WO2003012117A1 - Vaccin a adn - Google Patents

Vaccin a adn Download PDF

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Publication number
WO2003012117A1
WO2003012117A1 PCT/GB2002/003448 GB0203448W WO03012117A1 WO 2003012117 A1 WO2003012117 A1 WO 2003012117A1 GB 0203448 W GB0203448 W GB 0203448W WO 03012117 A1 WO03012117 A1 WO 03012117A1
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WO
WIPO (PCT)
Prior art keywords
dna construct
vaccine
construct according
domain
sequence
Prior art date
Application number
PCT/GB2002/003448
Other languages
English (en)
Inventor
Alice Marie Bennett
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
Priority claimed from GB0118475A external-priority patent/GB0118475D0/en
Application filed by The Secretary Of State For Defence filed Critical The Secretary Of State For Defence
Publication of WO2003012117A1 publication Critical patent/WO2003012117A1/fr

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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • the present invention relates to a DNA vaccine, in particular a vaccine which is applied as a "naked DNA” vaccine, or includes a naked DNA vaccine element, and which produces an immune response in a host to whom it is administered, which is protective against infection by Clostridium botulinum or intoxication by Clostridium botulinum neurotoxins .
  • Botulism is a potentially fatal intoxication caused by a neurotoxin produced by Clostridium botulinum.
  • Clostridium botulinum There are seven serotypes of botulinum neurotoxin (BoNT A, B,C,D,E,F, and G) and four of these, A,B,E and F are known to be toxic for humans.
  • a pentavalent vaccine which protects against types A, B,C,D and E is known.
  • the toxin consists of two protein chains (a heavy chain and a light chain) which are joined together. It is thought that the heavy chain binds to nerve cells and the light chain is then injected into the cells where it damages a protein involved in the transmission of nerve impulses.
  • the heavy chain consists of two regions, called the N domain and the C domain. The C domain is responsible for binding to target cells and the N domain functions in internalization of the toxin. It has been shown that the C domain of the heavy chain (He) is sufficient to generate protective immunity to the toxin type A.
  • Other vaccines based upon recombinant toxin fragments are described in WO 98/07864.
  • WOO/02524 describes Botulinum neurotoxin vaccines consisting of virus vectors which comprise the He domains of types A - G neurotoxins .
  • Nucleic acid vaccines are known in the art and they represent a powerful approach to the generation of vaccines . In particular, they represent a radical change in the way that antigens are delivered, involving the introduction of plasmid DNA encoding an antigenic protein which is then expressed in the cells of the vaccinated host. Nucleic acid vaccines have a number of advantages over conventional vaccines such as live attenuated, killed whole, or subunit vaccines. Although synthesised in the cells of the host like live-attenuated vaccines, nucleic acid vaccines are subunit vaccines, which encode selected components of a pathogen, rather than the entire pathogen. Therefore, nucleic acid vaccines place the vaccinated host at no risk of infection.
  • nucleic acid vaccines can raise both humoral and cellular immune responses . This is due to the ability of proteins that are synthesised within cells to access pathways for presentation by both class I and class II major histocompatibility antigens. Nucleic acid vaccines are potentially simple and inexpensive to manufacture and produce. In addition, DNA is heat stable, which would aid the transportation and subsequent storage of nucleic acid vaccines .
  • a DNA construct comprising a nucleic acid encoding a fusion of at least an antigenic part of an He domain of a botulinum neurotoxin and a signal sequence which is able to export protein from mammalian cells, operatively interconnected to a promoter which is active in mammalian cells, wherein the nucleic acid sequence encoding the antigenic part of an He domain is a recombinant sequence, wherein at least some codons are changed as compared to the wild-type sequence to codons which are preferred for expression in mammalian cells.
  • the botulinum neurotoxin is a BoNT Type A,B,C,D,E,F, or G toxin, more suitably a Type B,C,D,E,F, or G toxin, and preferably a Type F toxin.
