VACCIN SYNTHETIQUE CONTRE LA FIEVRE APHTEUSE ET PROCEDE DE PREPARATION CORRESPONDANT

SYNTHETIC VACCINE AGAINST FOOT AND MOUTH DISEASE AND A PROCESS FOR THE PREPARATION THEREOF

Abrégé anglais

A synthetic vaccine against foot and mouth disease is
produced by conjugating at least one membrane-anchoring
compound with at least one partial sequence of a protein
of the foot and mouth disease virus. The said vaccine has
the advantage that it can be stored for a very long time
even without cooling and that, by reason of its high
activity, it generates an adequate protection against
foot and mouth disease even after a single administra-
tion.

Revendications

- 12 -
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A synthetic vaccine against foot and mouth disease,
which vaccine comprises a conjugate of at least one
membrane-anchoring compound and at least one partial
sequence of a protein of the foot and mouth disease
virus.
2. A synthetic vaccine as claimed in claim 1, which
comprises a membrane-anchoring compound and a partial
sequence of foot and mouth disease virus, which are
linked together covalently.
3. A synthetic vaccine as claimed in claim 1, wherein the
membrane-anchoring compound is a bacterial membrane
lipoprotein.
4. A synthetic vaccine as claimed in claim 2, wherein the
membrane-anchoring compound is a bacterial membrane
lipoprotein.
5. A synthetic vaccine as claimed in claim 1, wherein the
membrane-anchoring compound has one of the formulae
below
<IMG> <IMG> <IMG>
I. II. III.
<IMG> <IMG>
<IMG>
IV. V. VI.

- 13 -
<IMG>
VII.
in which A can be sulfur, oxygen, disulfide
(-S-S-), methylene (-CH2-) or -NH-;
n = 0 to 5, m = 1 or 2;
C* is an asymmetric carbon atom with the R or S con-
figuration,
R, R' and R" are identical or different and each is
hydrogen or an alkyl, alkenyl or alkynyl group which
has 7 to 25 carbon atoms and which can be substitu-
ted by hydroxyl, amino, oxo, acyl, alkyl or cyclo-
alkyl groups, B in formula VI can have the meaning
of each of the -(CH2)n-(substituted alkyl) radicals
listed in formulae I-V, and R1 and R2 are identical
or different and have the same meanings as R, R' and
R" but can also be -OR, -O-COR, -COOR, NHCOR or
-CONHR, where X is a chain of 1 to 10 amino acids
to which the partial sequence of the virus is
bonded.
6. A synthetic vaccine as claimed in claim 1, wherein the
partial sequence of the foot and mouth disease virus
which is bonded to the membrane-anchoring compound is
selected from the group comprising
sequence-(134-154)
" -(135-154)
" -(134-158)
" -(134-160)
" -(141-160)
" -(141-158)

- 14 -
sequence-(200-213)
" -(200-210)
" -(161-180),
or the C-terminal amidated or alkylamidated forms
thereof, it being possible to use the sequences of
all known serotypes and subtypes.
7. A synthetic vaccine as claimed in claim 1, wherein the
partial sequence of the foot and mouth disease virus
VP 1 (135-154) is bonded to the membrane-anchoring
compound.
8. A synthetic vaccine as claimed in claim 1, which
comprises a mixture of peptides from various sero-
and/or subtypes of the foot and mouth disease virus,
each of which is covalently bonded to the membrane-
anchoring compound or membrane-anchoring compounds.
9. A synthetic vaccine as claimed in claim 1, which
comprises a mixture of sequences VP1 134-160 of
serotypes O, A or C bonded to the membrane-anchoring
compound N-palmitoyl-S-[2,3-(bispalmitoyloxy)propyl]-
cysteinyl-seryl-serine.
10. A synthetic vaccine as claimed in claim 1, which
vaccine comprises N-palmitoyl-S-[2,3-
(bispalmitoyloxy)propyl]-cysteinyl-seryl-seryl-VP 1
(135-154), it being possible for the membrane-
anchoring compound to be in the form of the R,S or R,R
diastereomer or of a mixture of diastereomers.
11. A synthetic vaccine as claimed in claim 1, wherein the
membrane-anchoring compound is in the form of the R,R
diastereomer.
12. A process for the preparation of a synthetic vaccine
as claimed in any one of claims 1 to 11, which
comprises the partial sequences of the foot and mouth
disease virus which have been prepared in a manner
known per se being bonded to the membrane-anchoring
compound by a conjugation reaction.

