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Document AM2 
Appl.No. 09/848,616 



Europaisches Patentamt 
Eur pean Patent Office 
Office eu rope en des brevets 



© Publication number: 



0 578 293 A1 



© 



EUROPEAN PATENT APPLICATION 



© Application number: 93201712.2 
© Date of filing: 15.06.93 



© intciAC12N 15/16, C07K 7/06, 
C07K 17/02, A61K 39/385, 
C12N 1/21, //(C12N1/21, 
C12R1:19) 



CO 

o> 

CM 
00 

in 



© Priority: 18.06.92 EP 92201775 

© Date of publication of application: 
12.01.94 Bulletin 94/02 

© Designated Contracting States: 

AT BE CH DE DK ES FR GB GR IE IT LI LU MC 
NL PT SE 



© Applicant: AKZO N.V. 
Velperweg 76 
NL-6824 BM Arnhem(NL) 

© Inventor: Van Der Zee, Anna 
Jasmijnlaan 1 
NL-3442 HZ Woerden(NL) 
Inventor: Van Die, Irma Marianne 
Waterlelie 124 
NL-2804 PZ Gouda(NL) 
Inventor: Hoekstra, Willem Pieter Martin 
Couwenhoven 61-46 
NL-Zeist(NL) 

Inventor: Gielen, Josephus Theodorus 

Kempensbos 10 

NL-5848 AM St Anthonis(NL) 

© Representative: Hermans, Franciscus G.M. et 
al 

P.O. Box 20 

NL-5340 BH OSS (NL) 



© E-coli P-fimbrine as immunogenic carrier system against GnRH. 

© The present invention is concerned with vaccination of mammals against GnRH. The vaccine comprises a 
GnRH peptide conjugate to E. coli fimbrial-filaments and elicits an immune response against GnRH. 



3NSDOCID: <EP 0578293A 1 J_> 



Rank Xerox (UK) Business Services 

13. 10/3.6/3.3- n 



EP 0 578 293 A1 



An immunogenic carrier system capable of eliciting an immune response against "Gonadotropin 
Releasing Hormone" GnRH, a recombinant DNA sequence coding for said carrier system and use of said 
carrier system for immunising a mammal against GnRH. 

The invention lies in the field of immunology. More specifically the invention is directed at an 
5 immunogenic carrier system capable of eliciting an immune response against the "Gonadotropin Releasing 
Hormone" (GnRH), also referred to as the "Luteinising Hormone Releasing Hormone" LHRH or an analogue 
or derivative of GnRH. 

A recombinant DNA sequence coding for said carrier system, a composition comprising the carrier 
system and use of said carrier system for immunising a mammal against GnRH also fall within the scope of 
10 the invention- The carrier system can for example be comprised in a vaccine or a medicinal preparation. 

GnRH is a decapeptide with hormonal activity with the following amino acid structure: pGlu-His-Trp-Ser- 
Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 , wherein the conventional three-letter code is used and pGlu is pyroglutamic 
acid and Gly-NH2 is glycine amide. The mRNA of GnRH comprises the GnRH sequence and a signal 
sequence which is cleaved of after translation followed by cyclization of the N-terminal Gin residue to form 
75 pyro-Glu. 

It is known that GnRH coupled to a carrier protein can be used to vaccinate mammals. Such a 
vaccination can be carried out for a variety of reasons, all connected with the natural function of the GnRH. 
The GnRH formed in the hypothalamus regulates the production and release of the sex hormones LH (i.e. 
"Luteinising Hormone") and FSH ("Follicle Stimulating Hormone") in the hypophysis. A reduction of the 

20 amount of gonadotrophic hormones in the blood results in a reduced stimulation of the gonads, which 
results in low levels of steroids in blood. A reduction of the blood steroid level to a level comparable to the 
level obtained after gonadectomy can be realized by effective immunization af the animal against GnRH. 

Upon administration of GnRH or its analogue as antigen, i.e. immunogen to a patient or animal, the 
GnRH or its analogue acts as a vaccine and the host generates antibodies to the GnRH or its analogue 

25 which also act against the body's own GnRH. Thus, the analogue's effect will persist after the analogue 
itself has been metabolised or excreted. This treatment is described for various GnRH analogues or GnRH 
itself byA. Arimura et al. in Endocrinology 93:1092-1103 (1973); by H.M. Fraser et al. in the Journal of 
Endocrinology 63:399406 (1974); by S.L. Jeffcoate et al. in Immunochemistry Vol. 11, p. 75-77 (1974); by 
I. J. Clarke et al. in the Journal of Endocrinology 78:39-47 (1978); by L Pique et al. in Immunochemistry Vol. 

30 15 pages 55-60 (1978); by V.C. Stevens et al. in the American Journal of Reproductive Immunology 1:307- 
314 (1981); and in U.S. Patent 3,963,691. 

In EP 0,181,236 a description is given of an immunogenic vaccine useful as an effective contraceptive 
agent, as an agent to treat sexual hyperactivity, for the treatment of cancers and other conditions stimulated 
by sexual hormones. Said vaccine comprises a conjugate between a carrier protein and one or more nona- 

35 and decapeptides derived from GnRH. Said immunisation is reversible which is an advantage over surgical 
methods. 

The International Patent Application WO 88/05308 proposes a method for immunoneutering mammals 
with a composition comprising an immunogenic protein such as bovine serum albumine, conjugated with a 
partial peptide of GnRH having a length of 5, 6 or 7 amino acids. 
40 Vaxstrate, the world's first commercially available contraceptive vaccine for cattle, is described as an 
anti-GnRH two-dose vaccine which has been shown to prevent pregnancy in about 80% of cull cows. Said 
vaccine comprises use of a synthetic GnRH conjugated to ovalbumin adjuvanted into an oil-based vaccine 
which then stimulates immunity against GnRH. Such a vaccine should further result in a higher body score 
and production efficiency. 

45 According to WO 90/11298 a more reliable vaccine than the previously described vaccines can be 
obtained, that is particularly suited for use in prevention of boar odour of meat. Said vaccine is based on a 
peptide having a GnRH tandem structure preferably conjugated to a protein such as KLH. Said peptide is 
initially used in combination with Complete Freund's Adjuvant (CFA), followed by a booster after 8 weeks. 
In WO 88/00056 a composition is described comprising two or more different carriers individually 
so coupled to GnRH or analogues of GnRH in amounts sufficient to elicit an immune response against GnRH. 
Usually a protein carrier and an adjuvant are used and one or more boosters are required. 

The known vaccines as described, using GnRH or its analogues after conjugation to protein carriers, are 
supposed to stimulate the immune system to produce anti-GnRH antibodies which should react with GnRH 
to effectively reduce its concentration in the body. This technique is however not effective in preventing 
55 conception for an initial period of variable length following injection. 

In WO 90/03182 a solution for this problem is given by use of a composition comprising (1) free GnRH 
or its analogue and (2) an immunogenic conjugate between GnRH or its analogue and a carrier protein. 
Free GnRH or its analogue acts to prevent conception in the mammal during the period from administration 

2 

BNSDOCID: <EP 0578293A1_L> 



EP 0 578 293 A1 



to about 6 weeks, until the GnRH antibodies formed in response to the conjugate are metabolised, generally 
after about 0.5-2 years. The polypeptide conjugates themselves, however, have so far been immunogenical- 
ly unsatisfactory. 

Procedures for the conjugation of GnRH to a polypeptide carrier, e.g. bovine or human serum albumin, 
5 or tetanus toxoid or thyroglobulin, have generally involved coupling methods resulting in a poorly defined 
immunogen unlikely to retain all the structural features of free GnRH in solution as considered desirable 
from the point of view of obtaining anti-GnRH antibodies capable of blocking functioning of GnRH in vivo. 
Furthermore there is a danger that the peptide is attached to the carrier through a region important for 
immunological recognition. 

10 Effective immunisation of mammals using such conjugates to provide a high titre of anti-GnRH 

antibodies capable of significantly reducing the biological efficacy of endogenous GnRH has indeed only 
been achieved in the presence of an adjuvant liable to cause undesirable side effects, most commonly 
Freund's Complete or Incomplete Adjuvant. Freund's Complete Adjuvant interferes with the tuberculin test 
in cattle. In addition this adjuvant as well as Freund's Incomplete Adjuvant cause a variable amount of 

75 chronic inflammatory reaction at the site of injection. 

In GB 2,196,969 a vaccine is described comprising analogues of GnRH with a short peptide extension 
at the C-terminus of the native amino acid sequence which has been predicted by potential energy 
calculations to have substantially the same conformation as native GnRH in solution and may be readily 
linked to a polypeptide carrier via the side chain of a cysteine or tyrosine residue provided at the C- 

20 terminus. 

The vaccines as described generally require large amounts of the vaccine concomitant with severe 
adjuvants to obtain any antibody response. They have little or no effect on biological activity connected with 
GnRH. Most of the vaccines described are capable of eliciting an immune response, said immune response 
merely comprising the formation of antibodies against GnRH and seldom comprising effect on the biological 
25 activity of GnRH in a vaccinated mammal. Those vaccines leading to a biological effect do not result in 
100% immunisation. 

The subject invention is directed at an immunogenic carrier system capable of eliciting a greatly 
improved immune response against GnRH or an analogue or a derivative of GnRH. The immune response 
obtainable from use of a carrier system according to the invention is high enough to affect the biological 

30 activity of GnRH in the immunised mammal. In particular the carrier system is suitable for use in effectively 
suppressing the oestrous cycle, spermatogenesis and/or sexual behaviour of an animal. The carrier system 
according to the invention can be used in a vaccine, said vaccine no longer requiring such agressive 
adjuvants as Freunds Adjuvant or Incomplete Freunds Adjuvant to cause an immune response. Less 
agressive adjuvants can be used. 

