EP1135409A1 - Proteines dbp a et b liant la decorine et gene codant pour elles - Google Patents

Proteines dbp a et b liant la decorine et gene codant pour elles

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Publication number
EP1135409A1
EP1135409A1 EP99954795A EP99954795A EP1135409A1 EP 1135409 A1 EP1135409 A1 EP 1135409A1 EP 99954795 A EP99954795 A EP 99954795A EP 99954795 A EP99954795 A EP 99954795A EP 1135409 A1 EP1135409 A1 EP 1135409A1
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EP
European Patent Office
Prior art keywords
sequence
dbpa
sequences
polypeptides
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP99954795A
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German (de)
English (en)
Inventor
Mark S. Hanson
Brian A. Mullikin
William Roberts
Raju Lathigra
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MedImmune LLC
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MedImmune LLC
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Publication of EP1135409A1 publication Critical patent/EP1135409A1/fr
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    • 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
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates generally to the field of bacterial antigens and their use, for example, as immunogenic agents in humans and animals to stimulate an immune response. More specifically, it relates to decorin binding proteins A and B and genes endcoding such proteins from bacterial species that cause Lyme disease which proteins are useful for the vaccination of a mammalian species as a mechanism for stimulating production of antibodies that help to protect the vaccine recipient against infection by pathogenic bacterial species that cause Lyme disease. Further, the invention relates to antibodies and antagonists against such polypeptides useful in diagnosis and passive immune therapy with respect to diagnosing and treating such Lyme disease bacterial infections. BACKGROUND OF THE INVENTION
  • Spirochetes classified as Borrelia burgdorferi, in a general sense are the causative agents of Lyme disease, the most commonly reported vector-borne infectious disease in the U.S. (Proc. Natl. Acad. Sci. USA 91 :2378-2383 (1 994)), and distributed throughout the Northern Hemisphere.
  • B. burgdorferi is transmitted to human and animal hosts primarily by Ixodes spp. ticks who deposit the spirochetes to the host while feeding upon the host. If diagnosed early, the disease can be effectively treated.
  • OspA outer surface protein A
  • OspC is fairly heterogenous in sequence (J. Clin. Microbiol. 33:1860- 1866 (1997); J. Bacteriol. 177:3036-3044 (1995) 1993 Infect. Immun. 61:2182-2191 (1993)), and predictions of at least 13 serogroups have been made using panels of OspC monoclonal antibodies (J. Clin. Microbiol. 33:1860-1866 (1997)). Additionally, OspC appears to be inaccessible to antibodies on some B. burgdorferi strains (Infect. Immun. 65:4661-4776 (1997); Proc. Natl. Acad. • Sci. USA 93:7973-7978 (1996)).
  • B. burgdorferi binds specifically (Infect. Immun. 63:3467- 3472(1995)) to the collagen associated proteoglycan called decorin (J. Biol. Chem. 264:2876-2884 (1989)), an activity that may promote colonization of host tissues.
  • the dbpBA locus is on the linear plasmid Ip54, which also encodes ospAB (Nature 390:580-586 (1997)).
  • DbpA and B are immunogenic and expressed in vivo, and DbpA is a target for antibody-mediated killing of B. burgdorferi during the early stages of infection (Protection of Borrelia burgdorferi infection by antibiotics to decorin-binding protein, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, p. 1 91 -1 95 (1 997).; Infect. Immun. 66:2143-21 53 ( 1 998)).
  • Active immunizations with recombinant DbpA (rDbpA) also protected mice from homologous, and in some cases heterologous, borrelial challenge. Hyperimmune antiserum against a single rDbpA immunogen had bactericidal activity against many B.
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al. , Nucleic Acids Res., 8:4057 (1 980).
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • HPS portion refers to an amino acid sequence as set forth in SEQ ID NO:2 for a choline binding protein ("CBP") of a pneumococcal bacteria that may be located amino terminal with respect to the proline rich portion of the overall amino acid sequence for such CBP.
  • CBP choline binding protein
  • identity refers to a calculation of differences between two contiguous sequences which have been aligned for "best fit” (to provide the largest number of aligned identical corresponding sequence elements, wherein elements are either nucleotides or amino acids) and all individual differences are considered as individual difference with respect to the identity.