  • antigenic part of the He domain is intended to cover deletion mutants, which retain the ability to produce a protective immune response. Such mutants can be determined using routine methods . Generally, these may be expected to encode one or more fragments of the full-length sequence, each fragment being at least 8 amino acids in length.
  • substantially all the He domain is encoded by the nucleic acid of the invention.
  • the He domains of the neurotoxins are fully described in the art (Atassi et al., (1999) . Structure, activity and immune (T and B cell) recognition of botulinum neurotoxins. Critical Reviews in Immunology, 19: 219-260) and the content of this document is incorporated herein by reference.
  • the nucleic acid sequence has at least some codons that are preferred for expression in mammalian cells, which generally means that the AT content of the sequence is reduced as compared to the wild-type sequence. Suitably this means that at least 40% and preferably at least 60% of the codons in the sequence correspond to those found at high frequency in mammalian genes (Wada et al (1992). Nucleic Acid Research, vol. 20, supplement; pp. 2111-2118) .
  • SEQ ID NO 1 This sequence, and variants, for example which show at least 60% identity, more preferably 90% identity to SEQ ID NO 1 form embodiments of the invention.
  • Such variants include SEQ ID NO 2.
  • Sequence identity in this case may be ascertained by any of the known algorithms .
  • a particular example is MegAlign found in the Lasergene sequence analysis software package (DNASTAR Inc., 1228 South Part Street, Madison WI 53715, US) .
  • variants will comprise sequences which hybridise SEQ ID NO 1 under stringent hybridisation conditions.
  • hybridisation occurs at, or between, low and high stringency conditions.
  • low stringency conditions can be defined as 3 x SSC at about ambient temperature to about 65°C
  • high stringency conditions as 0.1 x SSC at about 65°C.
  • SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate.
  • 3 x SSC is three times as strong as SSC and so on.
  • Preferred variants are those which hybridise under high stringency conditions.
  • Suitable signal sequences include signal sequences derived from immunoglobulins .
  • the V-J2-C region of the mouse Ig kappa chain is a preferred sequence.
  • Others include the 25 nucleotides which encode the signal peptide of herpes simplex virus type 2 (Higgins et al. (2000) . The Journal of Infectious Disease 182, 1311-1320) .
  • Another example is a sequence derived from the V H gene utilized by the IgM of the BCLl tumour (Rice et al. (1999). Vaccine 17, 3030-3038).
  • Promoter sequences which are active in mammalian cells are well known. They include the CMV major IE promoter.
  • the sequences of the invention are particularly suitable for use as "naked DNA” vaccines . Consequently they are suitably incorporated into plasmids or vectors, and particularly non- infectious nucleic acid vectors, suitable for use in vaccination.
  • vectors include the commercially available plasmid vector, pSECTAG-2C (Invitrogen) , as well as pVAX-1 (Invitrogen) , pcDNA vectors (Invitrogen) , pDisplay (Invitrogen), pCI and derivatives (Promega) .
  • the invention provides a prophylactic or therapeutic vaccine comprising a pharmaceutically acceptable plasmid or vector which includes a nucleic acid as described above .
  • Such vaccines may also include pharmaceutically acceptable carriers which may be liquid or solid.
  • they will be formulated such that they are suitable for parenteral administration, for example by combination with liquids such as saline.
  • the compositions of the invention are preferably formulated for parenteral administration and in particular intramuscular injection, although other means of application are possible as described in the pharmaceutical literature, for example administration using a Gene Gun, (Bennett et al . , (2000), Vaccine 18, 1893-1901). Oral or intra-nasally delivered formulations are also possible.
  • Such formulations include delivery of the plasmid DNA via a bacterial vector such as species of Salmonella or Listeria (Sizemore et al (1997) . Vaccine 15, 804-807) .
  • a synthetic version of the DNA encoding the He domain of type F neurotoxin has been used to construct a DNA vaccine. It is known in the art that altering the codon usage of a gene can increase its expression in a particular expression system. Although the intended expression system for this construct was mammalian cells in vivo, the cloned BoNT F gene used here had the codon usage for Pichia pastoris as this synthetic gene had been constructed previously and the codon usage was similar to mammalian codon usage.
