ACELLULAR VACCINE

Abstract: A vaccine suitable for use in inducing immunity to whooping cough in humans comprises a synergistic combination of the 69kDa antigen of Bordetella pertussis and toxoided lymphocytosis promoting factor of Bordetella pertussis.

Description: ACELLULAR VACCINE The present invention relates to an acellular Bordetella pertussis vaccine and to its use in medicine. B.pertussis causes a serious and debilitating disease in humans, especially children, which has been kept under control in the developed countries by large scale immunisation programmes. Generally, immunisation has been carried out using a whole cell vaccine derived from cell cultures of B.pertussis and has been found to be relatively effective in preventing the disease. However, concern over adverse reactions, such as fever, local reactions and persistent screaming, has led in recent years to a reduced acceptance of the vaccine and a debate about its continued use. With the low incidence of the disease in developed countries, the benefit/risk ratio associated with the use of whole cell vaccines is poorly defined. Many clinicians believe however that the risk of adverse reaction outweighs the benefit of immunity to infection. As a result, many children today have not been immunised thus increasing the risk of a whooping cough pandemic. Indeed, in recent years the incidence of whooping cough and resulting infant morbidity have increased as the use of the whole cell vaccine has decreased. Considerable research effort has, therefore, been directed towards the development of an improved vaccine which lacks the components responsible for the adverse effects of the whole cell vaccines, whilst retaining the components necessary to confer protection against the disease. The search for a safe and effective vaccine has been hampered by the paucity of information regarding the identity and mechanisms of action of the pathogenic, toxic and protective moieties of the bacterial organism contained in whole cell vaccines. Work has, therefore, focused on the isolation and characterisation of the twenty or more surface antigens of the organism and on their efficacy in inducing an immune reaction (J.Am.Med.Soc.. 1982, 248(1) 22-23). Examples of antigens that have been investigated include lymphocytosis promoting factor (LPF, otherwise known as pertussis toxin (PT) , filamentous haemagglutinin (FHA) , lipopolysaccharide (LPS), agglutinogens, dermonecrotic toxin (DNT) , heat labile and heat stable toxins, polymorphonuclear leukocyte inhibitor factor, adenylate cyclase, the outer membrane 69kOa antigen (F.69) and other surface components. None of these antigens on their own appears able to provide the basis of an effective vaccine. The present inventors have found that a combination of the 69kDa antigen and toxoided LPF is surprisingly more potent than the aggregate effect of the individual components. Moreover, the use of a synergistic combination of the 69kDa antigen and toxoided LPF provides a dose-dependent linear response which is parallel to that obtained with a whole cell vaccine (Seagroat, V. and Sheffield, F. , J.Biological Standard. 1981, £, 351-365). The synergistic combination therefore affords the basis of an effective vaccine for use in the prevention of whooping cough. Accordingly the present invention provides a vaccine containing synergistic combination of the 69kDa antigen of B.pertussis an toxoided LPF of B.pertussis. The 69kDa antigen is an N-terminal fragment of the precursor protein, P93, which is processed in vivo by removal of the signal peptide and C-terminal fragment. It has been previously characterised as an oute membrane protein of B. ertussis. In particular, it has a relativ molecular weight of 67 to 73kDa, as determined by 12% (w/w polyacrylamide gel electrophoresis, and a ratio of proline to glutami acid of substantially 1:1, as determined by amino acid analysis (EP- 162639). The amino acid sequence has also been deduced from th cloned DNA sequence (Charles et al. P.N.A.S.. 1989, £6., 3554-3558 although, in use with the present invention, this may be varie providing the resulting sequence is at least 90%, and preferably 95% homologous and has substantially similar biological properties in kind. The 69kDa antigen may be isolated from cultures of B. pertussis by conventional methods, for example as described by Novotny et al (1985) Infection and Immunity 50. 199-206 and European Publication No. 0162639. Alternatively, it may be obtained using recombinant DNA technology. In this regard, the DNA encoding the 69kDa antigen may be cloned, for example as described by Makoff et al. (Bio/Technology, 8., 1990, 1030-1033) , and expressed in an appropriate host. It is particularly preferred that bacteria, such as E. coli. or yeast, such as Pichia pastoris. are used as the host. The 69kDa antigen obtained in this way may be insoluble and thus may need to be refolded following the use of guanidinium hydrochloride as denaturant in conventional manner and in any event is preferably purified to a level consistent with its use in a human vaccine. LPF is a protein of 105 kDa that is released into the extracellular medium by virulent B. pertussis. (Ta ura, M et a_l, 1982, Biochemistry 2JL, 5516-5522). The DNA sequence of the operon encoding the five subunits has been elucidated (Nicosia et al.. , 1986 Proc. Natl. Acad. Sci. USA 82, 4631-4635; Iocht & Keith, 1986, Science. 