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Europaisches Patentamt 
European Patent Office 
Office europeen des brevets 



© Publication number: 



Document AN1 
Appl.No. 09/848,616 



0 425 082 A1 



® 



EUROPEAN PATENT APPLICATION 



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© Application number 90309614.7 
@ Date of filing: 03.09.90 



© int.Ci.5:C07K 13/00, C12N 15/31, 
A61K 39/10, C12N 15/62 



The title of the invention has been amended 


© Applicant: THE WELLCOME FOUNDATION 


(Guidelines for Examination in the EPO, A-lll, 


LIMITED 


7 3). 


Unicorn House 160 Euston Road 


London NW1 2BP(GB) 


© Priority: 04.09.89 GB 8919940 


@ Inventor: Charles, Ian George 


21.05.90 GB 9011358 




The Wellcome Research Laboratories, 


@ Date of publication of application: 


Langley Court 


02.05.91 Bulletin 91/18 


Beckenham, Kent, BR3 3BS(GB) 




Inventor: Fairweather, Neil Fraser 


© Designated Contracting States: 


The Wellcome Research Laboratories, 


AT BE CH DE DK FR GB IT LI LU NL SE 


Langley Court 




Beckenham, Kent, BR3 3BS(GB) 




© Representative: Datton, Marcus Jonathan 




William et al 




The Wellcome Research Laboratories Group 




Patents & Agreements, Langley Court 




Beckenham, Kent BR3 3BS(GB) 



© Bordetella vaccines. 

© A polypeptide suitable for use in a vaccine against Bordetella pertussis , B. parapertussis or B. bronchisep- 
tica presents an epitope comprising: 

(a) the amino acid sequence coded for by nucleotides 1885 to 1902 of the p.69 gene of B. pertussis CN2992; 

(b) the corresponding amino acid sequence of another strain of B. pertussi s or of a strain of B. parapertussis 
or B. bronchiseptjca ; or 

(c) lT said sequence (a) or (b) which has been modified such that the modified sequence has substantially the 
same antigenicity as that of the said sequence (a) or (b); the said polypeptide being no more than 50 amino 
add residues long or being a chimaeric protein having an amino acid sequence comprising the sequence of a 
carrier protein and a foreign sequence of no more than 50 amino acid residues which comprises the 
sequence of a said epitope. 



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VACCINES 



The present invention relates to vaccines against Bordeteila pertussis , Bordetella parapertussis or 
Bordetella bronchiseptica . 

Bordeteila pertussis is the causitive agent of whooping cough in man, although outbreaks of the disease 
have been associated with the related organism Bordetella parapertussis. Bordetella bronchiseptica is 

s primarily an animal pathogen, although it has been isolated from children with whooping cough-like 
symptoms. Immunisation programmes with whole-cell vaccine of B.pertussis have been relatively effective 
in controlling the disease, although currently the uptake of the vaccine is low in some developed countries 
because of the reactogenicity associated with vaccination. Clinical symptoms noted for the 1 in 10,000 
children suffering side-effects may include persistant screaming, fever and local reactions. 

io There is a need for a new pertussis acellular vaccine that lacks the components present in the whole 
cell vaccine associated with reactogenicity, but still comprises the protective epitopes. The search for the 
protective components has centered on a number of outer-membrane associated antigens. These include 
pertussis toxin (ptx Lymphocytosis promoting factor/LPF), filamentous haemagglutinin (FHA), cytotoxic 
adenylate cyclase (Adcase), dermonecrotic toxin (DNT), tracheal cytotoxin, the agglutinogens (Agg2, Agg3); 

75 the 69kDa outer membrane protein (omp) (P.69) and lipopolysaccharide (LPS). 

Much work has been carried out on the pertussis toxin (LPF), which is believed by many to be the most 
important part of any acellular pertussis vaccine (Bacterial Vaccines, 1984, Chapter 3, Manclark et al. Ed: 
Germainer). The results of a recent clinical trial in Sweden with an LPF/FHA vaccine showed that such a 
vaccine provided only about 69% protection (Lancet 1^ , 995, 1988). This is lower than the results expected 

20 for protection provided by a whole-cell vaccine, and resulted in the LPF/FHA vaccine not being licensed by 
the Swedish Health Authority. 

EP-A-01 62639 discloses a vaccine formulation for protection against B. pertussis which includes an 
antigenic preparation derived from B. pertussis comprising proteinaceous material associated with adenylate 
cyclase activity (ACAP) together with a pharmaceutical^ acceptable carrier therefor. An ACAP with a 

25 molecular weight of 69kDa is disclosed. 

In order to characterise which regions of this ACAP, termed the P.69 antigen, were important for 
protection the gene encoding the protein was cloned and sequenced (Charles et ajL PNAS, Vol 86 , 3554- 
3558, 1989). P.69 as extracted from B. pertussis appears to be the processed form of a 93kDa precursor 
(P.93). 

30 The present invention provides a polypeptide suitable for use in a vaccine which polypeptide presents 
an epitope comprising: 

(a) the amino acid sequence coded for by nucleotides 1885 to 1902 of the P.69 gene of B. pertussis 
CN2992; 

(b) the corresponding amino acid sequence of another strain of B. pertussis or of a strain of B. 
35 parapertussis or B. bronchiseptica ; or 

(c) a said sequence (a) or (b) which has been modified such that the modified sequence has 
substantially the same antigenicity as that of the said sequence (a) or (b); the said polypeptide being no 
more than 50 amino acid residues long or being a chim aerie protein having an amino acid sequence 
comprising the sequence of a carrier protein and a foreign sequence of no more than 50 amino acid 

40 residues which comprises the sequence of a said epitope. 

The polypeptides of the invention comprise a defined antigenically effective sequence. This sequence 

is 

PGPQPP 

based on the sequence of the P.69 gene of B. pertussis CN2992 disclosed by Charles <?t al (1989) and 
45 using the one letter code for amino acids (Eur. J. Biochem. 138 , 9-37, 1984). The sequence therefore 
essentially consists of amino acid residues 547 to 552 of the P.69 protein of B. pertussis CN2992. The 
corresponding sequence for other strains of B. pertussis and for strains of & parapertussis and B. 
bronchiseptica can be readily determined by lining up the amino acid sequence of the P.69 antigen, the 
P.70 antigen or the P.68 antigen respectively with the P.69 sequence shown by Charles et al (1989). 
50 Preferably the epitope comprises: 

(at) the amino acid sequence coded for by nucleotides 1876 to 1944 of the P.69 gen of a pertussis 
CN2992; 

(bi) the corresponding amino acid sequence of another strain of B. pertussis or of a strain of B. 
parapertussis or B. bronchiseptica ; or 

(ci) a said sequence (ai) or (bi) which has be n modified such that th modified s qu nee has 



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substantially the same antigenicity as that of the said sequence (ai) or (bi). 
Sequence (ai) is therefor : 

APQPGPQPPQPPQPQPEAPAPQP 

The sequence represents amino acid residues 544 to 566 of the P.69 protein of B. pertussis CN2992. 
s This sequence and the corr sponding sequence (bi) for the P.70 antigen of B. parapertussis and the P.68 

antigen of B. bronchiseptica can be aligned as follows. The PGPQPP epitope is underlined: 

P.70 APQPGPQ PGPQPP QPPQPPQPPQPPQPPQRQPEAPAPGP 

P.68 APQPGPQ PGPQPP QPPQPPQP PQRQPEAPAPQP 

P.69 APQPGPQP -P QPPQP QPEAPAPQP 

70 The sequences~can be modified in such a way that a modified sequence exhibits substantially the same 

antigenicity as the unmodified sequences. The modified sequence should therefore show a similar or 

greater degree of effectiveness in the Kendrick test than the corresponding unmodified sequence. A 

modified sequence has substantially the same amino acid sequence as an unmodified sequence but 

comprises one or more amino acid substitutions, insertions or deletions. 
75 One or more amino acid residues of a sequence (a), (ai), (b) or (bt) as above may therefore be 

replaced by one or more other amino acid residues which do not affect the antigenicity of the epitope. 

