WO2005123121A2 - Use of dna molecule as vaccine adjuvant - Google Patents
Use of dna molecule as vaccine adjuvant Download PDFInfo
- Publication number
- WO2005123121A2 WO2005123121A2 PCT/GB2005/002472 GB2005002472W WO2005123121A2 WO 2005123121 A2 WO2005123121 A2 WO 2005123121A2 GB 2005002472 W GB2005002472 W GB 2005002472W WO 2005123121 A2 WO2005123121 A2 WO 2005123121A2
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- WIPO (PCT)
- Prior art keywords
- vaccine
- antigen
- molecule
- organism
- dna
- Prior art date
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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
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/30011—Nodaviridae
- C12N2770/30034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- This invention relates to the identification of a DNA molecule encoding an antigen and in particular a rhabdovirus glycoprotein as new and effective adjuvant molecules and the use of said DNA molecules as adjuvants.
- the present invention further provides vaccine compositions comprising said adjuvant and one or more vaccine molecules, and the use of such compositions in vaccination.
- Vaccination of mammals and other organisms against various types of infection and disease is an important goal in many fields of biotechnology. In particular in fields of food production and farming it is vital to protect the reared organisms from infection and disease which can lead to illness or even death of organisms and thereby reduce the yield and quality of the food product.
- the development of new or improved vaccines which will protect against various types of infection and disease is desirable.
- the present invention provides the use of a DNA molecule encoding an antigen which is expressed on the surface of a host cell, or a variant, or a fragment thereof, as a vaccine adjuvant.
- the present invention provides the use of a DNA molecule encoding an antigen which is expressed on the surface of a host cell, or a variant, or a fragment thereof, to enhance the immune stimulating properties or vaccination properties of a vaccine molecule, or to enhance the immune response to a vaccine molecule.
- the term "antigen" is used herein to mean any foreign substance (i.e. a substance not normally present in a host organism) which is capable of triggering an immune response in an organism. Thus, molecules normally present in a particular host organism are excluded.
- antigens of the present invention are preferably antigens which are expressed on the surface of cells in the host organism in question. This means that molecules which encode antigens which remain in the cytoplasm of host cells are also preferably excluded.
- antigens which are included in the scope of the present invention are antigens which can be presented (expressed) on the surface of T cells in the host organism.
- appropriate antigens are antigens which, in their native form, are found on the surfaces of cells or other biological particles such as viruses or bacteria, and which are then expressed on the surface of cells in the particular host organism.
- preferred antigens are bacterial or viral proteins found on the surface of bacterial or viral particles, e.g. envelope proteins.
- glycoproteins are preferred antigens for use in all aspects of the present invention.
- the DNA molecule encoding an antigen which is expressed on the surface of a host cell is replaced by a DNA molecule encoding a glycoprotein.
- glycoproteins when expressed in the host organism, such glycoproteins are expressed on the surface of a host cell, for example such glycoproteins are anchored in the membrane and exposed on the surface of host cells.
- Such glycoproteins can be derived from any sources. However preferred sources are bacteria and viruses and thus bacterial and viral glycoproteins are preferred.
- the virus or bacteria from which the glycoproteins are derived is a virus or bacteria that can replicate in or can infect the organism to which the adjuvant is to be administered.
- the encoded glycoprotein is a virus glycoprotein, most preferably a rhabdovirus glycoprotein.
- the DNA molecule encoding the antigen and the vaccine molecule for which said DNA molecule acts as an adjuvant are expressed in the same cells in the host organism.
- the present invention further provides the use of a DNA molecule encoding an antigen which is expressed on the surface of a host cell, or a variant, or a fragment thereof, in the manufacture of a medicament for use as a vaccine adjuvant or to enhance the immune stimulating properties or vaccination properties of a vaccine molecule, or to enhance the immune response to a vaccine molecule.
- the present invention further provides a method of vaccination of an organism or a method of stimulating an immune response in an organism wherein a DNA molecule encoding an antigen which is expressed on the surface of a host cell, or a variant, or a fragment thereof, is used as a vaccine adjuvant or is used to enhance the immune stimulating properties or vaccination properties of a vaccine molecule, or to enhance the immune response to a vaccine molecule.
- DNA molecules encoding glycoproteins from any virus may be used in the present invention.
- Preferred viruses include rabes and/or rhabdovirus.
