WO1991007979A1 - Proteines chimeriques - Google Patents

Proteines chimeriques Download PDF

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Publication number
WO1991007979A1
WO1991007979A1 PCT/US1990/006811 US9006811W WO9107979A1 WO 1991007979 A1 WO1991007979 A1 WO 1991007979A1 US 9006811 W US9006811 W US 9006811W WO 9107979 A1 WO9107979 A1 WO 9107979A1
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protein
peptide
ctb
subunit
chimera
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PCT/US1990/006811
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English (en)
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Mark T. Dertzbaugh
Francis L. Macrina
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Center For Innovative Technology
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • 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
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the mucosal surfaces of the gut and oral cavity are constantly exposed to a large number of organisms which include pathogenic bacteria, fungi, viruses, and protozoa. Many of these pathogens use the mucosa as the principal portal of entry into the host, whereupon the systemic immune system eliminates the agent. Other pathogens do not have to invade the mucosa in order to be pathogenic, and systemic immunity is ineffective in these cases.
  • the mucosal- associated lymphoid tissue (MALT) probably developed in response to the need to protect the exterior surfaces of the host.
  • the concept of a common mucosal immune system is relatively new (McGhee and Michale , 1981). The predominant form of immunity at these sites is the release of dimeric secretory IgA (slgA).
  • Secretory IgA can prevent adherence of pathogens to the mucosa, neutralize viruses, and inactivate toxins (Tomasi, 1984). Induction of an immune response in the gut confers secretory immunity at distant sites of the mucosa such as the lungs, oral cavity, and the genitourinary tract. Antigen- primed lymphocytes are disseminated from the gut by the lymphatics to these sites where they mature into functional effector cells (McGhee and Michalek, 1981).
  • CT When co-fed, CT has been demonstrated to act as an adjuvant for other proteins which normally are not immunogenic when given orally (Lycke and Holmgren, 1986; Liang et al, 1988;' Nedrud et al, 1987).
  • Elson and Ealding (1984b) were able to abrogate tolerance to keyhole limpet hemocyanin (KLH) in mice that were fed this antigen mixed with CT.
  • KLH keyhole limpet hemocyanin
  • CT is produced by the enteric pathogen Vibrio cholerae and is composed of two subunits.
  • the toxinogenic A subunit is responsible for the ADP-ribosylation of eucaryotic adenylate cyclase. This modification increases cAMP activity, resulting in the diarrhea and fluid loss observed in patients with cholera (Betley et al, 1986).
  • the B subunit of cholera toxin (CTB) is composed of five identical subunits of 11.6 kDal joined together in a noncovalent association (Gill, 1976).
  • the CTB pentamer binds to the monosialoganglioside GM., (Cuatrecasas, 1973), which appears to be the natural ligand of the protein. This ganglioside is found in abundance on the surface of intestinal epithelial cells and probably facilitates entry of the A subunit into the cell.
  • CTB is non-toxic, it is still immunogenic when given orally (Lycke and Holmgren, 1987). This has encouraged attempts to use CTB, instead of the holotoxin, as a carrier or chemically created mucosal vaccines.
  • Bessen and Fischetti (1988) have induced protective immunity in mice that were intranasally immunized with an epitope from streptococcal M protein conjugated to CTB.
  • Some studies have suggested that CTB may act as more than just a simple carrier for antigens (McKenzie and Halsey, 1984; Ta ura et al, 1988). Moreover, it may be possible to increase the immunogenicity of the antigen even further by conjugating it to CTB.
  • Dental caries commonly occurs in people who consume sucrose as a part of their diet. Although the incidence of dental caries has been reduced in the general population, it is still a prevalent disease of man. For example, over 24 billion dollars was spent in the United States in 1984 to repair carious lesions (Loesche, 1986). Better oral hygiene has reduced the severity of the disease, but hygiene alone cannot eliminate the disease. The structure of the tooth surface contains fissures where S. mutans is more likely to accumulate and cause caries, even with good hygiene. Furthermore, proper mechanical debridement of the teeth is rather labor-intensive and, consequently, many people do not clean their teeth adequately. Currently, the only recourse to the problem of dental caries is to repair the damage after it has occurred. A more attractive approach is to prevent carious lesions from developing. A way in which to achieve this is to prevent colonization of the tooth surface by S. mutans.
  • S. mutans At present, two components of S. mutans have been identified as the best candidates for a subunit vaccine.
  • a component of the cell wall of S. mutans has been independently identified by two groups and designated either antigen I/II (Russell and Lehner, 1978) or SpaA (Holt et al, 1982).
  • Vaccination with this purified protein has been shown to reduce colonization and dental caries in monkeys (Lehner et al, 1980; Lehner et al, 1981), and it does not appear to induce heart cross-reactive antibodies (Bergmeier and Lehner, 1983).
  • the antibody response elicit was reported to be predominately serum IgG, which reaches the oral cavity via leakage into the gingival crevicular fluid.
  • the present inventors have surprisingly found that peptide sequences composed of only an epitope when fused to the N-terminal end of CTB elicit an antibody response, giving rise to a sustained antibody response with antibodies directed against the entire protein of interest.
  • peptide sequences composed of only an epitope when fused to the N-terminal end of CTB elicit an antibody response, giving rise to a sustained antibody response with antibodies directed against the entire protein of interest.
  • a 15 amino acid epitope of GtfB fused to CTB gives rise to antibodies against the GtfB protein which is over 1000 amino acids in composition.
  • Another object of the present invention is to provide chimeric peptides not toxic to a host which can be produced by recombinant DNA methods.
  • Yet another object is to provide a universal vaccine carrier that can be fused to form a chimeric peptide with any number of antigens or epitopes to elicit a secretory immune response to that specific antigen or epitope when the chimeric peptide is administered orally to a host. It is still another object to provide the recombinant methods, vectors and host organisms that produce peptides for effective oral vaccine compositions.
  • a chimeric peptide including the B subunit of cholera toxin and an epitope region of a desired antigen fused to the N-terminal end of the B subunit of cholera toxin.
  • the epitope region includes an antigenic determinant of the desired peptide.
  • Such chimeric peptides can be used for an oral vaccine according to certain preferred embodiments in which the epitope fused to the B subunit of cholera toxin gives rise to an immune response, producing antibodies directed against the peptide of interest.
  • Recombinant methods for producing a chimeric peptide, and the DNA sequences, vectors and hosts used in such recombinant methods are also provided.
  • an epitope of the GtfB peptide is fused to the CTB peptide to provide an oral vaccine for the prevention of dental caries.