232. 1258- 1264) . LPF may be obtained from cultures of B. pertussis following procedures described in the literature, for example the procedure described by Imaizu i et al .Infect. Immun.. 1983, 4_1, 1138-1143). It may be purified using column chromatography or other known purification procedures. It may be toxoided using a variety of different inactivating agents, such as formaldehyde (formalin) , glutaraldehyde, hydrogen peroxide (Sato et a . Lancet. 1984, (1), 122-126) or a combination of any of these reagents. In addition to chemical detoxification pertussis toxin may be inactivated by genetic manipulation. Pizza et al (1989) Science 246. 497-500, describe a number of genetically engineered alleles of the , pertussis toxin gene made by replacing 1 or 2 key amino acids within the enzymatically active SI subunit. The ratio of the 69kDa antigen to toxoided LPF may vary between broa limits, for example from 1:10 to 10:1, but is preferably approximatel 1:1 and in any event is such as to achieve a synergistic effect i vaccine potency. 'The ratio is by weight. The vaccine of the present invention may optionally contain additiona antigens of B.pertussis or other bacteria, such as tetanus an diphtheria. The vaccine of the present invention is normally associated with pharmaceutically acceptable vehicle which allows the synergisti combination to be administered to the patient. Administration i usually carried out via the oral or preferably parenteral route. I the case of the parenteral route, the vehicle is generally liquid an the synergistic combination is generally dissolved or suspended in it An example of a liquid vehicle is physiological saline solution. The vaccine may also contain an adjuvant for stimulating the immun response and thereby further enhancing the potency of the synergisti combination. Convenient adjuvants for use in the present inventio include, for example, aluminium hydroxide and aluminium phosphate. Conveniently the vaccine normally contains a final concentration o antigenic protein in the range of from 0.01 to 5mg/ml, preferably 0.0 to 2 mg/ml, most preferably 0.3 mg/ml. Aftef formulation the vacci may be incorporated into a sterile container which is then sealed a stored at a low temperature, for example 4 C, or is freeze-dried. The present invention also provides a method for inducing immunity whooping cough in humans, comprising the administration to the patie of an effective amount of the vaccine of the present invention. In order to induce immunity to whooping cough in humans one or more doses of the vaccine are normally administered. Each dose of the vaccine is 0.1 to 2 ml preferably 0.2 to 1 ml, most preferably 0.5 ml. The present invention will now be exemplified further by reference to the accompanying Figures and the Examples. Figure Leσends Fiσure 1 Construction of plasmids pPERtaσ 1. 2. 3. 4. 7 and 8 la. The DNA and amino acid sequences are SEQ ID N0S:1 and 2 respectively. Plasmid pPERtac2 made by cloning an Aval - Clal 1491bp fragment from pMLU6 into pPERtacl. Plasmid pPERtac3 made by cloning a S p hI - SacII 851bp fragment from pPERtac4 into pPERtac2. Plasmid pPERtac4 made by cloning an Aval - D^alll I875bp fragment from pMLU6 into pPERtacl. lb. Oligonucleotides 1-4 (SEQ ID NOS:3, 5, 6 and 8 respectively) were cloned between Apal and BamHI sites of pTETtac2 to form intermediate plasmid pPERtacl. The amino acid sequences shown above oligonucleotides l and 3 respectively are SEQ ID NOS.4 and 7. lc. Oligonucleotide 5 (SEQ ID NO:9) was cloned between £s£XI and Sa HI of pPERtac 4 to form pPERtac7. Id. Oligonucleotides 6 and 7 (SEQ ID NOS:10 and 12) were cloned between Bgll and BamHI sites of pPERtac7 to form pPERtacS. The amino acid sequence shown above oligonucleotide 6 is SEQ ID NO:11. Figure 2 a. Oligonucleotides 8, 9 and 10 are SEQ ID N0S:13, 16 and 14 respectively in which R denotes G or A and Y denotes C or T. Oligonucleotide 9 annealed to oligos 8 and 10, and then cloned between the Bglll and EcoRI sites of pPERtacδ provided transfor ants from which the expression plasmid pPERtac36 was isolated. The amino acid sequence above oligonucleotide 10 is SEQ ID NO:15. b. Top strand of above oligos after repair is vivo. shown as SEQ ID NO:17 in which R denotes G or A and Y denotes C or T. c. The changes in (b) present in pPERtacδ. d. The changes in (b) present in pPERtac36. Figure 3 SDS polyacrylamide gel of extracts of an induced culture of pPERtac36. Figure 4 Construction of the vector pPIC3-605K. Plasmid pPICl formed by cloning of synthetic oligonucleotides (upper strand-SEQ ID NO:18; lower strand-SEQ ID NO:19) between AsuII and EcoRI of pA0804. Plasmid pPIC2 formed from pPICl. Adaptor oligonucleotides (upper strand-SEQ ID NO:20, lower strand-SEQ ID NO:21) used to insert a 1.8 kb EcoRI-Nhel fragment of pPERtacδ into BamHI - Spel cut pPIC2 to form the expression vector pPIC3-60.5k. Figure 5 A plasmid map of the vector pPIC3-60.5K is shown in part A. Part B gives details of the DNA sequence of the P.69 gene that was used in the construction of