35 The invention is directed at an immunogenic carrier system capable of eliciting an immune response 
against GnRH or an analogue or a derivative of GnRH, said carrier system comprising at least a part of an 
E. coli P-fimbrial filament comprising at least a part of major subunit with an insert, said insert comprising a 
peptide with at least one antigenic determinant for GnRH or an analogue or a derivative of GnRK said 
insert being located in the major subunit at a position corresponding to a position in hypervariable region 4 

40 of the wild type major subunit. Wild type in this instance implying the form of the major subunit without the 
insert. 

Fimbrial filaments, also known as fimbriae, are long filamentous appendages that are frequently found in 
large amounts on many bacterial strains. Each filament is built up of approximately a thousand sub-units 
that are polymerised in an a-helical way (Korhonen, T.K. and Rhen, M. Am. Clin. Res. 1982, 14, 272-277; 

45 Giles, C.L. and Maas, W.K. Prog. Vet. Microbiol. Immuno 1987, 3, 139-158.) Fimbriae structure has been 
studied in great detail and recombinant fimbrial filaments, also known as hybrid fimbriae, comprising 
synthetic peptides have previously been described inthe literature. A description of the structure of fimbriae 
and a description of known hybrid fimbriae is given below. 

P-fimbriae that are mainly found associated with uropathogenic Escherichia coli and are involved in the 

so attachment of the bacteria to epithelial tissue comprise one type of major subunit and several different 
minor subunits. The localisation and biogenesis of the minor fimbrial components have been studied by 
Lindberg et al. (Nature (London) 1987 328:84-87 and Riegman et al 1988 Mol. Microbiol. 2: 73-80). 

The major subunit of E. coli P-fimbriae or P-fimbrillin is predominant and determines the antigenic 
properties. The serotypes F7-F13 have been alotted to P-fimbriae and said serotypes are known to be 

55 specified by the major subunit. Comparison of the amino acid sequences of the major subunit proteins from 
the various serotypes has revealed five hypervariable regions (HRs) among otherwise more homologous 
sequences. Said hypervariable regions contain the natural epitopes of the P-fimbriae (van Die et al (1987) 
Microbiol. Pathogen 3:149-154; van Die et al (1988) FEMS Microbiol. Let. 49: 95-100). 



3NSDOCID: <EP 0578293A1_I_ 



EP 0 578 293 A1 



In Mol. Gen. Genet. (1990) 222: 297-303 van Die et al have described how hypervariable regions 1 and 
4 (HR1 and HR4) of the major subunit of P-fimbriae with serotype F1 1 were exploited for insertion of foreign 
epitopes. Several oligonucleotides coding for antigenic determinants derived from different pathogens were 
cloned and the resulting recombinant major subunits were sometimes assembled in fimbriae. The assem- 
5 bling of fimbriae only occurred when the length of the inserted peptide did not exceed 14 amino acids. 

As already described in the introduction a lot of research has already been carried out on various 
systems suitable for eliciting an immune response against GnRH. Until now however no experiments with 
hybrid fimbriae comprising an epitope suitable for eliciting an immune response against GnRH have been 
described. 

io GnRH itself is not immunogenic and needs an immunogenic carrier. Due to their polymeric structure 

fimbriae are highly immunogenic and could possibly act as immunogenic carrier for GnRH or an analogue 
or derivative of GnRH. Furthermore the inventors presumed that an immunogenic carrier system for a 
foreign synthetic peptide comprising a P-fimbrial filament with an insert in the major subunit, said insert 
comprising an antigenic determinant against GnRH, a peptide known to be generally poorly immunogenic 

75 could possibly lead to an improved immunogenic response against said synthetic peptide. Furthermore the 
facts that fimbriated bacteria can be cultivated at low cost and that fimbriae are easily isolated and purified 
from said bacteria should result in a cheaper and more effective immunogenic carrier system for use in 
immunising against GnRH. 

This could however only be feasible if one of the epitopes of a fimbrial subunit could be replaced 

20 without interfering with the formation of the subunit and preferably without interfering with subsequent 
formation of a fimbrial filament comprising polymerised subunits in large numbers. Furthermore the foreign 
epitope would have to be present in the fimbrial subunit in a configuration in which the antigenic 
determinant is suitably exposed for eliciting an immune response and does not interfere with polymerisation 
of subunits. 

25 Investigations were subsequently carried out by the inventors incorporating a DNA sequence coding for 
GnRH in the HR1 of a major subunit gene coding for a fimbrial component. Contrary to the results of Van 
Die et al (1990, Mol. Gen. Genet. 222: 297-303) demonstrating an effective incorporation of peptides other 
than GnRH in HR1, a microorganism comprising DNA coding for a P-fimbrial filament with an insertion of 
GnRH in HR1 of the major subunit of a P-fimbrial filament was discovered to be practically incapable of 

30 fimbriae formation. 

Surprisingly however further investigations by the inventors revealed that a microorganism comprising 
DNA coding for an E. coli P-fimbrial filament with an insertion of a foreign peptide comprising at least one 
antigenic determinant for GnRH in the HR4 of the major subunit of a fimbrial filament results in good 
fimbriae formation. More importantly the resulting filaments apparently comprise at least one antigenic 
35 determinant for GnRH in a configuration giving good exposure of the determinant. Furthermore an animal 
injected with a composition comprising such filaments gives an unexpectedly high titre of antibodies against 
GnRH. In fact the resulting immune response is so high that a biological effect in a process connected with 
GnRH can be obtained. 

The subject invention is therefore directed at an immunogenic carrier system capable of eliciting an 
40 immune response against GnRH or an analogue or derivative of GnRH, said carrier system comprising at 
least a part of an E. coli P-fimbrial filament comprising at least a major subunit with an insert, said insert 
comprising a peptide with at least one antigenic determinant for GnRH or an analogue or derivative of 
GnRH and said insert being located in the major subunit at a position corresponding to a position in 
hypervariable region 4 (HR4) of the wild type major subunit. 
45 The peptide with at least one antigenic determinant for GnRH that is comprised in a carrier system 
according to the invention can be a decapeptide coding for GnRH with the sequence gln-his-trp-ser-tyr-gly- 
leu-arg-pro-gly (SEQ ID: NO: 19) or a derivative of said sequence comprising at least one antigenic 
determinant for GnRH. (The amino acid sequence is expressed in the conventional three letter code for 
amino acids). 

50 The nonapeptide kwsyglrpg is known to elicit an immune response against GnRH (US Patent 4,608,251) 
as are the partial peptides (with the following single letter amino acid sequences) #ehwsy, #ehwsyg, 
#ehwsygl. hwsyglr, wsyglr, syglrpg® and yglrpg© (WO 88/05308 International Patent Application). There- 
fore said derivatives are also suitable peptides for comprising a part of a recombinant fimbrial filament of a 
carrier system for eliciting an immune response against GnRH according to the invention. 

55 Other suitable peptides that can form a component of a carrier system according to the invention are 
analogues or derivatives of GnRH capable of eliciting an immune response against GnRH. In U.S. Patent 
3,963,691 and 4,608,251 GnRH analogues are disclosed useful for stimulating anti GnRH antibodies. 
Therefore a carrier system comprising any analogue described in said patents or any other analogue or 

4 

BNSDOCID: <EP 0578293A1 J_> 



EP 0 578 293 A1 



derivative of GnRH comprising at least one antigenic determinant capable of eliciting an immune response 
against GnRH as the peptide incorporated in a part of a major subunit of a P-fimbrial filament is an 
embodiment of a carrier system according to the invention. 

As already stated in this description the serotypes F7 through F13 have been distinguished for P- 
5 fimbriae (Orskov, L, and F. Orskov. 1985. Escherichia coli in extraintestinal infections. J. Hyg. 95: 551-575). 
The amino acid sequences of several P-fimbrillins with different serotypes are disclosed in van Die et al. t 
(Microbiol. Pathogen. 3, 149-154, 1987). A carrier system according to the invention can comprise 
recombinant major subunits derivable from any of the serotypes of the P-fimbriae. In the Examples serotype 
F1 1 has been used to illustrate a suitable embodiment of a carrier system according to the invention. 

io A carrier system as described above comprising a single recombinant major subunit as part of the P- 

fimbrial filament as well as a carrier system comprising polymerised recombinant major subunits as part of 
the P-fimbrial filament fall within the scope of the invention. 

Preferably the carrier system according to the invention will comprise parts of P-fimbrial filaments or 
complete P-fimbrial filaments comprising more than one major subunit. A complete recombinant P-fimbrial 

15 filament can comprise as many as one thousand recombinant major subunits. The preference for multicopy 
presence of the peptide capable of eliciting an immune response against GnRH or an analogue or derivative 
of GnRH in polymerised recombinant major subunits in a carrier system according to the invention stems 
from the fact that the multicopy presence of an epitope on a fimbria! filament can lead to extremely high 
immunogenic activity against such an epitope. 

20 A carrier system according to the invention can be obtained through expression of a recombinant DNA 

sequence coding for at least a part of a P-fimbrial filament comprising at least a major subunit with an 
insert, said insert comprising a peptide with at least one antigenic determinant for GnRH or an analogue or 
derivative of GnRH, said insert being located in the major subunit at a position corresponding to a position 
in hypervariable region 4 (HR4) of the wild type major subunit. Wild type being defined as stated previously 

25 in the description. Therefore such a recombinant DNA sequence falls under the scope of the invention. 

A recombinant DNA sequence according to the invention comprises at least a DNA sequence L, coding 
at least for a peptide with at least one antigenic determinant for GnRH or an analogue or derivative of 
GnRH, with said DNA sequence L being integrated in a DNA sequence S at a position corresponding to a 
position in the hypervariable region HR4 of the wild type major subunit of a P-fimbrial filament, said 

30 sequence S coding for at least a part of the wild type major subunit. 