  • Fig. 1 Construction of the ctxB fusion vectors. Plasmid pVA1662 which contains the ctxB gene (shaded area) was digested with EcoRI and Ndel to delete the 5' end of the gene. Then synthetic linkers with compatible ends were inserted and ligated to create the fusion vectors shown.
  • Fig. 2 Construction of pVA1555.
  • Plasmid pHK7 which contains a 1.9 kb insert encoding a truncated portion of the gtfB gene of Streptococcus mutans (stippled area), was inserted into pVA1542 as a Pstl-EcoRI fragment upstream of the ctxB gene (shaded area).
  • the resulting plasmid, pVA1555 expresses a gtfB::ctxB fusion protein of 58 kDal in E. coli.
  • Plasmid pVA1542 contained a promoterless version of the ctxB gene of Vibrio cholerae (stippled area) that lacked DNA encoding the first 17 amino acids of the leader sequence of the protein.
  • the synthetic oligonucleotide encoding gtfB.l was inserted into pVA1542 as an EcoRI-Ndel fragment to create pVA1599.
  • the resulting gtfB.1; ;ctxB gene fusion could be inserted easily into a number of expression vectors as an EcoRI-BamHI fragment, if desired.
  • the secretion vector pINIII omp_A2 contained DNA encoding the leader sequence of ompA (striped area) , an outer membrane protein of E___ coli, under control of the inducible lac promoter. Promoter activity is inhibited by repressor, the product of the laci gene, until induction by IPTG. Insertion of the gtfB.l: :ctxB gene into this vector resulted in expression of a chimeric protein of 14.4 kDal upon induction with IPTG.
  • Fig. 4 Linker sequences of the fusion vectors. These oligonucleotides were used as a translational junction between gtfB and ctxB. The number above each linker designates the plasmid vector containing the sequence. Bold ⁇ faced letters correspond to sequences actually encoded by the synthetic linkers. Important restriction endonuclease sites are shown above each linker; amino acid sequences are located below each linker.
  • Fig. 5 The sequence of the gtfB.l peptide.
  • the hydrophilicity plot is shown for a portion of the glucosyltransferase B enzyme of the cariogenic bacterium Streptococcus mutans.
  • the region encoding amino acid residues 345-359, corresponding to peptide gtfB.l, is shown in black.
  • the gtfB.l peptide (shaded area) was inserted between the leader sequence for the ompA gene of JL, coli and the B subunit gene of cholera toxin (jctxB) .
  • the cleavage site for ompA is indicated by the arrow.
  • the numbers above the amino acid sequence correspond to the residue number of the mature, secreted form of the chimeric protein.
  • the corresponding DNA sequence is listed above, along with important restriction sites.
  • Bold letters indicate the se uence encoded by the synthetic oligonucleotide used to construct the gene fusion.
  • Fig. 7 Immunogenicity of the chimera. Protein samples were fractionated by 10% SDS-PAGE and electrophoretically transferred to nitrocellulose sheets. Replicate blots were probed with antiserum to either CTB, GtfB, or chimera. Arrows denote immunoreactive proteins of interest. Lane 1, extracellular proteins form S_j_ mutans GS-5; VI792 chimera; lane 3, CTB.
  • Fig. 8 Kinetics of enzyme inhibition. Total glucan synthesis was measured over time (in hours). The enzyme was preincubated in either PBS (control) , normal rabbit serum (N.R.S.), anti-GtfB serum, or anti-Chimera serum before addition to the reaction mixture. All sampling was performed in triplicate.
  • Fig. 9 Differential inhibition of glucan synthesis. Protein samples preincubated in either PBS (control), anti-GtfB, or anti-chimera serum were assayed for differential glucan synthesis. The reactions proceeded for 8 h at 37°C before measuring activity. The samples were assayed in triplicate for both total glucans and water- insoluble glucans. Total glucans were precipitated with methanol. Water-insoluble glucans were precipitated with deionized water. The amount of water-soluble glucans produced was determined by subtracting insoluble glucans from total glucans. The activity of the samples in each assay was determined relative to the control. The activity of the control was normalized to 100%.
  • Fig. 10 Inhibition of fructan synthesis.
  • Extracellular protein from JjL. mutans GS-5 was preincubated for 1 h at 37°C in either PBS (control), normal rabbit serum
  • Fig. 11A The DNA sequence encoding the GtfB.l/CTB chimeric peptide.
  • Fig. 11B The amino acid sequence of the GtfB.l/CTB chimeric peptide.
  • the present invention shows that, using recombinant DNA techniques, plasmids can be constructed that can be transfected into hosts and chimeric, immunogenically active peptides are expressed which include an epitope of the peptide of interest fused to the N-terminal end of CTB.
  • the relative small size of the epitope amino acid sequence fused to the CTB reduces the chance that the structure or function of CTB will be altered. Large peptides fused to CTB are more likely to adversely affect the binding of CTB to mucosal tissue. Such alterations adversely affect the ability of the chimeric CTB peptide to elicit a secretory immune response.
  • an epitope should have the following characteristics.
  • An epitope is defined as an antigenic determinant or that portion of an antigenic molecule that interacts by molecular complementarity with the antigen combining site of an immunoglobulin molecule.
  • An epitope is a discrete portion of an entire antigen molecule which can trigger the immune response. As defined in the present invention, the epitope need not actually trigger an immune response by itself, but includes the particular region of the antigen which brings about the proper interaction to raise antibodies against that antigen when the epitope is fused to CTB.
  • the epitope should have little to no impact on CTB interaction with mucosal tissue when the epitope is fused to the CTB.
  • the epitope should be no greater than 100 amino acids long and no greater than 11 kDal in size.
  • the epitopes according to the present invention are in the range of about 10 to 30 amino acids long, or 15-20 amino acids long.
  • an antibody binding site or epitope By synthesizing an oligonucleotide corresponding to a domain of a protein with these properties, an antibody binding site or epitope can be identified.
  • Available algorithms such as those included in a software program known as MICROGENIE, from BECKMAN INSTRUMENTS, Palo Alto, California, may assist in finding epitope regions.
  • the hydrophilicity of regions of a protein can be determined using the algorithm of Hopp and Woods (1983). Secondary structure predictions of this region can also be made using the algorithm of Gamier et al. (1978). Domains that are identified which have both high segmental mobility and hydrophilicity can then be screened to ensure that these regions comprise an antigenic determinant or epitope.
  • such regions can be screened by fractionating proteins into peptides, isolating the peptides containing the putative epitope of interest, and determining its reactivity with antisera to the protein of interest.