pPIC3-60.5K. The DNA fragment used contains the region from Aval (nt.315) to Belli (nt. 1979) from the 93K precursor gene (Charles et al. f 1989, PNAS 8_6, 3554-3558). This region is flanked by the sequences shown which are derived from the vector pPERtacδ (Makoff e£ al. , Bio/Technology 8., 1990, 1030-1033). The sequences flanking the p69 gene are (left side) SEQ ID NO:22 and (right side) SEQ ID NO:23. Figure 6 DNA a dot blot screen of GS115/pPIC3-60.5K Mut S transformants. The filter shown had 56 transfoπnants, a positive control (position E10: multi-copy transformant 'clone 22', identified in a previous screen), and a negative control (Ell: GS115). Most of the transformants in the screen gave a similar weak signal, a very small proportion gave a much stronger signal indicative of multi-copy integration (A3, A4, B6: designated nos. 3, 4, and 18). A further four filters were screened in the same way, but no more multi-copy transformants were found. Figure 7 Southern blot antigens of pPIC-60.5K transformants probed with H154 DNA. Track 1-BglII-cut pPIC-60.5K; track 2-95115 DNA; tracks 3 and 8 - single-copy transplacements; tracks 4 to 7 - multi-copy clones nos. 3, 4, 18 and 22. Figure 8 Western blot of proteins (30 micrograms) from an induced single-copy transfor ant (track 5) and multi-copy transformants (tracks 6 to 9 - clones 3, 4, 18 and 22, respectively). Tracks 1 to 3 contain pure B.pertussis P.69 in amounts equivalent to 1, 2 and 5% of total cell protein (0.3, 0.6, and 1.5 micrograms), and track 4 contains size markers (not visible) . Figure 9 Western blot of fermenter samples taken at different times after induction of clone 22 (tracks 1 to 12: -1, 0, 2, 4, 6.75, 22.75, 24.75, 27.75, 30.25, 49.25, 57.25, 71.25 hrs, respectively). Track 13 contains an equivalent loading, 50 micrograms, of protein from a shake flask induction. Tracks 14 to 18 contain pure B.pertussis P.69 equivalent to 0.5, 1, 2, 4, and 8% expression levels. Figure 10 Coomassie blue-stained gel of solubilised P.69 preparation (tracks 1 to 3: 2, 5 and 10 microlitres) compared to pure B.pertussis P.69. Examples General information pertinent to the examples which follow : Media YPD, 1 litre : lOg yeast extract, 20g peptone, 20g glucose. YNBBG, 1 litre : 13.4g yeast nitrogen base w/o amino acids (Difco), 0.4mg biotin, 20ml glycerol. YNBBGCas : same as YNBBG plus lOg casamino acids. YNBBD : same as YNBBG but 20g glucose instead of 20ml glycerol. YNBBM : same as YNBBG but 5ml methanol instead of 20ml glycerol. YNBBDCas and YNBBMCas had lOg casamino acids per litre. Solid media : plus 20g of agar per litre. Example 1: Preparation of 69kDa Antigen of B.pertussis Production in E.coli (i) Construction of plasmids: The intermediate plasmid pPERtacl was constructed by cloning between the Apal and BamHI sites of pTETtac2 (Makoff et al. Bio/Technology. 1989, 2. 1043-1046), the four oligonucleotides shown in Figure lb (oligos 1-4) . Plasmids pPERtac2, 3, 4, 7 and 8 (Figure la) were all constructed by cloning various B.pertussis P93 coding sequence fragments into pPERtacl. The fragments encoding P93 were obtained from pMLϋβ, a subclone of pBPI60 (Charles et al. P.N.A.S.. 1989, ϋ. 3554-3558), using the following restriction sites: Aval (315), Sail (1065) fishl (1250), fl-ftl (1806), & (1979), £s£XI (2053), SacII (2101), fitftlll (2190). [The numbers in brackets refer t the nucleotide numbers in the published sequence of P93 (Charles et al. P.N.A.S.. 1989, 86, 3554-3558), part of which, from 1804 to 2217, is reproduced in Figure la]. Plasmid pPERtac2 was made by cloning into pPERtacl, an Aval-Clal 1491bp fragment from pMLUδ, while plasmid pPERtac4 was made by cloning an Aval-D^alll 1875bp fragment. Plasmid pFERtac3 was made by cloning a Sphl-SacII 851bp fragment from pPERtac4 into pPERtac2. Plasmid pPERtac7 was constructed by cloning the single stranded oligonucleotide (oligo-5, Figure lc) between the BstXI and BamHI sites of pPERtac4. Plasmid pPERtacδ was constructed by cloning the pair of oligonucleotides shown in Figure Id (oligos 6 and 7) between the Bgll and BamHI sites of pPERtac7. Comparison of the mobilities with native P.69 of the expression products of plasmids pPERtac 2, 3, 4, 7 and 8 plus the higher expressing two cistron derivatives (Makoff & Smallwood, 1990) are consistant with a C-terminus of P.69 at SerδOO. (Makoff et, ≤l 1990 Nucl. Acids Res. 18., 1711-1718. In order to increase the expression of P.69 (terminating at SerδOO), plasmid pPERtac36 was isolated from a number of transformants, obtained after annealing the mixed oligonucleotide oligo-9 to oligos 8 and 10 (Figure 2) and cloning between the Bglll and EcoRI sites of pPERtacδ, both of which originated in oligos 1 and 2 (Figure lb) . All oligonucleotides were synthesized, urified, kinased and the sequences confirmed as described previously (Makoff et al. Bio/Technology, 1989, 2, 1043-1046). E.coli strain MM294 (Meselson et al. Nature. 