- 15 -
13. A pharmaceutical or veterinary medical formulation,
which contains a synthetic vaccine as claimed in any
one of claims 1 to 11, where appropriate in addition
to customary auxiliaries and/or carriers, adjuvants
and/or other vaccines.
14. The use of a synthetic vaccine as claimed in any one
of claims 1 to 11 for raising antibodies against foot
and mouth disease viruses in mammals.

Description

1333563
Description
,
A ~ynthetic vaccine against foot and mouth disease and a
process for the preparation thereof
The present invention relates to a vaccine against foot
and mouth disea~e and a process for the preparation
thereof.
Foot and mouth disease (FMD) causes great losses in
cattle breeding, despite vaccines which have now been
available for a long time. One reason for the occurrence
of foot and mouth disease at present is the unreliability
of cla6sical vaccines which contain killed or inactivated
FMD virusess the inactivation of the ViN 8 is occasion-
ally incomplete 80 that "post-vaccination" outbreaks of
FMD may occur (cf. Bohm, Strohmaier, Tierarztl. Umschau
39, 3 - 8 (1984)). This danger does not exist with
synthetic FMD vaccines because in the latter only partial
sequences of certain viral protein~, which do not have
the function of an intact virus, are used.
Although synthetic FMD vaccines already exist (cf.
European Patent Application 0,204,480) they are still in
need of improvement.
It has now been found that particularly effective FMD
vaccines can be prepared using membrane-anchoring com-
pounds and certain partial sequences of the FMD virus.Although the preparation of synthetic vaccines is men-
tionQd in Canadian Patent Application Serial No. 512,181 as
one of many possible uses of membrane anchor/active
substance con~ugates, it was not to be expected that the
con~ugate~ of membrane-anchoring compounds and partial
sequences of the FMD virus (membrane anchor/active
substance con~ugates) would exhibit the exceptional
activity which has been found on administration of
relatively small amounts of vaccine.
A ~

133~7~63
-- 2 --
Furthermore, the said vaccines are distinguished, sur-
prisingly, by providing an adequate protection even after
a single administration of the vaccine. Moreover, they
have the advantage by comparison with conventional
vaccines that virtually unlimited storage without cooling
is possible.
Accordingly, the invention relates to a synthetic vaccine
against foot and mouth disease, which vaccine comprises
a conjugate of at least one membrane-anchoring compound
and at least one partial sequence of a protein of the
foot and mouth disease virus.
Membrane-anchoring compounds are compounds which can be
embedded in biological or synthetic membranes.
Further information on membrane-anchoring compounds is to
be found in the German Offenlegungsschrift 3,546,150
already cited and in G. Jung et al. in NPeptides,
Structure and Function", V.J. Hruby and D.H. Rich, pages
179 to 182, Pierce Chem. Co. Rockford, Illinois, (1983).
Preferred membrane anchor/active substance conjugates are
those in which a membrane-anchoring compound and an
active substance, i.e. a partial sequence of an FMD
virus, are linked together covalently.
Particularly suitable membrane anchor/active substance
conjugates have proven to be those in which the membrane-
anchoring compound is a lipoprotein. Very particularlysuitable are membrane-anchoring compounds of the follow-
ing formulae
R -C0-0-CH2 R -0-CH2 R -0-C0-CH2
R'-C0-0-CH* R'-0-CH* R'-0-C0-CH*
(CH2)n (CH2)n (CH2)n
A A
(CH2)m (~H2)m (CH2)m
R"-C0-NH-CH*-C0-X R"-C0-NH-CH*-C0-X R"-C0-NH-CH*-C0-X
1. II. III.