Consequently, one or more amino acid residue may be replaced by another which preserves the 

physiochemica! character of the original epitope, i.e. in terms of charge density, 

hydrophilicity/hydrophobicity, size and configuration, and hence preserves the immunological structure. 
20 Candidate substitutions are A for G and vice versa ; V by A, L or G; K by R; S by T and vice versa ; E for D 

and vice versa ; and Q by N and vice versa . 

A first type of polypeptide according to the invention is composed of up to 50 amino acid residues, for 

example up to 40 or up to 30 or up to 20 amino acid residues, which presents an epitope as defined above. 

Further amino acids may therefore be added to one or both ends of the epitope. One, two, three or four 
25 additional residues may be provided at the N-terminus or C-terminus or at both terminii of the defined 

epitope. 

Where additional residues are provided at either or both ends of the epitope, preferably these are the 
natural residues. These can be deduced from the sequence of the P.69 antigen of B. pertussis , of the P.70 
antigen of B. parapertussis or of the P.68 antigen of B. bronchiseptica . Preferred flanking sequences for the 

30 epitope aretherefore the flanking sequences which "naturally occur either side of the amino acid sequence 
for the epitope in the overall sequence of the P.69, P.70 or P.68 protein as the case may be. Also, a 
cysteine residue may be provided at the N-terminus or C-terminus. In particular, a cysteine residue may be 
added to the C-terminus alone. This is in order to facilitate carrier coupling and/or to enhance the 
immunogenicity of the polypeptide. 

35 The polypeptide may have a free Oterminal carboxy group. Alternatively, it may be in the form of a O 
terminal amide. Pharmaceutical^ acceptable salts of the polypeptide may be employed. The polypeptide 
may be coupled to a carrier in order to creature an immunogen which is antigenically active. Any 
appropriate physiologically acceptable carrier may be employed. A conjugate between the polypeptide and 
the carrier may be formed. The carrier may be for example bovine serum albumin, thyroglobulin, 

40 ovalbumin, keyhole limpet hemocyanin (KLH) or hepatitis B core antigen. 

A second type of polypeptide according to the invention is a chimaeric protein which presents the 
defined epitope. The chimaeric protein is typically a carrier protein which has been modified so that its 
amino acid sequence comprises a foreign sequence of up to 50 amino acids which includes the sequence 
of the desired epitope. Some amino acids of a protein may be replaced by the foreign amino acid 

45 sequence. Alternatively, the foreign amino acid sequence is fused to a protein. An intervening linker of up to 
10 amino acids, for example of up to 5 amino acids, may be provided between the epitope and the carrier. 
The foreign amino acid sequence may vary in length as described for the first type of polypeptide 
according to the invention. 

The epitope Is exposed on the surface of the chimaeric protein so that it is presented to the immune 

50 system. The chimaeric protein may take the form of a particle or form part of a particulate aggregation. 
Such an aggregation may comprise plurality of chimaeric proteins and/or may be a viral particle. A protein 
to which a foreign amino acid sequence comprising the epitope may be fused may be a particle-forming 
protein such as hepatitis B surface antigen (HBsAg, EP-A-0175261) or hepatitis B core antigen (HBcAg, JP- 
A-631 96299). The foreign sequence may be inserted into the sequence of a viral protein exposed on the 

55 surface of the virus (GB-A-21 25065). The viral protein may be a capsid protein of a virus. 

The foreign sequence may ther fore be provided at one of the antigenic sites of a picornavirus such as 
poliovirus (EP-A-0302801). The epitope may be presented at one of the antigenic sites, for example site 1, 
2 or 3, on a capsid protein of an attenuated strain of type 1 poliovirus, or at an antig nic site of type 2 or 3 

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pofiovirus. Other picomaviruses, suitably modified, may be used, e.g. Bovine enterovirus. 

Th amino acid sequence of an antig nic site of a picomavirus may be replaced completely or partly 
by th foreign amin acid sequence. Preferably th foreign amino acid sequenc is provided in place of 
some or all of antigenic sit 1 of an attenuated strain of type 1 poliovirus. The attenuated strain is typically 

s the Sabin 1 vaccine strain. Antigenic site 1 of a type 1 poliovirus is compos d of amino acid residues 91 to 
1 02 of the VP1 capside protein. 

The polypeptides of the invention are synthetic polypeptides. They may be prepared by chemical 
synthesis, in particular the first type of polypeptide of up to 50 amino acid residues long. Solid-phase or 
solution methods of peptide synthesis may be employed. A polypeptide can be built up therefore by a 

to process comprising condensing single amino acids and/or preformed peptides or two or more amino acids 
in the order in which amino acids occur in a polypeptide of the invention. The polypeptide may be 
synthesised so as to possess a free C-terminal carboxy group or a C-termlnal amide group. If desired, the 
polypeptide may be converted into a pharmaceutically acceptable salt. 

In solid-phase synthesis, the amino acid sequence of the desired polypeptide is built up sequentially 

is from the C-terminal amino acid which is bound to an insoluble resin. When the desired polypeptide has 
been produced, it is cleaved from the resin. When solution-phase synthesis is employed, the polypeptide 
may again be built up from the C-terminal amino acid. The carboxy group of this acid remains blocked 
throughout by a suitable protecting group, which is removed at the end of the synthesis. 

Whichever technique, solid phase or solution-phase, is employed each amino acid added to the 

20 reaction system typically has a protected a-amino group and an activated carboxy group. An amino group 
may be protected by the fluoren-9-ylmethoxycarbonyl (Fmoc) or t-butoxycarbonyl (Boc) group. A carboxy 
group may be activated as a pentafluorophenyl or 1-oxo-2-hydroxy- dihydrobenzotriazine ester. Each 
condensation step may be effected in the presence of dicyclohexylcarbodiimide or 1-hydroxybenzotria2ole. 
Side chain functional groups are typically protected too, for example the side chain amino group of a 

25 lysine, the side chain hydroxy group of a threonine or the side chain sulphydryl group of a cysteine. After 
each step in the synthesis, the a-amino protecting group is removed. 

However, any side-chain protecting groups are generally only removed at the end of the synthesis 
although they may be retained if desired. 