1968, 212. U10-1114) was used throughout. (ii) Induction and analysis of expressed protein: The expression plasmid, pPERtac36, contains a functional copy of the lad gene and can therefore be induced by isopropyl-0-D-thiogalactopyrano- side (IPTG). Cultures of E.coli MM294 containing pPERtac36 were grown overnight in L-broth (Maniatis et al. Molecular Cloning: A (Laboratory Manual, 1982, Cold Spring Harbor Laboratory) and diluted 1 in 5 into fresh medium containing 75μg/ml IPTG. Cells 10 were grown for a further four hours with rapid aeration and harvested by centrifugation (10,000 x g, lOmin) . The 69kDa antigen was detected by Western blotting using polyclonal antisera raised in rabbits against the purified antigen. Levels of expression were quantified by densitometer scanning of SDS-polyacrylamide gels using a Joyce-Loebl Chromoscan 3 (Makoff et al. Nucl.Acids Res.. 1989, 12, 10191-10202). This was determined for pPERtac36 as 36.0 ± 4.8% (Makoff e_t Bio/Technology, 1990, 8 , 1030-1033). (iii) Refolding and purification: After centrifugation, the cel pellet was resuspended in a buffer comprising 50mM Tris-HCl, pH7.5, 50mM NaCl and lysed by two passages through a Frenc .9 Pressure cell (15,000 lb in ). The pellet fraction of th lysate was washed in buffer (A) (50mM Tris-HCl, pH8.0, lOOm NaCl, ImM EDTA) comtaining 1% Triton X-100. The insolubl material (mostly comprising insoluble 69kDa antigen as inclusio bodies) was recovered by centrifugation and solubilized b resuspension in buffer (A) containing 6M guanidiniu hydrochloride (GuHCl). The protein solution was adjusted t 6mg/ml and the GuHCl concentration reduced to 1M by dilution The 69kDa antigen solution was placed in a dialysis bag an dialysed at 4 C for 24 hours with 3 changes against buffer (A so as to refold the protein. (Reviewed in Marston, Biochem.J. 1986, 2405. 1-12). The insoluble material that appears o dialysis was removed by centrifugation at 10,000 x g for 1 minutes. Figure 3, shows an SDS-polyacrylamide gel (10%) of extracts o an induced culture of pPERtac36, during the solubilisatio procedure. Lane 1, whole-cell extract; lane 2, firs supernatant fraction; lane 3, first pellet fraction; lane 4 Triton X-100 wash supernatant fraction; lane 5, total fractio after solubilisation in GuHCl and its removal by dialysis; la 6 supernatant fraction after dialysis; lane 7, pellet fraction after dialysis. Size markers are given in kDa. Soluble 69kDa antigen preparations were purified further by ion-exchange chromatography on Q-Sepharose. Samples were loaded on the column in 50mM Tris-HCl, pH8.0, washed and the antigen eluted with a 0-0.4M NaCl gradient. This purified material had below 0.1 endotoxin units per μg of protein by the Limulus amoebocyte lysate assay. Production in P.pas oris (i) Construction of expression vector: The vector pPIC3-60.5K, deposited at THE NATIONAL COLLECTION OF INDUSTRIAL AND MARINE BACTERIA LTD., P.O. Box 31, 135, Abbey Road, Aberdeen AB98DG, Scotland, UK on 30th March, 1990 under Accession No. 40270 and derived from pA0804 (K. Sreekrishna e£ sλ. , Biochemistry, 1989, 28. 4117-4125), was used for intracellular expression of 69kDa antigen in Pichia pastoris. This vector uses the promoter from the AOX1 gene to drive expression and can be integrated into the host chromosomal A0X1 locus. To facilitate insertion of the gene encoding the 69kDa antigen, the synthetic oligonucleotides shown in Fig. 4 were cloned between the AsuII and EcoRI sites of pA0804 to give pPICl. A derivative of this plasmid, pPIC2, which lacks the EcoRI site was then constructed. This was done by digesting with EcoRI followed by filling in of the protruding single stranded ends with the Klenow fragment of DNA pσlymerase I and ligating together the blunt ends. A 1.8kb EcoRI-Nhel fragment (see Fig. 5) from the plasmid pPERtacβ which contains most of the gene encoding the 69kDa antigen was inserted into BamHI-Spel cut pPIC2, using the adapter oligonucleotides shown in Fig. 4, to give pPIC3-60.5K. These oligonucleotides encode the 5' end of the gene including the initiator ATG codon. (ii) Transformation: Pichia pastoris strain GS115 fhis4 " ) was transformed with pPIC3-60.5K using the sphaeroplast method described by Cregg e£__≤l. , Bio/Technolopy. 1987, £, 479-485. Transformants were regenerated on minimal medium lacking histidine, so that His colonies would be selected. The transforming DNA was 10 or 20μg of Bglll-digested pPIC3-60.5K. This digest contains two DNA fragments, one of which (7.2 kb) has A0X1 sequences at either end, so that it is targetted to integrate at and replace ('transplace') the chromosomal A0X1 gene. The His transformants generated are mainly found to contai undisrupted AOX1. and transplacements (aoxl. may be isolate using a further screening procedure. Transplacements may b identified by their slow growth on methanol (Mut as opposed t Mut phenotype). The transformants can be picked directly of regeneration plates and tested for growth on minimal methano plates (YNBBM agar) . Alternatively, the regeneration top agar can be lifted and homogenised in water, and the yeast cell plated to about 300 colonies per plate on minimal glucose plate (YNBBD agar) . Mut colonies are then identified b replica-plating onto minimal methanol plates. In general, th proportion of Mut is found to be 10-20% of all th transformants. Occasionally Mut transformants might be score as Mut , and these may also prove to contain multiple copies o the vector. (iii) Screening: In order to screen large numbers of transformant rapidly, they were grown in individual wells in a 96-plac sterile microtitre plate (NUNCL0N) . 