTIGB94/02818 ~179639 SEQ ID No 2: is the sequence of a PCR primer ol~gnn~lrlpntide cuLLcL.~u~ldLng to the sequence of cafl which encodes a 'stem loop' downstream of the ~Prminnt,~nn codon with an added 5' region encoding SacI and AccI sites.
SEQ ID No 3: is that of a PCR primer nl~gnn-lrlPotide ~u.~ ,ul-dlng to ~n internal end region of the cafl gene starting 107 bases ' - ~LC
from the end of the first nl1gnmlrlpntide.
SEQ ID No 4: is that of the pFGAL2a construct showing the fusion of the first few bases of the ~-galactosidase sequence in the vector with cafl minus its signal sequence and having a 5' tail including a Sac I
restriction site; the sequence is shown up to the cafl MCC 3' end with some vector bases.
SEQ ID No 5: is that of the protein encoded by pFGAL2a.
SEQ ID No 6: is that of pFSIG3a: including cafl sequence encoding mature Fl expressed as a fusion with the F~,~ LT3 siOnal sequence encoded by the vector and having 5' tail including a SacI restriction site; the sequence shown to AACC at 3' end of cafl and its ad~oining vector bases.
SEQ ID No 7: is that of the protein encoded by pFSIG3a.
SEQ ID No ô: is that of pFORFlb: including the entire cafl gene and having a 5' tail including a SacI restriction site; the sequence shown to TATAG d~.m~j~Lc of the cafl open reading frame. The two series encoded at the 5 ' end of the sequence are produced separately to the Fl fusion.
SEQ ID No 9: is that of the end of the first protein encoded by pFORFlb.