  • Expression of the cloned DNA would produce a protein containing the same amino acid sequence as the native toxin He domain.
  • the DNA fragment was cloned into a commercially available plasmid vector, pSECTAG-2C (Invitrogen) , such that expression of the inserted DNA resulted in production of a fusion protein with the V-J2-C region of the mouse Ig kappa chain being fused to the amino terminus of the He domain of BoNT F. Expression of the fusion protein was under the control of the CMV major IE promoter.
  • the resulting plasmid was used to immunise Balb/c mice by intramuscular injection and the vaccinated mice survived a challenge with 10E+4 mouse lethal doses (MLD) BoNT F toxin. This is an outstanding result, indicating that the DNA vaccine might be useful for therapy of botulism as well as prophylaxis.
  • MLD mouse lethal doses
  • a method of treating an infection of Clostridium botulinum or a method of treating intoxication with Clostridium botulinum neurotoxins, which method comprises administering to a mammal in need thereof, a vaccine as described above.
  • Yet a further aspect of the invention provides a method of protecting a mammal against infection by Clostridium botulinum or intoxication by Clostridium botulinum neurotoxins, which method comprises, administering to said mammal, a vaccine as described above.
  • the invention provides a DNA construct as described above for use as a prophylactic or medicament.
  • the dosages used will vary depending upon the mammal being treated, the age and size of the mammal, and the disease status of the mammal. These will be determined using conventional clinical practice. Generally speaking however, for administration to a human as a prophylactic vaccine, dosage units of from 0.25 ⁇ g to 2.5mg may be employed. However, generally dosages of from 25 ⁇ g to 12.5mg may be employed.
  • the applicants have found that a dosage regime in which the DNA vaccine is used as a priming vaccine, followed by a boost with a purified protein having the amino acid sequence of the He domain of a botulinum toxin, is extremely effective at producing antibodies to the toxin.
  • the boost is administered some time after the administration of one or more doses of the DNA vaccine.
  • up to 4 doses of the DNA vaccine may be administered to a mammal, at intervals of 10- 20 days, and preferably about every 14 days, followed by a single dose of protein some time later, for example up to 100 days after the initial priming dose.
  • the dosage of the protein will be determined according to routine clinical practice, depending upon the nature of the patient, the severity of disease where present etc. Occasionally, dosages of 2.5 ⁇ g to 50mg may be employed. Generally, however, dosages of from l ⁇ g to 5mg may be employed and preferably dosages of from 2.5 ⁇ g to 5mg may be employed.
  • Such a dosage regime may be particularly useful in the prophylaxis or treatment of disease, and this forms yet a further aspect of the invention.
  • the invention further provides a combination of DNA construct as described above and a purified protein comprising the He domain of a botulinum toxin encoded by a nucleic acid within the construct, for use in prophylaxis or therapy of botulinum infection.
  • a further aspect of the invention comprises the use of the combination described above in the preparation of a medicament for use in the prophylaxis or therapy of botulism.
  • antisera or antibodies produced in this way using for example laboratory animals may be harvested and used for example in passive immunisation techniques, or in therapy.
  • the invention therefore further provides a method for producing antisera or antibodies against the He domain of a botulinum toxin, said method comprising administering to an animal, such as a mouse, or rabbit, a DNA construct as defined above, or a combination of a DNA construct and a protein as defined above, and recovering antisera or antibodies from the blood of said animal .
  • Antibodies obtained using this method together with their use in prophylaxis or therapy form yet further aspects of the invention.
  • a DNA vaccine is used as a priming vaccine, followed by a boost, with a purified protein having the amino acid sequence of the He domain of a botulinum toxin
  • the mix of antibodies produced is different to that produced using purified FHc protein alone.
  • the DNA vaccine predominantly induces the IgG2a subclass whereas FHc protein alone predominantly induces the IgGl subclass.
