WO1997028273A1 - Expression de la proteine de la membrane exterieure de neisseria meningitidis du groupe b (mb3) a partir de levures et de vaccins - Google Patents

Expression de la proteine de la membrane exterieure de neisseria meningitidis du groupe b (mb3) a partir de levures et de vaccins Download PDF

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WO1997028273A1
WO1997028273A1 PCT/US1997/001687 US9701687W WO9728273A1 WO 1997028273 A1 WO1997028273 A1 WO 1997028273A1 US 9701687 W US9701687 W US 9701687W WO 9728273 A1 WO9728273 A1 WO 9728273A1
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protein
yeast
group
porin
meningococcal
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PCT/US1997/001687
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WO1997028273A9 (fr
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Joseph Y. Tai
Mikhail Donets
Ming-Der Wang
Shu-Mei Liang
Maryellen Polvino-Bodnar
Conceicao A. S. A. Minetti
Francis Michon
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North American Vaccine, Inc.
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Priority to EP97906470A priority Critical patent/EP0877816A1/fr
Priority to AU21158/97A priority patent/AU2115897A/en
Priority to CA 2244989 priority patent/CA2244989A1/fr
Priority to JP52788197A priority patent/JP2001508758A/ja
Priority to IL12542097A priority patent/IL125420A0/xx
Publication of WO1997028273A1 publication Critical patent/WO1997028273A1/fr
Publication of WO1997028273A9 publication Critical patent/WO1997028273A9/fr
Priority to NO983474A priority patent/NO983474L/no

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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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 is in the field of recombinant genetics, protein expression, and vaccines.
  • the present invention relates to a method of expressing in a recombinant yeast host an outer membrane group B porin protein from Neisseria meningitidis.
  • the invention also relates to a vaccine comprising group A meningococcal polysaccharide (GAMP), group B meningococcal polysaccharide (GBMP) and group C meningococcal polysaccharide (GCMP) antigens, together with a pharmaceutically acceptable carrier.
  • GAMP group A meningococcal polysaccharide
  • GBMP group B meningococcal polysaccharide
  • GCMP group C meningococcal polysaccharide
  • the invention also relates to a method of inducing an immune response in a mammal, comprising administering the above-mentioned vaccine to a mammal in an amount sufficient to induce an immune response.
  • Meningococcal meningitis remains a worldwide problem, and occurs in both endemic and epidemic forms (Peltola, H., Rev. Infect. Dis. 5:71 -91 (1983);
  • Neisseria meningitidis is a gram-negative organism that has been classified serologically into groups A, B. 29e, W135, X, Y. and Z (Gotschlich. E.C., "Meningococcal Meningitis.” in Bacterial Vaccines. Germanier, E., ed..
  • group A polysaccharide is a partially O-acetylated (1 -6) linked homopolymer of 2-acetamido-2-detoxy-D-mannopyranosyl phosphate, and that both groups B and C polysaccharides are homopolymers of sialic acid.
  • meningitidis groups A, B, and C are responsible for approximately 90% of cases of meningococcal meningitis. Success in the prevention of group
  • a and C meningococcal meningitis was achieved using a bivalent polysaccharide vaccine (Gotschlich, E.C. et al, J. Exp. Med. 729: 1367-1384 ( 1 69); Artenstein. M.S. et al, N. Engl J. Med. 252:417-420 ( 1970)); this vaccine became a commercial product and has been used successfully in the last decade in the prevention and arrest of major meningitis epidemics in many parts of the world.
  • Vaccine 3:340-342 (1985) is the currently used meningococcal meningitis vaccine (Jennings, H.J., "Capsular Polysaccharides as Vaccine Candidates," in Current Topics in Microbiol and Immunol, Jann, D. and Jann, B., eds, Springer- Verlag.
  • the outer membranes of Neisseria species much like other Gram negative bacteria are semi -permeable membranes which allow free flow access and escape of small molecular weight substances to and from the periplasmic space of these bacteria but retard molecules of larger size (Heasley, F.A., et al, "Reconstitution and characterization of the N. gonorrhoeae outer membrane permeability barrier," in Genetics and Immunobiology of Neisseria gonorrhoeae, Danielsson and Normark, eds., University of Umea, Umea, pp. 12-1 (1980); Douglas, J.T., et al , FEMS Microbiol. Lett. 72:305-309 (1981)).
  • porins proteins which have been collectively named porins.
  • These proteins are made up of three identical polypeptide chains (Jones, R.B., et al. Infect. Immun. 30:773-780 (1980); McDade, Jr. and Johnston, J. Bacterial. 14I: ⁇ 183-1 191 (1980)) and in their native trimer conformation, form water filled, voltage-dependent channels within the outer membrane of the bacteria or other membranes to which they have been introduced (Lynch, E.C., et al, Biophys. J. -77:62 (1983); Lynch, E.C., et al, Biophys. J.
  • porin proteins were initially co-isolated with lipopolysaccharides (LPS). Consequently, the porin proteins have been termed "endotoxin-associated proteins" (Bjornson et al, Infect. Immun. 56:1602-1607 (1988)). Studies on the wild type porins have reported that full assembly and oligomerization are not achieved unless LPS from the corresponding bacterial strain is present in the protein environment (Holzenburg et al, Biochemistry 25:4187-4193 (1989); Sen and Nikaido, J. Biol. Chem. 266: 1 1295-1 1300 (1991 )).
  • meningococcal porins have been subdivided into three major classifications which in antedated nomenclature were known as Class 1 , 2, and 3 (Frasch, C.E., et al, Rev. Infect. Dis. 7:504-510 (1985)). Each meningococcus examined has contained one of the alleles for either a Class 2 porin gene or a Class 3 porin gene but not both (Feavers, I.M., et al . Infect. Immun. 60:3620-
  • Class 2 allelic type behave electrophysically somewhat differently than isolated gonococcal porins of the Class 3 type in lipid bilayer studies both in regards to their ion selectivity and voltage-dependence (Lynch. E.C., et al , Biophys. J. 41 :62 ( 1983); Lynch, E.C., et al , Biophys. J. 45: 104- 107 ( 1984)).
  • the ability of the different porins to enter these lipid bi layers from intact living bacteria seems to correlate not only with the porin type but also with the neisserial species from which they were donated (Lynch, E.C., et al, Biophys. J. -75:104-107 (1984)). It would seem that at least some of these functional attributes could be related to different areas within the protein sequence of the porin.
  • One such functional area previously identified within all gonococcal
  • Class 2-like proteins is the site of chymotrypsin cleavage. Upon chymotrypsin digestion, this class of porins lack the ability to respond to a voltage potential and close. Gonococcal Class 3-like porins as well as meningococcal porins lack this sequence and are thus not subject to chymotrypsin cleavage but nonetheless respond by closing to an applied voltage potential (Greco, F.. "The formation of channels in lipid bilayers by gonococcal major outer membrane protein.” thesis. The Rockefeller University, New York ( 1981 ); Greco, F.. et al. Fed. Proc. 39:1813 (1980)).
  • Neisseria porins are among the most abundant proteins present in the outer membrane of these organisms, and as they do not undergo antigenic shift during infection (unlike several other major surface antigens), their universal presence in both Neisseria meningitidis and Neisseria gonorrhoea, as well as their exposure at the surface, make them candidates for components of vaccines against these organisms.
  • Patients convalescing from meningococcal disease produce anti-porin antibodies, and antibodies elicited by immunization with porin proteins are bactericidal to homologous serotypes.
  • the antibody response to a polysaccharide in infants is limited to antibodies of the IgM isotype; the isotype switching necessary for production of non-IgM antibodies requires T-cell involvement.
  • Polysaccharide antigens present less of a problem in more mature humans (over age two), as they are able to induce antibodies of the IgG isotype as well as IgM (Yount el al, J. Exp. Med 727:633-646 (1968)).
  • the group B meningococcal polysaccharide is even less immunogenic in humans of all ages than other polysaccharides. Two major explanations have been proposed to account for this characteristic (Jennings, II. J., Adv.
  • the T-cell independent quality of polysaccharide antigens in infant humans can be overcome by conjugating (covalently coupling) the polysaccharide to a protein carrier.
  • the capsular polysaccharides of the bacteria primarily responsible for postneonatal meningitis have been conjugated to protein carriers; these include type b H. influenzae (Schneerson, R. et al, J. Exp. Med. 752:361 - 376 (1980); Anderson, P.W., Infect. Immun. 39:233-238 ( 1983); Marburg, S. et al, J. Am. Chem. Soc. 705:5282-5287 (1986)), group A (Jennings, H.J. and Lugowski. C., J. Immunol. 727:101 1 -1018 (1981 )) ; Beuvery, E.C. et al, Vaccine
  • a fusion protein which is a mature porin protein fused to a yeast secretion signal peptide; wherein said gene is operably linked to a yeast promoter; (b) transforming said plasmid containing said gene into a yeast strain;
  • MB3 Neisseria meningitidis outer membrane meningococcal group B porin protein
  • the yeast secretion signal peptide is selected from the group consisting of the secretion signal of the S. cerevisiae ⁇ -mating factor prepro-peptide and the secretion signal of the P. pastor is acid phosphatase gene (PHO).
  • codon changes are selected from the group of changes consisting of: (GTT to GTC at positions 4-6 of the native sequence), (ACC to ACT at positions 7-9 of the native sequence), (CTG to TTG at positions 10-12 of the native sequence), (GGC to GGT at positions 16-18 of the native sequence), (ACC to ACT at positions 19-21 of the native sequence), (ATC to ATT at positions 22-24 of the native sequence), (AAA to AAG at positions 25-27 of the native sequence), (GCC to GCT at positions 28-30 of the native sequence), (GGC to GGT at positions 31 -33 of the native sequence), (GTA to GTT at positions 34-36 of the native sequence), (GAA to GAG at positions 37-39 of the native sequence); wherein said positions are numbered from the first nucleotide of the native nucleotide sequence encoding said protein.
  • step (b) washing the insoluble material obtained in step (a) with buffers to remove contaminating yeast cellular proteins;
  • step (c) suspending and dissolving said insoluble fraction obtained in step (b) in aqueous solution of denaturant; (d) diluting the solution obtained in step (c) with a detergent, and
  • step (b) removing contaminating yeast culture impurities from the soluble secreted mate ⁇ al obtained in step (a) by precipitating said impurities with about 20% ethanol, wherein the soluble secreted mate ⁇ al remains in the soluble fraction, (c) precipitating the secreted mate ⁇ al from the soluble fraction of step (b) with about 80% ethanol,
  • step (d) washing the precipitated material obtained in step (c) with a buffer to remove contaminating yeast secreted proteins
  • step (e) suspending and dissolving the precipitated material obtained in step (d) in an aqueous solution of detergent
  • a yeast host cell that contains a gene coding for a protein selected from the group consisting of (a) a mature po ⁇ n protein (b) a fusion protein which is a mature porin protein fused to a yeast secretion signal peptide, wherein said gene is operably linked to a yeast promoter
  • yeast host cell as described above which is capable of expressing the Neisseria meningitidis mature outer membrane class 3 protein of serogroup B (MB3) It is still another specific object of the invention to provide a yeast host cell as described above wherein the yeast promoter is the AO 1 promoter.
  • It is another object of the invention to provide a vaccine comprising group A meningococcal polysaccharide (GAMP), group B meningococcal poly- saccharide (GBMP), and group C meningococcal polysaccharide (GCMP) antigens, together with a pharmaceutically acceptable carrier.
  • GAMP group A meningococcal polysaccharide
  • GBMP group B meningococcal poly- saccharide
  • GCMP group C meningococcal polysaccharide
  • Figure 1 A diagram showing the sequencing strategy of the PorB gene.
  • the PCR product described in Example 1 (Materials and Methods section) was ligated into the BamU ⁇ -Xho ⁇ site of the expression plasmid pET-17b.
  • the initial double stranded primer extension sequencing was accomplished using oligonucleotide sequences directly upstream of the BamW ⁇ site and just downstream of the Xhol site within the pET-17b plasmid. Additional sequence data was obtained by making numerous deletions in the 3' end of the gene, using exonuclease IH/mung bean nuclease reactions. After religation and transformation back into E. coli, several clones were selected on size of insert and subsequently sequenced. This sequencing was always from the 3' end of the gene using an oligonucleotide primer just downstream of the Bpu ⁇ 1021 site.