200μl of YPD broth in eac g well was inoculated with a Mut transformant, and the plate incubated for two days at 30 C without shaking, to ensure growt to stationary phase. Included on each microtitre plate was well containing GS115 (negative control) and one containing known pPIC3-60.5K integrant (positive control). Using multi-channel pipetter (Titertek) , 50 l samples of each micro-culture were transferred onto a nitrocellulose filter on a Schleicher & Schuell 'minifold' under vacuum. The filters were air dried, marked for orientation, then treated in the following way to lyse the cells: (i) 15min at room temperature with 50mM EDTA, 2.5% 2-mercaptoethanol pH9.0, (ii) 4hrs at 37°C with lmg/ml zymolyase (100T) in water, (iii) 5min at room temperature in 0.1M NaOH, 1.5M NaCl and (iv) twice for 5min at room temperature in 2 x SSC. Each treatment was performed by soaking 2 sheets of 3MM paper with the solution and placing the nitrocellulose filter on top. After these treatments the filters were baked at 80°C for lhr. The filters were probed by Southern hybridization using 69kDa antigen-specific DNA radiolabelled to high specific activity using random-primed labelling (Feinberg et al. Anal. Biochem. , 1989, 121, 6-13). Standard methods were used for prehybridization, hybridization, washing and autoradiography. Fig. 6 shows the results of such a screen of over 200 Mut transformants of pPIC3-60.5K. All the transformants reacted with the probe and most gave a similar weak signal (single-copy transformants) . However, a very small proportion gave much stronger signals for Example A3 and 4. These multi-copy transformants were tested further. The positive control in the filter shown is a transfor ant previously identified as multi-copy. The DNA from two selected single-copy transplacements and four multi-copy transformants identified by dot blots was further analysed by Southern blotting. DNA was prepared from 50ml cultures of Pichia cells according to Sherman, F..et al. , ( Methods in Yeast Genetics. 1983, Cold Spring Harbor, New York). JJglll digests were separated in a 0.6% agarose gel, transferred to nitrocellulose by Southern blotting, and hybridised with radio-labelled HIS4 DNA (Fig. 7). With the £glll-cut vector, pPIC3-60.5K (track 1), a 7.2 kb band corresponding to th expression cassette was observed, whereas the untransforme strain GS115 gave the chromosomal 2.7 kb HIS4 fragment (trac 2) . Single transformants gave both these bands (tracks 3 an 8) , while two of the multi-copy transformants had an increase intensity of the 7.2 kb band, indicating multiple insertion o the expression cassette (tracks 4 and 5). The other tw multi-copy transformants did not give a 7.2 kb band at all, bu showed higher molecular weight bands which are multimeric form lacking Bglll sites. (iv) Protein Analysis: Selected transformants were cultured i YNBBGCas for 2 days at 30°C to reach stationary phase. Thes starter cultures were diluted to an D- 00 of 0.25 in 5ml fres YNBBGCas and grown for 6hr. To induce these cultures the cell were collected by centrifugation, washed once in sterile water and resuspended in YNBBMCas. Inductions were carried out for days at 30°C. Cells were then harvested by low speed centrifugation, washe once in water and suspended 0.5ml in ice cold break buffer (20 sodium phosphate pH7.0, 0.1% triton X-100, 4mM phenylmeth sulphonylfluoride) . Acid washed glass beads (0.45mm) were adde and the cells were broken by vigorous vortexing. The protei concentration of the extracts was determined using the BioR protein assay (BioRad, according to manufacturer's instruction and the material was stored at -20 C. Proteins were separated by electrophoresis in 7. SDS-polyacrylamide gels (Laem li U.K., Nature. 1970, 22 680-785). The proteins were visualised in the gel staining wi Coomassie Brilliant Blue R. Alternatively the proteins we transferred to a nitrocellulose filter and reacted with t 69kDa antigen specific monoclonal antibody BB05 (Montar et al.. Infect. Immunity. 1985, 4 , 744-751); deposited at PHLS Public Health Laboratory Service Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, U.K. in the European collection of Animal cell cultures under Accession No. 90020103, on the 1st February, 1990 in accordance with the terms of the Budapest treaty), then with goat anti-mouse IgG conjugated to horse-radish peroxidase, and developed with H„0« and 4-chloronaphthol (BioRad). In this way, the expressed 69kDa antigen could be specifically detected. Fig. 8 shows a Western blot of induced extracts of 4 selected multi-copy transformants (clone nos. 3, 4, 18 and 22) and one typical single-copy transformant. By comparison to standard concentrations of pure 69kDa antigen, it can be estimated that single-copy transplacements express 69kDa antigen at 0.1-0.5% of total cell proteins (t.c.p.), clone no. 22 at approx. 2%, and clone 4 at approx. 