Specific favourable examples of the DNA sequence L in recombinant DNA sequences according to the 
invention are DNA sequences L comprising DNA coding for the following amino acid sequences: 
1) 

35 Leu-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Ser-Arg- 
Thr ; 

2) Leu-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Thr; 

40 3) 

Leu-Thr-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Asp- 
45 Pro-Thr ; 

4) 

Leu-Gly-Ser-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly- 

50 

Gly-Pro-Thr . 



Suitable DNA sequences coding for the above-mentioned amino acid sequences in their respective 
55 order are given: 
1) 



5 

lNSDOCID:<EP 057B293A1_I_> 



EP 0 578 293 A1 



TTG-CAG-CAC-TGG-AGC-TAC-GGC-CTG-CGT-CCA-GGA-TCC-CGA- 
ACC; 

5 2) TTG-CAG-CAC-TGG-AGC-TAC-GGC-CTG-AGG-CCT-GGA-ACC; 
3) 

TTG-ACT— CAG— CAC-TGG-AGC-TAC-GGC-CTG-CGT— CCA-GGG— GAT— 
7o CCA-ACC ; 

4) 

TTG— GGA— TCC— CAG— CAC— TGG— AGC— TAC— GGC— CTG-CGT— CCA— GGC— 
15 GGT-CCA-ACC . 

A suitable example of a recombinant DNA sequence according to the invention comprises the gene 
cluster of F1 1 with DNA sequence L integrated in hypervariable region 4 (HR4) of the major subunit. The 
20 genes responsible for synthesis of various serologically different P-fimbriae from uropathogenic E.coli, i.e. 
F7i, F7 2 , F8, F9, F11, and F13, have been cloned: 

- De Ree, J.M., P. Schwillens, L Promes, L van Die, H. Bergmans, and H. van den Bosch, 1985. 
Molecular cloning and characterization of F9 fimbriae from a uropathogenic Escherichia coli. FEMS 
Microbiol. Lett. 25:163-169; 

25 - De Ree, J.M., P. Schwillens, and J.F. van den Bosch, 1985, Molecular cloning of F11 fimbriae from a 
uropathogenic Escherichia coli and characterization of fimbriae with monoclonal antibodies. FEMS 
Microbiol. Lett. 29:91-97; 

- Hacker, J., M. Ott, G. Schmidt R- Hull, and W. Goebel, 1986, Molecular cloning of the F8 fimbrial 
antigen from Escherichia coli. GEMS Microbiol. Lett. 36: 139-144; 

30 - Hull, R.A., R.E. Gill, P. Hsu, B.H. Minshew, and S. Falkow, 1981. 

Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili 
from a urinary tract infection Escherichia coli isolate. Infect. Immun. 33: 933-938; 

- Rhen, M., J. Knowles, M.E. Pentilla, M. Sarvas, and T.K. Korhonen, 1983. P-fimbriae of Escherichia 
coli: molecular cloning of DNA fragments containing the structural genes. FEMS Microbiol. Lett. 1J5: 

35 119-123; 

- Van Die, I., G. Spierings, I. van Megen, E. Zuidweg, W.Hoekstra, and H. Bergmans, 1985. Cloning and 
genetic organization of the gene cluster encoding F7i fimbriae of a uropathogenic Escherichia coli 
and comparison with the F7 2 gene cluster. FEMS Microbiol. Lett. 28: 329-334; 

- Van Die, I., C. van den Hondel, H.-J. Hamstra, W. Hoekstra, and H. Bergmans, 1983. Studies on the 
40 fimbriae of an Escherichia coli 06:K2:H1:F7 strain: molecular cloning of a DNA fragment encoding a 

fimbrial anitgen responsible for mannose-resistant hemagglutination of human erythrocytes. FEMS. 
Microbiol. Lett. 19: 77-82, 

and any of these DNA~sequences or parts thereof can be used in a recombinant DNA sequence according 
to the invention. 

45 In some instances it is preferable that the insert, DNA sequence L, of a recombinant DNA sequence 
according to the invention not only codes for a peptide comprising at least one antigenic determinant for 
GnRH or an analogue or derivative of GnRH but also comprises DNA coding for further amino acids, 
flanking the DNA coding for said peptide. DNA sequence L can code for flanking amino acids present at 
one terminus or at both termini of the peptide comprising at least one antigenic determinant for GnRH or an 

so analogue or derivative of GnRH. Such a flanking amino acid sequence can comprise one or more amino 
acids. When DNA sequence L comprises such flanking amino acid sequences at both termini of the peptide 
comprising at least one antigenic determinant for GnRH or an analogue or derivative of GnRH the flanking 
amino acid sequences can be of equal length and/or of equal composition but may also differ in length 
and/or composition. The preference for a recombinant DNA sequence comprising a DNA sequence L 

55 coding for the presence of a flanking amino acid sequence at one terminus or at both termini of the peptide 
comprising at least one antigenic determinant for GnRH or an analogue or derivative of GnRH is due to the 
fact that when said recombinant DNA sequence is expressed a carrier system according to the invention 
can be obtained in which apparently at least one antigenic determinant against GnRH or an analogue or 

6 

BNSDOCID: <EP 0578293A1_I_> 



EP 0 578 293 A1 



derivative of GnRH has an improved configuration capable of eliciting a better immune response against 
GnRH or an analogue or derivative of GnRH than a carrier system in which such flanking amino acid 
sequences are absent. 

A suitable example of such a preferred recombinant DNA sequence according to the invention 
5 comprises a DNA sequence L coding for 15 amino acids, wherein the DNA coding for the peptide 
comprising at least an antigenic determinant for GnRH or an analogue or derivative of GnRH is a 
decapeptide flanked by two additional amino acids on the N-terminal side and three amino acids on the C- 
terminal side of said peptide. Another example of such a preferred recombinant DNA sequence according to 
the invention comprises a DNA sequence L coding for a decapeptide coding for at least one antigenic 
io determinant against GnRH or an analogue or derivative of GnRH flanked on both sides by the coding 
sequence of one amino acid. 

A recombinant DNA sequence according to the invention will preferably comprise a DNA sequence L 
coding for a peptide with a maximum length of 16 amino acids, as a recombinant microorganism comprising 
a recombinant DNA sequence according to the invention with an insert coding for more than 16 amino acids 
75 is severely restricted in its ability to form recombinant fimbriae. 

In a recombinant DNA sequence according to the invention the DNA sequence L can be integrated in 
DNA sequence S in such a manner that it either completely or partially replaces wild type hypervariable 
region 4 (HR4). 

A recombinant DNA sequence comprising DNA sequence S with DNA sequence L coding at least for a 

20 peptide comprising at least one antigenic determinant for GnRH or an analogue or derivative of GnRH 
integrated in a position corresponding to hypervariable region HR4 of the major subunit, with said DNA 
sequence S further comprising a mutation in hypervariable region 1 (HR1) and in the adjacent homologous 
region of the DNA sequence coding for the major subunit is a preferred recombinant DNA sequence 
according to the invention. This preference is due to the fact that expression of such a recombinant DNA 

25 sequence leads to a carrier system according to the invention comprising at least one antigenic determinant 
of GnRH or an analogue or derivative of GnRH capable of eliciting a better immune response against GnRH 
or an analogue or derivative of GnRH than an equivalent carrier system according to the invention in which 
the mutation in HR1 is absent. 

A suitable example of such a preferred recombinant DNA sequence comprises a Stul site in the 

30 hypervariable region 1 and the adjacent homologous DNA of DNA sequence S coding for at least a part of a 
major subunit. The mutated DNA sequence in hypervariable region 1 and the adjacent homologous region 
codes for amino acid sequence Gly-Leu-Gly. Particularly good results were obtained with a recombinant 
DNA sequence according to the invention wherein the DNA sequence L replacing the HR4 codes for 14 
amino acids and wherein a DNA sequence coding for Gly-Leu-Gly replaces nine nucleotides of the DNA 

35 sequence coding for the last amino acid of HR1 and the two subsequent amino acids of the adjacent 
homologous region. 

A recombinant DNA sequence according to the invention can also comprise further DNA such as DNA 

required for various steps in the procedure of biogenesis of fimbria! filaments by a microorganism. 

Biogenesis of fimbriae includes steps of translocation of the subunits of the fimbriae over the inner 
40 membrane of a microorganism, transport of the subunits in the periplasmic space and extrusion of the 

subunits to the outer membrane with subsequent polymerisation of the subunits into fimbriae. 

In the case of a recombinant DNA sequence according to the invention coding for at least a part of a P- 

fimbrial filament, such further DNA as mentioned in the previous paragraph can comprise one or more of 

the accessory genes that must be expressed for transportation of subunits and polymerisation of subunits 
45 into fimbrial filaments as carried out by a microorganism capable of biogenesis of fimbriae. The accessory 

genes can be coded by DNA obtained from a microorganism with the same serotype as the microorganism 

from which the DNA sequence S coding for the subunit can be derived. 

The further DNA coding for the accessory genes in a recombinant DNA sequence according to the 

invention can also be derived from a microorganism with a different serotype of P-filament than the 
so microorganism from which the DNA sequence encoding the recombinant major subunit has been derived 

due to the fact that the accessory genes derived from DNA coding for different serotypes of P-fimbriae can 

be exchanged in a microorganism without detrimental effect on the biogenesis of fimbriae. 

A further DNA sequence of a recombinant DNA sequence according to the invention can comprise any 

DNA sequence enabling a microorganism to secrete recombinant major subunit. For example a recombinant 
55 DNA sequence according to the invention could comprise a further DNA sequence coding for a signal 

peptide enabling the recombinant major subunit to pass through the membrane of the microorganism 

capable of expressing the recombinant DNA according to the invention. 