  • Figure 2 A gel electrophoresis showing the products of the PCR reaction
  • FIG. 3 A SDS-PAGE analysis of whole cell lysates of E. coli hosting the control pET-17b plasmid without inserts and an E coli clone harboring pET-17b plasmid containing an insert from the obtained PCR product described in the materials and methods section. Both cultures were grown to an O.D. of 0.6 at 600 nm, IPTG added, and incubated at 37°C for 2 hrs. 1.5 mis of each of the cultures were removed, centrifuged, and the bacterial pellet solubilized in 100 ⁇ l of SDS-PAGE preparation buffer.
  • Lane A shows the protein profile obtained with 10 ⁇ l from the control sample and Lanes B (5 ⁇ l) and C (10 ⁇ l) demonstrate the protein profile of the E. coli host expressing the PorB protein.
  • Fig. 3B Western blot analysis of whole cell lysates of £ coli harboring the control pET-17b plasmid without insert after 2 hrs induction with IPTG, Lane A, 20 ⁇ l and a corresponding E. coli clone containing a porB-pET-
  • Figure 5 A graph showing the Sephacryl S-300 column elution profile of both the wild type Class 3 protein isolated from the meningococcal strain 8765 and the recombinant Class 3 protein produced by BL21(DE3) -Ao pA E. coli strain hosting the r3pET-17b plasmid as monitored by absorption at 280nm and SDS-PAGE analysis.
  • the void volume of the column is indicated by the arrow. Fractions containing the meningococcal porin and recombinant porin as determined by SDS-PAGE are noted by the bar.
  • Figure 6 A graph showing the results of the inhibition EL1SA assays showing the ability of the homologous wild type (wt) PorB to compete for reactive antibodies in six human immune sera. The arithmetic mean inhibition is shown by the bold line.
  • Figure 7 A graph showing the results of the inhibition ELISA assays showing the ability of the purified recombinant PorB protein to compete for reactive antibodies in six human immune sera. The arithmetic mean inhibition is shown by the bold line.
  • Figure 8 A graph showing a comparison of these two mean inhibitions obtained with the wt and recombinant PorB protein.
  • Figure 9A and 9B The nucleotide sequence and the translated amino acid sequence of the mature class II porin gene cloned into the expression plasmid
  • Figure 10A and 10B The nucleotide sequence and the translated amino acid sequence of the fusion class II porin gene cloned into the expression plasmid pET-17b.
  • Figure 1 1 panels A and B: Panel A depicts the restriction map of the pET-17b plasmid. Panel B depicts the nucleotide sequence between the Bglll and Xhol sites of pET-17b. The sequence provided by the plasmid is in normal print while the sequence inserted from the PCR product are identified in bold print.
  • the level of MB3 expressed is depicted as mg of insoluble MB3 per gram of cell pellet per unit time.
  • Figure 13A The DNA sequence and translated amino acid sequence of pNV15 (MB3 in pET24a) before codon preference optimization.
  • Figure 13B The DNA sequence and translated amino acid sequence of
  • Figures 14A and 14B Graphs showing the elution of MB3 from a size exclusion column.
  • MB3 expressed in an intracellular form was purified by a denaturation/renaturation protocol, followed by gel filtration and ion exchange chromatography.
  • the resultant purified protein exhibited by size exclusion chromatography an elution profile which resembles the recombinant class 3 protein overexpressed in E. coli, and both give the same elution profile as the native wild-type counterpart. This indicates that MB3 refolds and oligomerizes, achieving full native conformation.
  • 14(A) the elution profile of MB3
  • 14(B) the elution profile of class 3 protein expressed and refolded from E. coli inclusion bodies.
  • Figure 15 A graph showing the size exclusion chromatography of purified MB3 on a Superose 12 (Pharmacia) column connected to an HPLC (Hewlett Packard model 1090). Based on the comparison of MB3 with the native wild-type counterpart, as well as calibration using molecular weight standards
  • the elution profile is indicative of trimeric assembly.
  • Figures 16A, 16B and 16C The DNA sequence of clone pnv 322.
  • This clone has the MB3 gene inserted into the EcoR ⁇ site of the Invitrogen expression vector pHIL-D2.
  • MB3 is thus inserted directly downstream from the AOXI promoter. This construct allows intracellular expression.
  • Vector sequences are shown in upper case letters, while the MB3 sequence is given in lower case letters.
  • Figures 17A, 17B and 17C The DNA sequence of clone pnv 318.
  • This clone has the MB3 gene inserted into the XhoI-BamHI sites of the Invitrogen expression vector pHIL-Sl .
  • MB3 is thus inserted directly downstream from the PHOl leader peptide, in frame with the secretion signal open reading frame for secretion of expressed protein.
  • Vector sequences are shown in upper case letters, while the MB3 sequence is given in lower case letters.
  • Figures 18A, 18B and 18C The DNA sequence of clone pnv 342.
  • This clone has the MB3 gene inserted into the EcoRl-Avrll sites of the Invitrogen expression vector pPIC-9.
  • MB3 is thus inserted directly downstream from the secretion signal of ⁇ -factor prepro peptide, for secretion of expressed protein.
  • Vecior sequences are shown in upper case letters, while the MB3 sequence is given in lower case letters.
  • Figures 19A, 19B and 19C The DNA sequence of clone pnv 350. This clone has the MB3 gene inserted into the EcoR ⁇ -Avrll sites of the Invitrogen expression vector pPIC-9K. MB3 is thus inserted directly downstream from the secretion signal of ⁇ -factor prepro peptide. for secretion of expressed protein. Vector sequences are shown in upper case letters, while the MB3 sequence is given in lower case letters.
  • Figure 20 A graph showing the absorbance spectra (electropherogram) of GAMP, TT-monomer, and GAMP-TT conjugate.
  • Figure 21 A graph showing the absorbance spectra (electropherogram) of GCMP, TT-monomer, and GCMP-TT conjugate.
  • Figure 22 A graph showing the A-specific IgG ELISA titer elicited by monovalent (A) and trivalent (A/B/C) meningococcal conjugate vaccines in mice.
  • Figure 23 A graph showing the B-specific IgG ELISA titer elicited by monovalent (A) and trivalent (A7B/C) meningococcal conjugate vaccines in mice.
  • Figure 24 A graph showing the C-specific IgG ELISA titer elicited by monovalent (C) and trivalent (A/B/C) meningococcal conjugate vaccines in mice.
  • Figure 25 A graph showing the A-specific bacteriocidal activity elicited by monovalent (A) and trivalent (A/B/C) meningococcal conjugate vaccines in mice.
  • Figure 26 A graph showing the B-specific bacteriocidal activity elicited by monovalent (A) and trivalent (A/B/C) meningococcal conjugate vaccines in mice.
  • Figure 27 A graph showing the C-specific bacteriocidal activity elicited by monovalent (A) and trivalent (A/B/C) meningococcal conjugate vaccines in mice. - 1 ⁇
  • Meningococcal B Class 3 porin protein may be expressed in yeast.
  • a preferred host is the methylotrophic yeast Pichia pastoris. which may be transformed with the Pichia
  • Yeasts are attractive hosts for the production of heterologous proteins. Unlike prokaryotic systems, their eukaryotic subcellular organization enables them to carry out many of the post-translational folding, processing and modification events required to produce "authentic" and bioactive proteins.
  • a eukaryote Pichia pastor is has many of the advantages of a higher eukaryotic expression system, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae.
  • yeast shares the advantages of molecular and genetic manipulations with Saccharomyces, and it has the added advantages of 10- to 100-fold higher heterologous protein expression levels and the protein processing characteristics of higher eukaryotes.
  • Pichia also provides advantages compared to expression in other yeast strains because Pichia does not tend to hyperglycosylate proteins as does S. cerevisiae. Further, proteins expressed and modified in Pichia may be more useful therapeutically than those produced by S. cerevisiae, as oligosaccharides added by Pichia lack the ⁇ l ,3 glycan linkages which are believed to be primarily responsible for the hyper-antigenic nature of proteins produced by S. cerevisiae.
  • Several vaccine antigens have been produced in yeast cells, including hepatitis B surface antigen which is in clinical use (Cregg et al ,
  • Carbonetti et al. were the first to clone an entire gonococcal porin gene into E. coli using a tightly controlled pT7-5 expression plasmid. The results of these studies showed that when the porin gene was induced, very little porin protein accumulated and the expression of this protein was lethal to the E. coli (Carbonetti and Sparling,
  • Feavers et al. have described a method to amplify, by PCR, neisserial porin genes from a wide variety of sources using two synthesized oligonucleotides to common domains at the 5' and 3' ends of the porin genes respectively (Feavers, I.M., et al, Infect. Immun. 60:3620-3629 (1992)).
  • the oligonucleotides were constructed such that the amplified DNA could be forced cloned into plasmids using the restriction endonucleases BglW and Xho ⁇ .
  • PorB protein was expressed, it was easily isolated, purified and appeared to reform into trimers much like the native porin.
  • the results of the inhibition ELISA data using human immune sera suggests that the PorB protein obtained in this fashion regains most if not all of the antigenic characteristics of the wild type PorB protein purified from meningococci.
  • This expression system lends itself to the easy manipulation of the neisserial porin gene by modern molecular techniques. In addition, this system allows one to obtain large quantities of pure porin protein for characterization. In addition, the present expression system allows the genes from numerous strains of Neisseria, both gonococci and meningococci, to be examined and characterized in a similar manner.
  • the Neisseria meningitidis outer membrane class 3 protein from serogroup B was also expressed in the methylotrophic yeast Pichia pastoris by placing the MB3 DNA fragment under the control of the strong P. pastoris alcohol oxidase promoter AOXI .
  • strains of P. pastoris transformed with the recombinant plasmids produced either a native or a fusion MB3 protein, which were reactive with mouse polyclonal J
  • the vector pHIL-S 1 which carries a native Pichia pastoris signal sequence from the acid phosphatase gene, PHOl
  • the vectors pPIC9 and pPIC9K which carry the secretion signal from the 5. cerevisiae ⁇ -mating factor prepro-peptide. Maps of the pHIL-Sl and pPIC9 vectors may be found on pp. 21 -22 of the Invitrogen Instruction Manual for the Pichia Expression Kit, Version E.
  • pHIL-Sl/MB3 construct provided the expression of a MB3- PHOl fusion polypeptide with an apparent molecular weight of 36.5 kDa. which was partly processed to generate mature 34 kDa MB3. About 5-10% of expressed MB3 was secreted to the yeast growth medium, and about 40-50% of the 36.5 kDa fusion polypeptide was cleaved (Table 4). Pichia recombinants transformed by pPIC9/MB3 or pPIC9K/MB3 constructs expressed only MB3 fused with ⁇ -factor, yielding a fusion polypeptide of approximately 45 kDa. There was no evidence of any cleavage or processing of that fusion protein.
  • An increase in the yield of expressed MB3 may be obtained by using strains of Pichia which contain multiple copies of the MB3 expression cassette.
  • strains harboring multiple copies exist naturally within transformed cell populations at ⁇ 10% frequency. These strains may be identified either by directly screening large numbers of transformants for levels of MB 3 expression via SDS- PAGE or immunoblotting, or indirectly screening by "dot blot" hybridization to select for clones containing multiple copies of the MB3 gene (Cregg et aF
  • Such multiple integrated clones may be constructed by using a new pAO815 vector (Invitrogen), which allows cloning of multiple copies of the gene of interest via repeated cassette insertion steps (Ibid, at p. 907).
  • Scale-up procedures using a fermenter will provide higher yeast cell densities and therefore improve the yields of the expressed proteins by at least 5-10 times. Optimization of protein expression (i.e., growth media composition, buffering capacity, casamino acids supplementation, increase of methanol concentration, etc.) may be carried out with routine experimentation.