The polypeptides may be prepared with a C-terminal carboxy or amide group as desired. In solid phase 

30 peptide synthesis, this may be determined by how the C-terminal amino acid is linked to the resin support 
and/or how the final peptide is cleaved from the resin. Typically the resin is a styrene and/or divinylbenzene 
polymer. The C-termina! amino acid may be linked to the resin via an ester linkage which can be cleaved 
by a strong acid such as HBr in trifluoroacetic acid or HF to give the peptide with a C-terminal carboxy 
group. Ammonolysis can give the corresponding amide instead. 

35 An alternative method of obtaining a polypeptide amide by solid phase synthesis is to arrange for the 
C-terminal amino acid of the polypeptide to be linked to the resin via a peptide aminobenzhydryl bond. This 
can be formed by coupling with dicyclohexylcarbodiimide and can be cleaved with HF, typically in the cold. 
For solution phase synthesis, whether a C-terminal carboxy or amide group is present may depend upon 
how the carboxy group of the C-termlnal amino acid is blocked and, at the end of the synthesis, unblocked. 

40 A polypeptide with a C-terminal carboxy group can be converted into one with a C-terminal amide group 
and vice versa . 

Both types of polypeptide according to the invention may be prepared by recombinant DNA methodolo- 
gies, in particular by: 

(i) preparing an expression vector which incorporates a DNA sequence encoding the said polypeptide 
45 and which is capable of expressing the said polypeptide when provided in a suitable host; and 

(ii) providing the said vector in the said host such as to enable expression of the said polypeptide to 
occur. 

Thus a DNA sequence encoding the desired polypeptide is provided. An expression vector is prepared 
which incorporates the DNA sequence and which is capable of expressing the polypeptide when provided in 

50 a suitable host The DNA sequence is located between translation start and stop signals in the vector. 
Appropriate transcriptional and translational control elements are also provided, in particular a promoter for 
the DNA sequence and a transcriptional termination site. The DNA sequence is provided in the correct 
frame such as to enable expression of the polypeptide to occur in a host compatible with the vector. 

In the case of a chimaeric protein, a DNA fragment encoding the foreign amino acid sequence is 

55 inserted into a vector at a location which enables the epitope of interest to be expressed, as part of the 
chimaeric protein, exposed on the surface of the protein. The chima ric prot in is then expressed. Cells 
harbouring the vector are cultured so as to enable expression to occur. Depending on the type of chimaeric 
protein, the protein may self-assemble into particles. 



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Any appropriate host-vector system may be employed. The vector may be plasmid. In that event, a 
bacterial or y ast host may b used for exampl E.coli or S. cerevisiae. Alternatively, the vector may be a 
viral vector. This may be used to transfect cells of a mammalian cell line, such as CHO cells, in order to 
cause polypeptide expr ssion. 

s An epitop according to the invention may b linked to one or more helper T-cell (Th-cell) epitopes. A 
Th-cell epitope is a site capable of eliciting help for antibody production. A Th-cell epitope is capable of 
binding class II major histocompatibility (MHC) molecules on the surface of host antigen-presenting cells 
and B-cells subsequently interacting with the T-cell receptor in the form of a trimolecular complex in order 
to induce B-cells to differentiate and proliferate. 

to A Th-cell epitope may be linked to the first type of polypeptide of the invention in a variety of ways. 
Glutaraldehyde polymerisation may be used, in which the polypeptide of the invention is copolymerised 
with a polypeptide which presents a Th-cel! epitope via their amino groups. The polypeptide of the invention 
and the polypeptide presenting the Th-cell epitope may be conjugated together via a heterobifunctional 
cross-linking agent such as m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). 

rs The polypeptide of the invention may alternatively by linked at its C-terminus or N-terminus to a 
polypeptide presenting a Th-ceil epitope via a peptide bond. This may be achieved by co-linear synthesis 
of the polypeptide of the invention and the polypeptide presenting the Th-ceil epitope or by use of 
recombinant DNA technology as above to express a fusion protein in which the two polypeptides are fused 
together. In any of the methods, any suitable Th-cell epitope may be used. 

20 A preferred polypeptide presenting Th-cell epitopes is hepatitis B core antigen (HBcAg). A first type of 
polypeptide of the invention may be chemically coupled to HBcAg. Recombinant DNA technology can be 
used to produce a fusion protein according to the second type of polypeptide of the invention, comprising 
HBcAg to the amino terminus of which is linked the sequence of a polypeptide of the invention. The epitope 
of interest may be fused directly to the amino terminus of HBcAg. Alternatively, the sequence may be fused 

25 to the HBcAg via an intervening linker. Such a linker may be composed of one or more, for example up to 
ten, amino acid residues. 

The polypeptides of the invention are useful as vaccines against B. pertussis , parapertussis or B. 

bronchiseptica . An effective amount of the polypeptide is administered to a host in need of vaccination. 

The polypeptide may be administered orally or parenteral^, for example subcutaneously or intramuscularly. 
so The polypeptide may be given in association with FHA. In this way, immunity to whooping cough may be 

induced in a human. Immunity to B. bronchiseptica may be induced in a mammal. 

Typically, a polypeptide is administered orally or parenteral^ in an amount of 1 to 1000 ug per dose. 

more preferably from 10 to 100 ug per dose. A single dose may be given or a plurality of doses may be 

administered over a period of time. When FHA is administered, it may be used in an amount of 20 to 75 ug 
35 per dose. The FHA is typically administered in the same vaccine formulation as the polypeptide of the 

invention. 

A vaccine of the invention comprises a polypeptide of the invention, optionally FHA, and a pharmaceuti- 
cal^ acceptable carrier or diluent. The carrier or diluent may be any liquid medium suitable for use as a 
vehicle to introduce an antigen into a patient, for example an isotonic saline solution. The polypetide may 
40 also be present with an adjuvant for stimulating the immune response and thereby enhancing the effect of 
the vaccine. A suitable physiologically acceptable adjuvant is aluminium hydroxide or aluminium phosphate. 

Conveniently the vaccine formulations are presented to contain final concentration of antigenic polypep- 
tide in a range of from 0.01 to 5mg/ml, preferably 0.03 to 2 mg/ml, most preferably about 0.3 mg/ml. After 
formulation the vaccine may be incorporated into a sterile container which is then sealed and stored at a 
45 low temperature, for example 4* C, or may be freeze-dried. 

One or more doses of such a formulation may be administered to a host to be immunised. It is 
recommended that each dose is from 0.1 to 2 ml. preferably from 02 to 1ml and most preferably about 
0.5ml of vaccine. Immunity can therefore be induced by the administration of an effective amount of a 
vaccine formulation. 

so The following Examples illustrate the present invention. In the accompanying drawings: 
Figure 1 shows the construction of vector pWYG7. 

Figure 2 shows the nucleotide sequence of the promoter region of GAL7 . The synthesised promoter 
corresponds to the Xhol to BamHI fragment Regions downstream of BamHI are present in native GAL7 
including the RNA start site (I) and the initiating ATG (underlined). The two basepalrs which were altered 
65 to give a BamHI site are underlined. 

Rgur 3 shows the construction of pWYG7HBP. 

Figure 4(a) shows the nucl otide sequence of synth tic oligo A containing the GAL7 untranslat d leader 
sequence and th s' region of the HBcAg gen . 



