This invention provides a DNA vaccine which comprises a naked DNA incorporating and expressing in vivo a nucleotide sequence encoding an antigenic polypeptide, preferably a gene of a pathogenic agent, and at least one adjuvant compound chosen from the polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and alkenyl derivative. The adjuvant compound is preferably a carbomer or an EMA.RTM.. This invention also provides a method of enhancing a DNA vaccine and/or the immunogenicity of a DNA vaccine.

SUMMARY OF THE INVENTION

The objective of the invention is to enhance the efficacy of DNA vaccines by providing new vaccine formulations which are simple and easy to prepare.

Its objective is also to provide such a solution which does not cause strong interactions between the DNA and the other ingredient, which are capable of leading to the formation of a complex.

Its objective is also to provide such a solution which makes it possible, either by simple mixing, to prepare stable vaccines, formulated in a liquid form, or to easily prepare a liquid vaccine by mixing immediately before use.

The applicant has found, surprisingly, that the carbomer class of compounds meet these various objectives and in particular are capable of acting as adjuvants for naked DNA vaccines in a simple manner but in very advantageous proportions.

DETAILED DESCRIPTION OF THE INVENTION

The subject of the of the present invention is therefore a DNA vaccine comprising a naked DNA, in particular circular vaccinal plasmid, supercoiled or otherwise, or a linear DNA molecule, incorporating and expressing in vivo a nucleotide sequence encoding an antigenic polypeptide, preferably a gene of a pathogenic agent, and at least one adjuvant compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative.

Naked DNA is understood to mean, as in nowadays commonly accepted, a DNA transcription unit in the form of a polynucleotide sequence comprising at least one nucleotide sequence encoding an antigentic polypeptide or an antigen of one valency and the elements necessary for its expression in vivo. These elements can include a promoter, origin of replication, and transcription termination sequence. The circular plasmid form, supercoiled or otherwise, is preferred. Valency in the present invention is understood to mean at least one antigen providing protection against a pathogen, it being possible for the valency to contain, as subvalency, one or more natural or modified genes, or one or more strains of the pathogen considered.

The preferred adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Pharmeuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 (incorporated herein by reference) which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol.RTM. (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among them, there may be mentioned Carbopol.RTM. 974P, 934P and 971P.

Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA.RTM. (Monsanto)=which are copolymers of maleic anhydride and ethylene, linear or cross-linked, for example cross-linked with divinyl ether, are preferred. Reference may be made to J. Fields et al., Nature, 186: 778 780, 4 Jun. 1960, incorporated herein by reference.

From the point of view of their structure, the polymers of acrylic or methacrylic acid and the copolymers EMA.RTM. are preferably formed of basic units of the following formula -- (see Original Patent).

The dissolution of these polymers in water leads to an acid solution which will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the vaccine itself will be incorporated. The carboxyl groups of the polymer are then partly in COO form.

Preferably, a solution of adjuvant according to the invention, especially of carbomer, is prepared in distilled water, preferably in the presence of sodium chloride, the solution obtained being at acidic pH. This stock solution is diluted by adding it to the desired quantity (for obtaining the desired final concentration), or a substantial part thereof, of water charged with NaCl, preferably physiological saline (NaCl 9 g/l) all at once or in several portions with concomitant or subsequent neutralization (pH 7.3 to 7.4), preferably with NaOH. This solution at physiological pH will be used as it is for mixing with the vaccine, which may be especially stored in freeze-dried, liquid or frozen form.

The polymer concentration in the final vaccine composition will be 0.01% to 2% w/v, more particularly 0.06 to 1% w/v, preferably 0.1 to 0.6% w/v.