  • MB3 takes advantage of the fact that the Pichia expression vector pPIC9K carries the kanamycin resistance gene which confers resistance to G418; in other respects, pPIC9K corresponds to pPIC9. Spontaneous generation of multiple insertion events can then be identified by the level of resistance to G418. Pichia transformants are selected on histidine-deficient medium and screened for their level of resistance to G418. An increased level of resistance to G418 indicates multiple copies of the kanamycin resistance gene.
  • the present invention relates to a method of expressing an outer membrane meningococcal group B porin protein, in particular, the class 2 and class 3 porin proteins.
  • the present invention relates to a method of expressing the outer membrane meningococcal group B porin protein in E. coli comprising:
  • a fusion protein comprising a mature porin protein fused to amino acids 1 to 20 or 22 of the T7 gene ⁇ l O capsid protein; wherein said gene is operably linked to the T7 promoter;
  • the meningococcal group B porin protein or fusion protein expressed comprises more than about 5% of the total proteins expressed in E. coli. In another preferred embodiment, the meningococcal group B porin protein or fusion protein expressed comprises more than about 10% of the total proteins expressed in E. coli. In yet another preferred embodiment, the meningococcal group B porin protein or fusion protein expressed comprises more than about 30% of the total proteins expressed in E. coli.
  • plasmids which contain the T7 inducible promotor include the expression plasmids pET-17b, pET-l la, pET-24a-d(+) and pET-9a, all of which are commercially available from Novagen (565 Science Drive, Madison,
  • E coli strain BL21 (DE3) AompA is employed.
  • the above mentioned plasmids may be transformed into this strain or the wild-type strain BL21(DE3).
  • E. coli strain BL21 (DE3) AompA is preferred as no OmpA protein is produced by this strain which might contaminate the purified porin protein and create undesirable immunogenic side effects.
  • the transformed E. coli are grown in a medium containing a selection agent, e.g. any ⁇ -lactam to which E. coli is sensitive such as ampicillin.
  • a selection agent e.g. any ⁇ -lactam to which E. coli is sensitive such as ampicillin.
  • the pET expression vectors provide selectable markers which confer antibiotic resistance to the transformed organism.
  • High level expression of meningococcal group B porin protein can be toxic in E. coli.
  • the present invention allows E. coli to express the protein to a level of at least almost 30% and as high as >50% of the total cellular proteins.
  • the present invention relates to a method of expressing an outer membrane meningococcal group B porin protein in yeast comprising:
  • a mature porin protein and (ii) a fusion protein comprising a mature porin protein fused to a yeast secretion signal peptide; wherein said gene is operably linked to a yeast promoter;
  • Transformation of the yeast host may be accomplished by any one of several techniques that are well known by those of ordinary skill in the art. These techniques include direct or hposome-mediated transformation of yeast cells whose cell wall has been partially or completely destroyed to form spheroplasts, treatment of the host with alkali cations and PEG, and freeze-thawing combined with PEG treatment (See Weber et al , Nonconventional ⁇ easts Their Genetics and Biotechnological Applications, CRC Crit Rev Biolechnol 7 281 , 317
  • the mature porin protein or fusion protein expressed comprises more than about 2% of the total protein expressed in the yeast host In yet another preferred embodiment, the mature porin protein or fusion protein expressed comprises about 3-5% of the total protein expressed in the yeast host
  • the mature porin protein is the Neisseria meningitidis mature outer membrane class 3 protein from serogroup B
  • the present invention relates to performing the above method of expressing the outer membrane meningococcal group B porin protein or fusion protein in yeast, wherein said yeast is selected from the group consisting of Saccharomyces cerevisiae Schizosaccharomyces pombe, Saccharomyces uvarum Saccharomyces carhbergensis Saccharomyces diastaticus, Candida tropicalis Candida maltosa, Candida parapsdosis Pichia pastoris, Pichia far inosa, Pichia pinus, Pichia vanrijii Pichia fermentans, Pichia guilliermondii, Pichia st ⁇ itis, Saccharomyces telluris, Candida utilis, Candida guilliermondii, Hansenula henricii Hansenula capsulala Hansenula polymorpha Hansen
  • nucleotide sequence of the gene encoding the mature porin protein or fusion protein incorporates codons which are optimized for yeast codon usage.
  • nucleotide sequence of the gene encoding the mature porin protein which has been optimized for yeast codon usage is the nucleotide sequence SEQ ID NO: 26.
  • the yeast secretion signal peptide is selected from the group consisting of the secretion signal of the S. cerevisiae - mating factor prepro-peptide and the secretion signal of the P. pastoris acid phosphatase gene.
  • the yeast secretes the protein or fusion protein.
  • the yeast promoter to which the gene is operably linked is selected from a group consisting of the AOXI promoter, the GAPDH promoter, the PHO5 promoter, the glyceraldehyde-3 -phosphate dehydrogenase (TDH3) promoter, the ADHI promoter, the MF ⁇ l promoter, and the GAL 10 promoter.
  • plasmids which contain the AOXI promoter include the expression plasmids pHIL-D2, pHIL-S 1 , pPIC9, and pPIC9K.
  • Plasmids comprise, in sequence, an AOXI promoter, restriction sites to allow insertion of the structural gene, an AOXI transcription termination fragment, an open reading frame encoding HIS4 (histidinol dehydrogenase), an ampicillin resistance gene, and a ColEl origin.
  • plasmids pPIC9 and pPIC9K comprise the ⁇ -factor secretion signal of S. cerevisiae
  • plasmid pHIL-S l comprises the PHOl secretion signal of P. pastoris.
  • pPIC9K also includes the kanamycin resistance gene, which confers resistance to G418 in Pichia.
  • the level of G418 resistance in Pichia transformants can be used to identify cells which have undergone multiple insertion events. This occurs at a frequency of 1 -10%. An increased level of resistance to G418 indicates the presence of multiple copies of the kanamycin resistance gene and of the gene of interest. See the Novagene catalogue, Version E, pp. 19-22 (1995).
  • yeast host strains having a mutation in a suitable marker gene which causes the strain to have specific nutritional requirements are employed.
  • Expression plasmids carrying a functional copy of the mutated gene as well as a copy of the meningococcal group B porin protein or fusion protein are then transformed into the yeast host strain, and transformants are selected on the basis of their ability to grow on medium lacking the required nutrient.
  • marker genes include the genes encoding imidazole glycerol phosphate dehydrogenase (HIS3), beta-isopropylmalate dehydrogenase (LEU2), tryptophan synthase (TRP5), argininosuccinate lyase (ARG4).
  • HIS3 imidazole glycerol phosphate dehydrogenase
  • LEU2 beta-isopropylmalate dehydrogenase
  • TRP5 tryptophan synthase
  • ARG4 argininosuccinate lyase
  • TRP1 N-(5'-phosphorilosyl) anthranilate isomerase
  • HIS4 histidinol dehydrogenase
  • UAA3 orotidine-5- phosphate decarboxylase
  • UAA1 dihydroorotate dehydrogenase
  • GALI galactokinase
  • LYS2 alpha-aminodipate reductase
  • This screening is performed by methods well known to those of ordinary skill in the art, for example, by selecting for transformants which grow slowly on medium which lacks the nutrient used to confirm transformation and includes methanol in order to induce expression of the outer membrane meningococcal group B porin protein or fusion protein from the AOXI promoter. These transformants are then grown up in glycerol-containing medium, and expression of the meningococcal group B porin protein or fusion protein is then induced by the addition of methanol.
  • P. pastoris host strains GS 1 15 or KM71 are employed. These strains have a mutation in the histidinol dehydrogenase gene (his 4) which prevents them from synthesizing histidine.
  • the expression plasmids pHIL-D2, pHIL-Sl , pPIC9, and pPIC9K carry the H1S4 gene which complements his4 in the host, allowing selection of transformants on histidine- deficient medium.
  • the cells are screened for integration of the meningococcal group B porin protein or fusion protein at the correct loci by selecting for transformants which grow slowly on his , methanoL plates.
  • These transformants which become mutated at the AOXI locus when the MB3 gene inserts into the host genome, are only capable of slow growth on methanol, as they are metabolizing methanol with the less efficient AOX2 gene product.
  • the transformants are then grown up in glycerol-containing medium, and expression of the meningococcal group B porin protein or fusion protein is then induced by the addition of methanol.
  • the present invention relates to performing the above method of expressing the outer membrane meningococcal group B porin protein in yeast, wherein said yeast is Pichia pastoris.
  • the present invention relates to a vaccine for inducing an immune response in an animal comprising the outer membrane meningococcal group B porin protein or fusion protein thereof, produced according to the above-described methods, together with a pharmaceutically acceptable diluent, carrier, or excipient.
  • the vaccine may be administered in an amount effective to elicit an immune response in an animal to Neisseria meningitidis.
  • the animal is selected from the group consisting of humans, cattle, pigs, sheep, and chickens.
  • the animal is a human.
  • the present invention relates to the above-described vaccine, wherein said outer membrane meningococcal group B porin protein or fusion protein thereof is conjugated to a meningococcal group B capsular polysaccharide (CP).
  • capsular polysaccharides may be prepared as described in Ashton, F.E. et al, Microbial Pathog. 6:455-458 ( 1989); Jennings, H.J. et al, J. Immunol 73-7:2651 ( 1985); Jennings. H.J. et al. J. Immunol 737:1708-1713 (1986); Jennings, H.J. et al, J. Immunol.
  • the invention also relates to a vaccine capable of simultaneously inducing an immune response against any one of several N meningitidis serogroups
  • the invention relates to a trivalent vaccine comprising the capsular polysaccharides from each of three different serogroups of N meningitidis
  • the vaccine of the invention comprises group A meningococcal polysaccharide (GAMP), group B meningococcal polysaccharide (GBMP), and group C meningococcal polysaccharide (GCMP) antigens, together with a pharmaceutically acceptable carrier
  • GAMP group B meningococcal polysaccharide
  • GCMP group C meningococcal polysaccharide
  • carrier proteins will be suitable to be used in the polysaccha ⁇ de-protein conjugates included in the vaccine of the invention
  • a suitable carrier protein will be one which is safe for administration to mammals, and which is immunologically effective as a carrier Safety includes absence of primary toxicity and minimal risk of allergic complications
  • any heterologous protein could serve as a carrier antigen
  • the protein may be, for example, native toxin or detoxified toxin (also termed toxoid)
  • genetically altered proteins which are antigenically similar to toxins and yet non-toxic may be produced by mutational techniques well-known to those of skill in the art
  • Such an altered toxin is termed a "cross reacting material," oi CRM CRM I97 is noteworthy, because it differs from native diphtheria toxin at only one ammo acid residue, and is immunologically indistinguishable from the native toxin (Anderson.
  • polysaccharide- protein carrier conjugates of the vaccine may be produced by several different methods.
  • the types of covalent bonds which couple a polysaccharide to a protein carrier, and the means of producing them, are well known to those of skill in the art. Details concerning the chemical means by which the two moieties can be linked may be found in U.S. Patent No. 5,371.197, and 4,902.506, the contents of which are herein incorporated by reference in their entirety.
  • One such method is the reductive amination process described in Schwartz and Gray (Arch. Biochim. Biophys. 757:542-549 (1977)).
  • This process involves reacting the reducing capsular polysaccharide fragment and bacterial toxin or toxoid in the presence of cyanoborohydride ions, or another reducing agent. Such a process will not adversely affect the toxin or toxoid or the capsular polysaccharide (U.S. Patent No. 4,902,506). Such a conjugation process is also described in Examples 12-14, below. While tetanus and diphtheria toxins are the prime candidates for carrier proteins, owing to their history of safety, there may be overwhelming reasons, well known to those of ordinary skill in the art. to use another protein. For example, another protein may be more effective as a carrier, or production economics may be significant.
  • a preferred carrier protein to which the group B meningococcal polysaccharide may be conjugated is the class 3 porin protein (PorB) of group B N. meningitidis.
  • a preferred protein carrier protein to which GAMP antigen and GCMP antigen may be conjugated is tetanus toxoid.
  • N-carbonyl groups (Jennings, H.J. et al, J. Immunol. 737:1708-1713 (1986)).