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EP 0 425 082 A1 

Rgur 4(b) shows the nucleotide sequence of synthetic oligo B, encoding the presumed BB05 epitope. 
Figure 5 shows th results of Western blot of soluble proteins in Example 2(6) from induced yeast cells 
transformed with (1) pWYG7HBF (expressing FMD peptide-HBcAg fusion), (2) pWYG7HBF, (3) no 
plasmid and (4) pWYG7HBC (expressing HBcAg). The blot was developed using a rabbit anti-HBcAg 
5 serum and a goat anti-rabbit IgG-peroxidase conjugate. The position in the gel of 30K, 21 .5K and 
14.3KDa size markers is indicated. 

Figure 6 shows the results of dot blot analysis in Example 2(7) of HBP fusion protein after sucrose 
density gradient centrifugation. Fraction 1 is the bottom fraction and fraction 20 the top. All the material 
reacting with the anti-HBcAg serum is in the middle fractions, indicating complete assembly of HBP into 
to particles. 

Figure 7 shows the region of P.69 from which peptides 683, 684 and 685 and the epitope presented by 
the HBP fusion protein are derived. BB05/CORE binds BB05. Peptide 683 binds BB05, BB07, E4A8 and 
E4D7. Peptide 684: no binding. Peptide 685: binds PBE3. The numbering of the amino acid sequence 
refers to the residues of mature (i.e. signal sequence processed) P.69. 
15 Figure 8 shows the analysis of peptides, covering residues 505 to 603 of the p.69 protein of B. pertussis 
, with monoclonal antibody. 

EXAMPLE 1: Preparation of peptides 

20 

The peptides shown below were synthesised using an adaptation of the Merrifield method (Merrifieid, 
JACS, 85 , 2149-2154, 1963) described by Houghten (Houghten, PNAS, 82 , 5131-5135, 1985): 
Peptide~683: APQPGPQPPQPPQPQPEAPAPQPPAGRELSC (peptide of invention) 
Peptide 684: AGRELSAAANAAVNTGGVGLASTLWYAEC 
25 Peptide 685: TLWYAESNALSKRLGELRLNPDAGGAWGRGC 

Peptide 683 is composed of the amino acids coded for by nucleotides 1876 to 1962 of the P.69 gene 
and has an additional non-natural carboxy-terminal cysteine residue. Peptide 684 is composed of the amino 
acids coded for by nucleotides 1948 to 2031 of the P.69 gene and has an additional non-natural carboxy- 
terminal cysteine residue. Peptide 685 is composed of the amino acids coded for by nucleotides 2014 to 
30 2100 of the P.69 gene and has an additional non-natural carboxy-terminal cysteine residue. 

Each peptide was synthesised on a p-methyi-benzhydrylamine divinylbenzene resin. The a-amino 
protecting group on each amino acid was t-butoxycarbonyl (Boc). Each coupling cycle was as follows: 

1. Wash resin with dichloromethane - 10 minutes 

2. Wash with 5% diisopropylethylamine in dichloromethane - 2 minutes x 3 
35 3. Dichloromethane wash - 1 minute x 2 

4. Couple t-butoxycarbonyl amino acid in dichloromethane, 0.3M diisopropylcarbodiimide - 60 minutes. 
For N and Q coupling was effected in dimethylformamide, 0.3M diisopropylcarbodiimide and 0.1 25M 
hydroxy benzotriazole. 

5. As -3 

40 6. Deprotect with 50% trifluoroacetic acid in dichloromethane - 20 minutes 

7. Dichloromethane wash - 1 minute x 6 

8. Return to 2. 

When coupling cycles were completed the peptide was cleaved off the resin using hydrogen fluoride for 
1 hour with an anisole scavenger 10%. The peptide was thus obtained with a carboxy-terminal amide group. 
45 It was then ether washed, dried, dissolved in 15% acetic acid and lyophilized. 



Example 2: Expression of B.pertussis epitope - HBcAg Fusion protein (HBP) in yeast 

50 

1. General 

An amino acid sequence corresponding to nucleotides 1855 to 1944 of the P.69 gene was fused 
g netically to the amino-terminus of HBcAg. The resulting fusion protein was expressed efficiently in yeast 
55 and reacted with monoclonal antibody (Mab) BB05 which was raised against the 68kDa protein of B. 
bronchiseptica but cross reacts with P.69 of B.pertussis . The fusion protein was shown to assemble into 
core particles and conferred protection against B.p rtussis challenge in the Kendrick t st, when used in 
conjunction with FHA. 



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2. Construction of yeast expression vector pWYG7 

The fusion protein was expressed in Saccharomyces cerevisiae from the multi-copy v ctor pWYG7 
which carried the regulated GAL7 promoter. The v ctor pWYG7, constructed at Wellcome, was used for th 
5 expression of HBP, i.e. of a fusion protein composed of a major epitope of P.69 fused to the amino- 
terminus of HBcAg. Vector pWYG7 is derived from the 2u vector pJDB219 (Beggs, Nature 275 , 104-109. 
1978) modified to contain a kanamycin-resistance marker (kan*) and the yeast galactose-regulated GAL7 
promoter. 

The construction of pWYG7 is outlined in Fig. 1. First the kan r marker (Hindi fragment from pUC4K; 

jo Vieira and Messing, Gene 19 t 259, 1982) was ligated into the unique Smai site of pJDB219 to give the kan 
r tet r vector pJDB219IC Secondly, a synthetic GAL7 promoter fragment (Xhol-BamHI fragment, sequence 
shown in Fig. 2) was cloned into the unique Sail and BamHI sites of pJDB219K. The resulting vector, 
pWYG7, has the GAL7 promoter with unique BamHI and Belli sites upstream of the yeast 2u plasmid FLP 
gene transcriptionallerminator (Sutton and Broach, Mol. Cell. Biol. 5 , 2770-2780, 1985). Foreign genes to 

15 be expressed from pWYG7 are inserted between the BamHI and Bell sites. The design of the GAL7 
promoter fragment is discussed below. 

The smallest fragment of DNA upstream of the GAL7 gene which exhibits full promoter activity has 
been defined by deletion mapping (Tajima et al Yeast 1_ , 67-77, 1985). Based on this information a 260 
bp GAL7 promoter fragment was synthesised (Fig. 2 for sequence). The 260bp promoter was synthesised 

20 as four overlapping oligonucleotides using a Pharmacia Gene Assembler (protocol supplied by Pharmacia). 
These oligonucleotides were phosphorylated and annealed using standard techniques, then ligated into 
Xhol-BamHI cut plC-20H (Marsh et al Gene 32 , 481-485, 1984). Positive clones were identified and their 
DNA sequenced using the double' : stranded DNA sequencing method with universal and reverse sequencing 
primers (Hong, BioscL Report 2 , 907, 1982). The sequence of the GAL7 inserts was confirmed, and then 

25 the Xhol-BamHI GAL7 insert was excised and cloned into pJDB2l9K as described above. 

The design oflhe GAL7 promoter fragment in pWYG7 is such that the natural GAL7 DNA sequence has 
been slightly modified "(2bp"changed) in order to make the BamHI cloning site upstream of the GAL7 mRNA 
start sites. The foreign gene to be expressed is then linked with synthetic DNA to the BamHI site, such that 
the GAL7 mRNA start sites are introduced, along with the GAL7 upstream untranslated sequences. Thus the 

30 first non-yeast DNA downstream of the promoter is the initiating ATG codon of the foreign gene, and the 
transcript produced will have a yeast GAL7 leader rather than a foreign leader which could reduce efficiency 
of translation. 

