HK1083763A - Polynucleotide vaccine formula against equine pathologies - Google Patents

Description

The present invention relates to a vaccine formula for the vaccination of equidae, particularly horses, and a corresponding vaccination method.

The pathology of horses is quite diverse: apart from the well-known respiratory diseases such as rhinopneumonia and influenza, horses are susceptible, especially in the Americas, to various encephalitis.

The conditions of exposure of horses to various pathogenic microorganisms have been increased by the long distance travel of many horses by land or air, so that the risk of infection tends to increase.

However, given the high cost of these animals, particularly for breeders, saddle and race horses, it is economically important to control as far as possible the risk of infection resulting from long absences or loss of the animal.

For example, inactivated or subunit vaccines have been developed for equine rhinopneumonia caused by the different strains of equine herpes virus (EHV), but all of them have a number of limitations, resulting in incomplete and short-lived protection and possibly safety concerns related to the adjuvants used.

Equine influenza is also an important disease, which is also being prevented by vaccination. The vaccines used are inactivated or subunit vaccines, which have some efficacy but are not without problems. Protection is often not complete and is usually relatively short-lived, requiring, as with rhinopneumonia, revaccination. Safety problems may also arise.

Vaccines made from tetanus antitoxin have also been developed and are proving to be highly effective.

There are also vaccines against encephalitis, some Eastern encephalitis, Western encephalitis and Venezuelan encephalitis, the effectiveness of which is still poorly understood.

For reasons of economics and rational management of equine vaccination, multivalent vaccine formulations have already been proposed for the prevention of several of these infectious diseases.

The combinations developed so far were made from inactivated or live vaccines and possibly mixtures of such vaccines. Their implementation poses problems of compatibility between valences and stability. Compatibility between the different valences must be ensured both in terms of the different antigens used and in terms of the formulations themselves, especially when inactivated and live vaccines are combined.

In addition, these formulations did not allow the association of three of the major valences, namely equine influenza valences, rhinopneumonia, in particular EHV-1 and EHV-4, and tetanus valences.

Patent applications WO-A-90 11 092, WO-A-93 19 183, WO-A-94 21 797 and WO-A-95 20 660 proposed the use of the recently developed technique of polynucleotide vaccines, which are known to use a plasmid capable of expressing the antigen inserted into the plasmid in the host cells.

All routes of administration have been proposed (intraperitoneal, intravenous, intramuscular, transcutaneous, intradermal, mucosale, etc.) Various means of vaccination may also be used, such as DNA deposited on the surface of gold particles and projected to penetrate the animal's skin (Tang et al., Nature 356, 152-154, 1992) and liquid jet injections to ensure transfection into both skin, muscle, fat, and breast tissue (Furth et al., Analytical Biochemistry, 205, 365-368, 1992).

Polynucleotide vaccines can use both bare DNA and formulated DNA, for example in liposomes or cationic lipids.

Polynucleotide vectors, incorporating either the HA or NP genes, have been tested for influenza virus in mice, ferrets and chickens.

In the case of tetanus, it has recently been reported that immunization of mice with a plasmid expressing, with fragment C, the non-toxic C-terminal region of the tetanus toxin, induced the production of serum-protective antibodies in mice.

However, it is not possible to directly translate the results obtained in these animals of short life span into other mammals, especially large mammals.

The protection of equidae and especially horses against infectious diseases therefore needs to be improved.

The invention is intended to provide a multivalent vaccine formula for vaccinating equidae, and in particular horses, against a number of pathogens.

Another objective of the invention is to provide such a vaccine formula combining different valences while presenting the required criteria for compatibility and stability of valences between them.

Another objective of the invention is to provide such a vaccine formula that allows different valences to be combined in the same vehicle.

Another objective of the invention is to provide such a vaccine formula that is easy to implement and inexpensive.

Another objective of the invention is to provide such a formula and method of vaccination of horses that provides protection, including multivalent protection, with a high level of efficacy and long-term, and good safety.