  • expression vectors could be prepared that produce chimeric peptides, including the regions to be screened fused to CTB, and testing the chimeric peptides to determine if such peptides raise antibodies directed against the protein of interest.
  • Such peptides could be fused to the CTB as described in the present application to form an oral vaccine.
  • Residues 141-158 and 200-213 of virus coat protein I form the Kaufbeuren strain of Foot and Mouth Disease Virus, a disease of cattle (DiMarchi, et al.. Science 232:639-641, 1986).
  • the conserved region of the M Protein of group A streptococci (residues 216-235, 248-269, 275-284), responsible for pharyngitis (Bessen and Fischetti, Infect. Immun. 56:2666-2672, 1988).
  • the pilin subunit of Hae ophilus influenza (Brinton, et al., Pediatrc. Infec. Dis. 8:(Suppl) S51-61. Fimbrial subunit of enterotoxic colibacillosis in neonatal piglets (Greenwood, et al., Vaccine 6:389-92, 1988). Active site residues of Pseudomonas aeruginosa exotoxin A (Lukac, et al., Infect. Immun. 56:3095-3098, 1988). Peptides corresponding to the cleavage region of VP3 protein of rotavirus which induce neutralizing antibodies (Streckert et al., J. Virol. 62:4265-4269, 1988).
  • Another nonexhaustive list below includes further antigens which include epitopes which can be fused to CTB to produce oral vaccines.
  • epitopes of the following list have not been characterized, one of ordinary skill in the art should be able to determine those regions using the methods described herein. Those methods include, but are not limited to, the use of algorithms to locate areas which are hydrophilic and which have high segmental mobility, preparing chimeric proteins with plasmids according to the present invention and screening those chimeric proteins for their ability to elicit an immune response.
  • peptide fragments identified by the algorithm method can be tested for cross-reaction with antisera to the protein of interest to screen for appropriate epitopes for use in the present chimeric peptide, oral vaccines.
  • gtfC the glucosyltransferase C enzyme produced by Streptococcus mutans, the causative agent of dental caries.
  • ftf the fructosyltransferase enzyme produced by Streptococcus mutans, the causative agent of dental caries.
  • Type I fimbrial subunit (an adherence protein) of Streptococcus sanguis a bacterium involved in early stages of dental plaque formation. Tetanus toxin of Clostridium tetani, the causative agent of tetanus.
  • Invasin protein of Yersina pseudotuberculosis The hemagglutinin (HA) antigen of influenza virus, the causative agent of flu, Tamura et al.. Vaccine 6:409-413 (1988).
  • the pilin subunit of Bacteroides nodosus the causative agent of foot rot of sheep.
  • Surface antigens of human papilloma virus the causative agent of genital warts.
  • Envelope proteins of herpes simplex virus Tumor-associated antigens in melanomas and carcinomas.
  • the utility of the fusion vectors for producing chimeric peptides has been demonstrated by the construction according to the present invention of the V1555 chimeric protein containing CTB and a portion of the GTF protein from S. mutans.
  • Construction of CTB fusion vectors, using synthetic oligonucleotide linkers, has been shown here to be a practical method of producing chimeric proteins for use as vaccines.
  • the use of synthetic linkers provides a convenient method of inserting a spacer of any length, rigidity, and composition between the two domains of the chimera.
  • the following examples show the use of vectors for the construction of gene fusions using DNA sequences from S. mutans. However, the vectors may be used to make fusions to other DNA as well.
  • an oligonucleotide specifying the immunologically important domains could be synthesized and inserted into the vectors.
  • the use of these peptide sequences would increase the specificity of the immune response, and impose minimal conformational changes on CTB which could affect its immunogenicity.
  • An ideal fusion vector would contain an inducible promoter anr ribosomal binding site, followed by a polylinker cloning site, and then the ctxB gene. This construct would allow almost any DNA sequence to be inserted into it and express a CTB fusion protein.
  • V1555 chimeric protein demonstrated the feasibility of constructing gene fusions with CTB, there were several problems which affected the usefulness of this protein.
  • immunoblotting analysis revealed the presence of 2 major species of 58 and 72 kDal, instead of the 58 kDal protein predicted by the nucleotide sequence data.
  • Sufficient DNA is located upstream of the start codon for gtfB to encode a protein of that size.
  • V1619 is an E_. coli strain that contains pHK7 and expresses the truncated gtfB gene used in the fusion. Second, the leader sequence of gtfB was not recognized in E . coli, which resulted in the protein being trapped in the cytoplasm. This made extraction of the protein difficult and contributed additional contaminants which had to be removed.
  • the protein could not be exported from the cytoplasm, it was degraded by intracellular proteases. The resulting degradation products were co-eluted on the GM.. affinity column, and produced a heterogeneous mixture of proteins which could not be easily resolved from one another.
  • the addition of the 46 kDal GtfB moiety to CTB appeared to affect the structure of the protein, based on the reduced affinity of the V1555 chimera for GM... This was not surprising, since the GtfB moiety is over 3 times the size of CTB. Genetic fusion of such a large protein to CTB appeared to interfere with its function which, in turn, could affect the putative adjuvant properties of CTB. For these reasons, it was concluded that the V1555 chimera was not an ideal model system for vaccine studies.
  • the present inventors constructed the V1782 chimera.
  • the present inventors believed that fusion of smaller peptides than those expressed from the V1555 construct to CTB would probably minimize the effect they had on the structure and function of the protein.
  • a putative antibody binding site of the GtfB enzyme had been identified by the present inventors as a result of the degradation of the V1555 chimera (see Example 11(D)).
  • the precise location of this site was unknown and was too large to synthesize a corresponding oligonucleotide to the entire degradation product.
  • regions of the protein being hydrophilic and having high segmental mobility were located. It was assumed that the epitope possessed these predicted characteristics.
  • the secretion vector system used to make V1782 chimera provided a convenient method of constructing and expressing small peptides fused to CTB. It improved an earlier system by using the inducible lac promoter and a signal peptide sequence to secrete the fusion protein into the periplasm of ____. coli. This system is similar to one which has been used to overproduce CTB for production of cholera vaccine (Sanchez and Holmgren, 1989). Placement of the chimera under control of an inducible promoter such as lac permitted overproduction of the protein upon induction with IPTG. By fusing the chimera to the peptide leader sequence of ompA, the protein could be transported to the periplasm where it was easier to isolate and purify.
  • the ompA leader was properly recognized in E ⁇ _ coli, even though it was fused to a foreign protein.