~ WO 95/18~31 21 ~ 9 6 3 9 PCT/GBg l/02818 SEQ ID No 10: i6 that of the Fl fusion encoded by pFORFlb.
SEQ ID No 11: i8 that of primer FIOU2 used to amPlify the Fl operon.
., SEQ ID No 12: is that of primer M4D used to amplify the Fl operon .
SEQ ID No 13: is that of primer M3U used to amplify the Fl operon.
SEQ ID No 14: is that of primer FIOD2 used to amplify the Fl operon .
SEQ ID No 15: is thst of a primer used with the primer of SEQ ID No 1 for preparation of pFSIG3a.
SEQ ID No 16: is that of a primer used with the primer of SEQ ID No 17 to produce pFORFlb.
SEQ ID No 17: is that of a primer used with the primer of SEQ ID No 16 to produce pFORFlb.
FIGURE:
Figure 1 shows schematic reprP~PntDt1nnq of the positions of Fl sequences in the cullaLlu~L~ pFGAL2a, pFSIG3a and pFORFlb.
Figure 2 shows to relative positions of the primers SEQ ID No 11 to 14, the retriction enzyme sites and the caflR, caflM, caflA and cafl subunits .
~ AMPLF!~.
General methods: Y. nPctiq was grown aerobically at 28C in Blood Agar Base broth, pH6.8, rr~nt~ininF 15g/l proteose peptone, 2.5g/l liver digest, 5g/l yeast extract, 5g/l NaCl 6--r~lPm ntP~l with 80ml 0.25% haemin in l/lOON NaO~. Strains of S. tynhiml-rl used were WO 95/18231 - ~ 1 7 9 6 3 9 PCTIGB94/02818 sL3261 and LB5010 as described by Hosieth & Stocker (1981) Nature (London) 291: p238-239 and Maskell et al (1987) Microb. Pathog. 2 pp211-221 respectively and these and E coli JM109 were cultured and stored as described by Sambrook et al (1989) Molecular Cloning Manual.
Cloning of cafl: DNA ~as isolated from Y. ~At~l 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).
nl;gnnl~rlpntideg were prepared with a Beckman 200A DNA synthesiser for use in the PCR.
FXAblPL~ 1: DFGAl ;lA cnnctrurt:
nl~enm-rlF.otide GATCGAGCTCGGCAGATTTAACTGCAAGCACC (SEQ ID No 1) was synth~ ,U.,G u..dlng to the first 21 bases of cafl i ''At~ly following the nucleotides encoding the signal sequence with an additional 5' region encoding a SacI gite and the ~ 1 im~ntAry nl ignn--rl ~.ntlde CAGGTCGAGCTCGTCGACGGTTAGGCTCAAAGTAG (SEQ ID No 2) C~LLG ~.,..~ing to the seouence which encodes a putative 'stem loop' structure ~' ~ L-G of the cafl t~rminAtinn codon with an added 5' region encoding SacI and AccI sites. A DNA frag_ent was obtained after 35 cycles of amplification (95C, 15 secs; 50C, 15 secs;
72C, 30 secs using a Perkin Elmer 9600 GeneAmp PCR system). The fragment was purified, digested with SacI and AccI, ligated into a similarly digested pUC18 plasmid and transformed into E. coli JM109 by elG~.Llu~u-..Llon. Ele~LLu~,u.~Llon was carried out using a Biorad Gene Pulser with 0.2 cm cuvettes at 1.25kV, 251~F, 8000hms with a time constant of 20.
A pFGAL2a colony rnnt~;nin~ the cloned cafl gene was identified by PCR
using an nl ignn~rl~ntide TGGTACGCTTA~ iow~:lAl (SEQ ID No 3) 1U~ v .ding to an internal region of the gene 128 to 153 nucleotides from the site identified as the signal sequence cleavage site (see WO 9~/18231 r~l,~,~, L'02818 ~17~39 Galyov et al (1990)~ and the SEQ ID No 2. A culture of the _s~21i rnntAinine the pFGAL2a was grown at 37C with shaking in Luria Broth rnntAinin~ lmM isopropyl-~-D-thiogal&~Lu~yL~,uslde (IPTG) for lô
'r hours. Whole cell lysates and periplasmic and cytoplasmic fractions of the bacteria were prepared as described by Sambrook et al (1989).
~nc-p,4n~ Pntl WPctPrn blnttin~: SDS-polyacrylamide gel ele~LLU~ UL~lS
(PAGE) and Western blotting were performed as described by Hunter et al (1993) Infec. Immun. 61. 3958-3965. 810ts were probed with polyclonal antisera raised in sheep (B283) against killed Y. ~ec~ic (EV76 strain grown at 37C) and bound antibody was detected with a horseradish peroxidase-labelled donkey anti-sheeP IgG (Sigma).
FYnrecainn of Fl in S. ty~hi : The pFGAL2a plasmid was isolated using general tPrhnirlllP~ described in Sambrook et al (1989) Molecular Cloning; a Laboratory Manual. 2nd Edition. Cold Spring Harbour Laboratory, New York. Purified plasmid was ele~LLu~-u--,Lt:~ into S.
tyrhi LB5010 (restriction~, modification~) and methylated pFGAL2a was s~~hceq~~Pntly isolated from the LB5ûlO for el~LLul,u.~.Lion into S. tv~hi~lrill"~ sL3261 (aro A-). Periplasmic and cytoplasmic fractions were prepared for SDS-PAGE and Western blotting as described above.
StAh~l~ty of ~ LIu L~: Five female Balb/c mice were innr~ll inLL~.LUU~ly with either 5xlOs or 5x107 cfu S. tv~hi rnntA~n~nE pFGAL2a in 200ul phosphate buffered saline. Control mice were innrlllAtP-l similarly with S. tynhil cnntAinin~ pUC18 with no insert. After 7 days the mice were killed by cervical tlinlnrntinn and their livers and spleens removed. The organs were ' ~ ced in lOml phosphate buffered saline using a stomacher on maximum setting for 2 minutes and the I ~, - te was serially diluted in phosphate buffered saline and placed onto L agar or L agar rnntAinin~ 55~g ml~
'rillin.