133~3
R -NH-CO-CH~ R -CO-CH2
R'-NH-CO-CH R'-CO-CH
(CH2)n (C,H2)n B
A A - A
1 ~2 m I *2 m I *2 m
R"-CO-NH-CH -CO-X R"-CO-NH-CH -CO-X R"-NH-CO-CH -CO-X
IV. V. VI.
Rl-CH2
R2-CH
(CH2)n
A
(CH2)m
R-CO-NH-CH*-CO-X
VII.
in which A can be sulfur, oxygen, disulfide (-S-S-),
methylene (-CH2-) or -NH-;
n = O to 5, m = 1 or 2;
C* is an asymmetric carbon atom with the R or S con-
figuration,
R, R' and R" are identical or different and each is
hydrogen or an alkyl, alkenyl or alkynyl group which has
7 to 25 carbon atoms and which can be substituted by

1333S63
hydroxyl, amino, oxo, acyl, alkyl or cycloalkyl groups,
B in formula VI can have the meaning of each of the
-(CH2)n-(substituted alkyl) radicals listed in formulae
I-V, and Rl and R2 are identical or different and have the
same meanings as R, R' and R" but can also be -OR,
-O-COR, -COOR, NHCOR or -CONHR, where X is a chain of 1
to 10 amino acids to which the partial sequence of the
virus i8 bonded.
Examples of these to be particularly emphasized are:
N-termini occuring in bacterial lipoprotein, such as, for
example: Y-Ser-Ser-Ser-Asn, Y-Ile-Leu-Leu-Ala, Y-Ala-Asn-
Asn-Gln, Y-Asn-Ser-Asn-Ser, Y-Gly-Ala-Met-Ser, Y-Gln-Ala-
Asn-Tyr, Y-Gln-Val-Asn-Asn, Y-Asp-Asn-Ser-Ser, where Y
can be one of the radicals listed under formula I to VII.
These lipopentapeptides can also be used in shortened
form (lipodi, lipotri or lipotetrapeptides) as membrane-
anchoring compound. Very particularly preferred is N-
palmitoyl-S-t2,3-(bispalmitoyloxy)propyl]-cysteinyl-
seryl-serine (Pam3Cys-Ser-Ser), N-palmitoyl-S-[2,3-(bis-
palmitoyloxy)propyl]-cysteinyl-seryl-glycine and N-
palmitoyl-S-[2,3-(bispalmitoyloxy)propyl]-cysteinyl-
alanyl-D-isoglutamine. Examples of other preferred
membrane-anchoring compounds are to be found in German
Offenlegungsschrift 3,546,150.
Many different partial sequences can be employed as the
partial sequences of the FMD virus which are bonded to
the membrane-anchoring compound. The following partial
~equences are preferred:
Partial sequence -(134-154)
" -(135-154)
-(134-158)
" -(134-160)
" -(141-160)
" -(141-158)
" -(200-213)
" -(200-210)
" -(161-180)
it being possible to use the sequences of all known