  • the secretion of foreign proteins using the ompA leader has been previously described (Ghrayeb et al, 1984). Because of the way in which the ompA leader was fused to the chimera, two additional amino acids were left at the N-terminus upon processing by E. coli. Although these additional residues could be removed by oliogonucleotide-directed mutagenesi ⁇ (Ghrayeb et al, 1984), they did not appear to interfere with the antigenicity or structure of the chimera, and were thus left intact.
  • vectors that can be used to express the chimeric peptide fusions once they are made in pVA1542, pVA1543 or pVA1544, with or without the ompA vector include pKK223-3 (J. Brosius, Gene 27:151-160, 1984), and pET translation vectors. These examples are in no way limiting and any number of vectors could be used for expressing the chimeric peptide.
  • the present invention shows that peptides can be fused to CTB with minimal effect on the structure of the protein.
  • chimeras can be constructed that retain the antigenicity of each moiety, which is important in the use of these proteins as subunit vaccines.
  • the ability to oligomerize and to bind to GM shows the liklihood that this protein will be selected out of the intestinal milieu and will induce an immune response in the Peyer's patches.
  • the V1782 chimera retains these properties, as well as much of the native structure of CTB.
  • the present examples are directed to the fusion of a portion of GtfB to CTB, the CTB vectors described herein can be used to fuse any number of suitable epitope regions to CTB to produce effective oral vaccines.
  • the particular 15 amino acid sequence of GtfB included in the chimeric peptide expressed by V1782 is in no way a limiting example. Extending the length of either end of that 15 amino acid sequence or shifting the span of that sequence on the GtfB sequence are also contemplated.
  • a vaccination regimen it is also contemplated to administer a combination of different chimeric peptides which include different portions of an antigen.
  • Spherosil XOC-005 was purchased from Supelco (Beliefonte, PA) .
  • Goat anti-CTB was purchased from Calbiochem (LaJolla, CA) .
  • Phosphatase-labeled second antibodies and BCIP/NBT substrate were purchased from
  • Plasmid pJBK30 containing a promoterless version of the ctxB gene from the El Tor strain of Vibrio cholerae, was obtained from J.B. Kaper, University of Maryland School of Medicine, Baltimore, MD (Kaper et al, 1984). Plasmid pHK7 was provided by H.K. Kuramitsu, Northwestern University, Chicago, IL. This plasmid encoded the amino terminal one-third of the gtfB gene of Si mutans GS-5 (Aoki et al, 1987).
  • Plasmid DNA was prepared by SDS- high salt lysis (Guerry et al, 1973) followed by dye-buoyant density equilibrium centrifugation (Welch et al, 1979).
  • E. coli HB101 was transformed with plasmid DNA by the CaCl 2 -heat shock method (Morrison, 1977). The cells were grown at 37°C in M-9 media (Miller, 1972) supplemented with 10 mg/ml casamino acids (Difco), 20 g/ml leucine and proline, and 2mg/ ml thiamine. V1555 was grown to an O.D.
  • 660 0.6, washed twice in phosphate-buffered saline (PBS, pH 7.3), and then lysed in a French pressure cell (SLM Aminco; Urbana, IL) . The lysate was clarified by centrifugation before use.
  • SLM Aminco Urbana, IL
  • Extracellular proteins of S ⁇ mutans were obtained as follows.
  • the remaining sample was diluted in PBS + 6 M urea and subjected to diafiltration through a YM-100 ultrafiltration membrane (Amicon; Danvers, MA).
  • the clarified sample was concentrated by ultrafiltration to 20 ml and then dialyzed in PBS (pH 7.3) in order to remove the urea.
  • the samples were aliquoted and stored at -70°C. This preparation was used as a control for immunoblotting analyses.
  • Rabbit antiserum to the glucosyltransferase B enzyme of S. mutans GS-5 was obtained from H.K. Kuramitsu, Northwestern University, Chicago, IL.
  • Antiserum to the purified, monomeric form of the V1782 chimeric protein was raised in female New Zealand White rabbits.
  • One mg of protein was emulsified in complete Freund's adjuvant and injected subcutaneously into the hind quarters of the animal.
  • the rabbit was boosted with the protein emulsifed in incomplete Freund's adjuvant. Then, one week later, the rabbit was bled.
  • the serum was collected, aliquoted, and stored at -20°C.
  • Oligonucleotides and their complementary strands were synthesized on an Applied Biosystems 380A synthesizer.
  • the linkers were isolated by thin-layer chromatography on Kieselgel 60 F -. silica gel plates (Merck; Darmstadt, W. Germany) in 1-propanol, ammonium hydroxide, and water (55:35:10).
  • the DNA bands were visualized by a hand-held UV light and then scraped off the plate.
  • the DNA was extracted by washing the silica gel three times with water, and then concentrated by evaporating the aqueous phase to dryness.
  • the larger oligonucleotides were separated by using a 20% polyacrylamide gel containing 8 M urea.
  • Plasmid pJBK30 containing the promoterless ctxB gene, was prepared for insertion of the linkers by deleting two restriction sites that could interfere with their placement.
  • the Ndel site, located near the origin of replication of the plasmid, and the EcoRI site located at the 3' end of the ctxB insert were both deleted from the plasmid (Fig. 1).
  • Plasmid pJBK30 was partially digested by restriction enzyme and the linearized DNA was isolated by electroelution from agarose gels (Maniatis et al, 1982). The 5' ends were filled-in using DNA polymerase I (Klenow fragment) and these blunt ends were ligated together using appropriate reaction conditions (Maniatis et al, 1982).
  • Plasmid pVA1662 was digested with Ndel and EcoRI and the larger of the two resulting fragments was isolated by electroelution from an agarose gel. The linkers were inserted into this fragment as described in Example 1(E) (also see Fig. 4). The plasmid was transformed into E___ coli HB101, and clones that were tetracycline (Tc)- and ampicillin (Ap)-resistant were selected and screened for insertion of the linker by restriction mapping. Plasmids containing each linker were isolated and designated pVA1542, pVA1543, and pVA1544. These vectors contained a truncated version of the ctxB gene in which the 5' end of the gene was deleted to remove the ribosomal binding site and DNA encoding the leader sequence of the protein. G. Gene Fusions
  • V1555 was constructed as shown in Figure 2. Plasmid pHK7 contained a 1.9 kb Pstl-EcoRI fragment from S___ mutans GS-5, Bratthall serotype c (Ha ada and Slade, 1980), that encoded a truncated portion of gtfB. The Pstl-EcoRI fragment from pHK7 was directionally cloned into each fusion vector. E. coli transformants that were Tc-resistant and Ap-sensitive were screened for expression of a protein that was recognized by antiserum to CTB. A clone was selected, containing a plasmid derived from pVA1542, and designated V1555.