JNSDOCID: <EP OS78293A1 J_> 



EP 0 578 293 A1 



The carrier system according to the invention can be obtained through expression of the above 
mentioned recombinant DNA sequences from an expression vector. Therefore an expression vector 
comprising at least a recombinant DNA sequence comprising at least a DNA sequence L coding at least for 
a peptide with at least one antigenic determinant for GnRH or an analogue or derivative of GnRH with said 

5 DNA sequence L being integrated in a DNA sequence S at a position corresponding to a position in the 
hypervariable region HR4 of the major subunit of a P-fimbrial filament, said sequence S coding for at least a 
major subunit and an expression vector comprising any of the various embodiments of the recombinant 
DNA sequence that have been mentioned in the subject description also form part of the invention. Such an 
expression vector according to the invention can be introduced into a host cell capable of expressing said 

w recombinant DNA sequence in a manner well known to the expert e.g. by transformation of a microorgan- 
ism. 

A host cell comprising at least a recombinant DNA sequence comprising at least a DNA sequence L 
coding at least for a peptide with at least one antigenic determinant for GnRH or an analogue or derivative 
of GnRH with said DNA sequence L being integrated in a DNA sequence S at a position corresponding to a 

is position in the hypervariable region HR4 of the major subunit of a P-fimbrial filament said sequence S 
coding for at least a major subunit and a host cell comprising any of the embodiments of a recombinant 
DNA sequence as given in the subject description also fall under the scope of the invention. The host cell 
can comprise said recombinant DNA sequence on an expression vector or integrated in its chromosome. 
The host cell will preferably be a micro organism such as a bacterial cell. 

20 A host cell comprising the recombinant DNA sequence is preferably capable of biogenesis of fimbriae. 

Biogenesis of fimbriae includes steps of translocation of the subunits of the fimbriae over the inner 
membrane of the host cell, transport of the subunits in the periplasmic space and the extrusion of the 
subunits to the outer membrane with subsequent polymerisation of the subunits. 

A host cell comprising a recombinant DNA sequence according to the invention, the recombinant host 

25 cell being incapable of biogenesis of recombinant fimbriae also falls under the scope of the invention. Such 
a fimbriae-recombinant microorganism can comprise DNA enabling the microorganism to secrete recom- 
binant major subunits or parts thereof into the culture medium of said recombinant microorganism. In such a 
recombinant microorganism the recombinant major subunits of the recombinant P-fimbrial filament can be 
transported to the periplasmic space without the subsequent polymerisation or transfer over the outer 

30 membrane to form recombinant P-fimbrial filaments comprising polymerised major subunits. In such a case 
the single recombinant major subunits comprising peptide comprising antigenic determinant for GnRH or an 
analogue or derivative of GnRH can be obtained from the microorganism in a manner well known to the 
expert. 

As already previously stated in the description a preferred carrier system according to the invention 

35 comprises polymerised recombinant subunits. A simple method for obtaining a carrier system according to 
the invention comprising polymerised subunits involves expression of a recombinant DNA sequence 
according to the invention by a microorganism capable of expressing said recombinant DNA, said 
microorganism also being capable of polymerisation of the resulting recombinant subunits. Said poly- 
merisation can take place during transfer of the subunits through the outer membrane as a step in 

40 biogenesis of fimbriae. 

The resulting recombinant fimbriae can be easily isolated from such a recombinant microorganism. 
Such isolation shall* preferably be carried out in non-denaturing circumstances in order to maintain the 
structure of the recombinant fimbrial filament. Riegman, N. et al describe a method lor obtaining purified 
fimbriae in J. Bacteriol. 1990 172:1114-1120. 

45 Preferably the recombinant microorganism will be easily discerned from the non-recombinant microor- 
ganism. This can be achieved by use of a microorganism that is incapable of biogenesis of fimbriae or by 
use of a microorganism that is incapable of producing fimbriae with a distinctive characteristic of the 
recombinant fimbriae. Any fimbriae" microorganism can be used as expression vehicle for recombinant 
DNA according to the invention. 

so Bacteria carrying P-fimbriae (usually found on uropathogenic Escherichia coli) bind to the c*-D-gal(l— 4)- 
£-D-gal moiety of P-blood group antigens. It is therefore simple to detect bacteria carrying P-fimbriae as 
said fimbriae will adhere to human erythrocytes in the presence of mannose which can be easily visualised 
as agglutination of the erythrocytes. A microorganism that cannot adhere to human erythrocytes before 
introduction of recombinant DNA according to the invention can be used as the expression vehicle for 

55 recombinant fimbrial filaments of the P-type. The resulting recombinant microorganism will be capable of 
adhesion to human erythrocytes in contrast to the original microorganism. 

A suitable example of an expression vehicle for recombinant DNA according to the invention is the E. 
coli K12 strain HB101 (Boyer H.W., Roulland-Dussoix D (1969) J.Mol.Biot. 41: 459-472). Until recently it was 



8 

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believed that HB101 is deficient in the production of type 1 fimbriae, however in Microbial Pathogenesis 
(1991) 10, 481-486 Elliott, S.J. et al describe that a standing culture of HB101 was able to develop type 11 
fimbriae. To be certain the developing fimbriae are in fact recombinant fimbriae derived from expression of 
recombinant DNA the HB101 cells should be grown in solid media or in broth media with agitation. 
5 Another example of an E. coli K12 strain that does not produce type 1 fimbriae is AM1727, a recA 

derivative of JE2571 (van Die et al (1983) FEMS Microbiol. Let. 19, 77-82). A further example of a useful 
microorganism for producing recombinant fimbriae for a carrier system according to the invention is JA221 
(Clark L; and Carbon J. (1978) J.Mol.Biol. 120:517-532). 

DNA that must be expressed in order to enable biogenesis of recombinant fimbria! filament can either 
io be partially available in the non-transformed microorganism or can be completely comprised on the 
recombinant DNA sequence according to the invention that is introduced into said microorganism. The DNA 
enabling the biogenesis can be introduced as part of the recombinant DNA sequence according to the 
invention but can also be comprised on a separate expression vector. 

In the Examples a description is given for obtaining various carrier systems according to the invention 
75 through preparation of several recombinant DNA sequences according to the invention and transformation of 
said sequences to fimbriae-deficient microorganisms from which the resulting recombinant fimbrial filaments 
can be isolated and purified. The recombinant DNA, the microorganisms comprising said recombinant DNA 
and compositions comprising a carrier system as described in the Examples also fall within the scope of the 
invention. 

20 The invention is also directed at a composition suitable for eliciting an immune response against GnRH 
or an analogue or derivative of GnRH, said composition comprising a carrier system as described in the 
subject description. A composition suitable for eliciting an immune response against GnRH or an analogue 
or derivative of GnRH comprising the expression product obtainable from a recombinant DNA sequence 
according to the invention, for example from a microorganism as described above, also falls under the 

25 scope of the invention. 

The invention is furthermore directed at use of such a composition for producing an immune response 
against GnRH or an analogue or derivative of GnRH. In particular at the use of such a composition in an 
amount and a manner that are sufficient to affect the biological activity of GnRH in an animal. A composition 
according to the invention is especially suited for use in suppressing the oestrous cycle, spermatogenesis 

30 and/or sexual behaviour of an animal sufficiently to prevent conception. Preferably a composition comprising 
a carrier system derived from a microorganism that was capable of biogenesis of P-fimbriae is used. The 
composition according to the invention can be used in a vaccine or any medicinal preparation suitable for 
eliciting an immune response to GnRH or an analogue or derivative of GnRH. 

In fact a composition according to the invention can be used for any of the applications described in the 

35 state of the art for the various known compositions, vaccines and medicinal preparations comprising 
antigenic determinants for GnRH or an analogue or derivative of GnRH. The examples of numerous possible 
applications that have been given in the introductory part of the subject description therefore serve as 
examples of various uses of a composition according to the invention. 

The composition according to the invention can be applied without the use of strong adjuvants such as 

40 Freunds Adjuvant and Incomplete Freunds Adjuvant, enabling the use of such a composition according to 
the invention in immunisation of animals including mammals without the concomitant negative effects of the 
mentioned adjuvants. Suitable adjuvants include for example aluminium salts (for example AI(OH) 3 , AIPO4, 
AI 2 (S0 4 )3), oil-in-water emulsions (Bayol F (R \ Marcol F (R) ), vitamin-E acetate solubilisate or saponins, if 
desired one or more emulsifiers such as Tween (R \ Span (R) are also incorporated into the vaccine. 

45 Above all use of such a composition according to the invention in immunisation of animals including 
mammals not only leads to the development of antibodies against GnRH or an analogue or derivative of 
GnRH, but in fact leads to altered biological activity due to neutralisation of GnRH by the antibodies elicited 
by use of such a composition. In particular the use of a composition according to the invention leads to 
suppression of reproductive activities in an animal on which said composition has been used. 

50 The composition according to the invention can be applied in the form of a vaccine. The vaccine can be 
applied subcutaneously or intramuscularly in a mammal that is to be immunised in a manner well known to 
the expert. Preferably one or more booster injections are given. Each injection will contain 0.01-1 mg of 
GnRH-antigen or GnRH-analogue-antigen or GnRH-derivative-antigen. 

In Examples 3 and 4 the use of a composition according to the invention for immunizing animals are 

55 further illustrated. 



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Example 1 

In this example a description is given of insertion of genetic information coding for GnRH in the gene 
encoding the major subunit of P-fimbriae with serotype F1 1 . 

5 

A) Preparation of Recombinant DNA 

Plasmid pPIL291-15 deposited at the CNCM of the Institut Pasteur under number I-709 was used to 
construct the plasmids pPIL291-1510 and pPIL291-1519. Plasmid pPIL291-15 comprises the genetic 
70 organisation of the F1 1 gene cluster. The BamHI-Cla1 fragment of pPIL291-15 comprises the gene coding 
for the F1 1 major subunit, the FelA gene. 

The construction of plasmids pPIL291-1510 and pPIL291-1519 was carried out as described in Van Die 
et al, Mol. Gen. Genet (1990) 222:297-303 and Van Die et al (1988) J. BacterioL 170: 5870-5876. 