  • Figure 2 is a graph showing survival of balb/c mice following challenge with 10 4 MLD botulinum neurotoxin F. Mice had been vaccinated according to the schedule in Table 1 below. Day 0 indicates the day of challenge.
  • Figure 3 is a graph showing survival of Balb/c mice following challenge with 10 4 MLD botulinum neurotoxin F. Mice had been immunised according to the schedule in Table 2 below.
  • Figure 4 shows the concentration (ng/ml) of IgG isotypes in serum collected 81 days after the primary vaccination from mice vaccinated according to the schedule in Table 1 below.
  • a synthetic version of the DNA encoding the He domain of type F neurotoxin has been used to construct a DNA vaccine.
  • the sequence of the synthetic BoNT FHc DNA is given (SEQ ID 1) .
  • the DNA fragment was cloned into the commercially available plasmid vectors, pVAX-1 and pSECTAG-2C (Invitrogen) to produce the plasmids pABFHcl and pABFHc2, respectively.
  • Expression of the FHc protein was under the control of the CMV major IE promoter in both plasmids.
  • Expression of the inserted DNA from pABFHc2 resulted in production of a fusion protein in which the V-J2-C region of the mouse Ig kappa chain was fused to the amino terminus of BoNT FHc.
  • mice 10 female, Balb/c mice (6-8 weeks old) were vaccinated according to the schedule in Table 1.
  • Groups 1-4 were immunised with 5 doses of lOO ⁇ g plasmid in 0.25% v/v bupivacaine hydrochloride by intra-muscular injection into the hind quarters (50 ⁇ g per leg) .
  • Group 5 was given three doses of 2.5 ⁇ g purified FHc protein in alhydrogel by intra-peritoneal (i.p.) injection. All animals were microchipped and the development of the antibody response was determined by immunoassay of serum samples taken at intervals.
  • Fig. 1 The results of the immunoassay are shown in Fig. 1 and demonstrate that the response to pABFHc2 increased with time following the first four doses. The fifth dose did not increase the level of anti-FHc IgG significantly. Serum from mice in groups 1, 3, 4 and 5 had undetectable levels of anti-FHc IgG using this assay.
  • mice All mice were challenged with 10 4 mouse lethal doses (MLD) of botulinum neurotoxin F by i.p. injection and the results are shown in Fig. 2.
  • MLD mouse lethal doses
  • the mice in group 1 had a 90% survival rate while all the animals in groups 2 and 5 survived the challenge.
  • the DNA vaccine consists of the plasmid pABFHc2 (100 ⁇ g/dose) formulated in bupivacaine hydrochloride (0.25%) .
  • the protein vaccine consists of a purified fusion protein of the maltose binding protein and botulinum neurotoxin type F binding domain, formulated in alhydrogel (20%) .
  • Serum samples were taken from mice two weeks after the final vaccination dose.
  • Antibodies to botulinum neurotoxin type F binding domain were measured in pooled serum samples using an enzyme-linked immunosorbent assay. Results are given in Table 1.
  • the results for group 1 and 2 in Table 3 are taken from separate experiments . A direct comparison of "DNA-prime+protein boost" with "vaccination using protein alone” has not been performed as yet within the same experiment. However the levels of antibodies raised using these vaccines follows a consistent pattern so the results given on Table 1 are indicative of what would be expected.
  • Figure 4 shows that immunisation with purified FHc protein induces predominantly IgGl, whereas immunisation with the DNA vaccine pABFHc2 induces a balanced profile with similar levels of IgGl and IgG2a.
  • the isotype profile following a protein boost is usually similar to that following the primary immunisation. Therefore boosting with FHc as described in Example 2 would not be expected to alter the profile of the pABFHc2 group significantly.

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  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
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  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une construction d'ADN comprenant un acide nucléique codant pour une fusion d'au moins une partie antigénique d'un domaine Hc d'une neurotoxine botulique et d'une séquence-signal permettant d'exporter une protéine à partir de cellules mammifères, en interconnexion fonctionnelle avec un promoteur actif dans les cellules mammifères. Ces constructions sont utiles dans la production de vaccins prophylactiques ou thérapeutiques.