  • V1782 The strategy used to construct V1782 is shown in Figure 3.
  • the oligonucleotides were inserted into the fusion vector pVA1542 at the 5' end of ctxB as previously described in part 1(E). Proper insertion of the oligonucleotides into pVA1542 was monitored by restriction enzyme digestion of plasmid DNA prepared from minilysates (Macrina et al, 1982). In order to express the translational fusion protein, the chimeric ctxB gene was transferred to the secretion vector pINIII ompA2. H. Screening
  • Plasmid DNA was obtained from cells by a rapid minilysis procedure (Macrina et al, 1982). The DNA was checked for plasmid content by agarose gel electrophoresis and restriction enzyme analysis. For protein analysis, a whole-cell lysate of the clones was prepared by a modification of the DNA minilysis procedure. Cells containing the secretion vector were screened for expression of a chimeric protein by first cultivating them on M-9 media containing 1 mM IPTG. After digestion in lysozyme, the cells were suspended in 0.5 ml of a hypotonic solution (10 mM Tris, 30 mM NaCl, pH 7.3) and then subjected to a rapid freeze- thaw.
  • a hypotonic solution (10 mM Tris, 30 mM NaCl, pH 7.3
  • a GM.. ganglioside affinity column was prepared and used to purify the CTB chimeras according to the method of Tayot et al (1981). Lyso-GM, was prepared from 50 mg of GM., and then covalently coupled to 15 g of DEAE-dextan coated Spherosil. The samples were recirculated through the column for 24 h at 4°C and at a linear flow rate of 1.5 ml/min. The column was washed with 10 volumes of wash buffer (10 mM NaPO. , 200 mM NaCl, pH 6.8) and then the chimera was released using elution buffer (50 mM citrate, 200 mM NaCl, pH 3.0).
  • the eluate was adjusted to pH 7.3 and then concentrated by diafiltration in PBS (10 mM NaP0 4 140 mM NaCl, pH 7.3) using a YM10 ultrafiltration membrane (Amicon; Danvers, MA).
  • the retentate was quantitated for total protein as well as for reactivity in the ELISA.
  • Native CTB was used as the reference standard for the ELISA.
  • a 1.6 x 200 cm Sephadex G-100 column was prepared and used to separate monomeric and oligomeric fractions of the V1782 chimera. A 5 mg sample was loaded onto the column and eluted in PBS (pH 7.3) at a linear flow rate of 0.13 ml/min.
  • the N-terminal sequence of the monomeric protein (40 g) was determined using an Applied Biosystems Model 470A Sequencer, with in-line PTH-amino acid analysis.
  • the peptides produced by cyanogen bromide cleavage were separated by high performance liquid chromatography on a C-18 reverse phase column (Varian), using a linear gradient of 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in 50% acetonitrile. A flow rate of 1 ml/min and a total time of 60 min was used. The effluent was monitored at 215 nm. Peaks were collected, and analyzed by amino acid analysis.
  • Samples of protein or peptides were hydrolyzed in sealed, evacuated tubes with constant boiling HCl for 24 hr at 110°C. Amino acid analyses were performed with a Durru MBF amino acid analyzer using o-phthaldehyde as the detection reagent. In some cases, the samples were alkylated with iodoacetamide prior to hydrolysis. Each sample was reacted with iodoacetamide (1 mole sample/10 moles iodoacetamide) in 50 mM Tris (pH 8.0) for 1 h at 25°C.
  • the monomer, oligomer, and CTB were analyzed by circular dichroism. Samples were extensively dialyzed against PBS (pH 7.3) using a Centricon 30 (Amicon) . The filtrate was saved for use as the buffer blank. A 1.5 ml sample (0.15 mg/ml) of each protein was loaded into a cell and analyzed on a Jasco J-500C spectropolarimeter.
  • Protein samples were separated by SDS-PAGE and then electrophoretically transferred to nitrocellulose sheets (Towbin et al, 1979). Nonspecific binding of antibody was prevented by blocking the sheets for 1 h at 25°C with a 5% solution of chicken serum in tris-buffered saline (TBS; 20 mM Tris, 500 mM NaCl, pH 7.5). The sheets were incubated for 6 h in primary antisera diluted 1:1000 in TBS with 5% chicken serum. The sheets were washed 3 times for 10 min in TBS with 0.05% Tween 20 (TTBS), and then incubated for 2 h in enzyme- labeled second antibody. After washing again 3 times in TTBS, the sheets were developed with substrate. L. Enzyme Assays
  • Glucosyltransferase activity was determined by measuring the amount of 14C-glucose converted into glucan polymer from specifically labeled sucrose.
  • Fructosyltransferase activity was measured by a similar means, except that sucrose labeled with 14C in the fructose moiety was used as the substrate.
  • the reaction mixture was incubated at 37°C and consisted of 10 ⁇ l enzyme,
  • mice Male C57B1/6 mice can be obtained from Charles River (Wilmington, MA) . The mice are used at 6-8 weeks of age, and should be fasted overnight before feeding the protein.
  • the chimera is administered using an intragastric feeding needle (G. Tiemann & Sons, Long Island, NY).
  • the GM..-purified chimera is diluted in 0.2 M Na ⁇ HCO-. and is delivered in a volume of 0.5 ml. Control animals receive saline solution.
  • the antigen is injected intraperitoneally into some animals. After the antigen is diluted in MAALOX, it was injected in a volume of 0.1 ml.
  • mice are immunized on day 0 and 14. On day 21, serum and intestinal secretions are collected and assayed to assay for specific antibody. Microtiter wells are coated with either CTB or synthetic peptide composed of the same 15 amino acid sequence as found in the chimera.
  • the assay for detecting peptide-specific antibody in intestinal washings should be sensitive enough such that 0.01 ng/ml of peptide-specific antibody may be detected.
  • a set of cloning vectors have been developed for constructing and expressing ctxB gene fusions in E ⁇ _ coli.
  • the plasmid pJBK30 was used as the basis of construction of the fusion vectors because it contained a promoterless ctxB gene and did not express this protein in E ⁇ coli.
  • an ELISA could be used to detect successful fusion, because the gene would only be expressed if it were translationally in-frame with sequences located upstream.
  • the linkers were designed to be compatible with any DNA that contained an EcoRI site within its open reading frame (Fig. 4) .