The 0.7 kb Hindlll-EcoRI fragment of pPIL291-151 (obtained from cloning the 3kb Clal-BamHI fragment 
75 of pPIL 291-15) was cloned into the bacterial phage vector m13mp8. This clone was used as a template for 
site directed mutagenesis. 

Site directed mutagenesis was performed by the gapped duplex method (Kramer V. et al (1984) Nucl. 
Acid Res. 12:9441-9456) essentially as described before (Van Die, I. et al (1988) J. BacterioL 170:5870- 
5876). 

20 The obtained double stranded DNA molecule was transformed to strain HB2154, white plaques were 
selected and restriction fragment DNA was isolated and checked. 

The mutagenic primer for HR1 had the following nucleotide sequence CAGC Mil AAAGGCCTTGGAG- 
CAGCTAAAA (SEQ ID NO: 20). In the mutagenesis experiment the bases 355-362 of the wild type F11 
sequence were replaced by different bases resulting in the modification of three amino acids in this region 

25 leading to the introduction of the Stul restriction site (AGGCCT) in the resulting DNA molecule. 

The mutagenic primer for HR4 had the following nucleotide sequence TTCTTTCGATGGGTTAACCCT- 
GAAAGATGG (SEQ ID NO: 21). In the mutagenesis experiment bases 502-520 of the wild type F11 
sequence were replaced by four new bases resulting in a deletion of 15 bases and the presence of a Hpal 
restriction site (GTTAAC) in the resulting DNA molecule. 

30 The Hindlll-EcoRI fragments were subsequently isolated and were used to replace the EcoRI-Hindlll 
fragment of plasmid pPIL291-151 resulting in the respective plasmids pPIL291-1510 (comprising a Stul 
restriction site in the hypervariable region 1 HR1) and pPIL291-1519 (comprising a restriction site for Hpal in 
the hypervariable region 4 HR4). Both cloning sites were constructed in the same reading frame. 

Several oligonucleotides of varying lengths were inserted into pPIL291-1510 and/or pPIL291-1519. The 

35 inserted oligonucleotides all code for the decapeptide GnRH with amino acid sequence gin his trp ser tyr 
gly leu arg pro gly. The oligonucleotides differ with respect to the length and the composition of amino acid 
sequences flanking the decapeptide. 

Figure 1 and SEQ ID NO: 1-8: show the oligonuclotides that were inserted into plasmids pPIL291-l5l0 
and pPIL291-1519. The coding strand of GnRH translated into the corresponding amino acids is underlined. 

40 After isolation of transformed cells comprising plasmids the plasmids containing inserts with linkers 1 
(SEQ ID NO:1, 2), 4 (SEQ ID NO: 5, 6) and 5 (SEQ ID NO: 7, 8) were selected by determination of the 
presence of a site for restriction endonuclease BamHL 

For detecting a plasmid comprising linker 3 (SEQ ID NO: 3, 4) as insert the Stul recognition site in the 
oligonuclotide could not be used as the Stul recognition site was immediately followed by two guanidine 

45 nucleotides forming a site that is recognised by E. coli methylase and is therefore protected from restriction 
because of methylated cytidine residues. For selection of incorporation of linker 3 the plasmids were 
therefore digested with Stul for detecting an insertion in HR1 and were digested with Hpal for detecting an 
insertion in HR4. Successful incorporation of an oligonuclotide in the respective hypervariable regions HR1 
and HR4 resulted in removal of the corresponding restriction sites. 

50 Subsequently the selected plasmids were sequenced in order to determine the presence of the linker in 
the correct orientation. The plasmids obtained as described with linkers in the desired orientation were 
designated pAI X.Y.0, whereby X indicates the presence of the hypervariable region, Y indicates the 
presence of the inserted linker and 0 indicates the presence of the accessory genes. 

The plasmids derived from pPIL291-1510 were therefore denoted as pAI 110, pAI 130, pAI 140, pAI 

55 150. In recombinant hypervariable region 1 HR1 i.e. after insertion into plasmid pPIL291-1510, the 
oligonucleotides are preceded by a codon for a glycine residue. 

The plasmids obtained from pPIL291-1519 comprising the linkers demonstrated in SEQ ID NO: 1-8 
were denoted pAI 410 (SEQ ID NO: 9, 10), pAI 430 (SEQ ID NO: 11,12), pAI 440 (SEQ ID NO: 13, 14) and 

10 

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pAI 450 (SEQ ID NO: 15, 16) respectively. The oligonucleotides inserted into hypervariable region 4 HR4 of 
plasmid PPIL291-1519 are preceded in the recombinant hypervariable region HR4 by a leucine residue. The 
last amino acid of the recombinant hypervariable region 4 is a threonine. 

In figure 2 and SEQ ID NO: 9-16 the DNA sequences and corresponding amino acid sequences of the 
5 recombinant hypervariable region 4 HR4 of the plasmids pAI 410 (SEQ ID NO: 9, 10), 430 (SEQ ID NO: 11, 
12), 440 (SEQ ID NO: 13, 14) and 450 (SEQ ID NO: 15. 16) are given as well as the DNA sequence and the 
amino acid sequence of the wild type HR4 of F11 (SEQ ID NO: 17). The coding regions of GnRH translated 
in according amino acids are indicated in the respective information chapters of the sequence listing under 
(ix) feature (B) location. 

70 In Table I a comparison is given of the flanking sequences of the decapeptide GnRH coding for at least 

one antigenic determinant for GnRH as well as a comparison of the lengths of the recombinant hyper- 
variable region 4 HR4 and the wild type hypervariable region 4 HR4. 

B) Analyses of fimbriae obtained from expression of the recombinant DNA 

15 

The Clal/BamHI restriction fragment, harbouring the FelA gene, of pPIL291-15 was replaced by the 
mutated Clal-BamHI fragments of the pAI-plasmids containing the linkers 1, 3, 4 and 5 respectively and the 
resulting four plasmids were transformed to competent cells of HB101. 

Haemagglutination positive clones were selected and checked by DNA restriction fragment analysis. 
20 Expression of the hybrid fimbriae by the transformed HB101 cells was also examined by electron 
microscopy. The results are summarised in Table 2. 

From these tests it was suggested that HB101/PAI 440 expressed fimbriae nearly as efficiently as 
HB101/pPIL291-15 carrying the normal F11-gene cluster. Fimbriae production was only slightly reduced in 
HB101 cells harbouring plasmids pAI 410 and 430. The insertion of linkers in hypervariable region 1 
25 appeared to severely disturb the biogenesis of the fimbriae as only a few fimbriae per cell could be 
detected. 

The expression of recombinant fimbriae was also examined through an ELISA assay with polyclonal 
antibodies placed against the complete bacteria comprising wild-type F11 fimbriae. This was carried out as 
the use of monoclonal antibodies was impossible due to distortion of the F11 specific epitopes through the 
30 new ligations (a phenomenon described in Van Die et al MGG222 (1990) biz. 297-303). The results are 
given in Table 3. 

Materials and methods. 

35 a) Bacteria 

Escherichia coli strain HB101 deficient in type 1 fimbriae formation was used as the host strain for 
morphogenetic expression of hybrid fimbriae (Boyer, H.W., and D. Roullard-Dussoix (1969); J. Mol. Biol. 41: 
459-472) HB101 was cultured on a rotary shaker to ensure that the non transformed cells were fimbriae". 
40 For DNA sequencing strain JM101 was used as the host for M13MP8 derivatives (Messing, J., and J. 
Vieira (1982); Gene 19: 269-276). 

In site directed mutagenesis experiments HB2154 was used as the host strain for M13mp18 derivatives 
(Carter, P., H. Bedouelle, and G. Winter (1985); Nucl.Acids Res. 13: 4431-43). 

Bacteria were grown on Brain Heart Infusion broth containing ampicillin (50 ug/ml). 

45 

b) Enzymes 

Restriction endonucleases were used according to instructions of the manufacturer. 
For ligation T4 DNA ligase was used according to instructions of the manufacturer. 

50 

c) DNA analyses 

Analysis of DNA fragments was performed by electrophoresis in 0.6% agarose gels. 
In DNA sequencing the dideoxy chain termination method of Sanger et al (Sanger, F., S. Nicklen, A.R. 
55 Coulson (1977); Proc. Natl. Acad. Sci. USA 74: 5463-67) was used. 



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d) Transformations 

Transformations were carried out according to Kushner (Kushner, S R. (1978); p. 17-23 In: H.W. Boyer 
and S. Nicosia (ed) Genetic Engeneering. Elsevier Biomedical Press, Amsterdam). 

5 

e) Localised mutagenesis 

Localised mutagenesis was performed by the gapped duplex method. The primer was hybridised with 
template DNA together with M13mp18 for one hour at 65 'C. Extension and ligation of this gapped duplex 
70 molecule was performed after addition of the required dNTP's, 10 units of T4 ligase and 3 units of Klenow 
fragment of DNA polymerase I followed by a 4 hour incubation at 16 e C. 

f) Linker synthesis 

75 The GnRH oligonucleotides were synthesised using a DNA synthesizer model 381 A of Applied 
Biosystems as is explained in the Users Manual. 

g) Insertion of linkers 

20 Plasmid pPIL291-1519 was digested with Hpal in the following manner: A mixture was prepared of 1 ug 

DNA, 5 units of restriction enzyme Hpal, 1.5 ul of a restriction buffer (10x) made up to 15 ul with TE buffer 
(10 mM tris 1mM EDTA) and this mixture was incubated at 37 *C for 1£ hours. The enzyme was 
subsequently inactivated by heating at 65 *C for 10 minutes and the mixture was subjected to a phenol 
extraction followed by alcohol precipitation. 

25 The linearised plasmid was ligated with GnRH linker in the following manner: 

A mixture was prepared of 5 units ligase, 1 ul ligase buffer (10x), vector DNA and linker DNA in a volume of 
10 uL The ligation was carried out O/N at 16* C. 

