EP0550558A1 - Vaccin acellulaire - Google Patents

Vaccin acellulaire

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Publication number
EP0550558A1
EP0550558A1 EP91917103A EP91917103A EP0550558A1 EP 0550558 A1 EP0550558 A1 EP 0550558A1 EP 91917103 A EP91917103 A EP 91917103A EP 91917103 A EP91917103 A EP 91917103A EP 0550558 A1 EP0550558 A1 EP 0550558A1
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EP
European Patent Office
Prior art keywords
seq
vaccine
antigen
69kda
vaccine according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP91917103A
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German (de)
English (en)
Inventor
Ian George Charles
Neil Fraser Fairweather
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Medeva Holdings BV
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Medeva Holdings BV
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Application filed by Medeva Holdings BV filed Critical Medeva Holdings BV
Priority to EP96110147A priority Critical patent/EP0764445A2/fr
Publication of EP0550558A1 publication Critical patent/EP0550558A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/235—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bordetella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • 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.
  • 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.
  • 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.
  • LPF lymphocytosis promoting factor
  • PT pertussis toxin
  • FHA filamentous haemagglutinin
  • LPS lipopolysaccharide
  • DNT dermonecrotic toxin
  • F.69 heat stable toxins
  • 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, 9, 351-365). The synergistic combination therefore affords the basis of an effective vaccine for use in the prevention of whooping cough.
  • the present invention provides a vaccine containing a synergistic combination of the 69kDa antigen of B . pertussis and 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 a C-terminal fragment. It has been previously characterised as an outer membrane protein of B.pertussis. In particular, it has a relative molecular weight of 67 to 73kDa, as determined by 12% (w/w) polyacrylamide gel electrophoresis, and a ratio of proline to glutamic acid of substantially 1:1, as determined by amino acid analysis (EP-A 162639). The amino acid sequence has also been deduced from the cloned DNA sequence (Charles et al. P.N.A.S. , 1989, 86, 3554-3558) although, in use with the present invention, this may be varied providing the resulting sequence is at least 90%, and preferably 95%, homologous and has substantially similar biological
  • the 69kDa antigen may be isolated from cultures of B. pertussis by conventional methods, for example as
  • 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
  • yeast such as Pichia
  • the 69kDa antigen obtained in this way may be insoluble and thus may need to be
  • 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.
  • LPF may be obtained from cultures of B. pertussis following procedures described in the literature, for example the procedure described by Imaizumi et al (Infect. Immun., 1983, 41, 1138-1143). It may be purified using column chromatography or other known purification
  • inactivating agents such as formaldehyde (formalin), glutaraldehyde, hydrogen peroxide (Sato et al, Lancet. 1984, (1), 122-126) or a combination of any of these reagents.
  • pertussis toxin may be inactivated by genetic manipulation.
  • the ratio of the 69kDa antigen to toxoided LPF may vary between broad limits, for example from 1:10 to 10:1, but is preferably approximately 1:1 and in any event is such as to achieve a synergistic effect in vaccine potency.
  • the ratio is by weight.
  • the vaccine of the present invention may optionally contain additional antigens of B.pertussis or other bacteria, such as tetanus and diphtheria.
  • the vaccine of the present invention is normally associated with a pharmaceutically acceptable vehicle which allows the synergistic combination to be administered to the patient. Administration is usually carried out via the oral or preferably parenteral route.
  • the vehicle In the case of the parenteral route, the vehicle is generally liquid and 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 immune response and thereby further enhancing the potency of the synergistic combination.
  • Convenient adjuvants for use in the present invention include, for example, aluminium hydroxide and aluminium phosphate.
  • the vaccine normally contains a final concentration of antigenic protein in the range of from 0.01 to 5mg/ml, preferably 0.03 to 2 mg/ml, most preferably 0.3 mg/ml.
  • Aftef formulation the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4°C, or is freeze-dried.