35 3. Construction of the HBP fusion protein yeast expression vector pWYG7HBP 



A BamHI-BamHI expression unit for HBP was assembled in an intermediate vector, pKGC-69K. This 
vector, which contains GAL 7 sequences upstream of the initiator ATG codon, was made by first 
constructing a HBcAg vectorTpKGC, with an Ncol (CCATGG) site engineered at the initiator ATG. The DNA 
40 encoding the predicted B.pertussis epitope was then inserted into this site as a synthetic Ncol-Ncol 
oligonucleotide linker. The overall scheme for the construction of pKGC, pKGC-69K, and the final expres- 
sion vector, pWYG7HBP, is shown in Fig. 3. 



45 (i) Construction of pKGC 

A synthetic Sall-EcoRI linker containing the 3' terminal sequences of HBcAg (Aval site to stop codon) 
was ligated between the Sail and EcoRI sites of pUC18 (Vieira and Messing, 1982) to give the plasmid 
pKGF. The rest of the HBcAg gene was then assembled In pKGF in a three-way ligation: the 2.7kb Hindlll- 

50 Aval fragment of pKGF was isolated and ligated to (i) an 87bp synthetic (phosphorylated) Hindlll-Maelll 
fragment (oligoA, Fig. 4a) containing yeast GAL 7 upstream sequences and the 5 end of the HBcAg gene, 
and (ii) a 500 bp Maelll-Aval fragment from~pEB208 (Clarke et al , Nature 330 f 381-384, 1987), which 
contains most of the central part of the gene. The resulting plasmid, pKGC, is an intermediate vector for 
expression of HBcAg from pWYG7. The sequence of the synthesised regions of pKGC was confirmed using 

55 the double-stranded method with universal and reverse sequencing primers (Hong, 1982). 



(Ii) Construction of pKGC-69K 



7 



EP 0 425 082 A1 



The HBcAg gen in pKGC had been modified to hav a unique Ncol (CCATGG) site at th initiator 
ATG. Therefore N-terminal peptide fusions could be mad by inserting suitable Ncol-Ncol DNA fragments 
at this site. The epitop of the P.69 antigen was predicted to occur at the Pro-rich repeat region, so a 
suitabi pair of oligonucleotides were synthesised to ncode this epitope. The oligonucleotides made and 

5 the corresponding amino acid sequence ar given in Fig. 4b; the B. pertussis DNA was altered to give a 
yeast-optimal codons. pKGC was digested with Ncol, and the annealed, unphosphorylated oligonucleotides 
were cloned in using the linker-tailing method (Lathe et al , BRL Focus 6 , issue 4, 1984). From the resulting 
plasmids with inserts, ones of the required orientation were selected by double-stranded sequencing of the 
inserts. The resulting vector, pKGC-69K could be used as a source of a BamHI-BamHI fragment for transfer 

w topWYG7. 



(iii) Construction of pWYG7HBP 

75 The BamHI-BamHI fragment from pKGC-69K was isolated and cloned into pWYG7 (dam ~ DNA) that 
had been digested with BamHI and Bell and then treaed with calf-mtestinal alkaline phosphatase. After 
transformation, Kan r colonies were tested for inserts of the correct orientation, and these plasmids were 
denoted pWYG7HBP. E. coll MC1061 containing pWYG7HBP was deposited on 28 July 1989 at the 
National Collection of industrial and Marine Bacteria (NCI MB), Aberdeen, GB under accession number 

20 NCIMB 40176. 



4. Transformation of yeast with pWYG7HBP 

25 The vector pWYG7HBF was introduced into the Saccharomyces cerevisiae strain S150-2B (a , Ieu2 , 
his3 , ura3 , trpl , McCleod et al ., Cold Spring Harbor Symp. Quant Biol. 49 , 779-787, 1984) using the 
HtiijmTansformation procedure of Ko et a! . (J. Bact 153 , 163-168, 1983). Transformed yeast cells were 
incubated in YPD broth (Sherman et a[ Methods in Yeast Genetics, Cold Spring Harbor, New York, 1983) 
at 30° C overnight prior to plating* out on selective medium (YPD plus 500 ug/ml G418). This allows 

30 expression of G418-resistance and increases transformation frequency. Colonies that came up as G418 r 
were checked on minimal medium lacking leucine (YNB + glucose + histidine + uracil + tryptophan, Sherman 
et al ., 1983) to check for the Leu* phenotype also conferred by pWYG7HBP. Positive transformants (G418 r 
Leu*) were used for expression analysis. 

35 

5. Galactose-inducfion of expression of HBP 

Transformants were grown to the mid-logarithmic stage (10 7 cells/ml) in YP broth containing 2% 
raffinose and 500ug/ml G418 at 30* C in an orbital shaker. An aliquot of 40% galactose was then added to 

40 a final concentration of 2%, and the culture was incubated for a further 48h. The cells were then harvested 
by low speed centrifugation, washed once in distilled water, and resuspended in ice-cold break buffer 
(20mM sodium phosphate pH7.0, 0.1% Triton X-100, 4mM phenylmethanesulphonyl fluoride, 4mM EGTA, 
and 2ug/ml each of pepstatin, antipain, leupeptin, and chymostatin; 5ml for cells from a 250ml culture) Acid- 
washed glass beads (0.45mm) were added and the cells were broken by vigorous vortexing. The crude ceil 

45 lysate was cleared by centrifugation for 15 mins at 10,000g. The protein concentration of the cleared 
supernatant was determined using the BioRad protein assay (BioRad, according to manufacturer's instruc- 
tions), and the material was stored at -20* C in aliquots. 



so 6. Analysis of cell lysates for expression 

The proteins in the induced yeast cell lysates were analysed by separation in SDS-polyacrylamide gels 
(Laemmli, Nature 227 , 680-685, 1970). 50ug of soluble protein were loaded per track, and as a negative 
control an extracToT induced S150-2B was loaded. A new protein band was detected by staining with 
55 Coomassie blu in the pWYG7HBP-transformed ceil extracts, migrating at about 24,000kDa (not shown). It 
was confirmed that this polypeptide is HBP by using Western blot analysis with either HBcAg or BB05 
antiserum (Fig. 5, r suit with HBcAg serum). ELISA quantitation data indicate expression levels of 10-30% 
of cell protein. 



8 



EP 0 425 082 A1 



7. HBP fusion protein expressed in yeast associates to form core particles 

In order to test whether or not the HBP protein made in yeast was present as core particles, the 
induced cell extract was layered over a 15%-45% w/v sucrose density gradient (in phosphate-buffered 
5 saline) and centrifuged in a Beckman SW28 rotor (28,000rpm, 4h). The gradient was fractionated and the 
fractions analysed for the presence of HBcAg- or B. pertussis epitope-reacting material by spotting aliquots 
from each gradient fraction onto a nitrocellulose gTTd and processing the filter as for Western blot (dot blot 
shown in Rg. 6). 