PCT/GB2002/003448 2001-07-28 2002-07-26 Vaccin a adn WO2003012117A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0118475A GB0118475D0 (en) 2001-07-28 2001-07-28 Dna vaccine
GB0118475.3 2001-07-28
GB0119271A GB0119271D0 (en) 2001-07-28 2001-08-08 DNA Vaccine
GB0119271.5 2001-08-08

Publications (1)

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WO2003012117A1 true WO2003012117A1 (fr) 2003-02-13

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2889066A1 (fr) * 2005-07-28 2007-02-02 Centre Nat Rech Scient Procede d'immunisation genetique par electrotransfert contre une toxine et antiserum susceptible d'etre obtenu par ledit procede
US8445650B2 (en) 2007-09-25 2013-05-21 Thomas Jefferson University Mutant botulinum neurotoxin serotype A polypeptide and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998034640A2 (fr) * 1997-02-07 1998-08-13 Merck & Co., Inc. Genes synthetiques du gag hiv
WO2000002524A2 (fr) * 1998-07-10 2000-01-20 U.S. Army Medical Research Institute Of Infectious Diseases Vaccin a base de neurotoxine botulinique
WO2000067700A2 (fr) * 1999-05-12 2000-11-16 United States Army Medical Research & Materiel Cmd Vaccin de recombinaison contre la neurotoxine botulique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998034640A2 (fr) * 1997-02-07 1998-08-13 Merck & Co., Inc. Genes synthetiques du gag hiv
WO2000002524A2 (fr) * 1998-07-10 2000-01-20 U.S. Army Medical Research Institute Of Infectious Diseases Vaccin a base de neurotoxine botulinique
WO2000067700A2 (fr) * 1999-05-12 2000-11-16 United States Army Medical Research & Materiel Cmd Vaccin de recombinaison contre la neurotoxine botulique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HIGGINS TERRY J ET AL: "Plasmid DNA-expressed secreted and nonsecreted forms of herpes simplex virus glycoprotein D2 induce different types of immune responses.", JOURNAL OF INFECTIOUS DISEASES, vol. 182, no. 5, November 2000 (2000-11-01), pages 1311 - 1320, XP002224536, ISSN: 0022-1899 *
RICE J ET AL: "Manipulation of pathogen-derived genes to influence antigen presentation via DNA vaccines", VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 17, no. 23-24, 6 August 1999 (1999-08-06), pages 3030 - 3038, XP004173614, ISSN: 0264-410X *
SHYU RONG-HWA ET AL: "DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice.", JOURNAL OF BIOMEDICAL SCIENCE, vol. 7, no. 1, January 2000 (2000-01-01), pages 51 - 57, XP009002343, ISSN: 1021-7770 *
WILLIAMSON E D ET AL: "Presentation of protective antigen to the mouse immune system: Immune sequelae.", JOURNAL OF APPLIED MICROBIOLOGY, vol. 87, no. 2, August 1999 (1999-08-01), 3rd International Conference on Anthrax;Plymouth, England, UK; September 7-10, 1998, pages 315 - 317, XP002224535, ISSN: 1364-5072 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2889066A1 (fr) * 2005-07-28 2007-02-02 Centre Nat Rech Scient Procede d'immunisation genetique par electrotransfert contre une toxine et antiserum susceptible d'etre obtenu par ledit procede
WO2007012671A3 (fr) * 2005-07-28 2007-04-26 Centre Nat Rech Scient Procédé d'immunisation génétique par électrotransfert contre une toxine et antisérum susceptible d'être obtenu par ledit procédé
JP2009502880A (ja) * 2005-07-28 2009-01-29 サントル ナショナル ドゥ ラ ルシェルシュ シアンティフィック（セーエヌエールエス） 毒素に対する電気移動による遺伝子免疫法および前記方法によって得られる抗血清
US8445650B2 (en) 2007-09-25 2013-05-21 Thomas Jefferson University Mutant botulinum neurotoxin serotype A polypeptide and uses thereof

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