  • the present invention also provides a method for inducing immunity to whooping cough in humans, comprising the administration to the patient of an effective amount of the vaccine of the present invention.
  • a method for inducing immunity to whooping cough in humans comprising the administration to the patient of an effective amount of the vaccine of the present invention.
  • 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
  • Plasmid pPERtac2 made by cloning an Aval - Clal 1491bp fragment from pMLU6 into pPERtacl.
  • Plasmid pPERtac3 made by cloning a SphI - SacII 851bp fragment from pPERtac4 into pPERtac2.
  • Plasmid pPERtac4 made by cloning an
  • Oligonucleotides 1-4 SEQ ID NOS:3, 5, 6 and 8
  • oligonucleotides 1 and 3 were cloned between Apal and BamHI sites of pTETtac2 to form intermediate plasmid pPERtacl.
  • the amino acid sequences shown above oligonucleotides 1 and 3 respectively are SEQ ID NOS.4 and 7.
  • Oligonucleotide 5 (SEQ ID NO: 9) was cloned between
  • 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.
  • Oligonucleotides 8, 9 and 10 are SEQ ID NOS: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 pPERtac8 provided transformants from which the expression plasmid pPERtac36 was isolated.
  • oligonucleotide 10 is SEQ ID NO: 15.
  • SEQ ID NO: 17 in which R denotes G or A and Y denotes C or T.
  • 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 pPERtac8 into BamHI - Spel cut pPIC2 to form the expression vector pPIC3-60.5k.
  • 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
  • DNA a dot blot screen of GS115/pPIC3-60.5K Mut S transformants.
  • the filter shown had 56 transformants, 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.
  • 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).
  • 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.
  • YPD 1 litre : 10g yeast extract, 20g peptone, 20g glucose.
  • YNBBG 1 litre : 13.4g yeast nitrogen base w/o amino acids (Difco),
  • YNBBGCas same as YNBBG plus 10g 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 10g casamino acids per litre.
  • Solid media plus 20g of agar per litre.
  • Plasmids pPERtac2, 3, 4, 7 and 8 were all constructed by cloning various B.pertussis P93 coding sequence fragments into pPERtacl.
  • the fragments encoding P93 were obtained from pMLU6, a subclone of pBPI60 (Charles et al, P.N.A.S., 1989, 86, 3554-3558), using the following restriction sites: Aval (315), Sall (1065) Sphl (1250), Clal (1806), BglI (1979), BstXI (2053), SacII (2101), Dralll (2190). [The numbers in brackets refer to 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 pMLU6, while plasmid pPERtac4 was made by cloning an Aval-Dralll 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 1c) between the BstXI and BamHI sites of pPERtac4.
  • Plasmid pPERtac8 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 Ser600. (Makoff et al 1990 Nucl. Acids Res. 18, 1711-1718.
  • 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 pPERtac8, both of which originated in oligos 1 and 2 ( Figure 1b). All oligonucleotides were synthesized, purified, kinased and the sequences confirmed as described previously (Makoff et al, Bio/Technology, 1989, 7, 1043-1046). E.coli strain MM294 (Meselson et al, Nature, 1968, 217, U10-1114) was used throughout.
  • 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, 17, 10191-10202). This was determined for pPERtac36 as 36.0 ⁇ 4.8% (Makoff et al Bio/Technology, 1990, 8 , 1030-1033).
  • the 69kDa antigen solution was placed in a dialysis bag and 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 on dialysis was removed by centrifugation at 10,000 x g for 10 minutes.
  • Figure 3 shows an SDS-polyacrylamide gel (10%) of extracts of an induced culture of pPERtac36, during the solubilisation procedure.
  • Lane 1 whole-cell extract; lane 2, first supernatant fraction; lane 3, first pellet fraction; lane 4, Triton X-100 wash supernatant fraction; lane 5, total fraction after solubilisation in GuHCl and its removal by dialysis; lane 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 ⁇ g of protein by the Limulus amoebocyte lysate assay.
  • 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 polymerase I and ligating together the blunt ends. A 1.8kb EcoRI-Nhel fragment (see Fig.
  • the transforming DNA was 10 or 20 ⁇ g of Bglll-digested pPIC3-60.5K.
  • This digest contains two DNA fragments, one of which (7.2 kb) has AOXl sequences at either end, so that it is targetted to integrate at and replace ('transplace') the chromosomal AOXl gene.