Peak reactivity was detected in the middle fractions of the gradient and none was detected at the top 
10 of the gradient. This indicates that all the HBP protein made in yeast associates to form core particles which 
sediment in a sucrose gradient For confirmation, an aliquot from a peak gradient fraction was sent for 
electron microscopy (phosphotungstic acid stain). A large number of viral core particles were clearly seen 
(not shown). 

15 

Example 3: Dot-blot antibody hybridisation 

Aqueous solutions of the three peptides prepared in Example 1 (2mg/ml) and the HBP fusion protein 
prepared in Example 2 were used in dot-blot antibody hydridization experiments. Briefly, 10ul samples of 
20 the peptide or fusion protein were applied to a nitrocellulose membrane and allowed to dry on. Filter 
hybridzation experiments with monoclonal antibodies were carried out by normal methods (see, for 
example, "Antibodies, a laboratory manual" p.178, Ed. E. Marlow and D. Lane 1988, Cold Spring Harbor 
Laboratory). The Mabs were: 

BB05 and BB07 (Montarez et al ., Infect. Immun.47 , 744, 1985) which were raised against the 68kDa 
25 protein from B. bronchisepticabut which cross-react with P.69 from B. pertussis ; and 

a panel of 7 Mabs raised against B. pertussis . 

The results are shown in TabTe 1 and Figure 7. Rgure 7 shows the region of P.69 around which the 

peptides were made. The three chemically synthesised peptides have an additional non-natural carboxy- 

terminal cysteine residue. The HBP fusion protein has a N-terminal methionine residue. The results 
30 establish that the epitope of interest Jies within the region with the following sequence: 

APQPGPQPPQPPQPQPEAPAPQP. 

Table 1 



35 




Peptide 






Monoclonal 


683 


684 


685 


HBP fusion 






antibody 








protein 


40 


Bordetella 


BB05 


+ 






+ 


bronchiseptica 


BB07 


T 










Bordetella 


BPE3 












pertussis 


BPD8 














BPE8, 










45 




D5E9 














F6E5 














E4A8 


+ 












E4D7 


+ 









50 



Example 4: Kendrick Test 
1. Coupling of peptides 

Th peptides of Example 1 were coupled to K yhol Limpet Haemocyanin (KLH) through an add d C- 

9 



EP 0 425 082 A1 



terminal cysteine residu using M-Maleimidobenzoyl-N-hydraxysulfosuccinimide ester (MBS) as a 
heterobifunctional cross-linker (Liu, F. et al , Biochemistry 18 , 690, 1979). Briefly, dialysed KLH is reacted 
with MBS dissolved in dimethylformamide (DMF) at room temperature, then run through a G-25 column. 
The KLH protein peaks were pooled, and added to free peptide. The pH was then adjusted to between 7 
s and 7.5, and after 3 hours stirring at room temperature the coupled peptide was stored at -20* C. The KLH 
concentration must not exceed 20mg/ml, MBS/KLH molar ratio is 40:1 , and the final concentration of DMF in 
the reaction should be below 30%. Peptides 683, 684 and 685 were coupled using 2.5mg KLH per 5mg 
peptide (a ratio of 1:2 wtfwt). 

10 

2. Preparation of Filamentous Heamagglutin (FHA) 

FHA can be prepared by methods well known in the art (see Arach and Munoz J.J. (1970), InfecL 
Immunology 25 764-767; Ashworth et a! (1982) Infect. Immun. 37 1278-1281). However FHA in the following 
75 procedure wasprepared in accordance" vith the following protocol. 



FHA purification: 

20 B. pertussis Tomaha or BP357 (Tn5 transposon mutant of A.A. Weiss et al (1983) which does not 
secrete the LPF) were grown in Stainer~& Scholte medium (0.05 Tris) in 650 ml Costar flasks (150 ml in 
each) for 5 days at 37° C (Sato et al , Infection and Immunity 41 , 313-320, 1983). Before centrifugation (30 
mins at 6000 x g) 50uM I.IO^henanthroline monohydrate as proteolysis inhibitor was added to the 
cultures. The cell free supernatant was applied to a 30 x 150 mm column of Hydroxylapatite (BD4) and 

25 washed (all at room temperature and a speed of pumping 500 ml/hr) sequentially with 10mM phosphate 
buffer, pH 8, 100 mM phosphate buffer, pH 7.1 until stable baseline. 

The retained material was eluted with 0.5M NaCI added to the 100mM phosphate buffer and the peak 
fractions agglutinating goose red blood cells were pooled. The pool was dialyzed overnight against 25-30 
volumes of 0.025 M Bis.-Tris/HCl buffer at 4*C. The precipitated FHA was collected on a centrifuge (20 

30 mins at 8000 x g). The next step was inspired by Cowell et al (in vol. IV, Bacterial Vaccine, 371-379, 
Seminars in Infectious Disease, Weinstein and Fields: editors, Thieme Verlag, New York, Stuttgart, 1982) 
who found that the FHA (as well as LPF) is soluble in 40mM beta-alanine buffer, pH 3.5. The precipitated 
FHA was solubilized in the smallest possible volume of 3-alanine buffer (3.57g 3-alanine and 0.35g formic 
acid per litre), insolubles removed by centrifugation and the clear supernatant was applied to a column (25 

35 x 500mm) of Ultrogel AcA 34 equilibrated and eluted with the same buffer. 

The haemagglutinating material appeared in a sharp peak followed by a shoulder. The shoulder material 
was discarded, the fractions from the sharp peak were pooled and kept frozen or re-precipitated by dialysis 
against 0.025M Bis-Tris buffer and dissolved in a smaller volume of 3-alanine buffer. The solubility is 
approx. 2.5 mg FHA/ml. The material frozen (at -20 or -40* C) at acid pH appeared stable as judged from its 

40 ELISA reactivity and appearance in the SDS-PAGE gels: it formed prevalently three strong bands in the 
region of 150-100KD. 



3. Kendrick Test 

45 

This was performed according to W.H.O. Requirements for Pertussis Vaccine using MF1 or NIH Mice 
(OLAC, category 3, free of most pathogens including B. brochiseptica ), weighing 14-16g. The antigens, in 
0.5ml volumes, were inoculated concomitantly intraperitoneally and comprised a top dilution and three four- 
fold serial dilutions. After two weeks the mice were challenged intracerebrally using the recommended 
so challenge strain 18-323 (100-200 LDso). The number of survivors in each group was used for calculation of 
the relative potency in respect to the British Pertussis Reference Vaccine 66/84 using a program of parallel 
line probit analysis. The results are shown in Table 2. 



55 



10 



EP 0 425 082 A1 



Table 2 



5 



70 



15 



20 



30 



Vaccine 


Peptides 


683 


684 


685 


+ FHA(ug) 


Survivors 


(ug) 












1 


10 


10 


10 


20 


6/16 


2 


3.3 


3.3 


3.3 


6.6 


3/16 


3 


1.11 


1.11 


1.11 


2.2 


7/16 


4 


0.37 


0.37 


0.37 


0.74 


5/16 


HBP fusion Protein (ug) + FHA(ug) 


1 


1 


20 






8/16 


2 


0.33 


6.6 






11/16 


3 


0.11 


2.2 






4/16 


4 


0.037 


0.74 






1/16 


FHA alone (ug) 


1 


20 








3/16 


2 


6.6 








2/16 


3 


22 








3/16 


4 


0.74 








1/16 


66/88 whole cell 








Survivors 


vaccine I.U. 