- a preferred viral glycoprotein is a rhabdovirus glycoprotein.
- Any enhancement or increase as described herein, particularly in connection with the immune stimulating properties or vaccination properties of a vaccine molecule or the immune response to a vaccine molecule includes any measurable enhancement or increase when the property in question in the presence of the DNA molecules (adjuvants) of the invention is compared with the equivalent parameters in the absence of the DNA molecules (adjuvants).
- the enhancement or increase is a statistically significant one.
- a parameter is generally regarded as significant if a statistical comparison using an appropriate statistical test such as a Student t-test shows a probability value of ⁇ 0.05.
- the methods and uses of the invention can be carried out by administration of appropriate DNA molecules encoding an antigen to any appropriate organism.
- Appropriate organisms may be mammalian or non-mammalian and include humans and all farmed or reared organisms.
- appropriate organisms include humans, avian species such as chickens or birds, cows, pigs, sheep, rodents (e.g. mouse, rat, guinea pig , hamster etc), fish, insects, crustaceans, etc.
- Farmed or reared organisms are particularly preferred and fish, in particular marine species offish such as Atlantic halibut, turbot, spotted wolfish, groupers, salmon, trout and cod, are especially preferred in this regard.
- marine species offish such as Atlantic halibut, turbot, spotted wolfish, groupers, salmon, trout and cod
- farmed fish which are preferred are salmonids, cadoids, groupers, flat fish (turbot, halibut), sea bream, trout, sea bass and striped jack.
- Flat fish are especially preferred.
- further preferred antigens for use as adjuvants or to enhance the immune stimulating properties, etc. are antigens, for example glycoproteins, derived from organisms or pathogens that can replicate or infect fish, e.g. are derived from fish pathogens.
- the glycoprotein is not derived from Hepatitis B, EBV, plant viruses (e.g. potato viruses) and/or cholera toxin
- adjuvant is used herein in its standard meaning known in the art and refers to substances which are added to antigens (or vaccine molecules) to enhance the immune response to such antigens (or vaccine molecules).
- the adjuvants are generally used to enhance the immune response to antigens which are distinct from the antigen which comprises the adjuvant.
- the adjuvant is DNA encoding a rhabdovirus glycoprotein, this is generally used as an adjuvant to enhance the immune response to antigens other than the rhabdovirus glycoprotein being used as the adjuvant.
- Preferred DNA molecules used in the present invention may encode a glycoprotein from any type of rhabdovirus and the DNA encoding the rhabdovirus glycoprotein for use in the present invention can be derived from any appropriate source.
- the rhabdovirus glycoprotein is a fish rhabdovirus glycoprotein, for example a glycoprotein derived from viral hemorrhagic septicemia virus (VHSV) or infectious hematopoietic necrosis virus (THNV).
- VHSV viral hemorrhagic septicemia virus
- THNV infectious hematopoietic necrosis virus
- a particularly preferred rhabdovirus glycoprotein is that derived from VHSV.
- a preferred DNA molecule in this regard is pVHSV-G as described in the Examples.
- DNA molecules encoding a full length antigen preferably a rhabdovirus glycoprotein
- variants functionally equivalent variants
- fragments functionally equivalent fragments of such full length molecules or variants
- fragments functionally equivalent fragments of such full length molecules or variants
- “Functionally equivalent” is used herein to define DNA molecules encoding proteins related to or derived from the native protein, where the amino acid sequence has been modified by single or multiple amino acid substitution, addition and/or deletion, but which nonetheless retain the ability to function as an adjuvant in the organism in question.
- Functionally equivalent variants also include degenerate sequences, i.e. nucleic acid sequences which contain base changes (i.e.
- nucleotide changes that do not cause a change in the encoded amino acid sequence.
- DNA molecules which are substantially homologous to the DNA molecule encoding the native protein or which encode sequences which are substantially homologous to the encoded native protein are also included.
- substantially homologous as used herein in connection with an amino acid or a nucleic acid sequence includes those sequences having a sequence homology or identity of approximately 60% or more, e.g. 70%, 80%, 90%, 95%, 98% or more with a particular sequence and also functionally equivalent variants and related sequences modified by single or multiple base or amino acid substitution, addition and/or deletion. Homology may be assessed by any convenient method.