Wo 95/18231 PCT1GB94102818 ~179639 f.hnll~n~ of i 7~ mir~: Male Balb/c mice were innrlllnted with -three doses of S.tvohim~lri containing pFGAL2a, intravenously (i.v,) with 5x107 cfu bacteria at 7 day intervals, or il~L._O_.,LL 1cally (i.g.) by ~ntllhntinn with l x lOI cfu on days l, 7 and 21. ~ 'r~llin was ,-given xul,~uL~1evusly for 5 days after each innrl~lnt~nn to stabilise the plasmid. Six weeks after the last dose five mice from both the i.g. and inLLc.._.~vuOly inrlr~11At~rl groups were bled then killed by cervical ~clnrntinn and their livers and spleens taken. Livers were ~ crt1 and plated onto L-agar to confirm that all r~nl~nn~lln had been cleared.
The remaining mice were rhnl1~ong~ u~uLtu1evu~ly with 50LD50 of Y.
~;i~i strain GB. Control mice were immunised i.v. and i.g. with S.
tv~h~ i containing pUCl8, i.~L. c~lnrly with formaldehyde-killed plague vaccine (Cutter USP) or intraperitoneally with lOug of purified Fl and survival rates assessed ( see Table l ) .
FXAMpLF 2: DF~T(~A cnnctrllrt:
Construct plasmids and S. tynhi transformed by them were obtained using the method of Example l altered in so far as the PCR
product was designed to be of SEQ ID No 2 after digestion with SacI.
The digested product was ligated into SacI digested pFSZ.2 giving the sequence SEQ ID No 5 located as shown in Figure I. This construct encodes for Fl which is expressed as a fusion with the ~ LTB
signal sequence. The primer usêd instead of SEQ ID No l was that of SEQ ID No 15.
r 1~ ~: DFnRF1 h cnnctrurt:
Construct plasmids and S. tV~hi llm transformed by them were obtained using the method of Example l altered in so far as the PCR
primers were selected to produce an amplification product which after digestion with SacI/AccI consisted of SEQ ID No 6.
Primers used were of sequences SEQ ID N0 16 and SEQ ID No 17.

217~63g This construct incuL~,c,.~L,,s the cafl si2nal sequence in addition the mature protein sequence as 3hown in SEQ ID No 6 and Fig l which shows all bases ~ of the Sac I site used for di~est Protection provided by intravenous injection and oral delivery of S tvrhi tra~sformed with plasmids rrn~Aining various ~u..~L-u,~s of the invention and comparative protection afforded by direct administration of Fl and Cutter vaccines. S tymhi transformed as ~ crrih~1 above with either pFGAL2a, pFSIG3a, pFORFlb or unmodified pUCl8 were administered to mice by intravenous or i11~L~$~LiC routes and compared in effect to intraperitoneal Fl and in~. 1 Ar Cutter vaccine as described above in Example l.
TABLE l C~IALLENGE OF MICE WITH 50 x MICE LD50 Y~ PESTIS
IV or IM"TREATMENT SURVIVORS ORAL TREATMENT SURVIVORS
S. typhimurium lO/lO S. typhimurium l/lO
/pFSIG3a IV /pFSIG3a S. typhimurium 9/lO S. typhimurium g/lO
/pFGAL2a IV /pFGAL2a S typhimurium lO/lO S. typhimurium 3/lO
/pFORFlb IV /pFORFib 5. typhimurium O/lO 5 . typhimurium 0/5 /pUCl8 IV /pUCl8 Fl protein IP 6/8 Cutter vaccine 9/lO
IM
IV = intravenous IM = in~ fr IP = intraperitoneally WO 95/18231 ~ 3 g PCT/GB94/02818 RX~MPLR 4: Fl nn-~rnn rnnc~n1~rt:
Attempts to PCR replicate the entire F1 operon as one piece were rUl~ 80 a gtrategy wag develoPed whereby it w_s amplifled using PCR to produce two di6crete fragments using primer pairs (A) of SEQ ID No 11 and 12 _nd (b) of SEQ No 13 and 14 respectively to produce fragments of 3 36kb and 1.89kb from Y. r~iq MP6 template DNA. Marmur extract of DNA was used without CsClz purification. The PCR cycle conditions used were 96C for 30 seconds, 57C for 30 seconds and 72C for i minute; total of 30 cycles.
These two fragments were digested using Nhel and ~oined together.
This fused fragment, encoding the full length operon (5.25kb), was digested with EcoR1 and Sall and then cloned into a number of vectors.
When this fragment was cloned into pBR322 and expressed in E. coli, S. tyohi LB5010 or SL3261 instability of the L~ nAnt plasmid was noted. To circumvent this problem the operon was cloned into plasmid pLG339, 8 low copy number plasmid kmR. The entire F1 operon was also been inserted into ~ gene on the ~ of tvohi '11~ using vector pBRD1084.
The positions of the primers, restriction sites and caflR to cafl are shown in Figure 2.
Full protection (4/4) of mice nhAll~ng~ with with 104 lethal doses of Y. oecti c W_8 provided on administration of these transformed organisms .