_ 5 _ 1333.~3
serotypes and subtypes. Examples of serotypes which may
be indicated in this connection are:
Se.~Ly~æ A: 134 160
A5 Westerwald NRYSTGGP--RRGDMGSAA~RAARQLP
161 180
ASFNYGAIRAITIHELLVRM
200 213
RHRQRIIAPARQLL
Al2 USA 134 160
NRYSASGSG-VRGDFGSLAPRVARQLP
161 180
ASFNY~A TRA~TIHELLVRM
200 212
~R RQR I IAPGRQL
Se-uLy~ C: 134 160
C~ Oberbayern TTY TAST ----RGnT~A~rr~TAT RAGHLP
161 180
TsFNFr~Au-RAF~TITGLLvAM
200 213
RHRQPLVAPARQLL
Serotype O:
134 160
l Raufbeuren CKYNnNAvpNLRGDLQvLAQRvARTLp
O~ LausanneCKYS~NAVPNLRGDLQVLAQRVARTLP
2 NormandyRKysKN-AvpNvRGDT~QArr7Q-RARTLp
O WuppertalCLYSDAKVSNvKGDLQVLAQRAERAL
O IsraelCRYGNVAVTNVRGDLQVrAQRA~RALP
200 213
O~ Raufbeuren ~URQRIVAPVRQTL
161 180
O~ Raufbeuren TSFNYr-ATRA--A~l~LYRM
Particularly suitable synthetic vaccines are those which
are composed of a mixture of peptides from various sero-

~ - 6 - 1333~63
and,/or subtypes of the foot and mouth disease virus, each
of which is covalently bonded to the membrane-anchoring
compound(s).
Particularly preferred synthetic vaccines are those which
are composed of a mixture of sequences VPl 134-160 of
serotypes O, A and C, bonded to the membrane-anchoring
compound N-palmitoyl-S-[2,3-(bispalmitoyloxy)propyl]-
cysteinyl-seryl-serine.
When the sequence 134-154 from serotype O and the se-
quence 134-155 from serotype A are used, the latter can,
as long as it contains C-terminal lysine, be linked
covalently via the ~-amino group to the membrane-anchor-
ing compound.
Particularly suitable synthetic vaccines according to the
invention have proven to be those which contain the
partial sequence of FMD virus VP 1 (135-154).
Additionally particularly preferred is a vaccine composed
of N-palmitoyl-S-[2,3-(bispalmitoyloxy)propyl]-cysteinyl-
seryl-seryl-VP 1 (135-154), i.e. the compound of the
formula below.
~5
Yi~ Al ~ \~ ;~\ \
S C :0 Ala
ÇH2-çH-c~2 CH2-CH
,0 0 N H Gln
C~O C O O C J
Ly s ~

~ ~ 7 ~ 133~3
The membrane-anchoring compounds can, in principle, be in
the form of R,S or R,R diastereomers or of a mixture of
diastereomers. However, it has emerged that the vaccines
which contain a R,R-diastereomeric membrane-anchoring
compound have particularly high activity.
The invention additionally relates to a process for the
preparation of a synthetic vaccine, which comprises
bonding partial sequences of the FMD virus by a conjuga-
tion reaction to the membrane-anchoring compound. The
conjugation reaction can be, for example, a condensation,
addition, substitution, oxidation or disulfide formation.
Preferred con~ugation methods are indicated in Example 1.
Further conjugation methods are described in German
Offenlegungsschrift 3,546,150 which has already been
cited.
The preparation of the membrane-anchoring compounds i8
likewise described in detail in the last-mentioned German
Offenlegungsschrift.
The separation of the diastereomers, which is necessary
where appropriate, can also be carried out by a variety
of methods as described, for example, in Hoppe-Seyler's
Z. Physiolog. Chem. 364 (1983) 593. A preferred separa-
tion process is described in Example 2.
The partiaI sequences of the particular FMD proteins can
be constructed in a variety of ways known from the
literature, cf., for example, Wunsch et al. in Houben-
Weyl, vol. 15/1,2, Stuttgart, Thieme-Verlag or Wunsch in
Angew. Chem. 83 (1971), 773, E. Gross and J. Meienhofer
(editors), The Peptides, vol. 1 (1979), 2 ~1979), 3
(1981) and 5 (1983), Academic Press, New York, or German
Offenlegungsschrift 3,546,150. A preferred process for
the preparation of a partial sequence and of a con~ugate
is explained in more detail in Example 3.
The invention additionally relates to pharmaceutical or