- sequences according to the present invention having 60%, 70%, 80%, 90%), 95% homology etc. may be determined using the ALIGN program with default parameters (for instance available on Internet at the GENESTREAM network server, IGH, adjoin, France).
- Such functionally equivalent variants mentioned above also include natural biological variations (e.g. allelic variants or geographical variations within a species) and derivatives prepared using known techniques.
- functionally equivalent molecules may be prepared either by chemical synthesis or in recombinant form using the known techniques of site directed mutagenesis (including deletion), random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acid.
- Functionally equivalent variants also include analogues in different genera or species.
- Preferred fragments for use in the present invention encode at least the transmembrane domain of an antigen, preferably the rhabdovirus glycoprotein.
- DNA molecules encoding rhabdovirus glycoproteins which may be used in the present invention are known in the art, for example DNA encoding the glycoproteins from VHSV and IHNV are known.
- DNA sequences encoding appropriate glycoproteins from any rhabdoviruses can be readily determined using standard and routine methods.
- a rhabdovirus can be taken and the glycoprotein isolated from the viral envelope, after which a peptide sequence and DNA sequence can readily be determined using standard methods.
- the DNA molecules encoding antigens, preferably rhabdovirus glycoproteins, for use in the present invention can take any appropriate form and can be produced by any appropriate method, e.g. by recombinant techniques.
- such DNA molecules take the form of plasmids or expression vectors and can be produced by any appropriate method, e.g.
- said DNA sequences encoding the antigen or variant, or fragment thereof are placed downstream of an appropriate promoter sequence, e.g. a eukaryotic promoter sequence such as the early CMV promoter, or another appropriate promoter which is recognised by the organism to which the DNA molecule is to be administered.
- an appropriate promoter sequence e.g. a eukaryotic promoter sequence such as the early CMV promoter, or another appropriate promoter which is recognised by the organism to which the DNA molecule is to be administered.
- promoters which are native to the organism which is being vaccinated can also be used and are sometimes preferred, e.g.
- Non-plasmid or non-vector forms of DNA molecules can also be used.
- DNA molecules encoding only the antigen without any vector components can be used. Plasmids or vectors containing appropriate promoter elements and other regulatory elements are readily available commercially or can readily be designed and made in the laboratory using standard skills and methods.
- an appropriate vector for use in this regard which contains the CMV early promoter is the pcDNA3 vector (Invitrogen).
- the DNA molecules encoding the antigen, preferably the rhabdovirus glycoprotein may optionally be modified so that they are more stable in vivo using methods which are well known and standard in the art.
- the DNA backbone may be modified to improve stability by methods well known and standard in the art.
- the DNA molecules encoding an antigen, preferably a rhabdovirus glycoprotein can be used as an adjuvant for any contemplated vaccine molecule (or indeed as an adjuvant for any antigen) and preferably a vaccine molecule against which a specific long lasting immune response is desired.
- the vaccine molecule may be any molecule wherein it is desirable to stimulate an immune response to that molecule or part thereof in the organism in question. Such vaccine molecules might be able to give rise to the stimulation of an immune response in their own right. However, the uses and methods of the invention are particularly appropriate for vaccine molecules which are not able to generate a strong or significant or effective or adequate immune response or protective response in their own right (e.g. when administered by themselves), in which case the adjuvant properties of the DNA molecule encoding the antigen are particularly valuable. Thus, in preferred embodiments the vaccine molecule does not itself act as an adjuvant. Thus, said vaccine molecule can be any known or new protein or DNA vaccine, including recombinant vaccines and peptide vaccines.
- vaccine molecules are known in the art and include all manner of bacterial or viral antigens or indeed antigens or antigenic components of any pathogenic species.
- Appropriate vaccine molecules may comprise whole organisms (whether live, dead or attenuated) or may be recombinant vaccine molecules, e.g. sub-unit vaccine molecules based on particular components of organisms, e.g. proteins, peptides or even carbohydrates.
- Such vaccine molecules may comprise the relevant protein, peptide or carbohydrate molecules or, if DNA vaccines are to be used, may comprise nucleic acid molecules which encode said relevant proteins or peptides.
- Vaccine molecules based on antigenic molecules associated with particular disease states e.g. cancer vaccines, can also be used.
- DNA vaccines are known and described in the art. However, in general such vaccines usually take the form of an expression vector (e.g. a plasmid) carrying an appropriate foreign DNA fragment (e.g. a fragment encoding a pathogenic protein or peptide) regulated by an appropriate promoter which is recognised by the organism being vaccinated (e.g. a eukaryotic promoter).