  • the 3' end of the linkers encoded a sequence compatible with an Ndel site located within the open reading frame of ctxB. Only one reading frame in each linker allowed translation of ctxB to occur. Although translation relative to the EcoRI site occurred in a different reading frame, the length of the linkers was adjusted so that the last codon terminated at the junction of the Ndel site. Sequencing data has shown that this junction separates codons translated within ctxB (Lockman and Kaper, 1983).
  • This fragment contained in pHK7 (Fig. 2), constitutively expressed a non- enzymatically active 48 kDal truncated polypeptide that cross-reacted with antiserum to GtfB.
  • the immunoblotting analysis of the V1555 chimera peptide was performed by purifying the peptide by GM.. ganglioside affinity chromatography. A clarified whole cell lysate from E ⁇ . coli strain V1555 was loaded onto the column (lysate).
  • Bound material was eluted from the column by decreasing the pH stepwise (pH 4, pH 3). The column was cleaned by washing it with 0.1 M NaOH. Duplicate blots were probed either with antiserum to glucosyltransferase B (anti-GTF) or to the B subunit of cholera toxin (anti-CTB) . Purified CTB and a lysate of V1619 were used as positive controls for the antisera. E_i. coli strain V1619 contains the plasmid pHK7 which expresses a 46 kDal truncated GTF protein.
  • V1555 revealed that the chimeric protein was trapped in the cytoplasm of !___. coli.
  • the leader sequence for GtfB was not recognized by the host, even though this protein is normally secreted by £___. mutans. This phenomenon has been observed with other extracellular streptococcal proteins that are expressed in EL_ coli (Aoki et al, 1986).
  • the cells had to be lysed in a French pressure cell. Once the cells were lysed, the protein was ready to be purified by affinity chromatography.
  • CTB has a high affinity for GM., ganglioside (Sillerud et al, 1980).
  • GM. ganglioside affinity column has been shown previously to be a rapid and efficient method of purifying cholera toxin (Tayot et al, 1981).
  • the lysate was recirculated through the column overnight at 4°C. The majority of the activity was eluted at pH 4, which was higher than that required to elute native cholera toxin from the column (Tayot et al, 1981).
  • Immunoblotting analysis of the column eluate revealed the presence of two novel proteins of 58 kDal and 72 kDal that cross-reacted with antisera to either GtfB or CTB. Based on the published DNA sequences of both genes, the chimera was predicted to be 58 kDal in size. The presence of the 72 kDal protein currently cannot be accounted for, but it does cross- react with antisera to both GtfB and CTB. Passage of the V1555 lysate through the affinity column was effective for removing many of the contaminating proteins from IS___ coli. Cleaning the column with 0.1 M NaOH only eluted a small amount of additional immunoreactive material, indicating that elution of the chimera was complete.
  • FIG. 5 One such domain, gtfB.1, is shown in Figure 5.
  • This domain was relatively hydrophilic and was devoid of a highly organized secondary structure.
  • a set of synthetic oligonucleotides were synthesized which were compatible with the amino acid sequence of this region.
  • the gtfB.l oligonucleotides were genetically fused to the promoterless ctxB gene in pVA1542 (Fig. 3). Insertion of gtfB.l into pVA1542 did not result in expression of ctxB, due to the lack of a promoter and ribosomal binding site.
  • V1782 chimera was able to express a chimeric protein that was immunoreactive with antiserum to CTB or to GtfB.
  • Immunoblotting analysis of the V1782 chimera was performed by preparing lysates from independently isolated clones from cells grown on M-9 agar plates containing 1 mM IPTG. The samples were separated by 15% SDS-PAGE under reducing conditions and then electrophoretically transferred to nitrocellulose sheets. Proteins that reacted with antiserum to the glucosyltransferase B enzyme are indicated by arrows.
  • the V1555 lysate contains two truncated versions of GtfB, and was used as a control. Immunoreactive proteins were detected in the lysates from V1555 and the clones expressing gtfB.l, but not from V1784 which was used as the background control.
  • the ability to overproduce the chimeric protein is a desirable trait for large-scale vaccine production.
  • the cells were allowed to express for 2 h before harvesting the contents of the periplasm by osmotic shock.
  • parallel cultures of each strain were grown in the absence of IPTG. Samples of the shock fluid were separated by 15% SDS-PAGE and then blotted to nitrocellulose. The blots were reacted with antiserum to CTB.
  • the chimeric protein was purified by GM. affinity chromatography, based on the affinity of the CTB moiety for GMi.
  • the column was used to concentrate the chimera from the shock fluid by recirculating the fluid through the column. Saturation at 4°C was achieved within 24 h of initial loading of the column. The chimera was eluted from the column only upon decreasing to pH 3.0. The use of a higher pH did not result in elution of the protein.
  • Table III lists the purification data from a typical experiment. Approximately 23 g of protein could be collected from one elution. Silver- staining of a sample from the column, separated by SDS-PAGE, revealed that most contaminating proteins were removed by this method. No proteolysis of the protein was observed as was seen in the V1555 chimera.
  • a sample of the affinity-purified chimera was loaded onto a gel filtration column (Sephadex G-100) and eluted in PBS. Gel filtration profile of the V1782 chimera.
  • the GM 1 column-purified chimera was fractionated through a 1.6 x 200 cm column containing Sephadex G-100. Samples of the column fractions were reduced and then separated by 15% SDS-PAGE. Proteins were visualized by silver-staining. Activity was measured by an ELISA assay, which depends on the ability of the chimera to bind to GM.-coated wells and to be recognized by antiserum to CTB. Total protein was measured using the BCA assay (Pierce). The sample was fractionated into two major size classes.
  • the monomeric form of the V1782 chimera was subjected to 25 cycles of Edman degradation.
  • the amino acid sequence was found to be identical to that predicted by the construction of such a fusion protein (Fig. 5).
  • the leader sequence of the ompA protein was absent from the periplasmic form of the protein. Fusion of the chimeric protein to the ompA leader did not appear to affect the ability of E_ ⁇ . coli to recognize and cleave the signal sequence.
  • the data also suggested the absence of any additional N-terminal sequences in the sample.
  • the amino acid composition of the C-terminal ends of monomeric and oligomeric forms of the chimera were also compared to that of native CTB. Protein samples were cleaved by reaction with cyanogen bromide and the peptide fragments were fractionated by HPLC. Elution of the protein was monitored by absorbance at 215 nm. The resultant peptides were then analyzed for their total amino acid composition (Table IV) . DNA sequence data for the ctxB gene predicted that cyanogen bromide cleavage would release an ala-asn dipeptide fragment if translation of the genes terminated properly in Ei coli (Lockman and Kaper, 1983). Table IV shows that peptide A had this composition. This dipeptide was isolated from all three samples.