1 


0.25 








11/16 


2 


0.08 








2/16 


3 


0.028 








2/16 


4 


0.009 








3/16 



The results show clearly that the combination of FHA with either a mixture of all three chemically 
synthesized peptides or with the HBP fusion protein is more potent that FHA alone. 

35 

EXAMPLE 5: Epitope mapping using synthetic peptides (Pepscan) 

. Hexameric peptides, overlapping by one amino acid residue, were synthesised on solid phase 
40 polyethylene pins as described by Geysen et ai (PNAS USA *H , 1984, 3998-4002). Ninety four hexameric 
peptides were synthesised, covering residues 505 to 603 of the P.69 antigen of B. pertussis . Pepscan 
peptide 1 was Thr(505>Asp(510). Pepscan peptide 94 was Ala(598)-Leu(603). 

Reactivity of the peptides to monoclonal antibody (mAb) BB05 was determined by incubation of the 
pins in antibody for 1-2 hrs or overnight, followed by washing and incubation in peroxidase conjugated goat- 
45 antimouse antibody. The mAb BB05 is an (lgG1) raised against the P.68 antigen from B. bronchiseptica 
(Montarez et al , Infect. Immun. 47 , 1985, 644-751). This mAb is a neutralising mAb which cross-reacts with 
P.69 from W. pertussis . 

Enzyme reactivity was determined by incubation of the pins in ABTS substrate solution (50 mg azino- 
di-3-ethyl-benzthiazodinsulphonate (ABTS) in 100ml buffer (0.1 molar disodium hydrogen orthophosphate. 
so 0.08 molar citric acid, pH 4.0 containing 30ul hydrogen peroxide)), and the A* 20 of the solution measured 
after 10-60 minutes using a Titertek Multiscan MC11Q0. Pins were sonicated for 30 min at 65* C in 0.1 M 
NaaHPOi, 0.1%SDS, 0.01 M B-mercaptoethanol to remove bound antibody and stain complexes prior to 
incubation in subsequent antibody. The results are shown in Figure 8. 

As can be seen from Rgure 8, mAb BB05 recognised only Pepscan peptide 43 (sequence PGPQPP). 
55 This is consistent with recognition of peptide 683 and located the BB05 epitope in the r gion of the (Pro- 
G!n-Pro)s repeats of P.69. 



11 



EP 0 425 082 A1 



Claims 

1. A polyp ptid which presents an epitope comprising: 

(a) th amino acid sequ nee coded for by nucleotides 1885 to 1902 of the P.69 gene of B. pertussis 
5 CN2992; 

(b) the corresponding amino acid sequence of another strain of B. pertussis or of a strain of B. 
parapertussis or B. bronchiseptica : or 

(c) a said sequence (a) or (b) which has been modified such that the modified sequence has 
substantially the same antigenicity as that of the said sequence (a) or (b); the said polypeptide being no 

to more than 50 amino acid residues long or being a chimaeric protein having an amino acid sequence 
comprising the sequence of a carrier protein and a foreign sequence of no more than 50 amino acid 
residues which comprises the sequence of a said epitope. 

2. A polypeptide according to claim 1, wherein the said epitope comprises 

(ai) the amino acid sequence coded for by nucleotides 1876 to 1944 of the P.69 gene of & pertussis 
15 CN2992; 

(bi) the corresponding amino acid sequence of another strain of B. pertussis or of a strain of B. 
parapertussis or B. bronchiseptica ; or 

(ci) a said sequence (ai) or (bi) which has been modified such that the modified sequence has 
substantially the same antigenicity as that of the said sequence (ai) or (bi). 
20 3. A polypeptide according to claim 1 or 2, which is composed of no more than 50 amino acid residues and 
which is provided with a cysteine residue at the N-terminus and/or the Oterminus. 

4. A polypeptide according to any one of the preceding claims, which is composed of up to 30 amino acid 
residues. 

5. A polypeptide according to claim 1 or 2, which is a fusion protein comprising hepatitis B core antigen to 
25 the amino terminus of which is linked the said foreign sequence. 

6. A polypeptide according to claim 1 , which has the amino acid sequence: 
APQPGPQPPQPPQPQPEAPAPGPPAGRELSC or 

PGPQPP; 

or which has the amino acid sequence: 
30 MAPPAPKPAPQPGPQPPQPPQPQPEAPAPQP 

fused to the amino terminus of hepatitis B core antigen. 

7. A process for the preparation of a polypeptide as defined in claim 1, which process comprises 
condensing single amino acids and/or preformed peptides of two or more amino acids in the order in which 
amino acids occur in the said polypeptide. 

35 8. A process for the preparation of a polypeptide as defined in claim 1 , which process comprises: 

(i) preparing an expression vector which incorporates a DNA sequence encoding the said polypeptide 
and which is capable of expressing the said polypeptide when provided in a suitable host; and 

(ii) providing the said vector in the said host such as to enable expression of the said polypeptide to 
occur. 

40 9. A conjugate comprising a polypeptide as defined in claim 1 linked to a physiologically acceptable carrier. 
10. A vaccine comprising a pharmaceutical^ acceptable carrier or diluent, a polypeptide as defined in claim 
1 and, optionally, filamentous haemagglutinin. 



12 



EP 0 425 082 A1 




EP 0 425 082 A1 



xhoi * i r 2 ? ? ? * ao 50 60 

CTCGAGACGT CTATACTTCG GAGCACTGTT GAGCGAAGGC TCATTAGATA TATTTTCTGT 
GAGCTCTGCA GATATGAAGC CTC6T0ACAA CTC5CTTCCG AGTAATCTAT ATAAAAGACA 

? 0 PQ 90 100 110 120 

* * * ♦ * * ♦ * + * ♦ * 

CATTTTCCTT AA?CC AAAAA TAAGGGAGAG GGTCCAAAAA GCGCTCGGAC AACTGTTGAC 
G7AAAAGGAA TTGGGTTTTT ATTCCCTTTC C^.CGTTTTT CGCGAGCCTG TTGACAACTG 

120 140 15C 160 170 180 

** * + * * • * * * * * 

CGTGATCCGA AGGACTGGCT ATACAGTGTT CACAAAATAG CCAAGCTGAA AATAATGTGT 
GCACTAGGCT TCCTGACCGA TATGTCACAA GTGTT7TATC GGTTCGACTT TTATTACACA 

'90 200 2IC 220 230 240 

** ** *♦ * * * * 

AGCCTTTAGC TATGTTCAGT TAGTTTGGCT AGCAAAGATA TAAAAGCAGG TCGGAAATAT 
TCGGAAATCG ATACAAGTCA ATCAAACCGA TCGTTTCTAT ATTTTCGTCC AGCCTTTATA 

2Z0 270 280 , . 290 300 

* * * * BamHt * • * w * * ♦ » 

TTATGGGCAT 7AT7ATGCAG AGGATCCACA TGATAAAAAA AACAGTTGAA TATTCCCTCA 
AATACCCGTA ATAATACGTr TOCTAGGTGT ACTATTTTTT TTGTCAACTT ATAAGGGAGT 



320 
* • 

AAAATGACTG 
TTTTACTGAC 



EP 0 425 082 A1 



Fig. 3. 
(a) 




15 



EP 0 425 082 A1 



Fig. 3. 