For the vaccination of pigs, the invention may apply in particular to vaccination against Aujeszky's disease virus (PRV or pseudorabies virus), porcine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRS virus), porcine parvovirosis virus (PPV virus), hog cholera virus (HCV virus) and bacterium responsible for actinobacillosis (A. pleuropneumoniae). The plasmids which can be used in the invention comprise, for each valency, one or more of the genes encoding major immunogens of the pathogenic agents considered. There may be mentioned in particular the gB and gD genes for the Aujeszky's disease virus, the HA, NP and N genes for the porcine influenza virus, the ORF5 (E), ORF3 and ORF6 (M) genes for the PRRS virus, VP2 for the parvovirosis virus, E2, E1+E2, E1+E2+C for the hog cholera virus and apxI, apxII and apxIII for A. pleuropneumoniae. In a particularly advantageous manner, reference may be made to the polynucleotide vaccine formulas described in patent application WO-A-98 03 658 (FR-A-2,751,224) which is incorporated herein by reference and relates to vaccines against pig reproductive and respiratory pathologies. This application describes in particular a number of plasmids which may be directly used by way of examples in the context of the present invention in combination with an adjuvant according to the invention. Persons skilled in the art will thus be able to combine, with the adjuvants in accordance with the invention, the plasmids specifically described in this previous application, namely pAB090 comprising the gB gene of the PRV virus, pPB098 comprising the gD gene of the PRV virus, pPB143 comprising the HA gene of porcine influenza, strain H1N1, pPB142 comprising the NP gene of porcine influenza, strain H1N1, pPB144 comprising the HA gene of porcine influenza, strain H3N2, pPB132 comprising the NP gene of porcine influenza, strain H3N2, pAB025 comprising ORF5 of the PRRS virus, strain Lelystad, pAB001 comprising ORF5 of the PRRS virus, strain USA, pAB091 comprising ORF3 of the PRRS virus, strain Lelystad, pAB092 comprising ORF3 of the PRRS virus, strain USA, pAB004 comprising the VP2 gene of the porcine parvovirus, pAB069 comprising the E1 gene of the hog cholera virus (HCV), pAB061 comprising the E2 gene of the hog cholera virus (HCV), pAB162 comprising the deleted apxI gene of A. pleuropneumoniae, pPB163 comprising the deleted apxII gene of A. pleuropneumoniae, pPB174', pPB189 and pPB190 comprising the deleted apxIII gene of A. pleuropneumoniae.

For the vaccination of horses, there may be mentioned in particular vaccination against equine rhinopneumonia virus (EHV), especially type 1 (EHV-1) and type 4 (EHV-4), against the equine influenza virus EIV, against tetanus (Cl. tetani), against the Eastern encephalitis virus (EEV), Western encephalitis virus (WEV) and Venezuelan encephalitis virus (VEV), as well as against Lyme disease (B. burgdorferi), against equine arthritis (EAV) and against rabies. Among the genes encoding major immunogens which can be used according to the invention, there may be mentioned gB and gD genes for the equine rhinopneumonia valency, especially types 1 and 4, the HA, NA and NP genes for equine influenza, the C subunit, optionally modified by mutation or deletion, for the tetanus valency, the C and E2 genes for encephalites, the OspA, OspB and p100 genes for Lyme disease, the E, M and N genes for equine arthritis and the G gene for rabies. Such polynucleotide vaccine formulas against horse pathologies are described in particular in patent application WO-A-98 03 198 (FR-A-2,751,226) which is incorporated herein by reference. This application describes a number of plasmids which can be directly used in the present invention in combination with an adjuvant in accordance with the invention. Persons skilled in the art will therefore be able to combine, with the adjuvant in accordance with the invention, a plasmid as precisely described in this application, namely pAB042 comprising the gB gene of the EHV-1 virus, pAB031 comprising the gB gene of the EHV-4 virus, pAB013 comprising the gD gene of the EHV-1 virus, pAB032 comprising the gD gene of the EHV-4 virus, pAB043 comprising the HA gene of equine influenza, Prague strain, pAB033 comprising the HA gene of equine influenza, Suffolk strain, pAB099 comprising the HA gene of equine influenza, Fontainebleau strain, pAB085 comprising the NP gene of equine influenza, Prague strain, pAB084 comprising the NP gene of equine influenza, Jillin strain, pAB070 comprising the gene for the C subunit of the tetanus toxin, pAB017 comprising the OspA gene of Borrelia burgdorferi, pAB094 comprising the E2 gene of the Eastern encephalitis virus, pAB093 comprising the C gene of the Eastern encephalitis virus, pAB096 comprising the E2 gene of the Western encephalitis virus, pAB095 comprising the C gene of the Western encephalitis virus, pAB098 comprising the E2 gene of the Venezuelan encephalitis virus, pAB097 comprising the C gene of the Venezuelan encephalitis virus and pAB041 comprising the G gene of the rabies virus.