Transformation was carried out by mixing 100 ul of competent cells (Kushner, S.R. (1978); p. 17-23 In: 
H.W. Boyer and S. Nicosia (ed) Genetic Engeneering. Elsevier Biomedical Press, Amsterdam) of HB101 

30 with the ligated plasmid for 30 minutes on ice. This mixture was subsequently subjected to a heatshock for 
5 minutes at 37 °C. LB-medium was added and after 1£ hours was plated out on Amp plates. The resulting 
colonies were isolated, cultivated and the plasmid DNA was isolated and sequenced from these colonies. 

The same procedure was followed for GnRH insertion in HR1 with the exception of digestion with Stul 
instead of Hpal. 

35 

h) Complete bacteria ELISA 

The antiserum that was used was an absorbed hyperimmune rabbit antiserum raised against F11 
fimbriae. 

40 In each assay a positive and a negative control strain were included. 

After washing the bacteria were seeded in flat bottom polystyrene microtitre plates. The bacterial 
suspensions were allowed to dry and after washing they were blocked with PBS/Tween-80/Newborn Calf 
Serum. Subsequently serial dilutions of absorbed serum were added and after 1 h incubation at 37 °C the 
plates were washed and peroxidase-conjugated goat-anti-rabbit lgG(H + L) was added. After washing and 

45 adding of TMB-substrate buffer, containing ureum-peroxide and S.S'^S'-tetramethylbenzidine the reaction 
was stopped by adding H2SO4 and colouring was measured with a Microelisa reader. Titres were 
determined as the highest antiserum dilution giving an A450 of at least 2 times the background A450. 

i) Production, isolation and purification of hybrid GnRH-Fl 1 fimbriae 

50 

E.Coli K-12 strains, transformed with plasmids pAI 410, pAI 440, pAI 10410 and pAI 10440 and 
maintained at -70 *C in 30% glycerol, were passed through two pre-cultures (overnight at 37 °C on plates 
with Blood Agar Base no.2 (Oxoid) + 100 ug/ml ampicillin and for 7 hours at 37° C in 100 ml Brain Hearth 
Infusion medium (BHI, Oxoid) + ampicillin (100 ug/ml) with agitation). 
55 For main-culture a fermentor, filled with 12 litre BHI, ampicillin (100 ug/ml) and 5 ml 10% PPG 
(antifoam), was inoculated with the preculture and grown for 17 hours (37 'C; 50% O2 saturation, adjusted 
with air; agitation 100-1000 rpm). 



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The fimbriae were removed from the cultured bacteria by heating at 65 °C (15 min), treatment at pHlO 
(1h, room temp., agitation) and centrifugation (15 min, 13000 rpm Sorvall RC-5B, rotor GSA). 

After concentration of the supernatant to 200-600 ml (XM 300 filter, Minitan System, Millipore) and 
washing with Tris/Glycine buffer (pH10), the pH was adjusted to 8.5 and the resulting precipitate was 
5 allowed to settle for at least 1 day at 4 • C. 

After harvesting the precipitate (20 min centrifugation at 48,000 g, 4*C) the pellet was dissolved in 100 
ml Tris/Glycine buffer pH8.5 with 2 M urea. This preparation was concentrated (YM100 filter, Amicon 
ultrafiltration cell), washed (2x200 ml Tris/Glycine buffer pH 8.5) and stored at -20 ° C until used for vaccin 
production. 

10 

Example 2 

A mutation in hypervariable region 1 and an insertion in hypervariable region 4 was also investigated. A 
Stul recognition site was constructed in the plasmids by localised mutagenesis as described in Example 1. 
75 Subsequently the formation of fimbriae and the determination of the antigenicity of these constructs was 
also determined in the same manner as is described for the constructs of Example 1 . 

Surprisingly it was discovered that the presence of a mutation in the hypervariable region 1, i.e. the 
presence of a Stul recognition site, gave recombinant DNA that resulted in the same amount of fimbriae 
formation by transformed cells as plasmids pAI 410, pAI 430 and pAI 440. The results are given in Table 2. 
20 Construction of the Stul site in hypervariable region 1 seemed to improve the exposure of linkers 

incorporated in hypervariable region 4. The mutants containing a Stul site in hypervariable region 1 showed 
a positive reaction in an immuno gold labelling experiment where no label could be detected on cells 
harbouring plasmid pAI 410 in the same experiment. This finding indicated that the two hypervariable 
regions under investigation may be in close contact. 

25 

Example 3 

Experiments were carried out with fimbriae preparations comprising recombinant fimbrial filaments 
obtained from microorganisms transformed with the constructs described in Examples 1 and 2. 

30 Immunisation tests were carried out to determine the neutralizing effect on GnRH. The immunisation 
tests led to the oestrous cycle of the immunised mammal being disturbed or even suppressed. 

In this Example mutant fimbriae carrying the amino acid sequence of GnRH were used for vaccination 
of female rats. The tests were used to ascertain any differences in activity between fimbriae in which only 
the GnRH-like peptide was constructed in Hypervariable Region 4, HR4 and fimbriae in which in addition 

35 another, but very small, change was performed (insertion of a Stu I site in HR1). 

After selecting the adult female rats bred from an initial Wistar strain for a regular oestrous cycle they 
were treated subcutaneously twice, 6 weeks apart, with 0.5 ml of an emulsion of fimbriae (50 tig) in an 
oil/water mixture (70/30, v/v) of which the oily phase contains Polysorbate 80 and Sorbitan mono-oleate in 
liquid paraffin (Marcol 52) and the water phase contains AI(OH) 3 in distilled water. The mutant fimbriae were 

40 pAI 10410, pAI 440 and pAI 10440. The fimbriae-preparations were obtained as described in Examples 1 
and 2. Before, between and after the injections daily vaginal smears were taken and at predetermined times 
blood was collected from the retro-orbital plexus and the sera were stored at -20 ° C until assayed. At the 
end of the experiment the animals were killed and the ovaries weighed. 

45 Vaginal smears 



Daily vaginal smears were made on microscope slides. After drying and fixing with methanol, they were 
stained for 20 min. with Giemsa solution (Merck, Darmstadt, W. Germany) diluted 1:10 with distilled water, 
washed thoroughly with tap-water and dried. Each smear was evaluated microscopically (100x) by 
so estimating the percentage of cornified and nucleated epithelial cells and of leucocytes. 
The vaginal sequence of normal rats with a 4-day oestrous cycle is: 
di-oestrus - pro-oestrus - oestrus. In the figures these oestrous phases are represented by scores: 
1 = di-oestrus, 2 = pro-oestrus and 3 = oestrus. 

55 Assay 

Binding of 125 l-GnRH by anti-GnRH antibodies in the serum samples was determined by a radioim- 
munoassay (RIA). 

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Before incubation the thawed serum samples were diluted in assay buffer (Na2HPC>4 2H 2 0, 0.01 mol/L, 
NaCI, 0.15 mol/L, 0.1% gelatin and 0.1% sodium azide pH 8.0). 

The RIA was performed by incubation of duplicates of 0.1 ml_ diluted serumsamples, 0.2 ml_ assay 
buffer and 0.05 mL of 125 l-GnRH for 16 hours at 4*C. Prior to the separation 0.05 mL human serum was 
5 added to the tubes as carrier protein. 

Separation of free and bound was achieved by adding 0.5 mL Peg solution (40% Peg-4000 in assay 
buffer without gelatin) to all tubes. The mixtures were centrifuged and the precipitate was counted in a 
gamma-spectrometer. 

The titer was calculated as the relative percentage of radioactivity bound as corrected for non-specific 
w binding vs total amount of radioactivity added. 

RESULTS 

Effects on anti-GnRH antibodies and oestrous cycle (fig. 3-5) 

75 

- Serum of animals treated with adjuvant only did not show Anti-GnRH antibodies. No suppression of 
oestrous cycle was observed in these animals. 

- All animals treated with the mutant fimbriae showed serum antibody binding which resulted in 
disruption and suppression of the oestrous cycles. 

20 

Body weights 

In particular the rats treated with pAI 10410 or pAI 10440 showed higher body weights than the 
placebo-treated animals from 3-5 weeks after booster injection onwards (fig. 6). 

25 

Ovarian weights 

The fimbriae of all mutants caused a reduction in ovarian weight (fig.7). 
30 Example 4 

In addition to the experiment in rats the same preparations were tested in bull calves. Four months old 
cross bred bull calves were treated subcutaneously twice, 8 weeks apart, with 2ml of an emulsion of 
fimbriae (200 ug) in an oil/water mixture (70/30, v/v) of which the oily phase contains Polysorbate 80 and 
35 Sorbitan mono-oleate in liquid paraffin (Marcol 52) and the water phase contains AI(OH) 3 in distilled water. 
The mutant fimbriae were pAI 410, pAI 10410, pAI 440 and pAI 10440. The fimbriae-preparations were 
obtained as described in Examples 1 and 2. Before, between and after the injections weekly scrotal 
circumference was determined and at predetermined times blood was collected from the jugular vein and 
the plasmas were stored at -20 °C until assayed. 

40 

Assay 

Binding of 125 l-GnRH by anti-GnRH antibodies in the plasma samples was determined by a radio- 
immunoassay (RIA) as described in Example 3. 

45 

RESULTS (fig.8 and 9) 

- Plasma of animals treated only with adjuvant did not contain Anti-GnRH antibodies. Scrotal circum- 
ference, increased regularly during the experimental period in the four bulls. 

so - Antibody binding was observed in plasma of bulls treated with pAI 410 which resulted in a reduction 
in scrotal circumference in comparison to the control animals. 

- Antibody binding was observed in plasma of bulls treated with pAI 440 being very high in one animal. 
This resulted in a reduction in scrotal circumference in comparison to control animals. 