- - 8 - i333~63
veterinary medical formulations which contain a con~ugate
of membrane-anchoring compound and partial sequence of a
FMD virus. Besides a solvent, there is normally no
additional need for additional auxiliaries and carriers
or adjuvants for the formulations according to the
invention. However, in some cases, it may be worthwhile
to add such auxiliaries and/or carriers as well as, where
appropriate, adjuvants to the formulations according to
the invention. The relevant substances are mixed and
dispensed by processes known to those ~killed in the art.
The amount of vaccine necessary for reliable immunization
of an animal depends on the species, on the membrane-
anchoring compound(s) and on the partial sequence(s) of
the FMD virus and should be determined empirically in the
individual case. For example, sufficient for reliable
immunization of a guinea pig against FMD virus serotype
O1R is a single administration of about 100 - 500 ~g of
vaccine according to the invention, without further
auxiliaries or carriers.
The invention additionally relates to the use of the
described vaccine for raising antibodies in mammals.
Example 1
Conjugation of peptides/protein~ with Pam3Cy~-Ser-Ser-OSu
or Pam3Cys-Ser-Ser-0~
1. Peptides and proteins soluble in DMF
2 ~mol of peptide/protein are dissolved in 0.5-1 ml
of DMF, and 8 ~mol (9.2 mg) of solid Pam3Cys-Ser-Ser-
OSu are added. A homogeneous solution is obt~ineA by
gentle heating and sonication, and 4 ~mol of organic
base (N-ethylmorpholine) are added. After stirring
for 12 h, 1 - 2 ml of chloroform: methanol (1:1) are
added, and the mixture is cooled in an ice bath for
2 h.
The sediment is taken up with 1 ml of cold chloro-

- 9 - 1333563
form:methanol (1:1) washed in tert.butanol/water
(3:1) (~onicate if necessary) and freeze-dried.
2. Peptides and proteins soluble in water
2 ~mol of peptide/protein are dissolved in 0.8 ml of
water, and 4 ~mol (4.5 mg) Pam3Cys-Ser-Ser-OH are
added. The mixture is thoroughly sonicated until an
emulsion is produced and a pH of 5.0 to 5.5 is set
up. After 5 mg of EDC (1-3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride) dissolved in 100 ~1
of H2O has been added the mixture is stirred at room
temperature for 18 h and then dialyzed twice against
1 1 of distilled H2O each time. The contents of the
dialysis tube are freeze-dried.
Example 2
Separation of the diastereomers of N-palmitoyl-S-[2,3-
(bispalmitoylo y)~lop~l]-cysteine tert.-butyl ester
(P~n3Cys-OBut):
2 g of Pam3Cys-OBut are dissolved in 10 ml of mobile
phase, dichloromethane/ethyl acetate (20:1), and loaded
onto a column (length 120 cm, diameter 4 cm) packed with
MN silica gel 60, 0.063-0.2 mm/70 - 230 mesh ASTM. At a
drop rate of 2 drops/sec, 350 fractions each of 10 ml are
collected, and an aliquot of each fraction is checked for
Pam3Cys-OBut after chromatography on silica gel 60 plates
in dichloromethane/ethyl acetate (20:1) and st~i~ing with
chlorine/TDM reagent.
Fractions 280 - 315 contain the R,R diastereomer, frac-
tions 316 - 335 contain a mixture of R,R and R,S, and
fractions 336 - 354 contain the R,S diastereomer of
Pam3Cys-OBut. After the solvent has been evaporated off in
a rotary evaporator and the residue has been taken up in
warm tert.-butanol and freeze-dried, 600 mg of R,R-,
370 mg of a mixture of R,R- and R,S-, and 540 mg of R,S-
Pam3Cys-OBut are obtained.