  • all individual element gaps (caused by insertions and deletions with respect to an initial sequence (“reference sequence")) over the length of the reference sequence and individual substitutions of different elements (for individual elements of the reference sequence) are considered as individual differences in calculating the total number of differences between two sequences.
  • isolated in the context of the present invention with respect to polypeptides and/or polynucleotides means that the material is removed from its original environment (e.g. , the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • the present invention relates to a vaccine for treating or preventing bacterial infections that cause Lyme disease, which vaccube utilizes as an immunogen at least one DBP A or B polypeptide according to SEQ ID NO:2, 4, 6, 8, 10, 1 2, 14, 16, 1 8, 20 and 22, or a truncate, analog, or variant having a highly conserved immunogenic portion with respect to different types of bacteria that cause Lyme disease.
  • vaccube utilizes as an immunogen at least one DBP A or B polypeptide according to SEQ ID NO:2, 4, 6, 8, 10, 1 2, 14, 16, 1 8, 20 and 22, or a truncate, analog, or variant having a highly conserved immunogenic portion with respect to different types of bacteria that cause Lyme disease.
  • Also preferred as vaccines are recombinantly-produced, isolated polypeptides corresponding to the suequences or immunogenic portion thereof according to SEQ ID NOS: 2, 4, 6, 8, 1 0, 1 2, 1 4, 1 6, 1 8, 20 and 22.
  • the present invention provides an isolated polypeptide comprising an amino acid sequence which has at least 90% identity to one of the amino acid sequences selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 1 2, 1 4, 1 6, 1 8, 20 and 22.
  • such isolated polypeptide comprises an amino acid sequence which has at least 95% identity, and more preferably 97% identity, to one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 1 0, 1 2, 14, 1 6, 1 8, 20 and 22.
  • the invention further relates to immunogenic fragments of such polypeptides.
  • the present invention provides a DBP A or B polypeptide encoded by a polynucleotide that will hybridize under highly stringent conditions to the complement of a polynucleotide encoding a polypeptide having an amino acid selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 1 2, 1 4, 1 6, 1 8, 20 and 22.
  • polypeptides comprising an amino acid sequence segment that is at least 95% identical to the amino acid sequence of SEQ ID NO:2.
  • polypeptides comprising a contiguous amino acid sequence that has at least 97% identity with respect to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20 and 22, or an immunogenic truncate or fragment thereof. And, even more preferred are polypeptides comprising an amino acid sequence that has at least 98% identity with respect to the amino acid sequence of SEQ ID NO:2.
  • the present invention provides polynucleotides which encode the hereinabove described polypeptides of the invention.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic
  • the DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the polynucleotides which encode polypeptides including the amino acid sequences of at least one of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18,
  • polypeptides 20 and 22 may be one of the coding sequences shown in SEQ ID NOS: 1 , 3, 5, ;7, 9, 1 1 , 1 3, 1 5, 17, 19 or 21 or may be of a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the DNA of SEQ ID NOS: 2, 4, 6, 8, 1 0, 1 2, 14,
  • NOS:2, 4, 6, 8, 1 0, 1 2, 14, 1 6, 1 8, 20 and 22 may include: only the coding sequence for the polypeptide; the coding sequence for the polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the polypeptide.
  • the invention further relates to a polynucleotide comprising a polynucleotide seqeunce that has at least 95% identity, preferably at least 97% identity, and even more preferably 98% identity to a polynucleotide encoding one of the polypeptides comprising SEQ ID NO:2, 4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20 and 22.
  • the invention further relates to fragments of such polynucleotides which include at least the portion of the polynucleotide encoding the polypeptide sequence corresponding to SEQ ID NO: 1 .
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides including the amino acid sequences of SEQ
  • the variants of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.
  • Complements to such coding polynucleotides may be utilized to isolate polynucleotides encoding the same or similar polypeptides.
  • such procedures are useful to obtain DBP A and B helical coding segments from different serotypes of bacterial species that cause Lyme Disease, which are especially useful in the production of "chain" polypeptide vaccines containing multiple immunogenic portions from different serotypes.
  • the present invention includes polynucleotides encoding polypeptides including the same polypeptides as shown in the Sequence Listing as SEQ ID NOS:2, 4, 6, 8, 10, 1 2, 14, 1, 6, 1 8, 20 and 22 as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20 and 22.
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in the Sequence Listing as SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9 and 21 .
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention.