  • the most preferred modification which satisfies the above criteria is a modification wherein the N-acetyl groups of the sialic acid residues of the B polysaccharide are removed by strong base and replaced by N-propionyl groups (see Examples 6 and 14).
  • the N-propionylated GBMP is subsequently conjugated to a carrier protein.
  • a carrier protein any carrier protein which enhances the immunogenicity of N-propionylated GBMP may be used, a preferred protein carrier is the class 3 outer membrane protein of group B N. meningitidis (MB3, or PorB).
  • GBMP antigen is conjugated to PorB after having been ⁇ -propionylated.
  • the capsular polysaccharide (CP) which may be group A, B or C meningococcal polysaccharide. is isolated according to Frasch. C.E., "Production and Control of Neisseria meningitidis Vaccines" in Bacterial Vaccines, Alan R. Liss, Inc., pages 123-145 (1990), the contents of which are fully incorporated by reference herein, as follows:
  • the crude CP is then further purified by gel filtration chromatography after partial depolymerization with dilute acid, e.g. acetic acid, formic acid, and trifiuoroacetic acid (0.01-0.5 N), to give a mixture of polysaccharides having an average molecular weight of 10,000-20,000.
  • dilute acid e.g. acetic acid, formic acid, and trifiuoroacetic acid (0.01-0.5 N
  • GBMP purified GBMP is then N-deacetylated with NaOH in the presence of sodium borohydride and N-propionylated to afford N-Pr GBMP.
  • the CP that may be employed in the conjugate vaccines of the present invention may be CP fragments, N- deacylated CP and fragments thereof, as well as N-Pr CP and fragments thereof, so long as they induce active immunity when employed as part of a CP-porin protein conjugate (see Examples 6 and 14).
  • the present invention relates to a method of preparing a polysaccharide conjugate comprising: obtaining the above-described outer membrane meningococcal group B porin protein or fusion protein thereof; obtaining a CP from a Neisseria meningitidis organism; and conjugating the protein to the CP.
  • the conjugates of the invention may be formed by reacting the reducing end groups of the CP to primary amino groups of the porin by reductive amination.
  • the reducing groups may be formed by selective hydrolysis or specific oxidative cleavage, or a combination of both.
  • the CP is conjugated to the porin protein by the method of Jennings et al, U.S. Patent No.
  • the vaccine of the present invention comprises the meningococcal group B porin protein, fusion protein or conjugate vaccine, or the trivalent GAMP.
  • the meningococcal group B porin protein, fusion protein or vaccine of the present invention can also be administered by an intraperitoneal or intravenous route.
  • the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Suitable amounts might be expected to fall within the range of 2 micrograms of the protein per kg body weight to 100 micrograms per kg body weight.
  • the vaccine comprises about 2 ⁇ g of the
  • GAMP GAMP, GCMP and GBMP polysaccharide antigen conjugates.
  • the vaccine comprises about 5 ⁇ g of the GAMP, GCMP and GBMP polysaccharide antigen conjugates.
  • the vaccine comprises about 2 ⁇ g of the GAMP and GCMP polysaccharide antigen conjugates, and about 5 ⁇ g of the
  • the vaccine of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions.
  • Any inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the meningococcal group B porin protein, fusion protein or conjugate vaccine have suitable solubility properties.
  • the vaccines may be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination programs. Reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, Osol (ed.) (1980); and New Trends and
  • the vaccines of the present invention may further comprise adjuvants which enhance production of porin-specific antibodies.
  • adjuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant (CFA), stearyl tyrosine (ST, see U.S. Patent No. 4,258,029), the dipeptide known as MDP, saponin, aluminum hydroxide, and lymphatic cytokine.
  • Freund's adjuvant is an emulsion of mineral oil and water which is mixed with the immunogenic substance. Although Freund's adjuvant is powerful, it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) or ST may be used for administration to a human.
  • the meningococcal group B porin protein or a conjugate vaccine thereof may be absorbed onto the aluminum hydroxide from which it is slowly released after injection.
  • the meningococcal group B porin protein or group A, B and C meningococcal polysaccharide conjugate vaccine may also be encapsulated within liposomes according to Fullerton, U.S. Patent No. 4,235,877.
  • the present invention relates to a method of inducing an immune response in an animal comprising administering to the animal the vaccine of the invention, produced according to methods described, in an amount effective to induce an immune response.
  • the invention relates to a method of purifying the above-described outer membrane meningococcal group B porin protein or fusion protein comprising: lysing the transformed E. coli to release the meningococcal group B porin protein or fusion protein as part of insoluble inclusion bodies; washing the inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; resuspending and dissolving the inclusion bodies in an aqueous solution of a denaturant; diluting the resultant solution in a detergent; and purifying the solubilized meningococcal group B porin protein by gel filtration.
  • the lysing step may be carried out according to any method known to those of ordinary skill in the art, e.g. by sonication, enzyme digestion, osmotic shock, or by passing through a mull press.
  • the inclusion bodies may be washed with any buffer which is capable of solubilizing the E. coli cellular proteins without solubilizing the inclusion bodies comprising the meningococcal group B porin protein.
  • buffers include but are not limited to TEN buffer (50 mM Tris HC1, 1 mM EDTA. 100 mM NaCl, pH
  • Denaturants which may be used in the practice of the invention include 2 to 8 M urea or about 2 to 6 M guanidine HC1, more preferably, 4 to 8 M urea or about 4 to 6 M guanidine HC1, and most preferably, about 8 M urea or about
  • detergents which can be used to dilute the solubilized meningococcal group B porin protein include, but are not limited to, ionic detergents such as SDS and cetavlon (Calbiochem); non-ionic detergents such as Tween, Triton X, Brij 35 and octyl glucoside; and zwitterionic detergents such as 3,14-Zwittergent, empigen BB and Champs.
  • ionic detergents such as SDS and cetavlon (Calbiochem)
  • non-ionic detergents such as Tween, Triton X, Brij 35 and octyl glucoside
  • zwitterionic detergents such as 3,14-Zwittergent, empigen BB and Champs.
  • the solubilized outer membrane meningococcal group B porin protein may be purified by gel filtration to separate the high and low molecular weight materials.
  • Types of filtration gels include but are not limited to Sephacryl-300, Sepharose CL-6B, and Bio-Gel A-l .5m.
  • the column is eluted with the buffer used to dilute the solubilized protein.
  • the fractions containing the porin or fusion thereof may then be identified by gel electrophoresis, the fractions pooled, dialyzed, and concentrated.
  • substantially pure (>95%) porin protein and fusion protein may be obtained by passing the concentrated fractions through a Q sepharose high performance column.
  • the present invention relates to expression of the meningococcal group B porin protein gene which is part of a vector which comprises the T7 promoter, which is inducible. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent.
  • the T7 promoter is inducible by the addition of isopropyl ⁇ -D- thiogalactopyranoside (IPTG) to the culture medium.
  • IPTG isopropyl ⁇ -D- thiogalactopyranoside
  • the Tac promotor or heat shock promotor may be employed.
  • the meningococcal group B porin protein gene is expressed from the pET-17 expression vector or the pET-1 1 a expression vector, both of which contain the T7 promoter.
  • the cloning of the meningococcal group B porin protein gene or fusion gene into an expression vector may be carried out in accordance with conventional techniques, including blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases.
  • blunt-ended or stagger-ended termini for ligation restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases.
  • the present invention relates to a method of refolding the above-described outer membrane protein and fusion protein comprising: lysing the transformed cells to release the meningococcal group B porin protein or fusion protein as part of insoluble inclusion bodies; washing the inclusion bodies with a buffer to remove contaminating cellular proteins: resuspending and dissolving the inclusion bodies in an aqueous solution of a denaturant; diluting the resultant solution in a detergent; and purifying the solubilized meningococcal group B porin protein or fusion protein by gel filtration to give the refolded protein in the eluant.
  • the present invention relates to a substantially pure refolded outer membrane meningococcal group B porin protein and fusion protein produced according to the above-described methods.
  • a substantially pure protein is a protein that is generally lacking in other cellular
  • Neisseria meningitidis components as evidenced by, for example, electrophoresis.
  • Such substantially pure proteins have a purity of >95%. as measured by densitometry on an electrophoretic gel after staining with Coomassie blue or silver stains.
  • the following examples are illustrative, but not limiting, of the method and compositions of the present invention.
  • Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in this art which are obvious to those skilled in the art are within the spirit and scope of the present invention.
  • Organisms The Group B Neisseria meningitidis strain 8765 (B: 15:P1 ,3) was obtained from Dr. Wendell Zollinger (Walter Reed Army Institute for
  • PI Transduction A PI,,,, lysate of E. coli strain DME558 was used to transduce a tetracycline resistance marker to strain BRE51 (Bremer, E., et al , FEMS Microbiol. Lett. 33: 173-178 (1986)) in which the entire ompA gene had been deleted (Silhavy, T.J., et al, Experiments with Gene Fusions, Cold Spring
  • 0.5 ml of chloroform was added and the phage culture stored at 4°C. Because typically 1-2% of the E. coli chromosome can be packaged in each phage, the number of phage generated covers the entire bacterial host chromosome, including the tetracycline resistance marker close to the ompA gene.
  • strain BRE51 which lacks the ompA gene, was grown in LB medium overnight at 37 °C.
  • the overnight culture was diluted 1 :50 into fresh LB and grown for 2 hr.
  • the cells were removed by centrifugation and resuspended in MC salts.
  • 0.1 ml of the bacterial cells were mixed with 0.05 of the phage lysate described above and incubated for 20 min. at room temperature. Thereafter, an equal volume of 1 M sodium citrate was added and the bacterial cells were plated out onto LB plates containing 12.5 ⁇ g/ml of tetracycline. The plates were incubated overnight at 37°C.
  • Tetracycline resistant ( 12 ⁇ g/ml) transductants were screened for lack of OmpA protein expression by SDS-PAGE and Western Blot analysis, as described below.
  • the bacteria resistant to the antibiotic have the tetracycline resistance gene integrated into the chromosome very near where the ompA gene had been deleted from this strain.
  • One particular strain was designated BRE-T R .
  • a second round of phage production was then carried out with the strain
  • BRE-T R using the same method as described above. Representatives of this phage population contain both the tetracycline resistance gene and the OmpA deletion. These phage were then collected and stored. These phage were then used to infect E. coli BL21(DE3). After infection, the bacteria contain the tetracycline resistance marker. In addition, there is a high probability that the
  • OmpA deletion was selected on the LB plates containing tetracycline.
  • Colonies of bacteria which grew on the plates were grown up separately in LB medium and tested for the presence of the OmpA protein. Of those colonies selected for examination, all lacked the OmpA protein as judged by antibody reactivity on SDS-PAGE western blots.
  • SDS-PAGE and Western Blot The SDS-PAGE was a variation of Laemmli's method (Laemmli, U.K., Nature 227:680-685 ( 1970)) as described previously (Blake and Gotschlich, J. Exp. Med. 759:452-462 (1984)). Electrophoretic transfer to Immobilon P (Millipore Corp. Bedford, MA) was performed according to the methods of Towbin et al. (Towbin, H., et al , Proc.
  • reaction components were as follows: Meningococcal strain 8765 chromosomal DNA ( 100 ng/ ⁇ l), 1 ⁇ l; 5' and 3 ' primers (1 ⁇ M) 2 ⁇ l each; dNTP (10 mM stocks), 4 ⁇ l each; 10 X PCR reaction buffer (100 mM Tris HC1, 500 mM KC1, pH 8.3), 10 ⁇ l; 25 mM MgCl 2 , 6 ⁇ l; double distilled H 2 0, 62 ⁇ l; and Taq polymerase (Cetus Corp., 5 u/ ⁇ l), 1 ⁇ l.
  • the reaction was carried out in a GTC-2 Genetic Thermocycler (Precision Inst. Inc,
  • the pET-17b plasmid (Novagen, Inc.) was used for subcloning and was prepared by double digesting the plasmid with the restriction endonucleases BamW ⁇ and Xho ⁇ (New England Biolabs, Inc., Beverly, MA). The digested ends were then dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis. IN). The digested plasmid was then analyzed on a 1% agarose gel, the cut plasmid removed, and purified using the GeneClean kit (Bio 101 , La Jolla, CA).