2~7g~39 SEQUENCE LISTING
l ) GENERAL INFORMATION:
( i ) APPLICANT:
(A) NAME: THE SECRETARY OF STATE FOR DEFENCE IN HER
BRITANNIC MAJESTY
(B) STREET: WHITEHALL
( C ) CITY: LONDON
(E) COUNTRY: UNITED KINGDOM
(F) POSTAL CODE (ZIP) :. SWlA 2HB
'A) NAME: RICHARD WILLIAM TITBALL
B ) STREET: CBDI~ PORTON DOWN
C) CITY: SALISBURY
D ) STATE: WILTSHIRE
'E) COUNTRY: UNITED KINGDOM (GB) ~F) POSTAL CODE (ZIP): SP4 OJQ
(A! NAME: ETHEL DIANE WTl.T.T~l'cON
(B STREET: CBDE PORTON DOWN
(C CITY: SALISBURY
(D I STATE: WILTSHIRE
( E COUNTRY: UNITED KINGDOM ( GB ) ( F l POSTAL CODE ( ZIP ): sP4 OJQ
(A l NAME: SOPHIE EMMA CLARE LEARY
B~ STREET: CBDE PORTON DOWN
C I CITY: SALISBURY
D STATE: WILTSHIRE
E, COUNTRY: UNITED KINGDOM ( GB ) F) POSTAL CODE (ZIP): sP4 OJQ
(A NAME: PETRA CLAIRE FARQUHAR OYSTON
(B I STREET: CBDE PORTON DOWN
( C ~ CITY: SALISBURY
(D,l STATE: WILTSHIRE
( E COUNTRY: UNITED KINGDOM ( GB ) (F, POSTAL CODE (ZIP): sP4 OJQ
(A I NAME: ANGELA HOWELLS
(B STREET: CBDE PORTON DOWN
(C : CITY : SALISaURY
(D ~ STATE: WILTSHIRE
(E COUNTRY: UNITED KINGDOM (GB) ~OS~L CoDs (ZIP): 51'4 OJ~

WO 95118231 PCT/GB9~102818 ~17~639 ( ii ) TITLE OF INVENTION: VACCINE u~ il'l'lUN~;
(iii)NUMBER OF SEQUENCES: l4 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B1 COMPUTER: IBM PC ~hl~
( C OPERATING SYSTEM: PC-DOS/MS-DOS
(D SOFTWARE: PatentIn Release @l.O, Version ~!1.25 (EPO) (vi PRIOR APPLICATION DATA:
(A APPLICATION NUMBER: GB 9326425.7 (B FILING DATE: 24-DEC-1993 (2) 1~... '~Tn~ FOR SEQ ID NO: l:
(i) SEQUENCE CHARAo~ 1lC~
' A, LENGTH: 32 base pairs B TYPE: nucleic acid ,C STR^ : double 1 D'1 TOPOLOGY: linesr ( :i 1 MOLECULE TYPE: DNA ( geno~ic ) (iii) BY~'U1rlh1lCAL: NO
( iii ) ANTI-SENSE: NO
( vi ) ORIGINAL SOURCE:
(A) ORGANISM: Yersinis pestis (xi) SEQUENCE I~U~ 1lUN: SEQ ID NO: l:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 35 base pairs B TYPE: nucleic acid C STP^~n~c: double ~ D 1 TOPOLOGY: linear ( i ~ MOLECULE TYPE: DNA (genomic) (ii' ) }iY~U1r~ ;AL: NO
( iii ) ANTI-SENSE: NO
( vi ) ORIGINAL SOURCE:
(A) ORGANISM: Yersinia pestis (xi) SEWENCE L)~U~l~1lUN: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARAu~ 11C~
A) LENGTH: 25 base pairs B) TYPE: nucleic acid I C ) STP ^ : double D ) TOPOLOGY: linear (:i) MOLECULE TYPE: DNA (genolDic) (iii)~1YNu1~1lCAL: NO
( iii ) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A)ORGANISM: Yersinis pestis (xi) SEQUENCE L~ U~l~1lUN: SEQ ID NO: 3:

~ Wo 95/18231 ~17 ~ ~ 3 9 PCT/GB9~02818 (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEWENCE CHARAUl~x~
A)LENGTH: 541 base pairs B)TYPE: nucleic acid I C)STRANn~nN~qC double ,'D)TOPOLOGY: linenr (i ) MOLECULE TYPE: DNA (genomic) (iii)~lYlJUlllh~ AL: NO
( iii ) 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: l . . 6 ( ix ) FEATURE:
( A ) NAME/KEY: misc _ recomb ( B ) LOCATION: 53 6 . . 541 ( xi ) SEQUENCE L~ U~ 111.1N: SEQ ID NO: 4:

Ser Ser Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu 1 ~ 5 lo 15 Val Glu Pro Ala Arg Ile Thr Ile Thr Tyr Lys Glu Gly Ala Pro Ile Thr Ile Met Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn m ACA GAT GCC GCG GGT GAT CCC ATG TAC TTA ACA m ACT TCT CAG 238 Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gln Asp Gly Asn Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys Asp Ser AGA GAT m GAT ATC TCT CCT MG GTA MC GGT GAG MC CTT GTG GGG 334 Arg Asp Phe Asp Ile Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly loo 105 llo GAT GAC GTC GTC TTG GCT ACG GGC AGC CAG GAT TTC m GTT CGC TCA 382 Asp Asp Val Val Leu Ala Thr Gly Ser Gln Asp Phe Phe Val Arg Ser WO 95/18231 2 1 7 9 6 3 9 1 ~ . ~ , 18 ~

Ile Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala GTA ACC GTA ACC GTA TCT AAC CAA TAATCCATAT AnATAATAnA TAAAr:f:Ann(: 484Val Thr Yal Thr Val Ser Asn Gln ~i-ALlAl~i~;C CTCCTTTAAT ATTTATGAAT TATCCTACTT TGAGCCTAAC CGTCGAC 541 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARAol~
(A`,LENGTH: 151 amino acids (B ITYPE: amino acid (D TOPOLOGY: linear (iilMOLECULE TYPE: protein (xi,lSEQUENCE ll~ 'llON: SEQ ID NO: 5:
er Ser Al~ Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val lu Pro Ala Arg Ile Thr Ile Thr Tyr Lys Glu Gly Ala Pro Ile Thr le Met Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe hr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gln Asp Gly Asn Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys Asp Ser Arg sp Phe Asp Ile Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp loO 105 110 Asp Val Val Leu Ala Thr Gly Ser Gln Asp Phe Phe Val Arg Ser Ile Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gln ( 2 ) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARAol~
,A) LENGTH: 542 base pair6 B) TYPE: nucleic acid C) sTRANnFnN~c~: double ,D) TOPOLOGY: linear ~ wo 95/lg231 ~ 1 7 9 ~ 3 9 F~l, .,,,, ~ '02818 (ii) MOLECULE TYPE: DNA (genomic) ( iii ) llYI~ul~lh~ AL: NO
( iii ) ANTI-SENSE: NO
(vi l ORIGINAL SOURCE:
(A ORGANISM: Yersinia pestis (ix FEATURE:
(A,~ NAME/KEY: CDS
( B I LOCATION: 3 . . 455 ( ix, FEATURE:
(A NAME/KEY: misc_recomb (B ~ LOCATION: 1. .6 (ix I F~ATURE:
(A NAME/KEY: misc _ recomb (Bj LOCATION: 536..541 (xi) SEQUENCE l)~i~Ul~l~llUN: SEQ ID NO: 6:

Ala Pro Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg Ile Thr Ile Thr Tyr Lys Glu Gly Ala Pro Ile Thr Ile Met Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ale Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gln Asp Gly Asn Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys Asp Ser Arg Asp Phe Asp Ile Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gln Asp Phe Phe Val Arg Ser Ile Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala 13û 135 140 GTA ACC GTA ACC GTA TCT AAC CAA TAATCCATA TAnATAATAn ATAAAnnAnG 484 Val Thr Val Thr Val Ser Asn Gln ~ ~ 7 ~ 6 3 9 ~CT/GB9~102818 ~

Gu1A1LA1~iu CCTCCmAA TATTTATGAA TTATCCTACT TTGAGCCTAA CCGTCQAC 542 (2) INFORMATION FOR SEQ ID NO: 7:
( i ) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 151 amino acids (B 1 TYPE: amino acid (D 1 TOPOLOGY: linear (ii1 MOLECULE TYPE: protein (xi SEQUENCE ~ U~l~1lUN: SEQ ID NO: 7:
la Pro Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val lu Pro Ala Arg Ile Thr Ile Thr Tyr Lys Glu Gly Ala Pro Ile Thr le Met Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe 50 55 . 60 hr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gln As Gly Asn Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys Asp Ser Arg ô5 90 95 sp Phe Asp Ile Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly As lOO 105 llO
Asp Val Val Leu Ala Thr Gly Ser Gln Asp Phe Phe Val Arg Ser Ile Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val Th Val Th V l S r A Gl 45r r a e sn u (2) INFOPIIATION FOR SEQ ID NO: 8: `
(i) SEQUENCE CHARACTERISTICS:
IA) LENGTH: 542 base pairs B) TYPE: nucleic acid C) ~ : double D) TOPOLOGY: linear OLECULE TYPE: DNA (genomic) (iii) hY~u~ lcAL: NO
( iii ) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Yersinia pestis ( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..7 WO 95/18231 . . PCTIGBg~/~28]8 (ix ~ FEAT~ffE:
(A l NAME/KEY: migc _ recomb (B LOCATION: 1..6 (ix I FEATll~E:
(A I NAME/KEY: misc recomb (Bl LOCATION: 536..54 (ix FEATUP,E:
(A NAME/KEY: CDS
(B ~ LOCATION: 21. .530 (Xi l SEQUENCE J~iff~ )N: SEQ ID NO: 8:
G AGC TCG TAnnAr~AA TAT ATG AAA AAA ATC AGT TCC GTT ATC GCC ATT 50 Ser Ser Met LYB Lys Ile Ser Ser Val Ile Ala Ile 5 lo Ala Leu Phe Gly Thr Ile Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg Ile Thr Leu Thr Tyr Lys Glu Gly Ala Pro Ile Thr Ile Met Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ala Gly Asp Pro 75 80 85 go Met Tyr Leu Thr Phe Thr Ser Gln Asp Gly Asn Asn His Gln Phe Thr 95 loo 105 Thr Lys Val Ile Gly Lys Asp Ser Arg Asp Phe Asp Ile Ser Pro Lys llo 115 120 Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu Ala Thr Gly AGC CAG GAT TTC m GTT CGC TCA ATT GGT TCC AAA GGC GGT AAA CTT 482 Ser Gln Asp Phe Phe Val Arg Ser Ile Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gln WO 95/18231 ~' 1 PCT1GB94/02818 TAATCCATAT AG 54z (2) lN~ ~TTnN FOR SEQ ID NO: 9:
(i) SEWENCE ~IARA~
(A~ LENGTH: 2 amino acids (B TYPE: a~ino ncid (D' TOPOLOGY: linear (ii MOLECULE TYPE: protein (xi SEQUENCE ll~;m~.luN: SEQ ID NO: 9:
Ser Ser (2) INFORMATION FOP. SEQ ID NO: lO:
( i ) SEWENCE CHARA~
(A) LENGTH: 170 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEWENCE Uh:~ lll)N: SEQ ID NO: lO:
et Lys Lys Ile Ser Ser Val Ile Ala Ile Ala Leu Phe Gl Thr Ile 5 lo y 15 la Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg Ile Thr Leu Thr Tyr Lys Glu Gly Ala Pro Ile Thr Ile Met Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val ly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr er Gln Asp Gly Asn Asn His Gln Phe Thr Thr Lyfi Val Ile Gly Lys lOo 105 110 Asp Ser Arg Asp Phe Asp Ile Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gln Asp Phe Phe Val Arg Ser Ile ~ly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gln ~ WO 95/18231 217 g ~ 3 9 pclr~GB9~m28l8 .