- All pAI 10410 treated bulls showed high plasma antibody binding, especially shortly after the booster 
55 injection, together with a considerable suppression of scrotal growth in comparison to the control 

animals. 

- The bulls treated with pAI 10440 showed plasma anti-GnRH antibody binding and a considerable 
reduction in scrotal growth in comparison to the control animals. 

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- Addition of the Stu1 site in HR 1 resulted in an important improvement of the induction of anti-GnRH 
antibodies as well as the activity, regardless of whether linker 1 or 4 was used. 

TABLE 1 

5 



linker 


HR 


length of 
recombinant HR4 


5'flanking length 


5'flanking sequence 


3'flanking length 


increase in 
HR4 length 


1 


HR4 


14 aa 


1 aa 


leu 


3 aa 


7 aa 


3 


HR4 


12 aa 


1 aa 


leu 


1 aa 


5 aa 


4 


HR4 


15 aa 


2 aa 


leu-thr 


3 aa 


8 aa 


5 


HR4 


16 aa 


3 aa 


leu-gly-ser 


3 aa 


9 aa 


wt 




7 aa 










aa = amino acid 

wt = HR4 of original microorganism, no insert 



TABLE 2 

20 





Plasmids 


Fimbriation 


Insertion site 


Hypervariable region 1 


110 


+ /- 


HR1 




130 


+ + 


HR1 




140 


+ /- 


HR1 




150 


+ 


HR1 


Hypervariable region 4 


410 


+ + + 


HR4 




430 


+ + + 


HR4 




440 


+ + + + 


HR4 




450 


+ /- 


HR4 




10410 


+ + + 


HR4 




10430 


+ + + 


HR4 




10440 


+ + + 


HR4 


Wildtype F11 


291-15 


+ + + + + 





Expression of fimbriae by HB101 cells carrying plasmids encoding the mutant fimbrillins. 

40 



45 



50 



55 



15 



INSDOCID: <EP 0578293A tj_> 



EP 0 578 293 A1 



TABLE 3 

Results whole-bacteria Elisa on Fll expression. 

Bacteria were grown in fermentors in Brain Heart Infusion broth. 



CONSTRUCT 


TITRE 


pAI 440 


1:64,000 


pAI 440 


1:32,000 


pAI 10410 


1: 8,000 


pAI 10430 


1:16,000 


pAI 1044 0 


1:32,000 


pPIL291-15 


1:32, 00O 



Legends 

25 

Fig. 1: 

Oligonucleotides inserted into plasmids pPIL291-1510 and pPIL291-1519. The encoded GnRH amino 
acid sequence is underlined. 
Fig. 2: 

30 DNA sequences of the recombinant HR4 region in plasmids pAI 410, 430, 440 and 450 and the 
corresponding amino acid sequences. The encoded GnRH amino acid sequence is underlined. 
Fig. 3: 

Development of anti-GnRH antibody titres in serum of female rats treated or not with mutant fimbriae 
carrying the amino acid sequence of GnRH. Relative binding at serum dilution of 5600x. 
35 Fig. 4: 

Oestrous cycle of rats treated adjuvant only. 
Score: 

1 = dioestrus 

2 = pro- or met-oestrus 
40 3 = oestrus 

Fig. 5: 

Oestrus cycle of rats treated with pAI 10410. 
Score: 

45 1 = dioestrus 

2 = pro- or met-oestrus 

3 = oestrus 
Fig. 6: 

Mean body weights of female rats treated or not with mutant fimbriae carrying the amino acid sequence 
50 of GnRH. 
Fig. 7: 

Ovarian weights of rats treated or not with mutant fimbriae carrying the amino acid sequence of GnRH. 
Fig. 8: 

Development of anti-GnRH antibody titres in plasma of young calves treated or not with mutant fimbriae 
55 carrying the amino sequence of Gonadotropin Releasing Hormone (GnRH). 
Relative binding at plasma dilution of 5600x. 
Fig. 9: 

Increase in scrotal- circumference in young calves either or not treated with mutant fimbriae carrying the 

16 



BNSDOCIO: <EP 0578293A1_I_> 



EP 0 578 293 A1 



amino acid sequence of GnRH. 



SEQUENCE LISTING 

5 

(1) GENERAL INFORMATION: 



(i) APPLICANT: 

(A) NAME: AKZO N.V. 

(B) STREET: Velperweg 76 

(C) CITY: Arnhem 

(E) COUNTRY: The Netherlands 

(F) POSTAL CODE (ZIP): 6824 BM 

(G) TELEPHONE: 04120-66223 

(H) TELEFAX: 04120-50592 

(I) TELEX: 37503 akpha nl 

(ii) TITLE OF INVENTION: Carrier system against GnRH 
(iii) NUMBER OF SEQUENCES: 21 



(iv) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

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

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) 



25 (2) INFORMATION FOR SEQ ID NO: 1: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 36 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : double 
30 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 

(iii) HYPOTHETICAL: NO 

35 (iii) ANTI-SENSE: NO 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 2.. 31 

40 

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



G CAG CAC TGG AGC TAC GGC CTG CGT CCA GGA TCCCG 36 

Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
1 5 10 

45 



(2) INFORMATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

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

(ii) MOLECULE TYPE: protein 



55 



17 



JNSDOCID: <EP 0578293A1 J_> 



EP 0 578 293 A1 



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

Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 

5 

(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
10 ( b) TYPE: nucleic acid 

(C) STRANDEDNESS : double 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 
15 (iii) HYPOTHETICAL: NO 

(iii) ANTI-SENSE: NO 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 2.. 31 

(ix) FEATURE: 

(A) NAME/KEY: misc_dif ference 

(B) LOCATION: replace (31, ) 

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



25 



30 



35 



40 



G CAG CAC TGG AGC TAC GGC CTG AGG CCT GGG 31 
Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 

(2) INFORMATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

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

(ii) MOLECULE TYPE: protein 

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

Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 



(2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 39 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: double 

(D) TOPOLOGY: linear 



55 



18 



BNSDOCID: <EP 0578293A1_ 



EP 0 578 293 A1 



(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iii) ANTI -SENSE: NO 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 5 . . 34 

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

GACT CAG CAC TGG AGC TAC GGC CTG CGT CCA GGG GATCC 39 
Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 

(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
20 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

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



75 



25 



50 



55 



Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 



(2) INFORMATION FOR SEQ ID NO: 7: 

30 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 42 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : double 

(D) TOPOLOGY: linear 

35 (ii) MOLECULE TYPE: DNA (genomic) 

(iii) HYPOTHETICAL: NO 
(iii) ANTI-SENSE: NO 



40 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 8.. 37 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 

45 GGGATCC CAG CAC TGG AGC TAC GGC CTG CGT CCA GGC GGTCC 4 2 

Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 



(2) INFORMATION FOR SEQ ID NO: 8: 



19 

INSDOCID: <EP 0578293A1_I_> 



EP 0 578 293 A1 



70 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

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

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 

Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 

(2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 48 base pairs 
J5 (B) TYPE: nucleic acid 

(C) STRANDEDNESS : double 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 
20 (iii) HYPOTHETICAL: NO 

(iii) ANTI -SENSE: NO 

(ix) FEATURE: 
25 (A) NAME/KEY: CDS 

(B) LOCATION: 7.. 36 

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

GGGTTG CAG CAC TGG AGC TAC GGC CTG CGT CCA GGA TCCCGAACCC 
„ Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 

30 1 5 10 

TG 

35 (2) INFORMATION FOR SEQ ID NO: lO: 

( i ) SEQUENCE CHARACTERISTICS : 

(A) LENGTH: 10 amino acids 

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

40 (ii) MOLECULE TYPE: protein 

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



45 



50 



55 



Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 



(2) INFORMATION FOR SEQ ID NO: 11: 
(i) SEQUENCE CHARACTERISTICS: 



20 

BNSDOCID: <EP 0578293A1J_> 



EP 0 578 293 A1 



10 



(A) LENGTH: 42 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: double 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iii) ANTI-SENSE: NO 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 7 . . 36 

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

GGGTTG CAG CAC TGG AGC TAC GGC CTG AGG CCT GGA ACCCTG 4 2 

Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 

(2) INFORMATION FOR SEQ ID NO: 12: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

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

25 (ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 



75 



20 



30 



35 



40 



Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 



(2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 51 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS; double 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iii) ANTI -SENSE: NO 



(ix) FEATURE: 

(A) NAME/KEY: CDS 
45 (B) LOCATION: 10.. 39 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 

GGGTTGACT CAG CAC TGG AGC TAC GGC CTG CGT CCA GGG GATCCAACCC 4 9 

50 



55 



21 

JNSOCCID: <EP 0578293A1 J_> 



EP 0 578 293 A1 



10 



15 



20 



25 



Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 

51 

TG 

(2) INFORMATION FOR SEQ ID NO: 14: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

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

(ii) MOLECULE TYPE: protein 

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

Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 

(2) INFORMATION FOR SEQ ID NO: 15: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 54 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : double 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL : NO 
(iii) ANTI-SENSE: NO 

( ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 13 -.42 

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

GGGTTGGGAT CC CAG CAC TGG AGC TAC GGC CTG CGT CCA GGC GGTCCAACCC 52 
Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 

TG 

(2) INFORMATION FOR SEQ ID NO: 16: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
45 (B) TYPE: amino acid 

(D) TOPOLOGY : linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 

50 



30 



35 



40 



55 



22 



BNSDOCID: <EP 057B293A1J_> 



EP 0 578 293 A1 



Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 



(2) INFORMATION FOR SEQ ID NO: 17: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 27 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : double 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iii) ANTI-SENSE: NO 



20 



25 



35 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..27 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 

GGG ACT GCA GGT GAC GCT TAT CCC CTG 27 
Gly Thr Ala Gly Asp Ala Tyr Pro Leu 
1 5 



(2) INFORMATION FOR SEQ ID NO: 18: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
30 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