  • the PCR product was prepared by extraction with phenol-chloroform, chloroform, and finally purified using the GeneClean Kit (Bio 101 ).
  • the PCR product was digested with restriction endonucleases Bglll and Xho ⁇ (New England Biolabs, Inc.).
  • the DNA was then extracted with phenol-chloroform, precipitated by adding 0.1 volumes of 3 M sodium acetate, 5 ⁇ l glycogen (20 ⁇ g/ ⁇ l), and 2.5 volumes of ethanol. After washing the DNA with 70% ethanol (vol/vol). it was redissolved in TE buffer.
  • the digested PCR product was ligated to the double digested pET- 17b plasmid described above using the standard T4 ligase procedure at 16°C overnight (Current Protocols in Molecular Biology, John Wiley & Sons, New York (1993)).
  • the ligation product was then transformed into the BL21 (DE3)- ⁇ ompA described above which were made competent by the method of Chung et al (Chung, C.T., et a , Proc. Natl Acad. Sci. USA 56:2172-2175 (1989)).
  • the transformants were selected on LB plates containing 50 ⁇ g/ml carbenicillin and 12 ⁇ g/ml tetracycline.
  • nucleotide Sequence Analysis The nucleotide sequences of the cloned Class 3 porin gene DNA were determined by the dideoxy method using denatured double-stranded plasmid DNA as the template as described (Current Protocols in Molecular Biology, John Wiley & Sons, New York (1993)). Sequenase II kits (United States Biochemical Corp., Cleveland. OH) were used in accordance with the manufacturer's instructions. The three synthesized oligonucleotide primers (Operon Technologies, Inc., Alameda, CA) were used for these reactions.
  • PorB gene product Using a sterile micropipette tip, a single colony of the BL21 (DE3)- ⁇ om/?A containing the PorB- pET-17b plasmid was selected and inoculated into 10 ml of LB broth containing 50 ⁇ g/ml carbenicillin. The culture was incubated overnight at 30 °C while shaking. The 10 ml overnight culture was then sterilely added to 1 liter of LB broth with the same concentration of carbenicillin, and the culture continued in a shaking incubator at 37 °C until the OD 6 reached 0.6-1.0.
  • IPTG IPTG
  • Rifampicin was then added (5.88 ml of a stock solution; 34 mg/ml in methanol) and the culture continued for an additional 2 hrs.
  • the cells were harvested by centrifugation at 10,000 rpm in a GS3 rotor for 10 min and weighed.
  • the cells were thoroughly resuspended in 3 ml of TEN buffer (50 mM Tris HC1, 1 mM Tris HC1, 1 mM EDTA, 100 M NaCl, pH 8.0) per gram wet weight of cells.
  • the pellet was then resuspended in freshly prepared TEN buffer containing 0.1 mM PMSF and 8 M urea and sonicated in a bath sonicator (Heat Systems, Inc., Plain view. NY).
  • the protein concentration was determined using a BCA kit (Pierce, Rockville, IL) and the protein concentration adjusted to less than 10 mg/ml using the TEN-urea buffer.
  • the sample was then diluted 1 : 1 with 10%
  • the human immune sera was diluted in PBS with 0.5% Brij 35 and added to the plate and incubated for 2 hr at room temperature. The plates were again washed as before and the secondary antibody, alkaline phosphatase conjugated goat anti-human IgG (Tago Inc., Burlingame, CA), was diluted in PBS-Brij, added to the plates and incubated for 1 hr at room temperature. The plates were washed as before and
  • the ELISA microtiter plate would be sensitized with purified wild type PorB protein and washed as before. In a separate V-96 polypropylene microtiter plate (Nunc, Inc.). varying amounts of either purified wild type PorB protein or the purified recombinant PorB protein were added in a total volume of 75 ⁇ l. The human sera were diluted in PBS-Brij solution to twice their half maximal titer and 75 ⁇ l added to each of the wells containing the PorB or recombinant PorB proteins.
  • This plate was incubated for 2 hr at room temperature and centrifuged in a Sorvall RT6000 refrigerated centrifuge, equipped with microtiter plate carriers (Wilmington, DE) at 3000 rpm for 10 min. Avoiding the V-bottom, 100 ⁇ l from each well was removed and transferred to the sensitized and washed ELISA microtiter plate. The ELISA plates are incubated for an additional 2 hr, washed, and the conjugated second antibody added as before. The plate is then processed and read as described. The percentage of inhibition is then processed and read as described. The percentage of inhibition is calculated as follows:
  • coli strain BL21 lysogenic for the DE3 lambda derivative (Studier and Moffatt, J. Mol. Biol. 759: 1 13-130 ( 1986)) was selected as the expression host for the pET-17b plasmid containing the porin gene. But because it was thought that the OmpA protein, originating from the E. coli expression host, might tend to co-purify with the expressed meningococcal porin protein, a modification of this strain was made by PI transduction which eliminated the ompA gene from this strain.
  • PorB protein expressed in the E. coli was insoluble in TEN buffer which suggested that when expressed, the PorB protein formed into inclusion bodies. However, washing of the insoluble PorB protein with TEN buffer removed most of the contaminating E. coli proteins. The PorB protein could then be solubilized in freshly prepared 8M urea and diluted into the Zwittergent 3,14 detergent. The final purification was accomplished, using a Sephacryl S-300 molecular sieve column which not only removed the urea but also the remaining contaminating proteins. The majority of the PorB protein eluted from the column having the apparent molecular weight of trimers much like the wild type PorB.
  • Inhibition ELISA Assays In order to determine if the purified trimeric recombinant PorB had a similar antigenic conformation as compared to the PorB produced in the wild type meningococcal strain 8765, the sera from six patients which had been vaccinated with the wild type meningococcal Type 15 PorB protein were used in inhibition ELISA assays. In the inhibition assay, antibodies reactive to the native PorB were competitively inhibited with various amounts of either the purified recombinant PorB or the homologous purified wild type PorB.
  • reaction conditions were as follows: BNCV M986 genomic DNA 200ng, the two oligonucleotide primers described above at 1 ⁇ M of each, 200 ⁇ M of each dNTP, PCR reaction buffer (10 mM Tris HC1, 50 mM KC1. pH 8.3), 1.5 mM MgC and 2.5 units of Taq polymerase, made up to 100 ⁇ l with distilled H-,0. This reaction mixture was then subjected to 25 cycles of 95°C for 1 min. 50°C for 2 min and 72 °C for 1.5 min.
  • the reaction mixture was loaded on a 1% agarose gel and the material was electrophoresed for 2h after which the band at 1.3 kb was removed and the DNA recovered using the Gene Clean kit (Bio 101 ).
  • This DNA was then digested with EcoRl, repurified and ligated to EcoRl digested pUCl 9 using T 4 DNA ligase.
  • the ligation mixture was used to transform competent E. coli DH5 ⁇ . Recombinant plasmids were selected and sequenced. The insert was found to have a DNA sequence consistent with that of a class 2 porin. See, Murakami, K. et al, Infect. Immun. 57:2318-2323 (1989).
  • the plasmid pET-17b (Novagen) was used to express the class 2 porin.
  • One plasmid was designed to produce a mature class 2 porin while the other was designed to yield a class 2 porin fused to 20 amino acids from the T7 gene ⁇ l 0 capsid protein.
  • the mature class 2 porin was constructed by amplifying the pUC19-class 2 porin construct using the oligonucleotides: 5'-CCT GTT GCA GCA CAT ATG GAC GTT ACC TTG TAC GGT ACA ATT AAA GC-3' and 5 '-CGA CAG GCT TTT TCT CGA GAC CAA TCT TTT CAG -3'.
  • This strategy allowed the cloning of the amplified class 2 porin into the Ndel and Xhol sites of the plasmid pET-17b thus producing a mature class 2 porin.
  • Standard PCR was conducted using the pUC19-class 2 as the template and the two oligonucleotides described above.
  • This PCR reaction yielded a 1.1 kb product when analyzed on a 1.0% agarose gel.
  • the D ⁇ A obtained from the PCR reaction was gel purified and digested with the restriction enzymes Nd and Xlio .
  • the 1.1 kb D ⁇ A produced was again gel purified and ligated to Ndel and ⁇ 7?ol digested pET-17b using T D ⁇ A ligase.
  • This ligation mixture was then used to transform competent E. coli DH5 ⁇ . Colonies that contained the 1.lkb insert were chosen for further analysis.
  • the D ⁇ A from the DH5 ⁇ clones was analyzed by restriction mapping and the cloning junctions of the chosen plasmids were sequenced.
  • the D ⁇ A obtained from the DH5 ⁇ clones was used to transform E. coli BL21 (DE3)- AompA.
  • the transformants were selected to LB-agar containing 100 ⁇ g/ml of carbenicillin.
  • the nucleotide sequence and translated amino acid sequence of the mature class II porin gene cloned into pET- 17b are shown in Figures 9 A and 9B.
  • the fusion class 2 porin was constructed by amplifying the pUC19-class 2 porin construct using the oligonucleotides: 5 '-CCT GTT GCA GCG GAT CCA
  • lkb product when analyzed on a 1.0% agarose gel.
  • the DNA obtained from the PCR reaction was gel purified and digested with the reaction enzymes BamWl and Xhol.
  • the 1.1 kb product produced was again gel purified and ligated to BamWl and .Y7 ⁇ oI digested pET-17b using T 4 DNA ligase.
  • This ligation mixture was then used to transform competent E. coli DH5 ⁇ . Colonies that contained the l .lkb insert were chosen for further analysis.
  • the DNA from the DH5 ⁇ clones was analyzed by restriction enzyme mapping and the cloning junctions of the chosen plasmids were sequenced.
  • the nucleotide sequence and translated amino acid sequence of the fusion class II porin gene cloned into the expression plasmid pET-17b are shown in Figures 10A and 10B.
  • the DNA obtained from the DH5 ⁇ clones was used to transform E. coli BL21 (DE3)-AompA.
  • the transformants were selected on LB-agar containing 100 ⁇ g/ml of carbenicillin.
  • Example 3 Cloning and Expression of the Mature class 3 porin from Group B Neisseria meningitidis strain 8765 in E. coli
  • Genomic DNA was isolated from approximately 0.5 g of Group B Neisseria meningitidis strain 8765 using the method described above (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press ( 1989)). This DNA then served as the template for two class 3 porin specific oligonucleotides in a standard PCR reaction.
  • the mature class 3 porin was constructed by amplifying the genomic DNA from 8765 using the oligonucleotides: 5'-GTT GCA GCA CAT ATG GAC GTT ACC CTG TAC GGC ACC-3' and 5'-GGG GGG ATG GAT CCA GAT TAG AAT TTG TGG CGC AGA CCG ACA CC-3'.
  • This strategy allowed the cloning of the amplified class 3 porin into the Ndel and BamHl sites of the plasmid pET-24a+ ( Figures 13A and 13B), thus producing a mature class 3 porin.
  • Standard PCR was conducted using the genomic DNA isolated from 8765 as the template and the two oligonucleotides described above.
  • the reaction conditions were as follows: 8765 genomic DNA 200 ng, the two oligonucleotide primers described above at 1 ⁇ M of each, 200 ⁇ M of each dNTP, PCR reaction buffer (10 mM Tris HCI. 50 mM KC1, pH 8.3), 1.5 M MgCl 2 , and 2.5 units of Taq polymerase, and made up to 100 ⁇ l with distilled water. This reaction mixture was then subjected to 25 cycles of 95 °C for 1 min. 50 °C for 2 min and 72 °C for 1.5 min.
  • This PCR reaction yielded about 930 bp of product, as analyzed on a 1% agarose gel.
  • the DNA obtained from the PCR reaction was gel purified and digested with the restriction enzymes Ndel and BamHl.
  • the 930 bp product was again gel purified and ligated to Ndel and BamHl digested pET-24a(+) using T4 ligase.
  • This ligation mixture was then used to transform competent E. coli DH5 ⁇ . Colonies that contained the 930 bp insert were chosen for further analysis.
