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WO2002018600A1 - Homologues groes et groel de francisella tularensis - Google Patents

Homologues groes et groel de francisella tularensis Download PDF

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Publication number
WO2002018600A1
WO2002018600A1 PCT/GB2001/003835 GB0103835W WO0218600A1 WO 2002018600 A1 WO2002018600 A1 WO 2002018600A1 GB 0103835 W GB0103835 W GB 0103835W WO 0218600 A1 WO0218600 A1 WO 0218600A1
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WO
WIPO (PCT)
Prior art keywords
tularensis
polypeptide
infection
pharmaceutical composition
nucleic acid
Prior art date
Application number
PCT/GB2001/003835
Other languages
English (en)
Inventor
Richard William Titball
Michael Green
Margaret Gillian Hartley
Kerri Anne Mack
Anders SJÖSTEDT
Original Assignee
The Secretary Of State For Defence
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The Secretary Of State For Defence filed Critical The Secretary Of State For Defence
Priority to AU2001282350A priority Critical patent/AU2001282350A1/en
Publication of WO2002018600A1 publication Critical patent/WO2002018600A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • 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 to a polypeptide useful in the prophylactic or therapeutic treatment of diseases caused by Francisella tularensis , and to vaccines and therapies using this .
  • Type A strains are fully virulent (e.g. Schu4), whereas Type B strains are less virulent in man, but cause tularemia in mice (e.g. HN63) .
  • the avirulent type B stain, live vaccine strain (LVS) causes disease in mice and is considered to be comparable to tularemia in man. It is therefore generally used as a model of the disease.
  • F. tularensis in common with many other bacteria produces heat shock-induced proteins including members of the highly conserved hsp ⁇ O family. These include proteins which are homologous to the GroEL and GroES chaperone proteins of Escherichia coli .
  • the sequence of F. tularensis GroEL protein (SEQ ID NO 1) and F. tularensis GroES protein (SEQ ID NO 2) is shown in Figure 1 hereinafter. These two proteins are understood to form an Hsp ⁇ O complex.
  • the present invention relates to polypeptides which are useful as prophylactic and therapeutic vaccines against F. tularensis .
  • the present invention provides a polypeptide comprising at least one component of an Hsp ⁇ O obtainable from Francisella tularensis , or a fragment thereof, or a variant of any of these, which polypeptide, when administered to a animal (such as a mammal), produces an immune response which is protective against F. tularensis infection, for use in prophylactic or therapeutic vaccination against F. tularensis .
  • the polypeptide of the invention comprises the GroEL protein of SEQ ID NO 1 or a fragment thereof, or a variant of either of these.
  • the polypeptide cross- reacts with anti-groEL antibody (available from Sigma) .
  • the polypeptide may comprise the GroES protein of SEQ ID NO 2 or a complex of GroEL and GroES proteins .
  • Hsp ⁇ O is constitutively expressed in F. tularensis and conserved between strains. It has been shown to be upregulated in response to stress (Ericsson M, et al., (1994). Infection & Immunity 62 (1) : 178-83) making it a useful vaccine against a range of F. tularensis strains.
  • Fragments of the polypeptides include deletion mutants and polypeptides where small regions of the polypeptide are joined together.
  • the fragments should contain at least one antigenic region however so that they continue to produce a protective immune response .
  • Suitable fragments of the polypeptide have one or more amino acids deleted from the sequence and may be as small as 6 amino acids in length, provided they contain at least one antigenic determinant of the Hsp ⁇ O protein and preferably the GroEL protein of F. tularensis .
  • the fragments will comprise at least 15, more suitably at least 30 and preferably at least 60 amino acids
  • variant refers to sequences of amino acids which differ from the base sequence from which they are derived in that one or more amino acids within the sequence are substituted for other amino acids .
  • Amino acid substitutions may be regarded as "conservative” where an amino acid is replaced with a different amino acid with broadly similar properties .
  • Non-conservative substitutions are where amino acids are replaced with amino acids of a different type.
  • Nonpolar Ala, Val, Leu, lie, Pro, Met, Phe, Trp
  • Uncharged polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin
  • altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.
  • Non-conservative substitutions are possible provided that these do not interrupt with the immunogenicity of the polypeptide.
  • variants will be at least 85% homologous, preferably at least 90% homologous to the base sequence.
  • variants include allelic variants as well as proteins which are encoded by nucleic acid sequences which hybridise to DNA sequences which encode the polypeptide of the invention under stringent conditions as explained hereinafter.