- an appropriate promoter which is recognised by the organism being vaccinated
- the DNA fragment of interest is expressed when the expression vector is taken up by cells and transported to the nucleus where the cellular transcription apparatus recognise the promoter.
- Such DNA vaccines are thought to be more safe than protein or peptide vaccines as they have no risk of reversion to virulence. Any appropriate promoter can be used in the DNA vaccines of the invention.
- Common promoters for use in this regard are eukaryotic promoters such as the CMV immediate early promoter.
- promoters which are native to the organism which is being vaccinated are also preferred, e.g. fish promoters such as the common carp ⁇ -actin promoter (Gomez-Chiarri et al., supra), or a trout interferon regulatory factor IA promoter could be used (Alonso et al., supra).
- Non-plasmid or non -vector forms of DNA vaccine can also be used.
- DNA molecules comprising only the foreign DNA fragments without any vector components can be used.
- said vaccine molecule is a DNA vaccine, more preferably a DNA vaccine which is designed to protect against viral or bacterial infection, more preferably viral infection.
- Such antiviral DNA vaccines generally comprise DNA encoding a viral component such as a glycoprotein or a capsid protein.
- a vast number of vaccine candidates have been proposed in the literature, for example in the treatment of viral or bacterial diseases and infections, or in the treatment of other disease states such as cancer, and DNA molecules encoding an antigen, preferably a rhabdovirus glycoprotein, may be used as an adjuvant for any of these in accordance with the present invention.
- DNA molecules encoding an antigen may also be used as adjuvants in conjunction with known vaccine molecules which have not been shown to stimulate an adequate immune response in their own right in order to improve or enhance said immune response, or in conjunction with other molecules which are candidates for use as vaccines but which have not previously been tested.
- the DNA molecule encoding the antigen preferably a rhabdovirus glycoprotein
- Such vaccine molecules are generally those which alone cannot induce protection against a subsequent challenge by the appropriate infective agent or disease.
- Nodaviruses are non- enveloped RNA viruses consisting of an icosahedral capsid 25-30 nm in diameter, which contains a genome of two single stranded positive-sense RNA segments, RNA1 (3.1 kb) and RNA2 (1.4kb).
- RNA1 encodes the putative RNA-dependent RNA polymerase while RNA2 encodes the capsid protein.
- nodavirus infections cause viral encephalopathy and retinopathy (VER) also termed viral nervous necrosis (VNN), and has emerged as a major constraint on the culturing of a number of marine fish species.
- VER viral encephalopathy and retinopathy
- VNN viral nervous necrosis
- Common external signs of nodavirus infection are related to the neuro-invasiveness of the virus and include hyperreactivity, abnormal swimming patterns with looping and spiral movements and reduced coordination and changes in pigmentation. Histopathological findings include vacuolation and necrosis of the central nervous system, the retina and the ganglia of the peripheral - nervous system.
- the vaccine molecules are derived from nodaviruses and, more preferably, the use of such nodavirus derived vaccine molecules in conjunction with the above described DNA molecules encoding an antigen, preferably a rhabdovirus glycoprotein, in accordance with the invention result in long term protection (70 days or more post vaccination) against a nodavirus challenge.
- the vaccine molecules may comprise nodavirus component proteins such as the RNA dependent RNA polymerase (referred to as nodavirus Protein A), the accessory proteins (referred to as nodavirus Proteins B, for example protein B 1 or protein B2) or the capsid protein (referred to as nodavirus Protein ⁇ ), or peptide fragments thereof.
- the vaccine molecules are DNA molecules encoding such nodavirus component proteins or variants, or fragments thereof (i.e. is a DNA vaccine).
- a particularly preferred vaccine molecule comprises a DNA molecule encoding the capsid protein of a nodavirus or a functionally equivalent variant, or a fragment thereof.
- said DNA molecule can take any appropriate form and can be produced by any appropriate method , e.g. by recombinant techniques.
- Said DNA molecule will generally be administered as part of an expression vector or plasmid, for example the open reading frame encoding the capsid protein (or other nodavirus derived protein), or variant, or fragment thereof, will be present downstream of an appropriate promoter sequence, e.g.