  • the DNA from the E. coli DH5 ⁇ clones was analyzed by restriction enzyme mapping and cloning junctions of the chosen plasmids were sequenced.
  • the DNA obtained from the E. coli DH5 ⁇ clones was used to transform E. coli BL21(DE3)- ⁇ w/ ⁇ 4.
  • the transformants were selected on LB-agar containing 50 ⁇ g/ml of kanamycin.
  • Several transformants were screened for their ability to make the class 3 porin protein. This was done by growing the clones in LB liquid medium containing 50 ⁇ g/ml of kanamycin and 0.4% of glucose at 30°C to OD WK)
  • E coli strain BL21(DE3) ⁇ om/?A [pNV-5] is grown to mid-log phase (OD
  • the resultant solution is then centrifuged at 13,000 rpm for 20 min and the supernatant discarded.
  • the pellet is then twice suspended in TEN buffer containing 0.5% deoxycholate and the supernatants discarded.
  • the pellet is then suspended in TEN buffer containing 8 M deionized urea (electrophoresis grade) and 0.1 mM PMSF (3 g/l Oml).
  • the suspension is sonicated for 10 min or until an even suspension is achieved.
  • 10 ml of a 10% aqueous solution of 3.14-zwittergen (Calbiochem) is added and the solution thoroughly mixed.
  • the solution is again sonicated for 10 min. Any residual insoluble material is removed by centrifugation.
  • Lysozyme is added (Sigma, 0.25 mg/ml) deoxycholate (Sigma, 1.3 mg/ml) plus PMSF (Sigma, ⁇ g/ml) and the mixture gently shaken for one hour at room temperature. During this time, the cells lyse and the released DNA causes the solution to become very viscous. DNase is then added (Sigma. 2 ⁇ g/ml) and the solution again mixed for one hour at room temperature. The mixture is then centrifuged at 15K rpm in a S-600 rotor for 30 minutes and the supernatant discarded. The pellet is then twice suspended in 10 ml of TEN buffer and the supernatants discarded. The pellet is then suspended in 10 ml of 8 M urea
  • the pooled fractions are precipitated with 80% ethanol and resuspended with the above-mentioned buffer.
  • Six to 10 mg of the material is then applied to a monoQ 10/10 column (Pharmacia) equilibrated in the same buffer.
  • the porin is eluted from a shallow 0.1 to 0.6 M NaCl gradient with a 1.2% increase per min over a 50 min period.
  • the Flow rate is 1 ml/min.
  • the peak containing porin is collected and dialyzed against TEN buffer and 0.05% 3,14-zwittergen.
  • the porin may be purified further by another S-300 chromatography.
  • Example 6 Purification and chemical
  • the capsular polysaccharide from both group B Neisseria meningitidis and E. coli Kl consists of ⁇ (2-8) polysialic acid (commonly referred to as GBMP or Kl polysaccharide).
  • High molecular weight polysaccharide isolated from growth medium by precipitation was purified and chemically modified before being coupled to the porin protein.
  • Example 14 Treatment with NaIO 4 followed by gel filtration column purification gave the oxidized N-Pr GBMP having an average molecular weight of 12,000 daltons.
  • Example 7 Coupling of oxidized N-Pr GBMP to the group B meningococcal class 3 porin protein (PP)
  • the oxidized N-Pr GBMP (9.5 mg) was added to purified class 3 porin protein (3.4 mg) dissolved in 0.21 ml of 0.2 M phosphate buffer pH 7.5 which also contained 10% octyl glucoside. After the polysaccharide was dissolved, sodium cyanoborohydride (7 mg) was added and the reaction solution was incubated at 37°C for 4 days. The reaction mixture was diluted with 0.15 M sodium chloride solution containing 0.01 % thimerosal and separated by gel filtration column chromatography using Superdex 200 PG. The conjugate (N-Pr GBMP-PP) was obtained as single peak eluting near the void volume.
  • the polysaccharide (2 ⁇ g)-conjugate was administered on days 1 , 14 and 28, and the sera collected on day 38.
  • the conjugates were administered as saline solutions, adsorbed on aluminum hydroxide, or admixed with stearyl tyrosine.
  • the sera ELISA titers against the polysaccharide antigen and bactericidal titers against N. meningitidis group B are summarized in Table 1.
  • Example 9 Expression of group B Neisseria meningitidis Outer Membrane (MB 3) Using Yeast Pichia pastoris Expression System
  • Pichia pastoris GS 1 15 (provided by Invitrogen) has a defect in the histidinol dehydrogenase gene (his4) which prevents it from synthesizing histidine. All expression plasmids carry the HIS4 gene which complements his4 in the host, so transformants are selected for their ability to grow on histidine- deficient medium. Until transformed, GS 1 15 will not grow on minimal medium alone.
  • his4 histidinol dehydrogenase gene
  • the vector pHIL-S l carries a native Pichia pastoris signal from the acid phosphatase gene. PHOl .
  • the vectors, pPIC9 and pPIC9K both carry the secretion signal from the S. cerevisiae ⁇ -mating factor pre-pro peptide.
  • the advantage of expressing secreted proteins is that P. pastoris secretes very low levels of native proteins.
  • the secreted heterologous protein comprises the vast majority of the total protein in the media and serves as the first step in purification of the protein (Barr et al, Pharm. Eng. 12(2) -4S-51 (1992)).
  • the genomic DNA of Group B Neisseria meningitidis served as the template for the amplification of class 3 porin (MB3) in a standard PCR.
  • the amplified DNA fragment (930 b.p. long) of the mature porin protein was ligated in Nde I - BamH I cloning sites of the pET-24a cloning/expression vector, originally constructed by Studier et al, J. Mol. Biol. 759: 1 13-130 (1986); Meth. Enzymol. 755:60-89(1990); J. Mol. Biol 279:37-44 (1991 ), and manufactured by Novagen.
  • the pET vectors were developed for cloning and for expressing target DNA fragments under the strong T7 transcription and translation signals. Expression from the T7 promoter is induced by providing the host cell with a source of T7 RNA polymerase. Newer, more convenient vectors utilizing the T7 expression system are now available from Novagen (Madison, WI 5371 1). The T7 expression system was successfully used for the expression of MB3 in E. coli (see Example 3).
  • Codon usage is known to affect the translational elongation rate, and therefore it has been considered an important factor in affecting product yields (Romanos et al, Yeast 5:423-488 ( 1992)). There is evidence that codon usage may affect both yield and quality of the expressed protein. A number of highly expressed genes show a strong bias toward a subset of codons (Bennetzen et al. ,
  • proteins containing amino acid misincorporations are difficult to purify and may have both impaired activity and antigenicity.
  • the presence of several rare codons has been shown to limit the production of tetanus toxin fragment C in E. coli (Makoff et al. Nucleic Acids Res. 77: 10191 -10201 (1989)).
  • yeast Hoekema et al. (Mol. Cell Biol. 7: 2914-2924 (1987)) showed that substitution of a large proportion of preferred codons for rare codons in the 5' portion of the PGK (phosphoglycerate kinase) gene caused a decrease in expression levels.
  • the expression of an immunoglobulin kappa chain in yeast has been shown to be increased 50-fold when a synthetic codon- optimized gene is used, although the level of kappa chain RNA remains the same.
  • Vector pHIL-Sl/MB3 containing the codon-optimized MB3 DNA. served as the template for the amplification of MB3 in a standard PCR. Oligomers were synthesized to serve as PCR primers. The PCR fragments of
  • MB3 were inserted into Pichia expression vectors either directly or by using the Original TA Cloning Kit (Invitrogen); details are given below.
  • Reverse primer (36 nt, having an engineered Avrll site and stop codon): 5'-CACCCTAGGTTAGAATTTGTGACGCAGACCGACACC-3'
  • Venf* DNA polymerase (NEB) was used for PCR amplification of the complete MB3 gene.
  • the fidelity of this polymerase is 5-15-fold higher than that observed for Taq DNA polymerase.
  • PCR fragments of MB3 full length and truncated fragments
  • Pichia expression vectors either directly or using the Original TA Cloning ® Kit (Invitrogen), which includes a pCRTMII vector for subcloning of PCR fragments.
  • Direct cloning of DNA amplified by either Vent ® DNA polymerase or Pfu DNA polymerase into the vector pCRTMII is difficult, as the cloning efficiency is often very low.
  • the Invitrogen protocol was modified as follows. Following amplification with Vent* or Pfu (see manual for The Original TA Cloning* Kit, protocol for the addition of 3'A-overhangs post amplification, p. 19), rather than placing the vial on ice, as recommended in the kit, the mineral oil in the PCR mixture was immediately removed using ParafilmTM. This was accomplished by pouring the PCR mixture onto the Parafilm, and zigzagging the drop down the surface of the Parafilm with a gentle rocking motion until all of the oil had adhered to the Parafilm surface. The reaction mixture, now free of oil. was then collected into a fresh tube. The Invitrogen protocol was then resumed with the addition of Taq polymerase. This method allowed the difficult cloning of PCR fragments into large expression vectors.
  • the expression cassette of the integrating vector contains the methanol-induced AOX I promoter and its terminator, flanked by stretches of nucleotides up- and downstream from the AOX 1 gene.
  • the P. pastoris His4 gene served as an auxotrophic marker.
  • These vectors do not contain a yeast ori, hence His + colonies must correspond to integration of the expression cassette.
  • All PCR fragments of MB3 were inserted in frame with a Pichia Kozak consensus sequence (CAAAAAACAA) (Cavenor et al. Nucleic Acids Res. 19:3185-3192 ( 1991 ); Kozak Nucleic Acids Res. 75:8125-8148 (1987); Kozak Proc. Natl. Acad
  • Other strains which may be suitable are DH5 ⁇ F , JM 109, or any other strain that carries a selectable F ' episome and is recA deficient (endA is preferable) (Pichia Expression Kit Instruction Manual, Invitrogen).
  • Colonies with an MB3 insert were used for the preparation of CsCl purified maxi-prep of a plasmid DNA for Pichia transformation (Sambrook, J. er al, Eds., Molecular Cloning: A Laboratory Manual. 2nd. Ed., Cold Spring Harbor Press ( 1989), pp. 1.42-1 .43). Restriction analysis and DNA sequencing (DNA Sequencing Kit, Version 2 (USB)) confirmed that these constructs were correct.
  • Modification of the starting MB3 sequence was especially useful for intracellular expression of the porin gene (pHIL-D2/MB3 construct). Because the other constructs (pHIL-Sl/MB3 and pPIC9/MB3) used for MB3 secretion contained codons optimal for Pichia in the leader peptide sequence upstream of the MB3 insert, the initiation of translation was not rate-limiting. In contrast, the pHIL-D2 vector does not include any leader sequence and the initiation of translation must be started from the rare codons of the MB3 insert. The optimization of this sequence is believed to be responsible for the fact that pHIL- D2/MB3 constructs gave the highest level of MB3 expression of any of the clones tested (Tables 3, 4).
  • Plasmid DNA was linearized with single or double (for higher integration efficiencies) digestion, and P. pastoris strain GS1 15 (his4 ) was transformed to the His + phenotype by the spheroplast method using Zymolyase followed by adsorption of transforming DNA and penetration of this DNA through the spheroplast pores into the Pichia cells in the presence of PEG and Ca " " 2 (Pichia Expression Kit manual, Invitrogen, pp.33-38).
  • the cells were harvested by centrifugation (4000 rpm for 10 minutes at room temperature) and were resuspended in methanol-containing Buffered Methanol- complex Medium (BMMY: 1 % yeast extract, 2% peptone. 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4xl 0 "s % biotin, 0.5% methanol) (Pichia Expression Kit manual, Invitrogen, p. 61 ) for the induction of the AOXI promoter. To replenish exhausted methanol, 0.5% of fresh methanol was added each day to induced cells.
  • BMMY 1 % yeast extract, 2% peptone. 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4xl 0 "s % biotin, 0.5% methanol
  • Cells were broken by agitation in breaking buffer (50 mM sodium phosphate, pH 7.4; 1 mM PMSF(phenylmethylsulfonyl fluoride), 1 mM EDTA and 5% glycerol). Equal volumes of acid-washed glass beads (0.5 mm in diameter) were added. The mixture was vortexed for a total of 4 min, 30 sec mixing each, followed by 30 sec on ice. The soluble fraction was recovered by centrifugation for 10 min at 14000 rpm at 4°C.