  • hybridisation occurs at high stringency conditions, for example those illustrated in "Molecular Cloning", A Laboratory Manual” by Sambrook, Fritsch and Maniatis, Cold Spring Habor Laboratory Press, Cold Spring Harbor, N.Y.
  • high stringency conditions include hybridisation in 0.1 x SSC at about 65°C. SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate.
  • homologous means that two sequences, when aligned, have similar (identical or conservatively replaced) amino acids in like positions or regions, and where identical or conservatively replaced amino acids are those which do not alter the activity or function of the protein as compared to the starting protein.
  • two amino acid sequences which are at least 85% homologous to each other have at least 85% similar (identical or conservatively replaced amino residues) in a like position when aligned.
  • Homology may be determined using methods well known in the art (see, for example, Deveraux et al . 1984, Nucleic Acids Research 12, 387-395, Wilbur, W.J. and Lipman, D.J. "Rapid Similarity Searches of Nucleic Acid and Protein Data Banks.” Proceedings of the National Academy of Sciences USA 80, 726- 730 (1983) and Myers E.and Miller W. "Optimal Alignments in Linear Space”. Comput. Appl. Biosci. 4:11-17(1988)).
  • MegAlign Lipman-Pearson one pair method (using default parameters) which can be obtained from DNAstar Inc, 1228, Selfpark Street, Madison, Wisconsin, 53715, USA as part of the Lasergene system.
  • polypeptide as used herein includes long chain peptides including proteins.
  • the polypeptide of the invention comprises a protein of SEQ ID NO 1.
  • Nucleic acids which encode a polypeptide as described above will be known or isolable from F. tularensis libraries, or readily deducible from a knowledge of the genetic code.
  • the nucleic acid may be DNA or RNA, and where it is aDNA molecule, it may comprise a cDNA or a genomic DNA. These nucleic acids may themselves be useful in therapy as elements of vaccine vectors, which when administered to an animal, result in expression of the desired polypeptide in vivo.
  • vaccines may comprise for example a DNA or cDNA vaccine which encodes a polypeptide as described above.
  • Such a vaccine may be constructed using cells .
  • the cell is a eukaryotic cell, such as J774 or a recombinant bacterial cell, such as recombinant Salmonella although other systems are available.
  • a nucleic acid as described above may be incorporated into a vector, which is used to transform a cell, preferably a eukaryotic eel.
  • the vector is one which is adapted to express the protein or peptide as described above in situ.
  • the vector may contain the usual expression control functions such as promoters, enhancers and signal sequences, as well as a selection marker in order to allow detection of successful transformants . The selection of these will depend upon the precise nature of the vector chosen and will be known to or readily determinable by a person skilled in the art.
  • the vaccine may alternatively be in the form of a so-called “naked DNA” vaccine or in DNA vaccine where the vector consists of a DNA plasmid which is adapted to express the protein or peptide in situ.
  • the nucleic acid in included in a viral vector as are well known in the art.
  • the vector is a viral vector, it is suitably attenuated to minimise any harmful effects associated with the virus on the host.
  • the viral vector is derived from vaccinia virus, as it has many properties which make it a suitable vector for vaccination, including its ability to efficiently stimulate humoral as well as cell-mediated immune responses .
  • polypeptides of the invention produce an immunogenic response in a animal to which it is administered which is protective against infection by Francisella tularensis making them useful as a basis for a prophylactic or therapeutic vaccine.
  • Vaccines are suitably in the form of pharmaceutically acceptable compositions.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising either (a) a polypeptide as defined above, or (b) a nucleic acid as described above which is incorporated into a vector, such that it is expressed in a host to which said vector is administered; in combination with a pharmaceutically acceptable carrier or diluent .
  • composition will comprise a polypeptide as defined above .
  • compositions may be in a form suitable for intra-muscular, intra-venous, mucosal or parenteral application.
  • Mucosal applications include intra-nasal or oral applications .
  • Suitable carriers are well known in the art and include solid and liquid diluents, for example, water, saline or aqueous ethanol.
  • the liquid carrier is suitably sterile and pyrogen free.
  • compositions may be in the form of liquids suitable for infusion or injection, or syrups, suspensions or solutions, as well as solid forms such as capsules, tablets, or reconstitutable powders .
  • the compositions of the invention may further additional other active components.
  • the other component may comprise an adjuvant which enhances the host's immune response, and/or the polypeptide may be combined with an antigen giving protective immunity against a different pathogen to form a multivalent vaccine in order to increase the benefit-to-risk ratio of vaccination.