For vaccination of dogs, the invention may apply in particular to vaccination against Canine Distemper (Carre's disease) virus (CDV), canine parvovirus (CPV), canine coronavirus (CCV), canine herpesvirus (CHV), Lyme disease and rabies. Among the genes encoding major immunogens which can be used in the context of the present invention, there may be mentioned most particularly the HA, F, M and N genes for the Canine Distemper virus, the VP2 gene for the canine parvovirus, the S and M genes for the canine coronavirus (CCV), the gB and gD genes for the CHV virus, the OspA and OspB and p100 genes for B. burgdorferi (Lyme disease) and the G gene for rabies. Such polynucleotide vaccine formulas are described in particular in patent application WO-A-98 03 199 (FR-A-2,751,227) which is incorporated herein by reference. Persons skilled in the art will therefore be able to refer to the plasmids described in this application, in combination with the adjuvants in accordance with the invention. Most particularly, they will be able to combine, with the adjuvants in accordance with the invention, the specific plasmids described in this application, namely pAB044 comprising the HA gene of CDV, pAB036 comprising the F gene of CDV, pAB024 comprising the VP2 gene of the canine parvovirus, pAB021 comprising the S gene of CCV, pAB022 comprising the M gene of CCV, pAB037 comprising the gB gene of CHV, pAB038 comprising the gD gene of CHV, pAB017 comprising the OspA gene of B. burgdorferi and pAB041 comprising the G gene of the rabies virus.

For vaccination of bovines, the invention may apply in particular to vaccination against the bovine herpesvirus type 1 or 5 (BHV-1 and BHV-5, responsible for the nervous form of the disease), the bovine respiratory syncytial virus (BRSV), the mucosal disease virus or bovine pestivirus (BVD), the bovine parainfluenza virus type 3 (BPI-3). Among the genes encoding the major immunogens allowing vaccination against these viruses, there may be mentioned in particular the gB and gD genes for the bovine herpesvirus, F and G for the bovine respiratory syncytial virus, E2, C+E1+E2 and E1+E2 for the mucosal disease virus, HN and F for the bovine parainfluenza virus type 3. Such vaccine formulas are described in particular in patent application WO-A-98 03 200 (FR-A-2,751,229) which is incorporated herein by reference. Persons skilled in the art will therefore be able to use the plasmids described in this application in combination with the adjuvants in accordance with the invention. In particular, they will be able to combine, with the adjuvants in accordance with the invention, the plasmids specifically described in this application, namely pPB156 comprising the gB gene of BHV-1, pAB087 comprising the gD gene of BHV-1, pAB011 comprising the F gene of BRSV, pAB012 comprising the G gene of BRSV, pAB058 comprising the C gene of BVD, pAB059 comprising the E1 gene of BVD, pAB060 comprising the E2 gene of BVD, pAB071 comprising the HN gene of BPI-3, pAB072 comprising the F gene of BPI-3.

For the vaccination of cats, the invention may apply in particular to vaccination against the feline leukemia virus FeLV, in particular subtypes A and B, the feline panleukopenia virus (FPV), the feline infectious peritonitis virus (FIPV), the coryza virus or feline herpesvirus (FHV), the feline calicivirosis virus (FCV), the feline immunodeficiency virus (FIV) and the rabies virus (rhabdovirus). Among the genes encoding major immunogens allowing vaccination against these pathogens, there may be mentioned in particular the env and gag/pol genes for feline leukemia, VP2 for panleukopaenia, M and modified S (FR-A-2,724,385 incorporated herein by reference) for infectious peritonitis, gB and gD for coryza, capsid for calicivirosis, env and gag/pro for feline immunodeficiency and G for rabies. Polynucleotide vaccine formulas are thus described in patent application WO-A-98 03 660 (FR-A-2,751,223) which is incorporated herein by reference. Persons skilled in the art will be able to combine plasmids as described in this application with the adjuvants in accordance with the invention. In particular, they will be able to combine, with the adjuvants in accordance with the invention, the plasmids specifically described in this application, namely pPB179 comprising the env gene of the FeLV-A virus, pPB180 comprising the env gene of the FeLV-B virus, pPB181 comprising the gag/pol gene of FeLV-A, pAB009 comprising the VP2 gene of FPV, pAB053 comprising the modified S gene (FR-A-2 724 385) of the FIPV virus, pAB052 comprising the M gene of FIPV, pAB056 comprising the N gene of FIPV, pAB028 comprising the gB gene of FHV, pAB029 comprising the gD gene of FHV, pAB010 comprising the C gene of FCV, pAB030 comprising the env gene of FIV, pAB083 comprising the gag/pro gene of FIV and pAB041 comprising the G gene of the rabies virus.