  • the marker sequence may be, for example, a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1 984)).
  • the present invention further relates to polynucleotides
  • hybridization target sequences which hybridize to the complements of the hereinabove-described sequences if there is at least 70% and preferably 80% identity between the target sequence and the complement of the sequence to which the target sequence hybridizes, preferably at least 85% identity. More preferred are such sequences having at least 90% identity, preferably at least 95% and more preferably at least 97% identity between the target sequence and the sequence of complement of the polynucleotide to which it hybridizes.
  • the invention further relates to the complements to both the target sequence and to the polynucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20 and 22.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the complements of the hereinabove-described polynucleotides as well as to those complements.
  • stringent conditions means hybridization will occur with the complement of a polynucleotide and a corresponding sequence only if there is at least 95%, preferably at least 97% identity, and even more preferably at least 98% identity between the target sequence and the sequence of complement of the polynucleotide to which it hybridizes.
  • polypeptides which hybridize to the complements of the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain an immunogenic portion that will cross-react with an antibody to at least one of the polypeptides having a sequence according to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22.
  • the present invention provides for the production of such polypeptides and vaccines as set forth above having a histidine label (or other suitable label) such that the full-length proteins, truncates, analogs or variant discussed above can be isolated due to their label.
  • the present invention relates to a method of prophylaxis and/or treatment of Lyme disease that is caused by or mediated by bacteria that have DBP A or B surface proteins.
  • the invention relates to a method for the prophylaxis and/or treatment of such infections in humans.
  • the present invention relates to a method of using one or more antibodies (monoclonal, polyclonal or sera) to the polypeptides of the invention as described above for the prophylaxis and/or treatment of Lyme disease.
  • antibodies monoclonal, polyclonal or sera
  • Particularly preferred are mixed antibodies for several serotypes of such bacteria.
  • Figure 1 is a diagram of a positional relationships among primers used for amplification of dbpA and dbpB genes. The annealing positionsof the primers relative to the dbpBA locus of B. burgdorferi sensu stricto isolate 297 are indicated. These primers are described further in Figures
  • Figures 2A-2D collectively, illustrate the alignment of deduced amino acid sequences of DbpA.
  • the sequences of DbpA from 30 B. burgdorferi sensu lato isolates were aligned using MEGALIGN. Residues that are identical with the reference DbpA 297 are boxed.
  • Figure 3 is a schematic molecular tree showing phylogenetic relationships among the different DbpA sequences, and comparison with DbpB.
  • the rectangular cladogram was generated from comparisons of 30 full length DpbA sequences by PHYLIP.
  • DbpB 297 was included for comparative purposes.
  • the numbers at the branch nodes indicate the result of bootstrap analysis.
  • B. burgdorferi sensu lato species names were abbreviated as follows: B. burgdorferi, B. bur. - B. afze/ii, B. afz.; B. garinii, B. gar.
  • Figure 4A is a plot of the identity score of the amino acid sequences shown in alignment in Figures 2A-2D.
  • the DbpA sequences were aligned by the program PileUp and the relative identity score among the DbpAs was derived by the program PlotSimilarity with a sliding window average of 10 amino acids (A).
  • Figure 4B shows the antigenic index for the aligned amino acid sequences shown in Figures 2A-2D.
  • the Jameson-Wolf antigenic index of the DbpA 297 was determined with the program PROTEAN (B). The position numbering is relative to the first Met codon of the DbpA 297 precursor protein; the position of the presumptive leader peptide region of this protein is indicated.
  • Figure 5 illustrates the DbpA sequence relatedness among isolates killed by DbpA 297 antiserum.
  • the molecular phylogram of the DbpA seuqences that were determined for 23 isolates evaluated for sensitivity to killing by DbpA 297 antiserum was generated by the distance matrix program, UPGMA. Branchlengths are proportional to the relative mutational distance.
  • the scale is in 0.1 nucleotide substitutions per site.
  • the growth inhibition endpoint of the DbpA 297 antiserum previously observed for each isolate is indicated.
  • a titer greater than or equal to 1 00 percent is defined as positive inhibition; which isolates are indicated by boxes.
  • the % amino acid similarity of each DbpA with DbpA 297 is shown for comparative purposes. The same abbreviations as used in Figure 3 are utilized in the comparison.