  • the monomeric and oligomeric forms of the chimera were analyzed for the presence of disulfide bonds by alkylation of the proteins with iodoacetamide.
  • the alkylated samples were hydrolyzed and then analyzed for their amino acid composition. After alkylation with iodoacetamide, 2.2 moles of carboxymethylcysteine per mole of protein were found for the monomer, while none was detected in the oligomer. This was consistent with the monomer having two free sulfhydryl groups, and suggested that the monomeric fraction contained a reduced form of the chimera which lacked the proper intramolecular disulfide bond.
  • Antiserum to the chimera was evaluated for its potential to inhibit glucosyltransferase activity in vitro. The specificity of the assay was verified before performing the inhibition studies. Extracellular proteins of S_ s _ mutans
  • GS-5 were assayed for glucosyltransferase activity by measuring the incorporation of 14C-(glu)-sucrose into glucan polymer. Total polymer was precipitated in methanol and collected on glass fiber filters. In order to determine if enzyme activity was actually being measured, the assay was verified in two ways: (A) , glucan synthesis was shown to increase linearly over 20 hours of incubation; (B), the amount of glucan synthesized was inversely proportional to the amount of protein used. Sampling was performed in triplicate. The mean counts per minute for each sample was subtracted from a background control containing PBS. It was demonstrated that the assay increased linearly over time and that dilution of the enzyme resulted in a decrease in polymer formation.
  • the enzyme preparation was diluted 1:2 in either PBS (control), pooled normal rabbit serum (N.R.S.), rabbit anti- GtfB serum, or rabbit anti-chimera serum.
  • the samples were then preincubated at 37°C for 1 h before performing the assay. Total glucan was measured after the reaction proceeded for 18 h at 37°C. All samples were performed in triplicate. Pre-incubation of the enzyme in undiluted antiserum to either the chimera or GtfB resulted in a reduction in total glucan synthesis, relative to the control. Pooled normal rabbit serum (NRS) did not inhibit enzyme activity. In order to assess the kinetics of inhibition, enzyme activity was measured over time.
  • the antisera were compared for their level of inhibition by dilution. Antiserum to either GtfB or the chimera was diluted in PBS prior to preincubation with the enzyme. The synthesis of total glucans was measured after the reaction had proceeded for 8 h at 37°C. Sampling was performed in triplicate. Results indicated that the ability of the anti-chimera antiserum to inhibit activity was approximately half that of the anti-GTF antiserum. Again, this was not surprising due to the limited specificity of the antiserum to the chimera. Water-insoluble glucans have been implicated as having a greater role in the accumulation of S. mutans to the tooth surface than water-soluble polymers.
  • the primary structure of the V1782 chimera was verified by sequence and compositional analysis.
  • the amino acid composition of the chimera agreed with that predicted by the nucleotide sequence data.
  • the sequence of the first 25 amino acid residues of the mature form of the chimera was determined, and verified that the ompA leader was cleaved at the site normally recognized by E_j_ coli (Ghrayeb et al, 1984). Cyanogen bromide cleavage provided a convenient method of isolating the C-terminal dipeptide fragment of the chimera and comparing it to that of CTB.
  • the C-terminal end of the chimera was no different from that of CTB, thus confirming that translation of the chimera was terminated at the stop codon normally recognized for CTB.
  • the monomer was still immunoreactive in a Western blot, suggesting that it lacked the disulfide bond required for GM. binding. This was verified by reacting samples of the monomer and oligomer with iodoacetamide. Only the monomer was found to contain reduced forms of cysteine. In order for the monomer to co-elute from the affinity column, it must have been able to bind to GM..
  • the subunits of CTB are reversibly dissociated by acidification (Hardy et al, 1988), and it may be that a fraction of the chimera was dissociated and reduced during elution from the column.
  • the monomer can be renatured by gradually dialyzing it away from 8 M urea under alkaline conditions. Renaturation resulted in the formation of oligomers and reactivity in ELISA.
  • the disulfide bridge of CTB appears to have a drastic effect on its conformation and function, since the absence of it precludes binding of chimera to GM. and forming oligomers.
  • the results reported here with the chimera are consistent with those reported for native CTB and suggest that addition of a peptide to the N- terminal end of CTB has minimal effect on its ability to bind to its ligand or to form oligomers.
  • gtfB.l peptide was immunogenic, and that antiserum to the peptide could recognize the native protein.
  • the gtfB.l peptide was derived from the sequence of gtfB enzyme, which catalyzes the formation of primarily water- insoluble glucans from sucrose. It was not known if antiserum to the gtfB.l peptide would inhibit enzyme activity. However, antiserum to gtfB.l not only inhibited the formation of water-insoluble glucans, but also the synthesis of soluble glucans as well.
  • Soluble glucans are produced by the product of the gtfD gene, which has recently been cloned (Hanada and Kuramitsu, 1989), but not yet sequenced. Another enzyme, the product of the gtfC gene, catalyzes the formation of both soluble and insoluble glucans. Comparison of the nucleotide sequence data for gtfB and gtfC demonstrated that the gtfB.l peptide was located in regions of both gene products that shared significant homology with one another. Although soluble glucan synthesis was somewhat reduced, compared to the control, insoluble glucan synthesis was almost completely eliminated.
  • the peptide may be more exposed on the surface of GtfB, where it is accessible to antibody, compared to the other enzymes. Alternatively, the peptide may play a greater role in formation of the active site of GtfB, compared to the other enzymes, in which case the antibody affects its structure.
  • Enzyme does not require dextran primer for activity. ⁇ Requires dextran primer for enzyme activity.
  • Table II Bacterial strains and plasmids.
  • the shock fluid was loaded onto a G j, ganglioside affinity column. After washing the column at pH 6.8, the chimera was eluted from the column by pH 3.0 buffer.
  • ⁇ Total protein was quantitated using the BCA assay (Pierce).
  • ⁇ Specific protein was calculated as the ⁇ g chimera per mg protein.
  • ⁇ This value represents the amount of chimera recovered from the first passage of the shock fluid through the column.