Hind II I Mae M Mae HI Aval 

loiigoA 1 pEB208 I 

• .* 



Aval 




16 



EP 0 425 082 A1 



Fig. 3. 
(c) 



Ncol 



HBcAg 



Ncol 
Bam HI. 




BB05 



Bam HI 



pKGC-69K 



pWYG 7 
Bam HI /Bet I-cut 
phosphatased 




pWYG 7 HBP 



17 



EP 0 425 082 A1 



Fig. 4a. 



Hind III BamHJ NcoI 

GAL7 leader 

5' AGCTTGGATCCACATG ATA A AAA A AACAGTTG AATATTCCCTC AACC fATGl G. 
3' ACCT AGGTGT ACT ATTTTTTTTGTCAACTT AT AAGGGAGT TGGT ACC . 

...AC ATT6ACCCTT ATAAAGAATTCGGAGCTACTGT6GA 3' 
...TGTAAC T GGGAATATTTCTT AAGC CT CG ATGAC ACCTC AATG 5' 

HBcAg gene 5' end Mae 111 



Fig. 4b. 



Nco! 

MAPPAPKPAPOPGPCIP P 
5' C|ATGJGCTCCACCAGCTCCAAA A C C A G C T C C A C A A C C A G G T C C A C A A C C A C C A . . 
3' CGAGGTGGTCGAGGTTTT^GGTCGAGGTGTTGGTC C AGGTGT TGGT GGT 

Q PPQPQPEAPAPQP 
...CAAC C ACC AC A ACC AC A ACC A G A AG C TC C AG C T C C AC A ACC 3* 
...GTTGGTGGTGTTGGTGTTGGTCTTC G AGGTC GAGGTGTT GGGT AC 5' 

NcoI 



18 



EP 0 425 082 A1 




12 3 4 



19 



EP 0 425 082 A1 



1 



10 



• • • 



11 



20 



20 



EP 0 425 082 A1 



Figure 7 



537 547 557 567 577 

• • • • • 

MAPPAPKPAPQPGPQPPQPPQPQPEAPAPQPPAGRELSAAAN 



[BB05/CORE] t- 



16831 

[684] 



587 597 607 617 

• • • • 

AAVNTGGVGLASTLWYAESNALSKRLGELRLNPDAGGAWGRG 

C 

[6851 C 



21 



EP 0 425 082 A1 



2i 



1.5- 



1 



0.5- 



BB05 



— i — ■ i ■ — i 1 1 — 

10 20 30 40 50 60 70 80 90 



22 



European 
Patent Office 



EUROPEAN SEARCH 
REPORT 



Application Number 
EP 90 30 9614 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 



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



Relevant 
to claim 



CLASSIFICATION OF THE 
APPLICATION (Int. CL5) 



D.X 



D,X 



D,Y 



DEVELOP. BIOL STANDARD, vol. 61 , 1985, pages 27-41, 
S. Karger, Basel, CH; P. NOVOTNY et al.: "Bordetella 
adenylate cyclase: A genus specific protective antigen and 
virulence factor" 

* The whole document * 

EP-A-0 162 639 (THE WELLCOME FOUNDATION LTD) 

* The whole document * 

PROCEEDINGS OF THE NATIONAL ACADEMY OF THE 
USA. vol. 86, no. 10, May 1989, pages 3554-3558. Washing- 
ton, DC, US; I.G. CHARLES et al.: "Molecular cloning and 
characterization of protective outer membrane protein P.69 
from Bordetella pertussis" 

* The whole document * 

IDEM 

PROC. NATL ACAD. SCL USA, vol. 81. July 1984, pages 
3998-4002; H.M. GEYSEN et al.: "Use of peptide synthesis 
to probe viral antigens for epitopes to a resolution of a single 
amino acid" 

* The whole document, especially page 4001, column 2, last 
paragraph * 

J. EXP. MED., vol. 168, October 1988, pages 1351-1362, 
The Rockefeller University Press; M.T. DE MAGISTRIS et 
al.: "Dissecting human T cell responses against Bordetella 
species" 

* The whole document * 

INFECTION AND IMMUNITY, vol. 50, no. 1, October 19851 
pages 199-206, American Society for Microbiology; P. 
NOVOTNY et al.: "Adenylate cyclase activity of a 
68,000-molecular-weight protein isolated from the outer 
membrane of Bordetella bronchiseptica" 

* The whole document * 



The present search report has been drawn up for al) claims 



1.2,10 



C07K 
13/00 

C 12 N 15/31 
A 61 K 39/10 
C 12 N 15/62 



1.2.10 



1,2,8-10 



3-7 



3-7 



TECHNICAL FIELDS 
SEARCHED (Int. CI.5) 



10 



A 61 K 

C07K 
C12N 



Place of search 

The Hague 



Date of completion of search 

07 January 91 



Examiner 

SKELLY J.M. 



CATEGORY OP CITED DOCUMENTS 
X : particularly relevant If taken alone 
Y : particularly relevant If combined with another 

document of the same catagory 
A: technological background 

: non-written disclosure 
P: Intermediate document 
T : theory or principle underlying the Invention 



E : earlier patent document, but published on, or after 

the filing date 
D : document cited In the application 
L: document cited for other reasons 

& : member of the same patent family, corresponding 
document 



European 
Patent Office 



EUROPEAN SEARCH 
REPORT 



Page 2 
Application Number 

EP 90 30 9614 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 



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



Relevant 
to claim 



CLASSIFICATION OF THE 
APPLICATION ant. C1.5) 



P,X 



INFECTION AND IMMUNITY, vol. 56, no. 12, December 
1988, pages 3189-3195, American Society for Microbiology; 
M.J. BRENNAN et al.: "Identification of a 69-kilodalton non- 
fimbrial protein as an agglutinogen of Bordetella pertussis" 

INFECTION AND IMMUNITY, vol. 47, no. 3, March 1985, 
pages 744-751, American Society for Microbiology; J.A. 
MONTARAZ et al.: "Identification of a 68-kilodalton protec- 
tive protein antigen from Bordetella bronchiseptica'' 

NATURE, vol. 330, no. 6146, 26th November - 2nd Decem- 
ber 1987. pages 381-384, London, GB; B.E. CLARKE et al.: 
Improved immunogenicity of a peptide epitope after fusion 
to hepatitis B core protein" 
* The whole document * 



5.6 



EP-A-0 338 170 
* Claims * 



(INSTITUT PASTEUR) 



1,2,10 



TECHNICAL FIELDS 
SEARCHED (Int. CI.5) 



The present search report has been drawn up for alt claims 



Place of saarcn 



Th Hague 



Date of completion of search 

07 January 91 



Examiner 



SKELLY J.M. 



CATEGORY F CITED DOCUMENTS 
X: particularly relevant If taken alone 
Y : particularly relevant H combined with another 

document of the same catagory 
A: technological background 

: non-written disclosure 
P: Intermediate document 
T: theory or principle underlying the Invention 



E : earlier patent document, but published on, or after 

the filing date 
D: document cited In the application 
L : document cited for other reasons 



& : member of the same patent family, corresponding 
document