  • Figure 6 shows an alignment of the 5' ends of selected dbpA genes which reveals potential mosaic structure in dbpA form B. afze/ii Pgau.
  • the sequence of the first 1 20 nucleotides of the coding region of dbpA PGau was aligned with the sequence of the first dpbA from three other B. afze/ii isolates, and with dbpA from three B. garinii isolates.
  • the positions at which the dbpA genes are heterogenous and of which dbpA PGau is identical to the intraspecies consensus, are indicated by black shading. Regions of putative mosaic structure in dbpA PGau , and their proposed species of origin, are indicated by the open boxes.
  • the positions of the codon TGT for cysteine, at the amino-terminus of the mature protein, is indicated for oreientation purposes.
  • Figures 7A-7E collectively show the origin and accession number of dbpA and dbpB gene sequences as well as the source of the isolate. Both the biological and geographic origins of the bacteria are indicated.
  • Figures 8A-8C collectively show an oligonucleotide list of primers that are useful for isolating the polynucleotides according to the invention.
  • the abreviation of the primer and its derivation are both indicated by the list.
  • Figure 9 shows the amino acid similarities of DbpA sequences among a number of B. burgdorferi sensu lato isolates.
  • the amino acid similarities, in percentages, are deduced for full-length DbpA sequences and were calculated by BestFit.
  • DbpAs of isolates B31 , HBNC, and 3029 are 100% identical and were considered as a group for simplicity.
  • isolates with > 90% similarity are boxed.
  • the alignments were perfomed as indicated above for identity.
  • a vaccine to produce a protective response, or improved protective response, against Lyme disease infections which vaccine employs a polypeptide which comprises a member selected from the group consisting of:
  • a particularly preferred embodiment of such an immunogenic composition is for use as a vaccine (or as an immunogen for producing antibodies useful for diagnostics or vaccines) wherein the active component of the immunogenic composition is an isolated polypeptide comprising at least one member selected from the group consisting of:
  • portion when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence.
  • the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide.
  • such terms refer to the products produced by treatment of said polynucleotides with any of the common endonucleases.
  • such an immunogenic composition may be utilized to produce antibodies to diagnose Lyme disease spirochete infections, or to produce vaccines for prophylaxis and/or treatment of such bacterial infections as well as booster vaccines to maintain a high titer of antibodies against the immunogen(s) of the immunogenic composition.
  • truncated polypeptides, functional variant analogs, and recombinantly produced truncated polypeptides of the invention are useful as immunogens for preparing vaccine compositions that stimulate the production of antibodies that can confer immunity against pathogenic species of bacteria. Further, preparation of vaccines containing purified proteins as antigenic ingredients are well known in the art.
  • vaccines are prepared as injectables, in the form of aqueous solutions or suspensions. Vaccines in an oil base are also well known such as for inhaling. Solid forms which are dissolved or suspended prior to use may also be formulated.
  • Pharmaceutical carriers are generally added that are compatible with the active ingredients and acceptable for pharmaceutical use. Examples of such carriers include, but are not limited to, water, saline solutions, dextrose, or glycerol. Combinations of carriers may also be used.
  • Vaccine compositions may further incorporate additional substances to stabilize pH, or to function as adjuvants, wetting agents, or emulsifying agents, which can serve to improve the effectiveness of the vaccine.
  • Vaccines are generally formulated for parenteral administration and are injected either subcutaneously or intramuscularly. Such vaccines can also be formulated as suppositories or for oral administration, using methods known in the art.
  • the amount of vaccine sufficient to confer immunity to pathogenic bacteria is determined by methods well known to those skilled in the art. This quantity will be determined based upon the characteristics of the vaccine recipient and the level of immunity required. Typically, the amount of vaccine to be administered will be determined based upon the judgment of a skilled physician. Where vaccines are administered by subcutaneous or intramuscular injection, a range of 50 to 500 ⁇ g purified protein may be given.
  • patient in need thereof refers to a human that is infected with, or likely, to be infected with, pathogenic bacteria that cause Lyme disease, or the like (however a mouse model can be utilized to simulate such a patient in some circumstances).
  • polypeptides of the present invention can be used as immunogens to stimulate the production of antibodies for use in passive immunotherapy, for use as diagnostic reagents, and for use as reagents in other processes such as affinity chromatography.
  • the polynucleotides encoding the immunogenic polypeptides described above may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention.