5%. These levels are for sub-optimal induction conditions. In fermenter inductions a 5-fold improvement was seen for clone 22, to 10% t.c.p.. However, clone 4, which turned out to be Mut , remained at 6% t.c.p. in the fermenter. In low-expressing transformants the 69kDa antigen is largely soluble, whereas at high expression levels it is mainly (90%) insoluble. This is preferable since the material can be readily isolated, renatured and purified from the insoluble fraction. (v) Fermentation : Production of 69kDa antigen by high cell density Pichia pastoris cultures was carried out using clone 22 in a 2L Braun fermenter equipped with monitors and controls for pH, dissolved 0„, stirring speed, temperature and air flow. A 10ml YNBBG overnight culture was used to inoculate the fermenter containing 1 litre of 5 x basal salts (phosphoric acid, 42mls/L; calcium sulphate 2H.0, 1.8g/L; potassium sulphate 28.6g/L; magnesium sulphate 7H-0, 23.4g/L; potassium hydroxide, 6.5g/L) with 4ml of PTM., salts (cupric sulphate 5H 2 0, 6g/L; potassium iodide, 0.08g/L; manganese sulphate H„0, 3g/L; sodium olybdate, 0.2g/L; ferrous sulphate 7H-0, 65h/L; biotin, 0.2g/L; sulphuric acid, 5ml L) and 5% (v/v) glycerol at 30°C. Dissolved oxygen was maintained above 20% by adjusting aeration and agitation, and the pH was maintained at pH5.0 by the addition of 50% (v/v) ammonium hydroxide. Growth was continued until the glycerol was exhausted (24-30hr). A limited glycerol feed (containing 50% v/v glycerol and 12ml/L PTM. salts) was then initiated at 12ml hr for 17-21 hr. After this period the culture was induced by replacing the glycerol feed with a methanol feed (100% methanol plus 12ml/L PTM^ salts) at lml hr for 2hr. Then the methanol feed rate was gradually increased over a period of 6 hr to 6ml/hr and the fermentation was continued using these conditions for a further 40hr. At this point the methanol feed rate was reduced to 2ml/hr. Samples were taken from the fermenter at different times after induction, and were analysed for 69kDa antigen expression as described above. The results of a Western blot analysis are shown in Fig, 9, indicating that levels of the 69kDa antigen of about 10% of cell protein were achieved. (vi) Renaturation: Fermenter samples from 28hr and 32hr after the start of induction, containing 30ml of cells at OD,-.. of 220, oUU were pooled for processing. The cells were harvested by low speed centrifugation, washed once in water, then taken up to 30ml with buffer A (50mM Tris pH8.0, 0.1M NaCl, ImM EDTA) containing 1% Triton X-100. The cells were then broken in a bead-beater (Biospec Products, Bartlesville, O.K.) with 0.45mm glass beads in ten one-minute pulses. The cell lysate was cleared by centrifugation at 15,000rpm for 30min (Sorvall, RC-5B, SS-34 rotor). Pellets were washed by suspending i buffer A and re-centrifuging. The pellet of insoluble protei was resuspended in buffer A (6ml) and 6 volumes of 7.0 guanidinium chloride, 50mM Tris pH8.0, ImM EDTA were added t solubilise proteins. This solution was diluted further with 7 guanidinium chloride to 110ml and dialysed extensively agains 5L of buffer A at 4 C. The material in the dialysis bag was then cleared by centrifugation (40min, 10,000rpm, RC-5B, SS-3 rotor). The final supernatant, containing solubilised 69kD antigen, was concentrated by dialysing against solid Sephade G-100. Equivalent amounts of fractions from each stage of this procedure were analysed by SDS-PAGE followed by Wester blotting. The results indicate that about 50% of the 69kD antigen was solubilised in this particular experiment but tha all of this remained soluble after dialysis. Coomassie-stained gel indicated that the purity of the fina material was high (60-70%; Fig. 10). Example 2: Preparation and Inactivation of Toxoided LPF Cultures of B.pertussis can be grown in flasks or in fermentors in modified Stainer-Scholte medium supplemented with heptakis-(2,6-0-di- methyl) - -cyclodextrin (MeBCD) (Imaizumi et al.. Infect, ftn-min.. 1983, 41: 1138-1143. LPF can be purified from the culture supernatant b column or batch adsorption on hydroxylapatite agarose (Sato et al. , "Seminars in Infectious Disease, 1982, vol IV" 380-5, Threme-Stratton, Inc. New York). The hydroxylapatite is first equilibrated with 0.01 phosphate buffer pH8.0. The culture supernatant can then be applie to the column and subsequently washed with a solution of 0.01 phosphate buffer pH6.0. Elution of LPF is carried out using 0.1 phosphate buffer pH7.0 containing 0.5m NaCl. The LPF may be furthe purified, for example, on a haptoglobin-Sepharose 4B column (Imaizumi et al.. Infect & Immun. 1983, 41, 1138-1143). Inactivation or 'toxoiding' of purified preparations of LPF may be carried out using a variety of different inactivating agents, such as formaldehyde, glutaraldehyde hydrogen peroxide or formalin (Sat et al. Lancet. 1984, (1), 122-126), or a combination of any of thes reagents. Example 3: Biological Potency in the Kendrick Test The test was performed according to W.H.O. requirements for Pertussi Vaccine using outbred NIH-S mice (OLAC, category 3, free of mos pathogens including B.bronchiseptica) . weighing 14-16g. The antigens in 0.5 ml volumes, were inoculated intraperitoneally as a mixture, an comprised a top dilution and three three-fold serial dilutions. Afte two weeks the mice were challenged intracerebrally using th recommended challenge strain 18-323 (-400 LD^) . The number o survivors in each group was used for calculation of the relativ potency in respect of the British Pertussis Reference Vaccine 66/8 (J.Biological Standard. 