(2) INFORMATION FOR SEQ ID NO: 11:
( i ) SEQUENCE CHARACTERISTICS:
;A' LENGTK: 38 base pairs B~ TYPE: nucleic acid C I STRA : double l D TOPOLOGY: linear (:i MOLECULE TYPE: DNA (genomic) (ii' ) ~Y~UlrlcllUAL: NO
( iii ) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Yersini~ pestis (xi) SEQUENCE l~ Hl~llUN: SEQ ID NO: 11:

(2) INFORMATION FOR SEQ ID NO: 12:
( i ) SEQUENCE CHARAu l ~;Kl~
A'I LENGTH: 29 base pairs B TYPE: nucleic acid C I STRA.~J~L..~::i: double D TOPOLOGY: linear (- i MOLECULE TYPE: DNA (genomic) (ii ' )HYPOTHETICAL: NO
( iii ) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Yersinia pestis (xi) SEWENCE IJ~::;UK1~11UN: SEQ ID NO: 12:
~ wlAllu~ TCGCTAGCM TGTTTMCG 29 (2) INFORMATION FOR SEQ ID NO: 13:
( i ) SEQUENCE CHARACTERISTICS:
~'A LENGTH: 31 base palrs B' TYPE: nucleic acid C ~ .~TRA ~: double 'D TOPOLOGY: linear ( i I MOLECULE TYPE: DNA (genomic) (ii' ) HYPOTHETICAL: NO
( iii ) ANTI -SENSE: ,NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Yersinia pestis (xi) SEQUENCE L'h~UKl~llUN: SEQ ID NO: 13:
ATCGTTAAAC ATTGCTAGCG Ann~ATA~'n~ C 31 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARAu1~;Kl~
(A) LENGTH: 39 base pairs ~B) TYPE: nucleic acid (C) sT~ANnFn~rc~: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ( iii ) HYPOTHETICAL: NO
( iii ) ANTI -SENSE: NO

WO 95/18231 PCTIGB9~1 ~179639 02818 ~

(vi) ORIGINAL SOURCE:
(A~ORGANISM: Yersinia pestis (xi) SEWENCE ~UK~ lUN: SEQ ID NO: 14:

(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARAu-rKl~llu~: ;
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) !;TP~ : double (D) TOPOLOGY: linear ii) MOLECULE TYPE: DNA (genomic) iii)ny~uelr~ AL: NO
iii ) ANTI-SENSE: NO
i) ORIGINAL SOURCE: (A) ORGANISM: Yersinia Pestis ,xi) SEWENCE l~uKll~llUN: SEQ ID NO: 15:
CTAGGAGCTC CCGCAGAm AACTGCAAGC 3û
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEWENCE CHA~Aul~Kl~llw (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STp^`~n~-cc: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)KYNu~ AL: NO
( iii ) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia Pestis (xi) SEQUENCE I~UKl~llUN: SEQ ID NO: 16:
GATCGAGCTC rTAr.nAr.nTA ATATATGAAA 30 (2) INFORMATION FOR SEQ ID NO: 17:
( i ) SEWENCE CHARAu 1 ~Kl~
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRA : double (D) TOPOLOGY: linear ii) MOLECULE TYPE: DNA (genomic) iii)~Y~U~ll~lll:AL: NO
iii ) ANTI-SENSE: NO
vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia Pestis xi) SEWENCE U~;~UKl~llUN: SEQ ID NO: 17:

Claims

23

1. Recombinant DNA that when incorporated into the DNA of a micro-organism is capable of transforming that micro-organism such that it is enabled to express aprotein in vivo which produces a protective immune response against Yersinia pestis in a human or animal body when the micro-organism is administered by oralroute.

2. A plasmid capable of transforming a micro-organism such that it is enabled toexpress a protein in vivo which produces a protective immune response against Yersinia pestis in a human or animal body when the micro-organism is administered by oral route.

3. A micro-organism comprising recombinant DNA, or a plasmid comprising recombinant DNA, whereby it is enabled to express a protein in viro which produces a protective immune response again Yersinia Pestis in a human or animalbody when administered by oral route.

4. A micro-organism as claimed in claim 3 characterised in that it is a human oranimal gut colonising micro-organism.

5. Recombinant DNA, a plasmid or a micro-organism as claimed in any one of claims 1 to 4 wherein the transformed micro-organism maintains its ability to colonise the human or animal gut.

6. Recombinant DNA, a plasmid or a micro-organism as claimed in claim 1, 2,3,4 or 5 wherein the protein which produces the immune response comprises all or part of the F1 protein of Yersinia pestis.

7. Recombinant DNA comprising a DNA sequence as described in SEQ ID No 4.

8. Recombinant DNA as claimed in claim 1 comprising the F1 operon of Yersinia Doctic including the caf1R, caf1M, caf1A and caf1 gene subunits .

9. Recombinant DNA as claimed in claim 8 wherein the F1 operon is derived by PCP, amplification of Yersinia pestis template DNA using primer pairs of SEQ ID No 11 and 12 and SEQ ID No 13 and 14 to produce two discrete fragments, these fragments digested using Nhel and then joined to provide a single F1 operon containing fragment.

10. A plasmid comprising recombinant DNA as claimed in claim 8 or 9.

11. A plasmid as claimed in claim 10 being a low copy number plasmid .

12. A plasmid as claimed in claim 10 or 11 wherein the plasmid is pLG339 or pBRD1084.

13. A plasmid as claimed in Claim 2 comprising a DNA sequence as described in SEQ ID No 4.

14. A plasmid as claimed in Claim 2 or Claim 13 characterised in that it comprises a lacZ promoter upstream of a sequence encoding for all or part of the F1 antigen.

15. A plasmid as claimed in Claim 14 characterised in that it comprises a pUC18 vector that has all or part of the caf1 sequence inserted downstream of the lacZ promoter.

16. A microorganism as claimed in Claim 3 or 4 containing recombinant DNA comprising a DNA sequence as described in SEQ ID No 4 or as claimed in claim 8 or 9.

17. A microorganism as claimed in Claim 3 or 4 containing a plasmid as claimed in any one of claims 7 to 15.

18. A microorganism as claimed in Claim 3, 4, 16 or 17 being an attenuated microorganism not capable of causing disease in humans or animals.

19. A microorganism as claimed in Claim 3, 4, 10, 11 or 12 being an aro A and/or aro C mutant.

20. A microorganism as claimed in Claim 18 or 19 being a Salmonella.

21. A microorganism as claimed in Claim 20 being a Salmonella typhimurium or a Salmonella typhi.

22. A microorganism as claimed in any one of claims 3, 4, 18, 19, 20 or 21 wherein a recombinant DNA of claim 8 or 9 is integrated directly into the microorganisms chromosomal DNA.

23. A vaccine comprising a microorganism as claimed in any one of Claim 3, 4 or 16 to 22 together with a pharmaceutically acceptable carrier.

24. A recombinant DNA, plasmid, microorganism or vaccine according to any one of claims 1 to 23 as described in Example 1 or Example 4.