- CMV early promoter or another appropriate promoter sequence is the pcDNA vector (Invitrogen).
- the DNA could be administered in non-plasmid or non -vector form.
- DNA molecules encoding only the vaccine molecules without any vector components can be used.
- the nodaviruses from which the vaccine molecules are derived may be of any type, e.g. may be either alphanodaviruses or betanodaviruses. Betanodaviruses are preferred, in particular in cases where the organism which it is desired to vaccinate is a fish, betanodaviruses are preferred.
- genotypes of betanodaviruses which may be used are striped jack nervous necrosis virus (S JNNV), tiger puffer nervous necrosis virus (TPNNV), barf ⁇ n flounder nervous necrosis virus (BFNNV) and red spotter grouper nervous necrosis virus (RGNNV).
- S JNNV striped jack nervous necrosis virus
- TPNNV tiger puffer nervous necrosis virus
- BFNNV barf ⁇ n flounder nervous necrosis virus
- RGNNV red spotter grouper nervous necrosis virus
- Some betanodaviruses do not belong to these four major groups, but the use of these is also included.
- An example of a nodavirus in this regard is turbot nodavirus (TNV) which is a preferred nodavirus from which the vaccine molecules of the invention can be derived.
- TMV turbot nodavirus
- Most preferred nodaviruses are genotypes of barfin flounder nervous necrosis virus (BFNNV) and in particular AHNV (Atlantic halibut nodavirus).
- BFNNV barfin flounder nervous necrosis virus
- AHNV Alkaolin halibut nodavirus
- a particularly preferred nodavirus molecule to be used as a vaccine molecule is the capsid protein derived from AHNV, and more particularly DNA encoding said capsid protein, or a fragment thereof.
- a preferred vaccine molecule in this regard is pAHNV-C as described in the Examples.
- Preferred organisms for use in embodiments of the invention where the vaccine molecules are derived from nodavirus are fish, insects or crustaceans, or any other organisms (for example salmon) which can be infected by nodaviruses.
- the adjuvant component i.e. the DNA molecule encoding the antigen, preferably a rhabdovirus glycoprotein, and the vaccine molecule can be administered at the same time (or substantially the same time), or sequentially. If administered at the same time, which is preferred, then optionally the DNA adjuvant component and the vaccine component may be administered as a mixture. Preferably both the antigen encoding DNA molecule and the vaccine molecule are administered at the same site, however it is also envisaged that different sites may be used.
- a single DNA molecule e.g. a single plasmid or vector
- the antigen and the vaccine molecule might be expressed under the control of the same or different promoter elements.
- a composition comprising (i) an adjuvant component which comprises a DNA molecule encoding an antigen which is expressed on the surface of a host cell (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof, and (ii) a vaccine molecule.
- Said compositions are suitable for use in the methods and uses described herein.
- compositions may be vaccine compositions which are used to vaccinate organisms or stimulate an immune response against a relevant bacterial or viral infection or against a relevant disease.
- the invention can also be seen to provide a vaccine composition for stimulating an immune response in an organism comprising (i) an adjuvant component which comprises a DNA molecule encoding an antigen and (ii) a vaccine molecule. Said adjuvant components and vaccine molecules and preferred examples thereof are as defined elsewhere herein.
- a method of stimulating an immune response in an organism comprising administering to said organism a vaccine composition as defined above.
- said immune response is generated against a relevant bacterial or viral infection or against a relevant disease depending on the nature of the vaccine molecule which is included in the vaccine composition.
- the invention further provides the use of a vaccine composition
- a vaccine composition comprising (i) an adjuvant component which comprises a DNA molecule encoding an antigen which is expressed on the surface of a host cell (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof, and (ii) a vaccine molecule in the manufacture of a medicament for use in therapy, for example for stimulating an immune response or in vaccination.
- a host cell preferably a rhabdovirus glycoprotein
- a vaccine molecule in the manufacture of a medicament for use in therapy, for example for stimulating an immune response or in vaccination.
- the above described compositions are pharmaceutically acceptable and may optionally contain a pharmaceutically acceptable carrier, excipient or diluent.
- a still further aspect of the invention provides a product comprising (i) an adjuvant component which comprises a DNA molecule encoding an antigen which is expressed on the surface of a host cell (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof, and (ii) a vaccine molecule as a combined preparation for simultaneous, separate or sequential use in stimulating an immune response or in vaccination.