  • the marker sequence may be, for example, a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1 984)).
  • the present invention also relates to vectors which include polynucleotides encoding one or more of the polypeptides of the invention that include the highly conserved alpha-helical amino acid sequence in the absence of an area encoding a choline binding amino acid sequence, host cells which are genetically engineered with vectors of the invention and the production of such immunogenic polypeptides by recombinant techniques in an isolated and substantially immunogenically pure form.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors comprising a polynucleotide encoding a polypeptide comprising the highly conserved alpha-helical region but not having a choline binding region, or the like of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the polynucleotides which encode such polypeptides.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • Vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter for example, LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the proteins.
  • bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or Bowes melanoma
  • adenoviruses plant cells, etc.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen, Inc.), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXT1 , pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are pKK232-8 and pCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and TRP.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription: Examples including the SV40 enhancer on the late side of the replication origin bp 1 00 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S.
  • heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 3701 7).
  • Such commercial vectors include, for example, pKK223-3
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, a french press, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • preferred are host cells which secrete the polypeptide of the invention and permit recovery of the polypeptide from the culture media.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:1 75 (1981 ), and other cell lines capable of expressing a compatible vector, for example, the C1 27, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the polypeptides can be recovered and/or purified from recombinant cell cultures by well-known protein recovery and purification methods. Such methodology may include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. In this respect, chaperones may be used in such a refolding procedure. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides that are useful as immunogens in the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, , by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Procedures for the isolation of the individually expressed polypeptides may be isolated by recombinant expression/isolation methods that are well- known in the art. Typical examples for such isolation may utilize an antibody to a conserved area of the protein or to a His tag or cleavable leader or tail that is expressing as part of the protein structure.
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1 975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1 983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1 985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • the appended Figures as describe above sets forth the bacterial source for the polypeptides of SEQ ID NOS:2, 4, ;6, 8, 10, 1 2, 14, 1 6, 1 8, 20 and 22 and the polynucleotides encoding them (SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 1 3, 1 5, 17, 19 and 21 , respectively).
  • the name and/or type of bacteria is specified and a credit or source is named.
  • the sequences from such types of bacteria are for illustrative purposes only since by utilizing probes and/or primers as described herein other sequences of similar type may be readily obtained by utilizing only routine skill in the art.
  • Genomic DNA preparation and molecular typing Total genomic DNA was extracted from various Borrelia isolates by standard methods (Sambrook, J., E.F. Fritsch, and T. Maniatis. 1 989. Molecular cloning - A Laboratory Manual. Cold Spring Harbor Laboratory Press,) or by the Puregene (Gentra Systems, Inc., Research Triangle Park, NO genomic DNA isolation kit according to the manufacturer's direction.
  • the rRNA rrf (5S)-/r/ (23S) intergenic spacer region was amplified from each isolate by PCR, and restriction fragment length polymorphism (RFLP) analysis was then performed to confirm classification into the B. burgdorferi sensu lato groups according to the methods described by Postic, et al.
  • RFLP restriction fragment length polymorphism
  • PCR was performed by using Taq DNA polymerase and buffers (50 mM KCl, 1 0 mM Tris pH 7.0, 2.5 mM MgCI 2 ) from Perkin-Elmer Cetus (Foster City, CA) and 250 ⁇ M deoxynucleoside triphosphates (dNTPs) from Boehringer-Mannheim (Indianapolis, IN).
  • buffers 50 mM KCl, 1 0 mM Tris pH 7.0, 2.5 mM MgCI 2
  • dNTPs deoxynucleoside triphosphates
  • Example 3 DNA blotting, hybridization and detection.
  • the dbpA ip90 DNA fragment was labeled with Digoxygenin using the Genius kit (Boehringer Mannheim); other gene fragments and oligonucleotides were labeled with fluorescein using the ECL labeling kit (Amersham).
  • Blots to be probed with fluorescein labeled fragments and/or oligonucleotides were prehybridized and hybridized at 50 °C in 5 x SSC, 0.5% SDS, 5 x Denhardts solution, 1 x ECL blocking solution, and 0.2 mg/ml heat denatured herring sperm DNA by standard protocols (Sambrook, J., E.F. Fritsch, and T. Maniatis. 1 989. Molecular cloning - A Laboratory Manual. Cold Spring Harbor Laboratory Press,).