lO- 1333563
Esample 3
SynthesisofN-palmitoyl-S-[2,3-(bispalmitoylosy)propyl]-
cysteinyl-seryl-seryl-VP 1 (135-154)
The VP 1 peptide sequence of FMD virus serotype OlR was
synthesised by solid-phase peptide synthesis. Fmoc-amino
acids were used. The following side-chain protective
groups were used: Lys(Boc), His(Fmoc), Arg(Mtr),
Ser(tBu), Asp(OtBu), Tyr(tBu). Starting from l g of (p-
benzoyloxybenzyl alcohol)- resin loaded with Fmoc-
Lys(Boc)-OH, (0.47 mmol/g), the following synthesis
cycles were performed:
N-Activation with 55 % piperidine in N-methylpyrrolidone
(1 x 2 min, 1 x 5 min), preactivation of Fmoc-A-A-OH
(1.5 mmol) in N-methylpyrrolidone (6 ml) with diiso-
propylcarbodiimide (1.5 mmol) and l-hydroxybenzotriazole
(1.5 mmol) with subsequent coupling for 1.5 h. Washing
with N-ethylmorpholine (5 % in N-methylpyrrolidone) was
followed by repetition of the preactivation and coupling.
The blocking of unreacted amino groups was carried out
with acetic anhydride (2.5 mmol) and diisopropylamine
(1.2 mmol) in N-methylpyrrolidone. After each ~tep the
peptide-resin was washed several times with N-methylpyr-
rolidone, dichloromethane and again with N-methylpyrrol-
idone.
After the resin-bound FMD virus sequence had been syn-
thesized, a part of the peptide was obtained by cleavage
with trifluoroacetic acid and checked by HPLC, MS, amino
acid analysis, chiral phase analysis and sequence analy-
sis. The bonding of 2 serine residues to the resin-bound
peptide was followed by coupling of the tripalmitoyl-S-
glycerylcysteine. After 4 hours 1 equivalent of N-methyl-
morpholine was added, and after another hour the lipopep-
tide-resin was wa~hed. The lipopeptide was separated from
the resin using 2 ml of trifluoroacetic acid (cont~ining
100 ~1 of thioanisole) within 4 1/2 hours. The filtrate

1333~63
-- 11
was evaporated, the residue was taken up with acetic
acid, and the solution was added to cold ether. The
precipitated lipopeptide was washed 3 x with ether.
Further purification was achieved by recrystallization
from trifluoroethanol/chloroform in the ratio 1:3 with
cold acetone and a few drops of water. The lipopeptide
was freeze-dried from tert.-butanol/water in the ratio
3:1.
Example 4
Activity test:
Guinea pigs with a weight of 450 to 500 g chosen at
random were inoculated intramuscularly or subcutaneously.
0.5 mg of the freeze-dried vaccine (N-palmitoyl-S-
[(2R,R)-2,3-(bispalmitoyloxy)propyl]-cysteinyl-seryl-
seryl-VP1(135-154)) was emulsified in 500 ~1 of a 1:1
mixture of 0.05 M phosphate buffer and Intralipid(R) (Rabi
Vitrum, Sweden). The mixture was sonicated for 10 s. Four
animals were infected with FMD virus by subcutaneous
injection into the left rear paw of at least 500 guinea
pigs units of a virulent 1~ FMD virus 21 days after the
inoculation. Control animals were in~ected with the
membrane-anchoring compound or phosphate buffer in place
of the vaccine. A high titer of neutralizing antibodies
loglOSN50 of 0.36 was found in all the inoculated animals.
The control animals had no antibody titer (blank 0.17).
The titer of neutralizing antibodies was determined as
the logarithm of the serum dilution necessary to neutra-
lize 50 % of the virus cells in a monolayer of BHR (baby
hamster kidney) cells. It was possible to detect anti-
bodies in the inoculated animals by means of an anti-
peptide ELISA (A4~2), which was not possible for the non-
inoculated animals. Inoculated animals showed no secon-
dary lesions, whereas all the non-inoculated animals
showed the complete picture of foot and mouth di~ease
infection.