  • the other active component comprises an adjuvant which enhances the host's immune response and in particular promotes a cellular immune response, such as a CD8+, a CD4+ and/or a Thl response.
  • Adjuvants which may achieve these effects include cytokines such as interleukins and interferons.
  • the other component comprises a cytokine such as an interleukin, which acts as a Th-1 adjuvant.
  • cytokine such as an interleukin
  • Th-1 adjuvant a particularly preferred interleukin for inclusion in the vaccines of the invention is IL-12, which has been shown to drive the expansion of a protective Th-1 cell response during early murine tularemia (Golovliov I, et al.. (1995). Infection and Immunity 63 (2) -.534-8) .
  • Th-1 adjuvant IL-12 to include protection against the more virulent type B strains.
  • a fully virulent type B strain HN63 was used as the challenge strain to demonstrate protection beyond that provided by a humoral response. It is believed that this improved survival is due to stimulation of cell mediated immunity.
  • Polypeptides as described above may be prepared by various means including chemical synthesis, isolation from natural sources followed by any chemical modification if required, or using recombinant DNA technology.
  • the polypeptide is prepared by a method which comprises incorporating a nucleotide sequence which encodes said polypeptide into a recombinant expression vector, transforming a host cell with said vector, and culturing said cell and recovering the peptide from the culture .
  • the host cell may be eukaryotic or prokaryotic, but is conveniently a prokaryotic cell such as E. coll.
  • the invention provides the use of a polypeptide as described above in the prevention or treatment of infection by F. tularensis.
  • polypeptide may be used in the preparation of a medicament for the prevention or treatment of infection by F. tularensis.
  • the invention provides the use of a nucleic acid as described above in the prevention or treatment of infection by F. tularensis .
  • the invention provides a method for preventing or treating infection by F. tularensis, said method comprising administering to an animal, a polypeptide as described above.
  • the polypeptide may be administered directly in the form of a pharmaceutical composition.
  • a nucleic acid encoding said polypeptide is administered to an animal in a form in which it is expressed in situ . Suitable forms or vectors are described above.
  • Figure 1 shows the amino acid sequences of the F. tularensis GroEL and F. tularensis GroES proteins
  • Figure 2 shows an ECL western blot of A) eluted Hsp ⁇ O B) LVS lysate C) Schu 4 lysate D) molecular weight marker after probing with anti-groEL antibody;
  • Figure 3 is a graph showing the survival of mice immunised as described in Example 2 below following intraperitoneal challenge with 10 4 MLD's of live vaccine strain;
  • Figure 4 is a graph showing the survival of mice immunised as described in Example 2 below following intraperitoneal challenge with 100 MLD's of the more virulent HN63 strain.
  • Figure 5 shows a silver-stained SDS PAGE showing: lane A and E molecular weight markers, lane B eluted GroEL, lane C LVS and lane D HN63.
  • Example 1 Francisella tularensis LVS was grown on blood cysteine glucose agar for 72 hours, harvested into PBS, washed and heat killed at 65°C for 1 hour. The bacteria were lysed by boiling in Laemmli denaturing sample buffer and the proteins were separated using SDS-PAGE. GroEL was identified by western blotting using a monoclonal antibody to GroEL (Sigma) and by molecular weight determination. Once located GroEL was excised from large format unstained gels and electroeluted into buffer using the Hoefer gel eluter (Amersham Pharmacia) set at 70 volts for 2 hours and following manufacturers instructions .
  • Hoefer gel eluter Amersham Pharmacia
  • the eluted product was pooled and purified by diluting (more than x20) in volatile buffer (ammonium bicarbonate 3.95g/l, SDS lg/1) and centrifuged over a dialysis membrane (MWC 10,000 Vivascience) .
  • the protein was then diluted (x20) in sterile water and centrifuged further, until significant concentration was achieved. Any excess SDS was removed by cold precipitation followed by microcentrifugation.
  • Sections of gel above 60kDa, known not to contain protein bands were excised, eluted, purified and concentrated in a similar manner; and used to provide control vaccinations.
  • the samples were protein assayed by the BCA protein assay using BSA as a standard.
  • mice were immunised on days 0 and 14 as detailed in Table 1. Groups were challenged on day 28 with either 10 4 MLD's of LVS intraperitoneally or 100 MLD's of HN63 subcutaneously. Mice were monitored for 14 days and survival to a humane end point was recorded. Table 1: immunisation regime
  • Figures 3 and 4 show the survival of mice immunised with 50 ⁇ g (in total) of this protein and challenged with either LVS or the more virulent HN63. In both challenges all of the control mice died (groups 3) , and there was partial protection from immunisation with Hsp ⁇ alone (groups 2) .