  • breaking buffer 50 mM sodium phosphate, pH 7.4; 1 mM PMSF(phenylmethylsulfonyl fluoride), 1 mM EDTA and 5% glycerol.
  • Proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were stained with Coomassie Brilliant Blue R250, or were transferred to polyvinylidene difluoride (PVDF) membrane using a Transblotl apparatus (BioRad Laboratories) according to the company specification.
  • Immunostaining of proteins was carried out as follows.
  • the transfer membrane was rinsed with TBS ( l OmM Tris-HCl/.09% NaCl, pH 7.2).
  • the membrane was then incubated in 1 % non fat dried milk PBS solution (M-PBS) with .02% sodium azide at 37°C for 3 hours (or at 4°C overnight).
  • the membrane was then washed 3 times with TBS/0.5% BSA (BS A/TBS) and once with TBS.
  • the membrane was then incubated with the primary mouse anti-MB3 antibody (mouse polyclonal antisera against purified OMP class 3) diluted to about 1 :4000 in PBS/1 %BSA (BSA/PBS).
  • the strategy used to insert the cDNA encoding the mature MB3 into expression vectors and the steps using this construct for the transformation of P. pastoris are outlined below.
  • the MB3 gene is cloned into one of the 4 Pichia expression vectors.
  • the resulting construct is linearized by digestion with Notl or Bgl ⁇ l, and his4 Pichia spheroplasts are transformed with the linearized construct.
  • a recombination event occurs in vivo between the 5 ' and 3 'AOXI sequences in the vector and in the genome, resulting in replacement of the AOXI gene with the MB3 gene.
  • the Pichia transformants are selected on histidine-deficient medium, on which only cells that have undergone gene replacement can grow.
  • the one-step gene replacement method described for S. cerevisiae (Rothstein. Meth. Enzymol 707:202-21 1 (1983)) was successfully used by Cregg et al (Biological Research on Industrial Yeast, Vol. II, Stewart et al. eds ,CRC Press. Boca Raton, pp.1-18 (1987)) for the replacement of the P. pastoris AOX I structural gene.
  • Transformation of GS1 15 with 10 ⁇ g of linearized expression vectors (pHIL-D2, pHIL-Sl, pPIC9, and pPIC9K) with MB3 insert gave more than 100 colonies in each experiment. Thus, the procedure yielded >10 2 His colonies per ⁇ g DNA. which is comparable to that reported for the best results of P pastoris transformations. These transformants have the ability to grow on histidine- deficient medium (MD-minimal dextrose), and so arc His . About 10-40% of these recombinants were "methanol slow” (Muf — "methanol utilization slow”). i.e., demonstrated impaired growth on media such as MM (minimal methanol). which contains methanol as the sole carbon and energy source.
  • HisVMuf transformants are a result of the replacement of the AOXI structural gene with the MB3 expression cassette containing the His' gene via a double crossover event. Recombination events may also occur as integration or insertion (single crossover events) of the expression cassette into the 5' or 3' AOXI region, which leaves the AOXI gene intact.
  • His7Mut s clones 25-35% were positive, MB3-expressing transformants (Table 2). The reason that the AOX1- deleted transformants grow at all on methanol medium is due to low-level expression of alcohol oxidase activity by the AOX2 gene product.
  • the amount of expressed MB3 was determined by densitometric scanning of the Coomassie brilliant blue stained protein bands fractionated by SDS-PAGE using a Model GDS-7500 scanning densitometer (UVP Life Sci.) or Model IS-1000 densitometer (Alpha Innotech Corp.). Purified OMP class 3 extracted wild type of N. meningitidis was used as a standard. Based on the results (summarized in Table 3), the level of protein expression was estimated to be moderate to high.
  • the level of MB3 expression by the best clones was in the range of 0.1-0.6 g per IL of cell suspension, or 1-3 mg per g of cell pellet (Table 3, Fig. 12).
  • Such efficiency of expression in yeast has been reported for many of the following manufactured proteins: hepatitis B surface antigen (0.3 g/L), superoxide dismutase (0.75 g/L).
  • bovine and human lysozyme (0.3 and 0.7 g/L, respectively), human and mouse epidermal growth factors (0.5 and 0.45 g/L respectively), human insulin-like growth factor (0.5 g/L), human interleukin-2 (1.0 g/L), aprotinin analog (0.8 g/L), Kunitz protease inhibitor (1.0 g/L), etc. (Cregg et al, Biotechnology. 77.903-906, Table 1 ( 1993)). It should be emphasized that all of the previously listed levels of expression for manufactured proteins are the result of production of these proteins during fermentation in high cell density fermentors. MB3 was expressed utilizing only shake flask cultures which, as a rule, provide much lower expression levels than does fermentation.
  • PEST sequences contain proline (P), glutamic acid (E), serine (S) and threonine (T), and are found in all rapidly degraded eukaryotic proteins of known sequence; such proteins have been implicated as favored substrates for calcium-activated proteases (Rogers et al, Science 23-7:364-369 (1986)).
  • MB3 also does not contain the highly conserved pentapeptide sequences mentioned above.
  • the sequence ROSFI (75-79aa) is present in MB3: this sequence displays some homology to the degradation pentapeptide QRXFX. but is not believed to greatly destabilize MB3.
  • the nature of the NH 2 -terminal amino acid residue can also be an important factor in the susceptibility of a protein to degradation. Varshavsky el al. have demonstrated that the presence of certain amino acids at the NH 2 - terminus provide a stabilizing effect against rapid degradation by ubiquitin- mediated pathways (the N-end rule pathway) (Varshavsky et al. Yeast Genetic-
  • proteins that are expressed at high levels in yeast have a stabilizing amino-terminus amino acid residue (A, C, G, M, S, T or V).
  • examples of such proteins include human superoxide dismutase, human tumor necrosis factor, phosphoglycerate kinase from S. cerevisiae, invertase from 5. cerevisiae, alcohol oxidase from P. pastoris, and extracellular alkaline protease from ⁇ '. Hpolytica (Sreekrishna et al, Biochemistry 25:41 17-4125 (1989)).
  • MB3 is expressed well in yeast, the NH 2 -terminal aspartic acid (D) of MB3 does not provide a stabilizing effect against rapid degradation by ubiquitin-mediated pathways. It is possible that the NH 2 -terminal aspartic acid of MB3 will play a role in the level of MB3 produced from Pichia in large scale production. Replacing the first amino acid of MB3 with one of the amino acids known to stabilize the NH 2 -terminus of proteins, mentioned above, could improve the level of MB3 production. It was decided to proceed with experiments attempting to express MB3 in yeast, as most of the factors known to reduce expression levels were not present in MB3.
  • MB 3 The best expression of MB 3 was provided by Pichia clones transformed with the pHIL-D2/MB3 expression cassette (Tables 3 and 4).
  • This pHIL-D2 vector generated intracellular expression of complete, monomeric, non-fusion, non-secreted MB3 with an expected MW of about 34 kDa.
  • These clones provided the highest level of expression of MB3. up to 600 mg/L or 3 mg per g of wet cell pellet (Table 4).
  • About 90-95% of this product was insoluble, membrane-associated material, i.e., material which sediments upon centri- fugation for 5 min at l O.OOOg, and that can be extracted by treatment with SDS- containing buffer (PAGE sample buffer) followed by boiling.
  • the protein can then be renatured to a conformation that can be easily recognized by an anti- meningococcal OMP class 3 antibody.
  • the pHIL-D2/MB3-containing Pichia recombinant is the most promising for commercial production.
  • This clone provides relatively high levels of expression which could be significantly improved by using multiple-copy recombinants, and by producing the protein in a fermentor.
  • the fact that MB3 is rapidly produced also provides an advantage for large scale manufacture.
  • MB3 expressed in an intracellular form was purified by a denaturation/renaturation protocol, followed by gel filtration and ion exchange chromatography.
  • the resultant purified protein exhibits an elution profile on size exclusion chromatography that resembles the recombinant class 3 protein overexpressed in E. coli.
  • MB3 expressed by either E. coli or P. pastoris co-elutes with the native wild-type counte ⁇ art. indicating that MB3 expressed by either E. coli or P. pastoris refolds and oligomerizes, achieving full native conformation (Figs. 14A and 14B).
  • the pHIL-S l Pichia transfer vector includes a sequence encoding the 2.5 kDa PHOl leader peptide, a secretion signal peptide of P. pastoris.
  • the sequence encoding MB3 was inserted downstream of the PHOl leader sequence.
  • the pPIC9 and pPIC9K Pichia transfer vectors include a sequence encoding the 10 kDa alpha-factor leader derived from S. cerevisiae. Pichia clones transformed by pPIC9/MB3 or pPIC9K/MB3 did not secrete porin.
  • Example 10 Isolation, purification and characterization of MB3 protein expressed as a secretory protein
  • Yeast cells cultures harboring the expression vector containing the gene for MB3 were configured to isolate the protein as soluble secreted material).
  • the supernatant was clarified by precipitation with 20% ethanol (v/v) to remove contaminating yeast culture impurities.
  • the supernatant was then precipitated with 80% ethanol (v/v).
  • the resulting pellet was washed with TEN buffer (Tris HC1, pH 8.0, 100 mM NaCl and 1 mM EDTA), in order to remove other hydrosoluble contaminating secreted proteins.
  • the pellet containing MB3 was dissolved in an aqueous solution of detergent (solubilizing buffer), comprised of TEN buffer with 5% Z 3-14.
  • Example 11 Isolation, purification and characterization of MB3 protein expressed as an insoluble- membrane bound protein
  • Yeast cells cultures harboring the expression vector containing the gene for MB3 (pHILD-2-pNV322) (see Table 3) were resuspended in breaking buffer (i.e., 50 mM sodium phosphate buffer, pH 7.4, 1 mM EDTA, and 5% glycerol), to a concentration equivalent to 50-100 ODs.
  • breaking buffer i.e., 50 mM sodium phosphate buffer, pH 7.4, 1 mM EDTA, and 5% glycerol
  • the suspension was added to the same volume of acid treated glass beads.
  • the suspension was lysed using a Minibead-Beater (Biospec Products, Bartlesville, OK), in 8 consecutive cycles of 1 min each, followed by 1 min on ice, between each cycle.
  • the lysis process was facilitated by the addition of Zymolase to the breaking buffer.
  • the suspension was transferred to a glass sintered filter to separate the glass beads, and the cell suspension was collected in the filtrate. The beads were further washed and the filtrates combined. The suspension was then centrifuged at 12,000 ⁇ m for 15 min at 4°C. A series of consecutive washing steps was applied to the resultant pellet, consisting of the following: (a) TEN (Tris HC1, pH 8.0, 100 mM NaCl, and 1 mM EDTA) containing 0.5% deoxycholate; (b) TEN containing 0.1% SDS and 1% Nonidet, after which the suspension was rotated for 30 min at 25 °C; (c) washing with TEN buffer; and (d) washing with TEN buffer containing 5% Z 3-14. under rotation overnight at
  • each washing step was followed by centrifugation at 12,000 ⁇ m for 10 min at 4°C to collect the pellet for the following step.
  • the suspension was passed through an 18 gauge needle in lieu of rotation in steps (b) and (d).
  • the MB3 was extracted with 8M urea, or 6M guanadinium HCI, and the extract was sonicated for 10 min, using a water bath sonicator.
  • the extract was clarified by centrifugation (12,000 rpm, for 10 min at 4°C), the same volume of a 10% aqueous solution of 3, 14-zwittergen (Calbiochem) was added and the solution thoroughly mixed. The solution was again sonicated for 10 min.
  • FIGS. 14A, 14B and 15 depict the elution profile of purified MB3 in a Sepharose 12 (Pharmacia) connected to an HPLC (Hewlett Packard, model 1090). Based on the comparison with the native wild-type class 3 protein, as well as calibration using molecular weight standards, the elution profile is indicative of trimeric assembly.