  • the His transformants generated are mainly found to contain undisrupted AOXl, and transplacements (aoxl) may be isolated using a further screening procedure. Transplacements may be identified by their slow growth on methanol (Mut as opposed to Mut phenotype). The transformants can be picked directly off regeneration plates and tested for growth on minimal methanol plates (YNBBM agar). Alternatively, the regeneration top agars can be lifted and homogenised in water, and the yeast cells plated to about 300 colonies per plate on minimal glucose plates (YNBBD agar). Mut colonies are then identified by replica-plating onto minimal methanol plates. In general, the proportion of Mut is found to be 10-20% of all the transformants. Occasionally Mut transformants might be scored as Mut s , and these may also prove to contain multiple copies of the vector.
  • 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, 132, 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 s 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 transformant previously identified as multi-copy.
  • 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).Bglll digests were separated in a 0.6% agarose gel, transferred to nitrocellulose by Southern blotting, and hybridised with radio-labelled HIS4 DNA (Fig. 7).
  • Proteins were separated by electrophoresis in 7.5% SDS-polyacrylamide gels (Laemmli U.K., Nature, 1970, 227: 680-785). The proteins were visualised in the gel staining with Coomassie Brilliant Blue R. Alternatively the proteins were transferred to a nitrocellulose filter and reacted with the 69kDa antigen specific monoclonal antibody BB05 (Montaraz et al., Infect. Immunity, 1985, 47, 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.
  • 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.
  • 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..
  • 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 2 O, 1.8g/L; potassium sulphate 28.6g/L; magnesium sulphate 7H 2 O, 23.4g/L; potassium hydroxide, 6.5g/L) with 4ml of PTM 1 salts (cupric sulphate 5H 2 O, 6g/L; potassium iodide, 0.08g/L; manganese sulphate H 2 O, 3g/L; sodium molybdate, 0.2g/L; ferrous sulphate 7H 2 O, 65h/L; biotin, 0.2g/L; sulphuric acid, 5ml/L) and 5% (v/v) glycerol at 30°C.
  • 5 x basal salts phosphoric acid, 42mls/L; calcium sulphate 2H 2
  • 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% w/v glycerol and 12ml/L PTM 1 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 1 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.
  • Renaturation Fermenter samples from 28hr and 32hr after the start of induction, containing 30ml of cells at OD 600 of 220, 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.
  • buffer A 50mM Tris pH8.0, 0.1M NaCl, ImM EDTA
  • the cell lysate was cleared by centrifugation at 15,000rpm for 30min (Sorvall, RC-5B, SS-34 rotor). Pellets were washed by suspending in buffer A and re-centrifuging. The pellet of insoluble protein was resuspended in buffer A (6ml) and 6 volumes of 7.0M guanidinium chloride, 50mM Tris pH8.0, ImM EDTA were added to solubilise proteins. This solution was diluted further with 7M guanidinium chloride to 110ml and dialysed extensively against 5L of buffer A at 4°C.
  • the material in the dialysis bag was then cleared by centrifugation (40min, 10,000rpm, RC-5B, SS-34 rotor).
  • the final supernatant, containing solubilised 69kDa antigen, was concentrated by dialysing against solid Sephadex G-100.
  • Cultures of B.pertussis can be grown in flasks or in fermentors in a modified Stainer-Scholte medium supplemented with heptakis-(2,6-0-di- methyl)- ⁇ -cyclodextrin (MeBCD) (Imaizumi et al., Infect, fm mun., 1983, 41: 1138-1143.
  • LPF can be purified from the culture supernatant by 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 0.01M phosphate buffer pH8.0.
  • the culture supernatant can then be applied to the column and subsequently washed with a solution of 0.01M phosphate buffer pH6.0.
  • Elution of LPF is carried out using 0.1M phosphate buffer pH7.0 containing 0.5m NaCl.
  • the LPF may be further 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 (Sato et al, Lancet, 1984, (1), 122-126), or a combination of any of these reagents.
  • inactivating agents such as formaldehyde, glutaraldehyde hydrogen peroxide or formalin (Sato et al, Lancet, 1984, (1), 122-126), or a combination of any of these reagents.
  • the test was performed according to W.H.O. requirements for Pertussis Vaccine using outbred NIH-S mice (OLAC, category 3, free of most pathogens including B.bronchiseptica). weighing 14-16g.
  • the antigens in 0.5 ml volumes, were inoculated intraperitoneally as a mixture, and comprised a top dilution and three three-fold serial dilutions.
  • the mice were challenged intracerebrally using the recommended challenge strain 18-323 (-400 LD 50 ). The number of survivors in each group was used for calculation of the relative potency in respect of the British Pertussis Reference Vaccine 66/84 (J.
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTTON SEQ ID NO:6:
  • MOLECULE TYPE DNA
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 18:

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Abstract

Vaccin propre à immuniser les êtres humains contre la coqueluche, comprenant une combinaison synergique de l'antigène 69kDa du Bordetella pertussis et du facteur d'activation de la lymphocytose/atoxine du Bordetella pertussis.
EP91917103A 1990-09-27 1991-09-27 Vaccin acellulaire Withdrawn EP0550558A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP96110147A EP0764445A2 (fr) 1990-09-27 1991-09-27 Vaccin acellulaire

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB909021004A GB9021004D0 (en) 1990-09-27 1990-09-27 Acellular vaccines
GB9021004 1990-09-27

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP96110147.4 Division-Into 1996-06-24

Publications (1)

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EP0550558A1 true EP0550558A1 (fr) 1993-07-14

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EP91917103A Withdrawn EP0550558A1 (fr) 1990-09-27 1991-09-27 Vaccin acellulaire
EP96110147A Withdrawn EP0764445A2 (fr) 1990-09-27 1991-09-27 Vaccin acellulaire

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Application Number Title Priority Date Filing Date
EP96110147A Withdrawn EP0764445A2 (fr) 1990-09-27 1991-09-27 Vaccin acellulaire

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EP (2) EP0550558A1 (fr)
JP (1) JP3140776B2 (fr)
GB (1) GB9021004D0 (fr)
WO (1) WO1992005798A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000039302A2 (fr) 1998-12-31 2000-07-06 Chiron Corporation Expression amelioree de polypeptides hiv et production de particules de type viral

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8412207D0 (en) * 1984-05-12 1984-06-20 Wellcome Found Antigenic preparations
GB8512972D0 (en) * 1985-05-22 1985-06-26 Univ Glasgow Vaccine production
EP0267998A1 (fr) * 1986-11-17 1988-05-25 Institut Pasteur Moyens de protection contre l'infection et l'intoxication par bordetella
GB8807860D0 (en) * 1988-04-05 1988-05-05 Connaught Lab Pertussis vaccine
JP2706792B2 (ja) * 1988-11-29 1998-01-28 財団法人化学及血清療法研究所 百日咳毒素のトキソイド化法
GB8910570D0 (en) * 1989-05-08 1989-06-21 Wellcome Found Acellular vaccine

Non-Patent Citations (1)

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Title
See references of WO9205798A1 *

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Publication number Publication date
JP3140776B2 (ja) 2001-03-05
EP0764445A3 (fr) 1997-04-23
JPH06502159A (ja) 1994-03-10
GB9021004D0 (en) 1990-11-07
EP0764445A2 (fr) 1997-03-26
WO1992005798A1 (fr) 1992-04-16

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