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Polypeptide comprenant au moins un composant d'un Hsp60 pouvant être obtenu à partir de Francisella tularensis, et notamment la protéine GroEL, ou un fragment de celle-ci, ou une de leurs variantes; lorsque ce polypeptide est administré à un animal, il produit une réponse immune qui protège contre l'infection par F. tularensis, ce qui peut être utilisé pour la vaccination prophylactique ou thérapeutique contre F. tularensis. Le polypeptide peut être administré directement à l'hôte, ou l'acide nucléique codant le polypeptide peut être administré sous une forme sous laquelle il peut être exprimé in vivo. Les vaccins à base de ces polypeptides comprennent de façon appropriée un composant supplémentaire et notamment un additif qui favorise une réponse immune cellulaire.
PCT/GB2001/003835 2000-08-26 2001-08-24 Homologues groes et groel de francisella tularensis WO2002018600A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001282350A AU2001282350A1 (en) 2000-08-26 2001-08-24 Groes and groel homologs of francisella tularensis

Applications Claiming Priority (2)

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GB0021008.8 2000-08-26
GBGB0021008.8A GB0021008D0 (en) 2000-08-26 2000-08-26 Protein and nucleic acid sequence

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WO2002018600A1 true WO2002018600A1 (fr) 2002-03-07

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004003009A2 (fr) * 2002-06-28 2004-01-08 The Secretary Of State For Defence Proteines immunogenes et adn les codant
US20050089509A1 (en) * 2003-09-12 2005-04-28 Board Of Regents, The University Of Texas System Treatment of Francisella infection with an IFN-gamma inducer and a chemotherapeutic agent
US8198430B2 (en) 2002-05-31 2012-06-12 The Secretary Of State For Defence Immunogenic sequences
US8323664B2 (en) 2006-07-25 2012-12-04 The Secretary Of State For Defence Live vaccine strains of Francisella
US8609108B2 (en) 2009-04-14 2013-12-17 The Secretary Of State For Defence Gamma-glutamyl transpeptidase attenuated Francisella

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ERICSSON M ET AL: "Increased synthesis of DnaK, GroEL, and GroES homologs by Francisella tularensis LVS in response to heat and hydrogen peroxide.", INFECTION AND IMMUNITY, vol. 62, no. 1, 1994, pages 178 - 183, XP002184370, ISSN: 0019-9567 *
ERICSSON MATS ET AL: "Characterization of the nucleotide sequence of the groE operon encoding heat shock proteins chaperone-60 and -10 of Francisella tularensis and determination of the T-cell response to the proteins in individuals vaccinated with F. tularensis.", INFECTION AND IMMUNITY, vol. 65, no. 5, 1997, pages 1824 - 1829, XP002184369, ISSN: 0019-9567 *
ERICSSON MATS ET AL: "Long-lasting recall response of CD4+ and CD8+ alphabeta T cells, but not gammadelta T cells, to heat shock proteins of Francisella tularensis.", SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES, vol. 33, no. 2, 2001, pages 145 - 152, XP001041558, ISSN: 0036-5548 *
WEIGL E ET AL: "Heat shock proteins in immune reactions.", FOLIA MICROBIOLOGICA, vol. 44, no. 5, 1999, pages 561 - 566, XP001041540, ISSN: 0015-5632 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8198430B2 (en) 2002-05-31 2012-06-12 The Secretary Of State For Defence Immunogenic sequences
WO2004003009A2 (fr) * 2002-06-28 2004-01-08 The Secretary Of State For Defence Proteines immunogenes et adn les codant
WO2004003009A3 (fr) * 2002-06-28 2004-02-26 Secr Defence Proteines immunogenes et adn les codant
US20050089509A1 (en) * 2003-09-12 2005-04-28 Board Of Regents, The University Of Texas System Treatment of Francisella infection with an IFN-gamma inducer and a chemotherapeutic agent
US8323664B2 (en) 2006-07-25 2012-12-04 The Secretary Of State For Defence Live vaccine strains of Francisella
US8790910B2 (en) 2006-07-25 2014-07-29 The Secretary Of State For Defence Live vaccine strain
US8609108B2 (en) 2009-04-14 2013-12-17 The Secretary Of State For Defence Gamma-glutamyl transpeptidase attenuated Francisella

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AU2001282350A1 (en) 2002-03-13

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