  • NMA polysaccharide for conjugation.
  • NMA meningitidis group A
  • ethanol for fractional precipitation with ethanol.
  • the high molecular weight polysaccharide was further purified by ultra filtration. Partial hydrolysis of the polysaccharide with 100 mM sodium acetate buffer pH 5.0 at 70°C yielded a low molecular weight polysaccharide in the range of 10,000-20,000 daltons. The free reducing terminal residue of the polysaccharide was reduced with
  • Tetanus toxoid (Serum Statens Institute, Denmark) was first purified to its monomeric form (mw 150,000) by size exclusion chromatography using a Superdex G-200 column (Pharmacia). Freeze-dried tetanus toxoid monomer ( 1 part by weight) and oxidized GAMP (2.5 part by weight) were dissolved in 0.2 M phosphate buffer pH 7.5. Recrystallized NaBH 3 CN (1 part) was added and the reaction mixture incubated at 37°C for 4 days.
  • the conjugate was purified from the free components by size exclusion chromatography using a Superdex G-200 column (Pharmacia), and PBS containing 0.01% thimerosal as an eluent. Purified GAMP-tetanus toxoid conjugate was stored at 4°C in this buffer. The polysaccharide content of the conjugate based on phosphorus analysis (Chen assay) was about 18-20% by weight.
  • NMC polysaccharide for conjugation.
  • the capsular polysaccharide was isolated from the growth medium of Neisseria meningitidis group C (NMC) strain C 1 1. This strain was grown in modified Franz medium.
  • the NMC polysaccharide (group C meningococcal polysaccharide (GCMP)) was isolated from the culture medium by cetavlon precipitation as described for the GAMP.
  • Native GCMP was O-deacetylated with base and depolymerized by oxidative cleavage with NaIO 4 to an average molecular weight of 10,000-20,000.
  • the cleaved polysaccharide was sized and purified by gel filtration chromatography to provide a highly purified product of average molecular weight about 12,000 daltons and having aldehyde groups at both termini.
  • Neisseria rPorB Expression of class 3 N meningitidis porin protein (PorB) in E. coli and purification of porin gene products is described supra.
  • the recombinant rPorB protein was purified by using a sephacryl S-300 molecular sieve column equilibrated with 100 mM Tris- HCl, 200 nM ⁇ aCl, 10 mM EDTA, 0.05% Zwittergen 3, 14 (Calbiochem. La Jolla, CA), 0.02% sodium azide pH 8.0.
  • the protein fractions as measured by their OD 28(1 eluting with an apparent molecular weight of trimers were pooled and diafiltered against 0.25 M HEPES. 0.25 M ⁇ aCl, 0.05% Zwittergen 3, 14 pH 8.5. to a concentration of 10-12 mg/ml.
  • N-Pr-GBMP-rPorB conjugate To 10 mg of oxidized N-Pr-GBMP of average molecular weight 12,000 was added 33 ⁇ l of a 12 mg/ml of rPorB protein in 0.25 M HEPES. 0.25% M NaCl. 0.05% Zwittergen 3. 14, pH 8.5. The solution was mixed until all solid dissolved and 6.5 mg of recrystallized NaBH : ,CN was added. The solution was incubated at 37°C for 4 days and the conjugate was purified from the mixture by using a Superdex G-200 column (Pharmacia) equilibrated with PBS -0.0%) thimerosal. Protein fractions were combined and stored at 4°C. The conjugates were analyzed for their sialic acid content by the resorcinol assay and for protein with the Pierce Coomassie Plus assay. The resulting conjugate had a polysaccharide content of about 20-
  • the capillary was conditioned between runs with a high pressure rinse for 2.0 minutes with 0.1 M sodium hydroxide followed by 2.0 minutes with deionized water. All samples were pressure injected. All buffer and sample media were filtered through an appropriate 0.2 ⁇ m membrane filter and degassed prior to use.
  • Fig. 20 shows the GAMP-TT conjugate spiked with GAMP and TT-monomer conjugate components
  • Fig. 21 shows the GCMP-TT conjugate spiked with GCMP and TT-monomer conjugate components.
  • the lower limit of detection (LLD) for the free form polysaccharide and protein components for the method was determined to be in the subnanogram level.
  • a lower limit of quantitation (LLQ) of approximately 0.6 ng was obtained for the free form of each component.
  • a linear response was obtained for the selected total mass of each component.
  • a linear response was obtained for the selected total mass range of 0.6-2.6 ng and 0.6-2.4 ng for the polysaccharide and protein, respectively, with a coefficient of determination of 0.99 for both curves.
  • Trivalent conjugate vaccine formulation Each individual conjugate component (A, B, C) was absorbed onto Aluminum hydroxide (Al(OH) 3 ) Alhydrogel (Superfos, Denmark) at a final Al concentration of 1 mg/ml of the trivalent vaccine. Three vaccines were formulated in which the doses of each conjugated polysaccharide varied.
  • Formulations had either about 2 ⁇ g of each A, B, and C conjugated polysaccharide; or about 2 ⁇ g A conjugated polysaccharide, about 5 ⁇ g B conjugated polysaccharide and about 2 ⁇ g C conjugated polysaccharide; or about 5 ⁇ g of each A, B, and C conjugated polysaccharide per dose of 0.2 ml of PBS, 0.01 % thimerosal.
  • ELISAs Antibody titers to each A, N-propionylated B and C polysaccharides were determined by ELISA using the corresponding HSA conjugates as coating antigen (Figs. 22, 23, and 24). Antibody titer was defined as the x-axis intercept of the linear regression curve of absorbance vs. absorbance x dilution factor.
  • Bactericidal Assays Bactericidal assays were performed using baby rabbit serum as a source of complement and N.
  • meningitidis strains H 44/76 (Serotype 15), C l l and Al respectively used as group B meningococcal, group C meningococcal, and group A meningococcal organisms in this assay (Figs. 25, 26, and 27).
  • Bactericidal titer was defined as the serum dilution producing 50% reduction in viable counts.
  • Table 4 The expression of MB3 by recombinant clones with different expression cassettes. The main characteristic of the best clones.

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Abstract

La présente invention porte, en général, sur une méthode d'obtention des protéines de porine de groupe B méningococciques des protéines de membrane extérieure, en particulier MB3, ainsi que leurs protéines de fusion. En particulier, la présente invention concerne une méthode d'expression des protéines de porine de groupe B méningococciques de protéines de membrane extérieure dans la levure. Elle concerne également une méthode d'expression à un niveau élevé des protéines mentionnées, dans laquelle le taux d'expression de protéines est renforcé par la substitution d'une séquence de nucléotide pour la région 5' du gène codant cette protéine, cette séquence ayant été optimisée pour l'utilisation comme codon de la levure. L'invention concerne également un vaccin comprenant des antigènes au polysaccharide méningococcique de groupe A (GAMP), au polysaccharide méningococcique de groupe B (GBMP) et au polysaccharide méningococcique de groupe C (GCMP), ainsi qu'un véhicule pharmaceutiquement acceptable. L'invention concerne également une méthode permettant de susciter une réponse immunitaire chez un mammifère, consistant à administrer le vaccin cité à un mammifère en quantité suffisante pour provoquer une réaction immunitaire.
PCT/US1997/001687 1996-02-01 1997-01-31 Expression de la proteine de la membrane exterieure de neisseria meningitidis du groupe b (mb3) a partir de levures et de vaccins WO1997028273A1 (fr)

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EP97906470A EP0877816A1 (fr) 1996-02-01 1997-01-31 Expression de la proteine de la membrane exterieure de neisseria meningitidis du groupe b (mb3) a partir de levures et de vaccins
AU21158/97A AU2115897A (en) 1996-02-01 1997-01-31 Expression of group b neisseria meningitidis outer membrane (mb3) protein from yeast and vaccines
CA 2244989 CA2244989A1 (fr) 1996-02-01 1997-01-31 Expression de la proteine de la membrane exterieure de neisseria meningitidis du groupe b (mb3) a partir de levures et de vaccins
JP52788197A JP2001508758A (ja) 1996-02-01 1997-01-31 グループｂナイセリア・メニンギチジス外膜（ｍｂ３）タンパク質の酵母からの発現およびワクチン
IL12542097A IL125420A0 (en) 1996-02-01 1997-01-31 Expression of group b neisseria meningitidis outer membrane (mb3) protein from yeast and vaccines containing said protein
NO983474A NO983474L (no) 1996-02-01 1998-07-28 Ekspresjon av gruppe B Neisseria meningitidis ytre membran (MB3) protein fra gjµr og vaksiner

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WO2000011182A1 (fr) * 1998-08-18 2000-03-02 Smithkline Beecham Biologicals S.A. Les basb024, proteines de la membrane exterieure du neisseria meningitidis
WO2000069456A2 (fr) 1999-05-13 2000-11-23 American Cyanamid Company Preparations de combinaisons d'adjuvants
WO2000071725A2 (fr) * 1999-05-19 2000-11-30 Chiron S.P.A. Compositions a base de combinaisons de neisseria
JP2001008690A (ja) * 1998-07-06 2001-01-16 Tosoh Corp Ｉｌ−６レセプター・ｉｌ−６直結融合蛋白質
EP1069133A1 (fr) * 1999-07-13 2001-01-17 Institut National De La Sante Et De La Recherche Medicale (Inserm) Compositions a base de Neisseria meningitidis et leur utilisation comme agents anti-infectieux
WO2001038350A2 (fr) * 1999-11-29 2001-05-31 Chiron Spa ANTIGENE DE NEISSERIA A 85kDa
WO2001064922A2 (fr) * 2000-02-28 2001-09-07 Chiron Spa Expression heterologue de proteines issues du gonocoque
US6451317B1 (en) 1997-07-17 2002-09-17 Baxter International Inc. Immunogenic conjugates comprising a Group B meningococcal porin and an H influenzae polysaccharide
JP2003514868A (ja) * 1999-11-29 2003-04-22 カイロン エセ．ピー．アー． 血清型ＢおよびＣ由来のＮｅｉｓｓｅｒｉａｍｅｎｉｎｇｉｔｉｄｉｓ抗原、ならびにさらなる抗原を含む組成物
JP2009114211A (ja) * 2001-06-20 2009-05-28 Chiron Srl 莢膜性多糖類の可溶化および組合せワクチン
US7604810B2 (en) 1999-04-30 2009-10-20 Novartis Vaccines And Diagnostics Srl Conserved Neisserial antigens
JP2010142247A (ja) * 1998-07-06 2010-07-01 Tosoh Corp Ｉｌ−６レセプター・ｉｌ−６直結融合蛋白質
EP2261352A2 (fr) 1998-05-01 2010-12-15 Novartis Vaccines and Diagnostics, Inc. Antigènes de Neisseria meningitidis et compositions
EP2275129A2 (fr) 2000-01-17 2011-01-19 Novartis Vaccines and Diagnostics S.r.l. Vaccin omv complèté contre la méningocoque
EP2289546A2 (fr) 2003-01-30 2011-03-02 Novartis Vaccines and Diagnostics S.r.l. Vaccins injectables contre les multiples serogroupes du meningocoque
US8663656B2 (en) 2002-10-11 2014-03-04 Novartis Ag Polypeptide-vaccines for broad protection against hypervirulent meningococcal lineages
WO2015073801A1 (fr) * 2013-11-14 2015-05-21 Duke University Amélioration de vaccins à base de cellules dendritiques par le toxoïde tétanique et ccl3
EP3085711A1 (fr) * 2015-04-23 2016-10-26 Institute of Medical Biology Chinese Academy of Medical Sciences & Peking Union Medical College Purification de polysaccharide capsulaire bactérien par système micellaire aqueux à deux phases
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PL328096A1 (en) 1999-01-04
AU2115897A (en) 1997-08-22
HUP9901039A2 (hu) 1999-07-28
JP2001508758A (ja) 2001-07-03
KR19990082265A (ko) 1999-11-25
NO983474D0 (no) 1998-07-28
NO983474L (no) 1998-09-30
EP0877816A1 (fr) 1998-11-18
IL125420A0 (en) 1999-03-12

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