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



Gly Thr Ala Gly Asp Ala Tyr Pro Leu 
1 5 



(2) INFORMATION FOR SEQ ID NO: 19: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: NO 

(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 



55 



23 

1NSDOCID: <EP 057B293A1_L> 



EP 0 578 293 A1 



70 



15 



20 



Gin His Trp Ser Tyr Gly Leu Arg Pro Gly 
15 10 

(2) INFORMATION FOR SEQ ID NO: 20: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 30 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : double 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iii) ANTI-SENSE: NO 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: 
CAGCTTTTAA AGGCCTTGGA GCAGCTAAAA 30 

(2) INFORMATION FOR SEQ ID NO: 21: 



( i ) SEQUENCE CHARACTERISTICS : 

(A) LENGTH: 30 base pairs 

(B) TYPE: nucleic acid 

25 (C) STRANDEDNESS: double 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 

(iii) HYPOTHETICAL: NO 

30 

(iii) ANTI-SENSE: NO 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: 
TTCTTTCGAT GGGTTAACCC TGAAAGATGG 30 

35 



Claims 

40 

1. An immunogenic carrier system capable of eliciting an immune response against GnRH, an analogue or 
derivative of GnRH, said carrier system comprising at least a part of an E. coli P-fimbrial filament 
comprising at least a part of a major subunit with an insert, said insert comprising a peptide with at 
least one antigenic determinant for GnRH, an analogue or derivative of GnRH, said insert being located 

45 in the major subunit at a position corresponding to a position in hypervariable region 4 of the wild type 
major subunit. 

2. An immunogenic carrier system according to claim 1, wherein the peptide containing at least one 
antigenic determinant for GnRH, an analogue or derivative of GnRH is derived from the amino acid 

so sequence gin his trp ser tyr gly leu arg pro gly . 

3. An immunogenic carrier system according to any of the previous claims wherein the major subunit is 
derived from a P-fi lament with serotype F-1 1 . 

55 4. An immunogenic carrier system according to any of the previous claims, wherein the insert has a 
maximum length of 16 amino acids. 



24 



BNSDOCID: <EP 0578293A1_I_> 



EP 0 578 293 A1 



. An immunogenic carrier system according to any of the previous claims wherein the insert further 
comprises at least one amino acid flanking the peptide containing at least one antigenic determinant for 
GnRH, an analogue or derivative of GnRH. 

. An immunogenic carrier system according to any of the previous claims, wherein the major subunit with 
an insert further comprises a mutation in the amino acid sequence corresponding to a part of the 
hypervariable region 1 and the adjacent homologous region of the wild type major subunit. 

. A recombinant DNA sequence coding for at least a part of the E. coli P-fimbrial filament major subunit 
with an insert, said insert comprising a peptide with at least one antigenic determinant for GnRH, an 
analogue or derivative of GnRH, said insert comprising a peptide with at least one antigenic deter- 
minant for GnRH, an analogue or derivative of GnRH, said insert being located in the major subunit at a 
position corresponding to a position in hypervariable region 4 of the wild type major subunit. 

. An expression vector comprising a recombinant DNA sequence according to claim 7. 

. A microorganism comprising a recombinant DNA sequence according to claim 7 and/or comprising an 
expression vector according to claim 8 said microorganism being capable of expressing said recom- 
binant DNA sequence. 

0. A microorganism according to claim 9 capable of biogenesis of fimbriae. 

1- Vaccine capable of eliciting an immune response against GnRH, an analogue or a derivative of GnRH 
in an animal, said vaccine comprising an effective amount of an immunogenic carrier system according 
to any of claims 1-6 or comprising an expression vector according to claim 8 or comprising a 
microorganism according to claim 9 or 10 and optionally comprising one or more adjuvantia or other 
compounds commonly used in a vaccine. 



25 



EP 0 578 293 A1 



FIG. 1 

linker oligonucleotide sequence 
nr 



BamHI 



G CAG CAC TGG AGC TAC GGC CTG CGT CCA GGA TCC CG 
gin his trp ser tyr qlv leu arg pro gly ser arg 

StuI 



G CAG CAC TGG AGC TAC GGC CTG AGG CCT GG 
gin his trp ser tvr qlv leu arg pro qlv 

BamHI 



G ACT CAG CAC TGG AGC TAC GGC CTG CGT CCA GGG GAT CC 

thr gin his trp ser tvr gly leu arg pro gly asp pro 

BamHI 

G GGA TCC CAG CAC TGG AGC TAC GGC CTG CGT CCA GGC GGT CC 

gly ser gin his trp ser tyr qlv leu arg pro gly gly pro 



BNSDOCID: <EP 0578293A 1 _l_> 



26 



EP 0 578 293 A1 



GGG 
gly 



GGG 
gly 



GGG 
gly 



GGG 
gly 



GGG 
gly 



FIG. 



pAI 410 



RECOMBINANT HR4 



BamHI 



TTG CAG CAC TGG AGC TAC GGC CTG CGT CCA GGA TCC CGA ACC 
leu gin his trp ser tyr alv leu arq pro qlv ser arg thr 



pAI 430 



gtui; 



TTG CAG CAC TGG AGC TAC GGC CTG AGG CCT GGA ACC 
leu gin bis trp ser tyr qlv leu arg pro gly thr 

pAI 440 



BamHI 



TTG ACT CAG CAC TGG AGC TAC GGC CTG CGT CCA GGG GAT CCA ACC 
leu thr gin his trp ser tvr glv leu arg pro gly asp pro thr 



pAI 450 

BamHI 



TTG GGA TCC CAG CAC TGG AGC TAC GGC CTG CGT CCA GGC GGT CCA ACC 

leu gly ser gin his trp ser tvr glv leu arg pro glv gly pro thr 
Wild Type 

ACT GCA GGT GAC GCT TAT CCC 
thr ala gly asp ala tyr pro 



CTG 
leu 



CTG 
leu 



CTG 
leu 



CTG 

leu 



CTG 
leu 



1NSDOCID: <EP 0578293A1 J_> 



27 



EP 0 578 293 A1 



FIG. 3 




Treatment: □ Placebo o pAI 440 a pAJ 10410 x pAJ 10440 



BNSDOCID: <EP 0578293A1_L> 28 



EP 0 578 293 A1 



FIG. 4 

RAT LV1 




RAT RV1 




RAT St1 




RAT LV11 



5, 




RAT RV1 1 




RAT St11 




3NSDOCID: <EP 0578293A1_I_> 



29 



FIG.5 



EP 0 578 293 A1 



RAT LV4 




? 

booster 



Day after primary injection 



BNSDOCID: <EP 0578293A1_I_> 



30 



EP 0 578 293 A1 




3NSDOCID: <EP 0578293A1_I_> 



31 



EP 0 578 293 A1 



FIG. 7 



60 " 




Placebo pAI440 pA1 10410 pA1 10440 



BNSDOCID: <EP 0578293A1_I_> 32 



EP 0 578 293 A1 



FIG. 8 



30 




Booster Day after first injection 

Treatments Blank + pAI 410 o pAI 440 a pAJ 10410 x pAI 10440 



3NSDOCID: <EP 0578293A1J_> 



33 



EPO 578 293 A1 



FIG. 9 




-24 -4j 16 36 56 76 96 116 136 156 176 196 216 236 

PrimJnj. Booster ^ Day* aft* firmt injpcton 

Treatment: □ Blank + pAI 410 o pAI440 a pA1 10410 x pAI 10440 



BNSDOCID: <EP 0576293A1_L> 



34 



J 



European Patent 
Office 



EUROPEAN SEARCH REPORT 



Application Number 

EP 93 20 1712 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 



Citation of document with indication, where appropriate, 
of relevant passages 



Relevant 
to I 



CLASSIFICATION OF THE 
APPLICATION (Int. CX.5 ) 



D.Y 

X 
Y 
Y 

D.Y 



WO-A-9 004 963 (DANBIOSYST LTD, UK) 
17 May 1990 

* The claims * 

WO-A-9 003 182 (PITMAN-MOORE INC, US) 
5 April 1990 

* the whole document * 

EP-A-0 314 224 (AKZ0, NV, NL) 

3 May 1989 

* the whole document * 

WO-A-9 011 298 (STICHTING CENTRAAL 
DIERGENEESKUNDIG INSTITUUT, NL) 

4 October 1990 

* Claims 1-9 * 

WO-A-8 800 056 (THE STATE OF VICTORIA, AU) 
14 January 1988 

* Claims 1-10 * 

GB-A-2 228 262 (NATIONAL INSTITUTE OF 
IMMUNOLOGY, INDIA) 
22 August 1990 

* Claims 1-15 * 

WO-A-8 606 635 (BIOTECHNOLOGY AUSTRALIA 
PTY. LTD. AU) 
20 November 1986 

* The claims * 



1,2,4-6 

1-6 

10 

1-6 

1-6 

1,2,4-6 
1-6 

1-6 



The present search report has been drawn up for all claims 



C12N15/16 

C07K7/06 

C07K17/02 

A61K39/385 

C12N1/21, 

//(C12N1/21, 

C12R1:19) 



TECHNICAL FIELDS 
SEARCHED (IM. Q.3 ) 



C12N 

C07K 
A61K 



Hac* •fwsta 

THE HAGUE 



Dau of cM«ilciiM of tfe aewca 

17 SEPTEMBER 1993 



NAUCHE S.A. 



i 

O 



CATEGORY OF CITED DOCUMENTS 

X : particularly relevant if taken alone 

Y : particularly relevant if combined with another 

document of the sane category 
A : technological background 
O : non-written disclosure 
P : intermediate document 



: theory or principle underlying the in vend on 
earlier patent document, out published on, or 
after the filing date 

: document cited in the application 
document dted for other reasoes 



A : member of the same patent family, corresponding 



NSDOCID: <EP 057B293A1_I_>