- a kit for use in stimulating an immune response or in vaccination said kit comprising: (i) a DNA molecule encoding an antigen which is expressed on the surface of a host cell (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof; and (ii) a vaccine molecule.
- a pharmaceutically acceptable excipient or diluent may be present in either or both component (i) or (ii) of said kits, or may be supplied as a separate component.
- Components (i) and (ii) may be presented in the kit as a mixture or as separate components.
- Components (i) and (ii) may be part of the same molecular entity, e.g. a single DNA molecule (e.g. a single plasmid or vector) might encode both components (i) and (ii).
- the invention provides a DNA molecule encoding an antigen which is expressed on the surface of a host cell (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof, together with a vaccine molecule for use in therapy, for example for stimulating an immune response or in vaccination.
- a host cell preferably a rhabdovirus glycoprotein
- a vaccine molecule for use in therapy, for example for stimulating an immune response or in vaccination.
- the present invention provides compositions of the invention for use in therapy, for example for stimulating an immune response or in vaccination.
- the invention also provides the use of a DNA molecule encoding an antigen which is expressed on the surface of a host cell (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof, together with a vaccine molecule in the manufacture of a medicament for use in therapy, for example for stimulating an immune response or in vaccination.
- a host cell preferably a rhabdovirus glycoprotein
- a variant, or a fragment thereof together with a vaccine molecule in the manufacture of a medicament for use in therapy, for example for stimulating an immune response or in vaccination.
- the present invention provides the use of compositions of the invention in the manufacture of a medicament for use in therapy, for example for stimulating an immune response or in vaccination. Processes for preparing compositions of the invention are also provided.
- compositions of the invention may be formulated in any convenient manner according to techniques and procedures known in the art, e.g. using one or more appropriate carriers, excipients or diluents.
- the nature of the compositions and carriers or excipients or diluents may be selected in a routine manner according to for example the choice and desired route of administration, nature of molecules to be administered, etc.
- Appropriate effective doses of the DNA molecule encoding an antigen and the vaccine molecule to be administered can be readily determined in a routine manner according to for example the choice and desired route of administration, the nature of molecules to be administered, the purpose of vaccination, type and weight of organism being vaccinated, nature of vaccine molecule.
- Doses, carriers, excipients and diluents are selected such that when administered to the organism in question, the DNA molecule encoding the antigen (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof, can act as an adjuvant to stimulate an enhanced or increased immune response to the particular vaccine molecule in the composition.
- the immune response is such that protection against a challenge by the appropriate infective agent or disease is conferred.
- suitable doses of adjuvants and vaccine molecules might be in the order of nanogram amounts or may be higher doses such as 1-50 ⁇ g or 2-20 ⁇ g or 5-20 ⁇ g.
- Preferred DNA molecules (adjuvant molecules) and vaccine molecules for use in the above described embodiments are as described elsewhere herein. It should also be mentioned that the adjuvants as described herein could be administered in conjunction with multiple different vaccine molecules. Thus, in all aspects of the invention discussed herein, one or more types of vaccine molecule may be used. Any appropriate mode of administration may be used to administer the DNA molecules (adjuvant molecules), vaccine molecules, or compositions in accordance with the invention, e.g. injection (e.g.
- intramuscular, intravenous or intraperitoneal injection infusion, topical administration, gene gun (particle bombardment of epidermis), scarification of the skin, immersion using DNA coated beads or liposomes, use of ultrasound etc.
- gene gun particle bombardment of epidermis
- scarification of the skin immersion using DNA coated beads or liposomes, use of ultrasound etc.
- injection and in particular intramuscular injection is preferred.
- preferred methods and uses of the invention are methods of vaccination or uses of the above described molecules in vaccination.
- the ultimate aim of any such vaccination method or use is to provide protection against a challenge by the relevant infective agent or disease, which is determined by the vaccine molecule component of the composition used.
- Such a protective effect can be conferred prophylactically, e.g. by administering the active agents or vaccine compositions to organisms in advance of a challenge by the pathogenic organism or disease.
- such a protective effect can be conferred by administering the active agents or vaccine compositions once the pathogenic organism or disease is present in the organism to be vaccinated.
- the protection provided by the methods and uses of the invention is long term protection (for example protection which is still effective at least 70 days after vaccination and preferably for the life time of the organism in question) against challenge by the relevant disease or infective agent.