1981, £, 351-365) using a program of paralle line probit analysis. The results are shown in Tables 1, 2 and 3 Examination of these data shows limited protection for either th 69kDa antigen or toxoided LPF when used alone as vaccines. I contrast, the combination of the 69kDa antigen and toxoided L exhibits a surprising synergy giving rise to levels of protection ve similar to the whole cell reference vaccine. TaHe Name and Description Survivors Dose Male Female Total Table 2 Name and Description Survivors Dose Male Female Total Table 3 Name and Description Survivors Dose Male Female Total Toxoid 20μg LPF Toxoid 20μg LPF 69kDa 20μg(Pichia) Ref vaccine Challenge SEQUENCE LISTING (l) -mFxaac-Ticw FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) I-ENGIH: 414 base pairs (B) TYIE: nucleic acid (C) STRANDEIKESS: double (D) TOPOIOGY: linear (ϋ) MDI-ECϋl-E TΪEE: ENA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: l: TAIOGAITGG COGCCACCGG CBATCGGCAG TGGftGCCIGG TGGGGGCGAA GGOGCCGCCG 60 GCGCCCftAGC OD-K-GCOGCA GCOGGGTCCC (--AGOOGCOGC AGCCGCCGCA GCCGCftGC03 120 GAWX-GCOGG CGCCGCAACC GCOGGOΞGGC AGGGAGTIGT <--OXX_GC-α--C CAACGCGGCG 180 GTCAACAOGG G GGGGTGGG CCIGGCCAGC ACGCTCCGGT AOSC-CGAAAG CAATCCGTIG 240 TCCAAGCGCC TGGGGSAGIT GOGOCTCAAT COGGAOSCCG GOC-IGCGCCIG GGGCCGCGGC 300 TIOGGGCAAC GCTAGC-AGCT GGACAACCGC GAAGGGCGGC GCITOGACCA GAAΞGTCGCC 360 GGCITCGAGC TGGGCGCCGA CCACGCGGIG GCGGIGGCCG GOGGAGGCIG GCAC 414 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHaRACIERISTICS: (A) I_E2*3IH: 138 a-rair-o acids (B) TYPE: amino acid (D) TOPOIOGY: linear (ϋ) MDLECUI-E TYPE: peptide ( i) SEQUENCE EESCRIPnON: SEQ ID NO:2: Tyr Arg Leu Ala Ala Asn Gly Asn Gly Gin Trp Ser Leu Vail Gly Ala 1 5 10 15 Lys Ala Pro Pro Ala Pro Lys Pro Ala Pro Gin Pro Gly Pro Gin Pro 20 25 30 Pro Gin Pro Pro Gin Pro Gin Pro Glu Ala Pro Ala Pro Gin Pro Pro 35 40 45 Ala Gly Arg Glu Leu Ser Ala Ala Ala Asn Ala Ala Val Asn Thr Gly 50 55 60 Gly Val Gly Leu Ala Ser Thr Leu Trp Tyr Ala Glu Ser Asn Ala Leu 65 70 75 80 Ser Lys Arg Leu Gly Glu Leu Arg leu Asn Pro Asp Ala Gly Gly Ala 85 90 95 Trp Gly Arg Gly Fhe Ala Gin Arg Gin Gin leu Asp Asn Arg Ala Gly 100 105 110 Arg Arg Rie Asp Gin Lys Val Ala Gin Ehe Glu Leu Gly Ala Asp His 115 120 125 Ala Val Ala Val Ala Gly Gly Arg Trp His 130 135 (3) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 102 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ii) MDIEOJIE TYPE: EKA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 : α --»TCITAA TCATCCACAG GAGACITTCT GAIGGATTGG AACAACCACT Cπ-ATTCTTAA 60 AACTGGIGAA C-GTCAGCACG GAAITCACAT GCAGGGTTCT GA 102 (4) INFORMATION FOR SEQ ID N0:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOIOGY: linear (ii) MDI-ECUIE TYPE: peptide ( i) SEQUENCE DESCRIPTION: SEQ ID NO:4 : Met Asp Trp Asn Asn Gin Ser lie Val Lys Thr Gly Glu Arg Gin His 1 5 10 15 Gly lie His lie Gin Gly Ser Asp 20 (5) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE C_HARACTE_RISTICS: (A) LENGTH: 116 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ii) MDIECUIE TYPE: ENA ( i) SEQUENCE D-SCRIPTION: SEQ ID NO: 5: CαSGGTCIAG GATIAGTAGG TGTOCTC GA AAGACTACCT AACCTICTIG GTCAGAIAAC 60 AAITITGACC ACTIGCAGTC GTGCCITAAG TGTAAGTCCC AAGACIGGGC CCAAIA 116 (6) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) IENGTH: 118 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ii) MDIECUIE TYPE: ENA (xi) SEQUENCE I-ESC-R-t-PITON: SEQ ID NO:6: CCCGGGΠAT OGAΠAGCIG CTAAOΞGIAA CGGΓCAGIGG TCΓCIGGTTG G GCIAAAGC 60 TOCACCAGCT CCAAAACCAG C EACCGCG GTTAACACGC GGTGTAAGCA TG ITAAG 118 (7) INFORMATION FDR SEQ ID NO:7 : (i) SEQUENCE CHARACTERISTICS: (A) I-EN7IH: 27 amino acids (B) TYPE: amino acid (D) TOPOIOGY: linear (ii) MDLEOILE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Pro Gly Tyr Arg I-eu Ala Ala Asn Gly Asn Gly Gin Trp Ser leu Val 1 5 10 15 Gly Ala Lys Ala Pro Pro Ala Pro Lys Pro Ala 20 25 (8) INFORMATION FOR SEQ ID N0:8: (i) SEQUENCE CHARACTEKESTTCS: (A) LENGTH: 112 base pairs (B) TYPE: nucleic acid (C) STRANDEIlϊESS: single (D) TOPOIOGY: linear (ii) MDI-ECUIE TYPE: ENA (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 8 : GCTAATCGAC GATIGOCAIT GCCACTCACC AGAGACCAAC CAGCATITΑ-. AGGTGGTCGA 60 GGTITΓGGTC GAAΠGGOGC CAATIΌTGCG CCAGAΓΓCGT ACAAAT CCΓ AG 112 (9) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 12 base pairs (B) TYPE: nucleic acid (C) --rn?ANDE___NESS: single (D) TOPOIiDGY: linear (ii) MDI-ECUIE TYPE: ENA (xi) SEQUENCE EESCRIPπON: SEQ ID NO:9: CGCGATTCCT AG 12 (10) INFOEMAπON FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 72 base pairs (B) TYFE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ii) MDIECUIE TYPE: ENA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CAGCIGTTAA CACTGGTGGT GTAGGCCTIG CAAGTACTCT GIGGTAOGCT GAAICIAAIG 60 CA-π-ATCTTA AG 72 (11) INFORMATION EOR SEQ ID NO:11: (i) SEQUENCE CHARAC--ΕKISTICS: (A) IENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOIOGY: linear (ii) MDIECUIE TYPE: peptide ( i) SEQUENCE EESCRIPITON: SEQ ID NO:11: Ala Ala Val Asn Thr Gly Gly Val Gly su Ala Ser Thr leu Trp Tyr 1 5 10 15 Ala Glu Ser Asn Ala leu Ser 20 (12) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) IENGK: 