Abstract

On décrit un peptide chimérique comprenant au moins une partie de la sous-unité B de la toxine du choléra et une région épitope d'un antigène donné fusionné à l'extrémité à terminaison N de la sous-unité B de la toxine du choléra. La région épitope comprend un déterminant antigénique du peptide voulu. Selon certaines réalisations préferées, de tels peptides chimériques sont utilisés en tant que vaccin pour produire dans un sujet une réponse immunitaire à un antigène donné. Des méthodes à médiation par ADN recombinant pour la production d'un peptide chimérique, des séquences ADN, des vecteurs ainsi que les organismes hôtes utilisés dans ces méthodes sont également décrits.
PCT/US1990/006811 1989-11-29 1990-11-28 Proteines chimeriques WO1991007979A1 (fr)

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WO1993022341A1 (fr) * 1992-05-01 1993-11-11 Forsyth Dental Infirmary For Children Vaccins de peptides de synthese pour prevenir les caries dentaires
WO1995010301A1 (fr) * 1993-10-08 1995-04-20 Duotol Ab Agent d'induction d'immunotolerance
WO1995031556A1 (fr) * 1994-05-11 1995-11-23 Unilever Nv Proteines se liant au glucan et leur utilisation
WO1996016178A1 (fr) * 1994-11-17 1996-05-30 Maxim Pharmaceuticals, Inc. Immunogenes pour stimuler l'immunite des muqueuses
WO1997005267A2 (fr) * 1995-07-26 1997-02-13 Maxim Pharmaceuticals Apport de polynucleotides dans les muqueuses
WO1998023763A1 (fr) * 1996-11-29 1998-06-04 The General Hospital Corporation Antigenes heterologues exprimes dans des souches de cellules vivantes v. cholerae
WO1999041402A2 (fr) * 1998-02-11 1999-08-19 Maxygen, Inc. Ciblage de vecteurs de vaccins genetiques
US6043057A (en) * 1988-09-16 2000-03-28 Vitec Aktiebolag Recombinant systems for expression of the cholera B-sub-unit with the aid of foreign promoters and/or leader peptides
US6287566B1 (en) * 1995-05-19 2001-09-11 The United States Of America As Represented By The Secretary Of The Army Protective peptides neurotoxin of C. botulinum
US7101562B1 (en) 1998-04-10 2006-09-05 The Forsyth Institute Conjugate vaccines for the prevention of dental caries
WO2013026445A1 (fr) * 2011-08-24 2013-02-28 Bioserv Analytik Und Medizinprodukte Gmbh Procédé pour l'isolement ou la purification rapide de protéines de recombinaison comprenant un domaine de la cholératoxine (non toxique)

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US5876727A (en) * 1995-03-31 1999-03-02 Immulogic Pharmaceutical Corporation Hapten-carrier conjugates for use in drug-abuse therapy and methods for preparation of same

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FR2636842B1 (fr) * 1988-09-27 1994-06-10 Liege Universite Etat Proteine de fusion d'une sequence derivee de la sous-unite b de la toxine cholerique et d'un antigene heterologue doue de proprietes immunogenes, compositions de vaccins les contenant et acides nucleiques recombinants contenant une sequence de nucleotides codant pour ladite proteine de fusion

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FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETIES LETTERS, Volume 241, No. 12, issued December 1988, J. SANCHEZ et al., "Genetic Fusion of a Non-Toxic Heat Stable Enterotoxin-Related Decapeptide Antigen to Cholera Toxin B-Subunit", pages 110-114. *
JOURNAL OF BACTERIOLOGY, Volume 169, Number 9, issued September 1987, T. SHIROZA et al., "Sequence Analysis of the alphatfB Gene from Streptococcus Mutans", pages 4263-4270. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 83, issued September 1986, V. MEHRA et al., "Efficient Mapping of Protein Antigenic Determinants", pages 7013-7017. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 86, issued January 1989, J. SANCHEZ et al., "Recombinant System for Overexpression of Chlorea Toxin B Subunit in Vibrio Cholerae as a Basis for Vaccine Development", pages 481-485. *
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6043057A (en) * 1988-09-16 2000-03-28 Vitec Aktiebolag Recombinant systems for expression of the cholera B-sub-unit with the aid of foreign promoters and/or leader peptides
WO1993022341A1 (fr) * 1992-05-01 1993-11-11 Forsyth Dental Infirmary For Children Vaccins de peptides de synthese pour prevenir les caries dentaires
EP1266662A3 (fr) * 1992-05-01 2003-05-28 Forsyth Dental Infirmary For Children Vaccins de peptides de synthése pour prevenir les caries dentaires
US5686075A (en) * 1992-05-01 1997-11-11 Forsyth Dental Infirmary For Children Synthetic peptide vaccines for dental caries
WO1995010301A1 (fr) * 1993-10-08 1995-04-20 Duotol Ab Agent d'induction d'immunotolerance
WO1995031556A1 (fr) * 1994-05-11 1995-11-23 Unilever Nv Proteines se liant au glucan et leur utilisation
WO1996016178A1 (fr) * 1994-11-17 1996-05-30 Maxim Pharmaceuticals, Inc. Immunogenes pour stimuler l'immunite des muqueuses
US6287566B1 (en) * 1995-05-19 2001-09-11 The United States Of America As Represented By The Secretary Of The Army Protective peptides neurotoxin of C. botulinum
WO1997005267A2 (fr) * 1995-07-26 1997-02-13 Maxim Pharmaceuticals Apport de polynucleotides dans les muqueuses
WO1997005267A3 (fr) * 1995-07-26 1997-04-24 Maxim Pharmaceuticals Apport de polynucleotides dans les muqueuses
US6036953A (en) * 1996-11-29 2000-03-14 The General Hospital Corporation Heterologous antigens in live cell V. cholerae strains
WO1998023763A1 (fr) * 1996-11-29 1998-06-04 The General Hospital Corporation Antigenes heterologues exprimes dans des souches de cellules vivantes v. cholerae
WO1999041402A3 (fr) * 1998-02-11 1999-11-11 Maxygen Inc Ciblage de vecteurs de vaccins genetiques
WO1999041402A2 (fr) * 1998-02-11 1999-08-19 Maxygen, Inc. Ciblage de vecteurs de vaccins genetiques
US7101562B1 (en) 1998-04-10 2006-09-05 The Forsyth Institute Conjugate vaccines for the prevention of dental caries
WO2013026445A1 (fr) * 2011-08-24 2013-02-28 Bioserv Analytik Und Medizinprodukte Gmbh Procédé pour l'isolement ou la purification rapide de protéines de recombinaison comprenant un domaine de la cholératoxine (non toxique)

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EP0502099A4 (en) 1993-02-17
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JPH05503420A (ja) 1993-06-10
CA2069106A1 (fr) 1991-05-30

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