For vaccination of avian species, the invention may apply in particular to vaccination against the Marek's disease virus (MDV), the Newcastle disease virus (NDV), the Gumboro disease virus (IBDV or Infectious Bursal Disease Virus), the infectious bronchitis virus (IBV), the infectious anemia virus (CAV), the infectious laryngotracheitis virus (ILTV), the encephalomyelitis virus (AEV or avian leukosis virus ALV), the pneumovirosis virus or pneumovirus, and the avian influenza virus. Among the genes encoding the major immunogens which can be used in the present invention, there may be mentioned most particularly the gB and gD genes for the Marek's disease virus, HN and F for the Newcastle disease virus, VP2 for the Gumboro disease virus, S, M and N for the infectious bronchitis virus, C+NS1 for the infectious anemia virus, gB and gD for the infectious laryngotracheitis virus, env and gag/pro for the encephalomyelitis virus, F and G for the pneumovirosis virus and HA, N and NP for avian influenza. Such polynucleotide vaccine formulas are described in patent application WO-A-98 03 659 (FR-A2,751,225) which is incorporated herein by reference. Persons skilled in the art will therefore be able to refer to the plasmids described in this application in order to combine them with the adjuvants in accordance with the invention. Most particularly, persons skilled in the art will be able to combine, with the adjuvants in accordance with the invention, the plasmids described specifically in this application, namely pAB045 comprising the gB gene of MDV, pAB080 comprising the gD gene of MDV, pAB046 comprising the HN gene of NDV, pAB047 comprising the F gene of NDV, pAB048 comprising the VP2 gene of IBDV, pAB049 comprising the S1 gene of IBV, pAB050 comprising the M gene of IBV, pAB051 comprising the N gene of IBV, pAB054 comprising the VP1 gene of CAV, pAB055 comprising the VP2 gene of CAV, pAB076 comprising the gB gene of ILTV, pAB089 comprising the gD gene of ILTV, pAB086 comprising the env gene of AEV, pAB081 comprising the gag/pro gene of AEV, pAB082 comprising the G gene of the pneumovirus, pAB077 comprising the HA gene of avian influenza, strain H2N2, pAB078 comprising the HA gene of avian influenza, strain H7N7, pAB088 comprising the NP gene of avian influenza, strain H1N1, pAB079 comprising the N gene of avian influenza, strain H7N1.

Each naked, in particular plasmid, DNA comprises a promoter capable of bringing about, in the host cells, the expression of the gene inserted under its control. It will be in general a strong eukaryotic promoter and in particular a cytomegalovirus early promoter CMV-IE, of human or murine origin, or alternatively possibly of another origin such as rat, pig or guinea pig. In a more general manner, the promoter may either be of viral origin, or of cellular origin. As viral promoter other than CMV-IE, there may be mentioned the SV40 virus early or late promoter or the Rous sarcoma virus LTR promoter. It may also be a promoter coming from the virus from which the gene is derived, for example the actual promoter of the gene. As cellular promoter, there may be mentioned the promoter of a cytoskeleton gene, such as for example the desmin promoter (Bolmont et al., Journal of Submicroscopic Cytology and Pathology, 1990, 22, 117 122; and Zhenlin et al., Gene, 1989, 78, 243 254), or alternatively the actin promoter. When several genes are present in the same naked, in particular plasmid, DNA they may be present in the same transcription unit or in two different units.

Of course, a vaccine may combine, for each of the valencies described above, several genes within the same naked, in particular plasmid, DNA and/or several naked, in particular plasmid, DNAs each comprising one or more genes of the same virus.

The subject of the invention is also multivalent recombinant vaccines, that is to say containing one or preferably two or more naked, in particular plasmid, DNAs expressing antigens for two or more diseases, in the form of a mixture in an adjuvant solution in accordance with the invention.

In the ready-for-use vaccine, the naked DNA, in particular the vaccinal plasmid, is present in the quantities normally used and described in the literature.

The subject of the invention is also a method of vaccination consisting of administering by the parenteral, preferably intramuscular, intradermal, route or by the mucosal route a DNA vaccine in accordance with the invention at the rate of one or more administrations.

The subject of the invention is also the use of the adjuvant compounds in accordance with the invention for the production of adjuvant-containing DNA vaccines as described here.
 

Claim 1 of 6 Claims

1. A DNA vaccine comprising (i) a naked DNA plasmid containing and expressing in vivo a polynucleotide encoding an antigenic polypeptide, wherein the antigenic polypeptide comprises an antigen of equine rhinopheumonia virus; and (ii) at least one adjuvant which is a polymer of acrylic or methacrylic acid.

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