- long term protection for example protection which is still effective at least 70 days after vaccination and preferably for the life time of the organism in question
- the provision of long term protection by the adjuvants of the invention when combined with vaccine molecules is surprising.
- Short term or early protection for example protection for less than 70 days or less than 35 days or less than 8 days after vaccination
- short term protection for example protection for less than 70 days or less than 35 days or less than 8 days after vaccination
- the adjuvants of the invention are also effective to induce short term protection (preferably as early as 8 days after vaccination or earlier), against disease or infection. This is extremely advantageous as it means that the organisms in question are protected from disease from a very early time period and thus are protected from infection or disease for a proportion of the time during which long term protection is being established.
- vaccines have been developed in some fields in order to protect organisms from various types of infection and disease.
- vaccines often contain or encode part of the infective organism and the vaccines may persist in the organism in the long term after administration, then this gives rise to concerns with regard to human safety, particularly in farming applications where it is preferable that the final food product should not contain significant amounts of foreign molecules such as vaccine molecules.
- a further important advantage of the DNA molecules encoding an antigen, particularly a rhabdovirus glycoprotein, contemplated for use in the present invention is that such molecules can stimulate quicker elimination of coadministered molecules such as vaccine molecules.
- a DNA molecule encoding a rhabdovirus glycoprotein in the form of pVHSV-G
- a coadministered DNA molecule encoding the luciferase gene in the form of pCMV-luc
- DNA could still be detected at the site of injection 160 days post injection.
- a yet further aspect of the invention relates to the use of a DNA molecule encoding an antigen which is expressed on the surface of a host cell (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof, to improve or increase the elimination of other foreign DNA molecules from an organism.
- Such other foreign DNA molecules for example vaccine molecules, will preferably have been coadministered with the DNA molecule encoding said antigen, however they may also be administered before or after the administration of the DNA molecule encoding said antigen.
- both the antigen encoding DNA molecule and the foreign DNA molecule are administered at the same site, however it is also envisaged that different sites may be used.
- Preferred DNA molecules encoding the antigens for use in this further aspect of the invention are as discussed above.
- the other foreign DNA molecule is a vaccine molecule as described above.
- a further advantage is that, after the protective effect has been conferred, the elimination of said vaccine molecule is improved or enhanced.
- Preferred compositions, organisms, modes of administration, etc for this aspect of the invention are as described above.
- Preferred doses are also as described above, although higher doses, for example in the order of milligram amounts (e.g. 1- 50 mg or 5-30 mg) can also be used for this aspect of the invention.
- the invention thus further provides the use of a DNA molecule encoding an antigen which is expressed on the surface of a host cell (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof, in the manufacture of a composition which can improve or increase the elimination of other foreign DNA molecules from an organism.
- the invention further provides a method of improving or increasing the elimination of foreign DNA molecules from an organism, said method comprising the administration of a DNA molecule encoding an antigen which is expressed on the surface of a host cell (preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof.
- the DNA molecule encoding an antigen is preferably co-administered with said foreign DNA molecules which are to be eliminated.
- Said improvement, enhancement or increase in the elimination of foreign DNA molecules in the presence of a DNA molecule encoding an antigen refers to an improvement, enhancement or increase in the elimination of such molecules as compared to the elimination observed when said foreign DNA molecules are administered alone, i.e. in the absence of the DNA molecule encoding an antigen.
- said improvement, enhancement or increase is statistically significant. Methods of determining the statistical significance of differences in parameters are well known and documented in the art. For example herein a parameter is generally regarded as significant if a statistical comparison using an appropriate statistical test such as a Student t-test shows a probability value of
- Figure 1 shows cumulative mortality curves of DNA vaccinated turbots (diamonds) and control turbots (squares) challenged by an intra-muscular injection of AHNV at 70 days post vaccination.
- Example 1 DNA encoding a rhabdovirus glycoprotein can act as an adjuvant for a nodavirus DNA vaccine.
- AHNV challenge strain (AH95NorA) was propagated in the SSN-1 cell line as obtained from the European Collection of Animal Cell Cultures (ECACC), Salisbury, UK as described by Dannevig et al., (Dis. Aquat. Org. 2000; 43: 183 -9).