  • the blots were washed as follows: 2 washes with 2 x SSC/0.1 % SDS for 2 minutes each at room temperature (RT), a wash with 2 x SSC/0.1 % SDS for 2 min at 50°C, and then a wash with 0.5 x SSC/0.1 % SDS at 50°C). Bound probe was visualized on film (Kodak X-AR, ECL Hyperfilm) following development with the ECL detection kit.
  • DIG-labeled fragment used to identify and clone dbpA p9Q
  • prehybridization and hybridization were done in the same solution of 5 x SSC, 2% DIG blocking solution, 0.1 % Sarkosyl, 0.02% SDS, and 50% formamide at 42 °C).
  • Probes were again used at a concentration of 1 0-20 ng/ml. Blots were then washed (2 washes for 5 minutes each at RT using 2 x SSC/0.1 %SDS, then 2 washes for 1 5 minutes each at 65 °C with 0.5 x SSC/0.1 % SDS) and visualized according to the manufacturer's protocol.
  • Genomic sublibrary construction and gene cloning Genomic sublibrary construction and gene cloning.
  • DNA fragments containing dbpA genes were identified and cloned from restriction enzyme digests of total genomic DNA by hybridization with dbpA probes.
  • Primers 2 + 1 2 ( Figures 1 and 7A-7E), derived from dbpA 297 , were used to PCR amplify a 280 bp fragment from genomic DNA of B. garinii Ip90. Sequencing this fragment confirmed that it was a portion of the dbpA ip30 gene. This fragment was again PCR-amplified, incorporating DIG-UTP label, and used to probe blots of digested Ip90 DNA. A 1 .3 kb Hind III fragment hybridized to the probe.
  • Plasmid DNA was prepared from the presumptive clones using the QIAprep plasmid miniprep kit (Qiagen, Santa Clarita, CA), and the Borrelia DNA inserts were sequenced to identify dbpA containing clones.
  • PCR fragments containing full-length or partial dbpA and dbpB genes were amplified with primer pairs described in Figures 7A-7E using standard PCR conditions: 96 °C denaturing for 30 seconds, 52°C annealing for 30 seconds, and 72°C extension for 60 seconds. The annealing temperature was lowered to 45 °C when amplifying with degenerate oligonucleotides.
  • the amplified fragments were purified from the PCR reaction mix over a Qiaquick PCR purification column (Qiagen). The majority of dbpA and dbpB sequences were obtained by direct double stranded cycle sequencing of the purified PCR product.
  • PCR amplified fragments were cloned into the pCRII vector of the TA Cloning kit (Invitrogen, Carlsbad, CA) and subsequently sequenced with vector primers (primers 20, 21 ; Table 1 ) and dbpA specific primers. Gaps were bridged using genomic DNA as template and primers derived from the first round of sequencing. The complementary positions of the various primers used for PCR amplification are illustrated schematically in Figure 1 . Multiple independent sequence confirmation was available on both strands for most of the genes cloned by PCR. Sequencing in all cases was performed using the Dye Terminator Cycle sequencing kits (Applied Biosystems, Inc., Foster City, CA). All sequencing reactions were run on the ABI Model 373A Sequencer.
  • Relative similarity among the DbpA sequences by amino acid position was derived using the program PlotSimilarity (GCG) with a sliding window average of 1 0 residues.
  • the Jameson-Wolf antigenic index of the DbpA 297 sequence was determined using the program PROTEAN (Lasergene). Screening for multiple and direct repeats in the dbpBA locus and flanking genomic sequence was performed using DNA Strider v1 .2.
  • the phylogenetic relatedness among the DbpA sequences was estimated using the PHYLIP (Felsenstein, J. 1 989. PHYLIP-phylogeny inference package (version 3.2). Cladistics 5:1 64-1 66.) analysis package, version 3.5c.
  • a distance matrix was generated from the full length amino acid sequences of the 30 DbpAs, and DbpB 297 , by the PHYLIP program PROTDIST using the Dayhoff PAM (percent accepted mutations) matrix option.
  • NEIGHBOR PHYLIP
  • phylogenetic trees were generated from the PROTDIST output by the distance matrix UPGMA (unweighted pair-group method using arithmetic averages) method.
  • the sequence data was resampled into 100 replicate sets by the bootstrap analysis option of SEQBOOT (PHYLIP) to estimate the reproducibility of the tree branch nodes.