79 base pairs (B) TYPE: nucleic acid (C) S--RANDEENESS: single (D) TOPOIOGY: linear (ii) MDIECUIE TYPE: ENA (xi) SEQUENCE D--SCRIP--TON: SEQ ID NO:12: TGCGTOGACA AΠGTGAOCA CCACAICOGG AAOGITCAIG AGACACCATG CGACTTAGAT 60 ACX-TAATAG AATTCCTAG 79 (13) INFORMATION FOR SEQ ID N0: 13 : (i) SEQUENCE CHARACTERISTICS: (A) IJENGIH: 20 base pairs (B) TYPE: nucleic acid (C) STRANEEENESS: single (D) TOPOLOGY: linear (ii) MDIECUIE TYPE: ENA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GATΌTTAATC AIOCACAGGA 20 (14) INFORMAπON FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) IE GIH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOLOGY: linear (ii) MDIECUIE TYPE: ENA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TCTATIGTTA AAACIGGIGA ACGTCAGCAC GG 32 (15) INFORMATION FDR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: e-raino acid (D) TOPOIOGY: linear (ii) MDIECUIE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Met Asp Trp Asn Asn Gin Ser lie Val Lys Thr Gly Glu Arg Gin His 1 5 10 15 Gly lie (16) INFORMATICN FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) IENGIH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOFOLDGY: linear (ii) MDIECUIE TYPE: ENA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AATTAGTAGG TGTCCIYTGA AAGAYTACCT CCITRTIR GIYAGAIAAC AA-rTTTGACC 60 ACTTGCAGTC GTGCCITAA 79 (17) INFORMAITON FOR SEQ ID NO:17: (i) SEQUENCE CHARACTE-ISTTCS: (A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIiDGY: linear (ii) MDIECUIE TYPE: ENA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GATCITAAIC AICCACAGGA RACITTCIRA TGGATIGGAA YAAYCARTCT AITGITAAAA 60 CIGGTGAACG TCAGCAOGG 79 (18) INFORMATION EOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LEW-JIH: 75 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIJDGY: linear (ii) MDIECUIE TYPE: ENA - 29 - (Xi) SEQUENCE --ESCRΣFTTON: SEQ ID NO: 18: OGAAGGATOC AAA03A-PGAG A1T1U.T11A ATΠTTACIG AGACTAGTCC OGGGGTAAGT 60 AAGTAAGCGG COGOG 75 (19) -COTOEMAITON FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOLOGY: linear (ii) MDIECUIE TYPE: ENA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TTCCTAGGIT TGCTACTCT-A AAGGAAGTTA AAAATGACTC TGATCAGGGC CCCATTCATΓ 60 C-AT1O--CCGG CGCITAA 77 (20) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) I-ENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOLOGY: linear (ii) MDIECUIE TYPE: ENA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GAIOCAAACG ATGGACIGGA ACAACCAATC CAIOGTCAAG ACCGGTGAAA GACAACACGG 60 (21) INPOFMAIION FOR SEQ ID NO:21: (i) SEQUENCE (_HftRACIERI-_rn.es: (A) IENGJIH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ii) MDIECUIE TYPE: ENA (xi) SEQUENCE D--SC-RIPITON: SEQ ID NO:21: tflTHJUIiftOC TGACCTIGTT GGITAGGTAG CAl-rTl 'lU C C&CITTCIGA ACTGCCITAA 60 (22) INFORMATICN FDR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANEEENESS: single (D) TOPOIOGY: linear (ii) MDIECUIE TYPE: ENA (Xi) SEQUENCE DESCRIPΠQN: SEQ ID NO: 22: GAAITCACAT TCAGGGTTCT GACCOGGG 28 (23) INFORMATION FOR SEQ ID NO:23 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 90 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ii) MDIECUIE TYPE: ENA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23 : GCCAAOGCAG CTGTTAACAC TGGIGGIGTA GGCCT-TGCAA GTACTCTGTG GTΑCGCTGAA 60 TCTAATGCAT TIAICTTAAGG A_K3-_GCIAGC 90

Claims: CLAIMS 1. A vaccine containing a synergistic combination of the 69kDa antigen of Bordetella pertussis and toxoided lymphocytosis promoting factor of Bordetella pertussis. 2. A vaccine according to claim 1, in which the 69kDa antigen has been obtained using recombinant DNA technology. 3. A vaccine according to claim 1 or 2, in which the lymphocytosis promoting factor has been toxoided using an inactivating agent selected from formaldehyde, glutaraldehyde, hydrogen peroxide or a combination thereof. 4. A vaccine according to any one of the preceding claims, in which the weight ratio of the 69kDa antigen to the toxoided lymphocytosis promoting factor is approximately 1:1. 5. A vaccine according to any one of the preceding claims, which further comprises a pharmaceutically acceptable liquid vehicle suitable for parenteral administration. 6. A vaccine according to claim 5, in which the concentration of antigenic protein is from 0.03 to 2 mg/ml. 7. A vaccine according to any one of the preceding claims, which further comprises an adjuvant. 8. A vaccine according to any one of the preceding claims, which further comprises another antigen of Bordetella pertussis or an antigen of diphtheria or tetanus. 9. A process for the preparation of a vaccine as claimed in claim 1, which process comprises mixing the 69kDa antigen of Bordetella pertussis and toxoided lymphocytosis promoting factor of Bordetella pertussis in such amounts as to provide a synergistic combination thereof. 10. A method for inducing immunity to whooping cough in a human patient, which method comprises administering thereto an effective amount of a vaccine as claimed in any one of claims 1 to 8.

Inventors: CHARLES IAN GEORGE, FAIRWEATHER NEIL FRASER, CHARLES, IAN, GEORGE, FAIRWEATHER, NEIL, FRASER
Applicants: WELLCOME FOUND, THE WELLCOME FOUNDATION LIMITED
Application year: 1991
Main classification: A61K39
IPC codes: A61K39, A61P31, C07K14