- the virus titer was determined based on cytopathologic observation by end-point dilution (10-fold serial dilution) on SSN-1 cells cultured in 96-well cell culture plates (Falcon Primera), and TCID50 was determined using eight parallel wells for each dilution.
- the DNA vaccine containing the capsid-encoding region of AHNV was prepared as described in Sommerset et al., (Vaccine 2003; 21:4661-4667). Briefly, the capsid- encoding region and part of the 3' UTR of AHNV RNA2 (Grotmol et al., Dis. Aquat. Org. 2000; 39:79-88) was subcloned behind the early cytomegalovirus promoter of pcDNA3.1 (+) (Invitrogen). The recombinant pcDNA3.1 -AHNV-C plasmid was transformed into Top 10 E. coli cells (Invitrogen) and verified by DNA sequencing.
- pAHNV-C Large-scale preparations of the vaccine plasmid (called pAHNV-C) were purified from overnight cultures by anion exchange chromatography (EndoFreeTM Plasmid Kit, Qiagen). The presence of supercoiled plasmid was verified by agarose gel electrophoresis, and DNA quality and quantity were determined spectrophotometrically (A260/A280). The pAHNV-C was resuspended in PBS to final concentrations of 0.5 mg/ml and 2 mg/ml respectively, and stored at -20°C until used.
- the DNA molecule encoding the VHSV glycoprotein (G), herein called pVHSV-G, contains the G gene inserted downstream of the CMV promoter in the pcDNA3 vector (Invitrogen) as described in detail elsewhere (Lorenzen et al., Fish Shellfish Immunol, 1998; 8:261-270 and Heppell et al. Fish Shellfish Immunol, 1998;
- the fish vaccinated with pVHSV-G and pAHNV-C show a significantly lower mortality rate (4/60 fish died, i.e. 6.7%) than the control fish which are not vaccinated (41/60 died, i.e. 68.3%).
- the vaccinated fish display almost complete protection against nodavirus infection when challenged at 70 days post vaccination.
- Example 2 A DNA molecule encoding a rhabdovirus glycoprotein can enhance the elimination of a co-administered DNA molecule.
Abstract
Description
Claims
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CA 2572171 CA2572171A1 (en) | 2004-06-22 | 2005-06-22 | Use of dna molecule as vaccine adjuvant |
EP20050757731 EP1784208A2 (en) | 2004-06-22 | 2005-06-22 | Use of a dna molecule as vaccine adjuvant |
NO20070345A NO20070345L (en) | 2004-06-22 | 2007-01-18 | Use of DNA molecule as a vaccine adjuvant |
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CN100436599C (en) * | 2006-05-12 | 2008-11-26 | 中国水产科学研究院黄海水产研究所 | Female-specific AFLP fragment of Cynoglossus semilaevis Gunther and PCR method for identification of genetic sex |
CN101914529A (en) * | 2010-07-29 | 2010-12-15 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method |
-
2005
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- 2005-06-22 CA CA 2572171 patent/CA2572171A1/en not_active Abandoned
- 2005-06-22 EP EP20050757731 patent/EP1784208A2/en not_active Withdrawn
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2007
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Non-Patent Citations (3)
Title |
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BOUDINOT P ET AL: "Combined DNA Immunization with the Glycoprotein Gene of Viral Hemorrhagic Septicemia Virus and Infectious Hematopoietic Necrosis Virus Induces Double-Specific Protective Immunity and Nonspecific Response in Rainbow Trout" VIROLOGY, ACADEMIC PRESS,ORLANDO, US, vol. 249, no. 2, 30 September 1998 (1998-09-30), pages 297-306, XP004445647 ISSN: 0042-6822 * |
NIELS LORENZEN ET AL.: "Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens" DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, vol. 26, 2002, pages 173-179, XP002347949 * |
SOMMERSET I ET AL: "A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot" VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 21, no. 32, 1 December 2003 (2003-12-01), pages 4661-4667, XP004469682 ISSN: 0264-410X cited in the application * |
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CN100436599C (en) * | 2006-05-12 | 2008-11-26 | 中国水产科学研究院黄海水产研究所 | Female-specific AFLP fragment of Cynoglossus semilaevis Gunther and PCR method for identification of genetic sex |
CN101914529A (en) * | 2010-07-29 | 2010-12-15 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method |
CN101914529B (en) * | 2010-07-29 | 2012-03-14 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method |
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CA2572171A1 (en) | 2005-12-29 |
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