  • a 50% majority-rule consensus tree was constructed by the program CONSENSE (PHYLIP) and displayed by the program TreeView (Page, R.D.M. 1 996.
  • the UPGMA method was also used to construct a phylogram from a subset of 23 DbpA sequences.
  • Genbank accession numbers for the dbpA and dbpB sequences determined in this study are listed in Figures 7A-7E and will be released after filing of this application.
  • Sequencing of dbpA and dbpB genes Determined were the nucleotide sequences of dbpA genes from 29 isolates, and of dbpB genes from 1 5 isolates, of B. burgdorferi sensu lato.
  • a PCR-based approach initially using primers derived from the original sequence of the dbpBA locus of isolate 297 (Genbank U75866 and U75867), was employed to amplify and sequence alleles of dbpA and dbpB from multiple B. burgdorferi sensu lato isolates.
  • dbpA In the case of dbpA, only a limited number of new genes could be amplified with the original primer sets, in contrast to similar methods used for amplification and sequencing of multiple alleles of the ospA (Will, G., S. Jauris-Heipke, E. Schwab, U. Busch, D. Robler, E. Soutschek, B. Wilske, and V. Preac-Mursic. 1 995. Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species. Med. Microbiol. Immunol.
  • ospC Jauris-Heipke, S., G. Liegl, V. Preac-Mursic, E. Schwab, E. Soutschek, G. Will, and B. Wilske. 1 997.
  • OspC outer surface protein C
  • DbpA from isolate 2501 5 the sole member of its phylogenetic group (Casjens, S., M. Delange, H.L. Levy, III, P. Rosa, and W.M. Huang. 1995. Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order. J. Bacteriol. 1 77:2769-2780.; Mathiesen, D.A., J.H. Oliver, C.P. Kolbert, E.D. Tullson, B.J. Johnson, G.L. Campbell, P.D. Mitchell, K.D. Reed, S.R. Telford, III, J.F. Anderson, R.S. Lane, and D.H. Persing. 1 997.
  • afzelii isolates had an identical presumptive leader peptide 20 amino acids in length. All seven B. garinii isolates had a nearly identical leader peptide, again 20 amino acids long, but differing from the conserved B. afzelii sequence in five positions. Two different leader peptide sequences, of 24 and 27 amino acids, were seen among the 1 5 B. burgdorferi sensu stricto isolates. Isolate 2501 5 has been placed in a separate phylogenetic group from B. burgdorferi sensu stricto by RFLP analysis of the rrf (5S)-/v7 (23S) intergenic spacer (Postic, D., M.V. Assous, P. A. Grimont, and G.
  • the DbpA multiple sequence alignment was analyzed by the distance matrix program, UPGMA, from the Phylip package to further investigate potential phylogenetic relationships among the various proteins.
  • DbpB from B. burgdorferi sensu stricto isolate 297 was included in this analysis as a potential sequence outlier.
  • the inferred DbpA molecular phylogeny (Fig. 3) shows a major division between the group including most of the B. afzelii sequences and the rest of the DbpAs, with a further major division between the B. burgdorferi sensu stricto and B. garinii sequences. With higher numerical representation, the B. burgdorferi sensu stricto branch was further divisible into smaller subgroups.
  • DbpA cladogram had high bootstrap values, indicating that the overall topology of this phylogenetic tree was well supported.
  • DbpB sequences The similarity of the amino acid sequences of DbpB from 1 5 B. burgdorferi sensu stricto isolates, and one B. garinii isolate, was very high, ranging from 96.3% to 1 00% (95.1 % to 100% identity, data not shown).
  • the dbpB gene of Ip90 was divergent in sequence from the 1 6 PCR-amplified dbpB genes (data not shown) indicating that the full extent of dbpB heterogeneity is yet to be determined. Southern hybridization of genomic DNA from several different B. burgdorferi sensu lato species with a probe derived from dbpB 297 gave further evidence of the interspecies heterogeneity of this gene (data not shown).
  • B. burgdorferi sensu lato isolates killed by monovalent DbpA antiserum include those with divergent DbpA sequence phylogeny.
