Note: Descriptions are shown in the official language in which they were submitted.
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.
WO 95/18231 PCT/GB94/02818
217~63g
11
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
12
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 .
WO 95/18231 PCT/GB94/02818
2~7g~39
13
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
14
( 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:
GATCGAGCTC GGCAGATTTA ACTGCAAGCA CC 32
(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:
CAGGTCGAGC TCGTCGACGG TTAGGCTCAA AGTAG 35
(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:
TGGTACGCTT ACTCTTGGCG GCTAT 25
~ 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:
G AGC TCG GCA GAT TTA ACT GCA AGC ACC ACT GCA ACG GCA ACT CIT 46
Ser Ser Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu
1 ~ 5 lo 15
GTT GM CCA GCC CGC ATC ACT ATT ACA TAT MG GM GGC GCT CCA ATT 94
Val Glu Pro Ala Arg Ile Thr Ile Thr Tyr Lys Glu Gly Ala Pro Ile
20 25 30
ACA ATT ATG GAC MT GGA MC ATC GAT ACA GM TTA CTT GTT GGT ACG 142
Thr Ile Met Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val Gly Thr
35 40 45
CTT ACT CTT GGC GGC TAT MM 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
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
65 70 75
GAT GGA MT MC CAC CM TTC ACT ACA MA GTG ATT GGC MG GAT TCT 286
Asp Gly Asn Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys Asp Ser
80 85 90 95
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
115 120 125
WO 95/18231 2 1 7 9 6 3 9 1 ~ . ~ , 18 ~
16
ATT GGT TCC AAA GGC GGT AAA CTT GCA GCA GGT AAA TAC ACT GAT GCT 430
Ile 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 AnATAATAnA TAAAr:f:Ann(: 484Val Thr Yal Thr Val Ser Asn Gln
145 150
~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
5 10 15
lu Pro Ala Arg Ile Thr Ile Thr Tyr Lys Glu Gly Ala Pro Ile Thr
20 25 3
le Met Asp Asn Gly Asn Ile 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
hr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gln Asp
65 70 75 80
Gly Asn Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys Asp Ser Arg
85 90 95
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
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 Gln
145 150
( 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
17
(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:
GA GCT CCC GCA GAT TTA ACT GCA AGC ACC ACT GCA ACG GCA ACT CTT 47
Ala Pro Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu
5 10 15
GTT GAA CCA GCC CGC ATC ACT ATT ACA TAT MG GM GGC GCT CCA ATT 95
Val Glu Pro Ala Arg Ile Thr Ile Thr Tyr Lys Glu Gly Ala Pro Ile
20 25 30
ACA ATT ATG GAC AAT GGA AAC ATC GAT ACA GAA TTA CTT GTT GGT ACG 143
Thr Ile Met Asp Asn Gly Asn Ile 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 191
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 239
Phe Thr Asp Ala Ale Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gln
65 70 75
GAT GGA AAT AAC CAC CAA TTC ACT ACA AAA GTG ATT GGC AAG GAT TCT 287
Asp Gly Asn Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys Asp Ser
80 85 90 95
AGA GAT TTT GAT ATC TCT CCT AAG GTA MC GGT GAG AAC CTT GTG GGG 335
Arg Asp Phe Asp Ile 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 383
Asp Asp Val Val Leu Ala Thr Gly Ser Gln 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 43
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
145 150
WO 95/18231
~ ~ 7 ~ 6 3 9 ~CT/GB9~102818 ~
18
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
5 10 15
lu Pro Ala Arg Ile Thr Ile Thr Tyr Lys Glu Gly Ala Pro Ile Thr
20 25 30
le Met Asp Asn Gly Asn Ile 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
hr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gln As
65 70 75 80
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
115 120 125
Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val
130 135 140
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
2179639
WO 95/18231 . . PCTIGBg~/~28]8
19
(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
GCA TTA TTT GGA ACT ATT GCA ACT GCT AAT GCG GCA GAT TTA ACT GCA 98
Ala Leu Phe Gly Thr Ile Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala
15 20 25
AGC ACC ACT GCA ACG GCA ACT CTT GTT GAA CCA GCC CGC ATC ACT CTT 146
Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg Ile Thr Leu
30 35 40
ACA TAT MG GAA GGC GCT CCA ATT ACA ATT ATG GAC AAT GGA AAC ATC 194
Thr Tyr Lys Glu Gly Ala Pro Ile Thr Ile Met Asp Asn Gly Asn Ile
45 50 55
GAT ACA GAA TTA CTT GTT GGT ACG CTT ACT CTT GGC GGC TAT AAA ACA 242
Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr
60 65 70
GGA ACC ACT AGC ACA TCT GTT MC TTT ACA GAT GCC GCG GGT GAT CCC 290
Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ala Gly Asp Pro
75 80 85 go
ATG TAC TTA ACA TTT ACT TCT CAG GAT GGA AAT AAC CAC CAA TTC ACT 338
Met Tyr Leu Thr Phe Thr Ser Gln Asp Gly Asn Asn His Gln Phe Thr
95 loo 105
ACA AAA GTG ATT GGC AAG GAT TCT AGA GAT TTT GAT ATC TCT CCT AAG 386
Thr Lys Val Ile Gly Lys Asp Ser Arg Asp Phe Asp Ile Ser Pro Lys
llo 115 120
GTA AAC GGT GAG AAC CTT GTG GGG GAT GAC GTC GTC TTG GCT ACG GGC 434
Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu Ala Thr Gly
125 130 135
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
140 145 150
GCA GCA GGT AAA TAC ACT GAT GCT GTA ACC GTA ACC GTA TCT AAC CAA 530
Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gln
155 160 165 170
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
20 25 30
Thr Leu Val Glu Pro Ala Arg Ile Thr Leu Thr Tyr Lys Glu Gly Ala
35 40 45
Pro Ile Thr Ile Met Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val
50 55 60
ly 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
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
115 120 125
Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gln Asp Phe Phe Val
130 135 140
Arg Ser Ile ~ly 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 Gln
165 170
~ WO 95/18231 217 g ~ 3 9 pclr~GB9~m28l8
.
21
(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:
TCGCCCGGGA ATTCCGMCA TMMTCGGTT CAGTGGCC 38
(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 ~
22
(vi) ORIGINAL SOURCE:
(A~ORGANISM: Yersinia pestis
(xi) SEWENCE ~UK~ lUN: SEQ ID NO: 14:
GATAGATCTG TCGACTGAAC CTATTATATT GCTTCGCGC 39
(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:
CAGGTCGAGC TCGTCGACCT ATATGGATTA TTGGT 35