  • the sequence of DbpA has been now determined for 23 of the isolates evaluated in our earlier study. Among the 23 isolates that were sequenced, the 1 2 that were highly sensitive to killing by DbpA 297 antiserum (inhibition endpoint titer >1 00) were distributed among all major branches of the DbpA phylogram, and were not restricted to those isolates with the highest DbpA 297 sequence similarity (Fig. 5). Inspection of the alignment of these 1 2 sequences (not shown) reveals that the longest run of contiguous amino acids identical among all isolates is three residues (TTA; DbpA 297 residues 148-1 50).
  • TTA tripeptide and several conserved flanking residues, are also found in isolates not killed by DbpA 297 antiserum.
  • antibody binding sites on proteins are generally considered to be larger than three amino acids, it is likely that the epitope, or epitopes, targeted by the borreliacidal antibodies in the DbpA 297 antiserum is (are) composed of non-contiguous amino acids. Further, these putative epitope(s) appear(s) to be conserved among DbpA proteins that are divergent in their primary structure.
  • garinii strains gave further support for the possibility of lateral exchange of dbpA sequence between these species (Fig. 6).
  • the dbpA gene in B. afzelii isolate PGau is nearly identical to B. afze/ii IPF, M7, and VS461 between nucleotides 1 to 57 encoding the putative leader peptide region, but the region just downstream of the Cys (nucleotides 63 to 1 20) shows greater similarity to the corresponding region of B. garinii VSBP, G25, and PBr.
  • the dbpA gene from B. afzelii U01 has more stretches of nucleotides in common with B.
  • garinii dbpA genes throughout its entire length, including the leader peptide (data not shown), and DbpA U01 has > 90% overall similarity with six B. garinii DbpAs (Table 3). These data, in conjunction with the phylogenetic analysis (Fig. 3) suggest that the entire dbpA gene of B. afzelii U01 may have been acquired from a B. garinii isolate.

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Abstract

La présente invention concerne des agents bactériens immunogènes destinés à être administrés à des humains et à des animaux pour stimuler la réponse immunitaire. Elle concerne notamment la vaccination d'espèces mammaliennes au moyen de polypeptides dérivés à partir d'espèces bactériennes qui provoquent la maladie de Lyme, ladite vaccination fonctionnant comme un mécanisme stimulant la production d'anticorps qui protègent le receveur du vaccin contre l'infection par ces espèces bactériennes pathogènes ou le rendent plus résistant à ce type d'infections. Dans un autre aspect, l'invention concerne des anticorps dirigés contre ces protéines et des complexes protéiques pouvant être utilisés pour le diagnostic et/ou comme des agents de protection/de traitement contre l'action des espèces bactériennes pathogènes.
EP99954795A 1998-10-09 1999-10-08 Proteines dbp a et b liant la decorine et gene codant pour elles Withdrawn EP1135409A1 (fr)

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WO2009003492A1 (fr) 2007-07-03 2009-01-08 Dako Denmark A/S Procédés améliorés pour produire, marquer et utiliser des multimères du cmh
EP2361930A3 (fr) 2007-03-26 2011-10-26 Dako Denmark A/S Multimeres du complexes peptide-cmh et leur utilisation dans des maladies infectieuses de borrelia
EP2197908A2 (fr) 2007-09-27 2010-06-23 Dako Denmark A/S Multimères cmh dans le diagnostic, le vaccin et le traitement de la tuberculose
DK2254592T3 (da) 2008-02-28 2019-09-09 Dako Denmark As MHC-multimerer til Borrelia-diagnostik og sygdom
EP2254592B1 (fr) * 2008-02-28 2019-06-05 Dako Denmark A/S Multimères de mhc dans le diagnostic et le traitement de la borréliose
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US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease
WO2010037402A1 (fr) 2008-10-02 2010-04-08 Dako Denmark A/S Vaccins moléculaires contre les maladies infectieuses
SG11202104136YA (en) 2018-10-23 2021-05-28 Dragonfly Therapeutics Inc Heterodimeric fc-fused proteins
AU2021260960A1 (en) 2020-04-22 2022-11-24 Dragonfly Therapeutics, Inc. Formulation, dosage regimen, and manufacturing process for heterodimeric Fc-fused proteins

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DE19632862B4 (de) * 1996-08-14 2006-08-03 Mikrogen Molekularbiologische Entwicklungs-Gmbh Immunologisch aktive Proteine von Borrelia burgdorferi, dafür kodierende Nukleinsäuren sowie deren Verwendung in Testkits und als Impfstoffe

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