The invention therefore concerns a vaccine formulation for equine diseases, including equine disease, comprising at least 3 valences of a polynucleotide vaccine each containing a plasmid incorporating, in vivo in cells, a valence gene for the equine pathogen, selected from the group consisting of equine rhinopneumonia virus EHV, equine influenza virus EIV and tetanus (C1. tetanus), these valences comprising, for each valence, one or more genes selected from the group consisting of the equine rhinopneumonia virus gB and gD, HA, NP, N for the equine influenza virus and one or more genes coding for the whole or part of the equine influenza virus tetanus.

Valence, as used in this invention, means at least one antigen providing protection against the virus of the pathogen in question, the valence of which may contain, as a subvalence, one or more natural or modified genes from one or more strains of the pathogen in question.

A pathogen gene is not only the complete gene, but also the different nucleotide sequences, including fragments, that retain the ability to induce a protective response. The term gene covers nucleotide sequences that are equivalent to those described in the examples, i.e. different but coding for the same protein. It also covers nucleotide sequences from other strains of the pathogen in question, providing cross-protection or strain or strain-specific protection. It also covers nucleotide sequences that have been modified to facilitate expression in vivo by the host animal but coding for the same protein.

Thus, preferably, the vaccine of the invention includes in the equine rhinopneumonia valence at least one antigen of the EHV-1 strain and at least one antigen of the EHV-4 strain, and preferably the same type of antigen.

Therapeutically effective amounts of polynucleotide valences shall be contained or intended to be contained in a vehicle suitable for animal administration, preferably an aqueous vehicle free of oily constituents.

For equine valence rhinopneumonia, the combination of gB and gD genes is preferred, preferably from EHV strains, in particular strains 1 and 4.

In equine influenza valence, the gene coding for HA or the combination of genes coding for HA and NP is preferred. Preferably, the HA sequences of influenza viruses, especially from different strains found in the field, are combined in the same vaccine. NP, on the other hand, provides cross-protection and can therefore be used to sequence a single strain of the virus.

For the tetanus valence, subunit C, possibly modified by mutation or deletion, is preferred.

The association of genes coding for several antigens of the same valence, or of the same strain in a valence, can be achieved by mixing a plasmid expressing a single antigen, or vice versa by inserting several genes into a single plasmid.

The combination of the different valences of the vaccine according to the invention may preferably be carried out by mixing polynucleotide plasmids expressing one or more antigens of each valence, but it may also be envisaged to express several antigens of several valences by a single plasmid-type vector.

In an enhanced form of the invention, the formulation may also contain one or more other valences of other equine pathogens, including valences of Eastern EEV, Western WEV and Venezuelan VEV encephalitis viruses, preferably all three simultaneously.

These valences may also include the valence of Lyme disease, B. burgdorferi, equine arthritis (EAV) and rabies.

Among the genes for encephalitis mentioned above, the genes for C and E2 antigens are used, preferably the E2 gene alone or a combination of both E2 and C genes.

In Lyme disease valence, one chooses from the genes OspA, OspB and p100, preferably OspA.

For equine arteritis, we'll stick to the E, M and N genes, alone or in combination.

For rabies, we'll stick with the G gene.

A vaccine formula according to the invention may be presented in a volume of doses between 0.1 and 10 ml and in particular between 1 and 5 ml.

The dose will generally be between 10 ng and 1 mg, including 100 ng and 500 μg, and preferably between 1 μg and 250 μg per plasmid type.

It is preferable to use bare plasmids simply placed in the vaccination vehicle which will generally be physiological water (NaCl 0.9%), ultrapure water, TE buffer, etc. Of course all forms of polynucleotide vaccine described in the previous art.

Each plasmid contains a promoter capable of ensuring the expression of the gene inserted under its control in the host cells. This is usually a strong eukaryotic promoter, and in particular an early promoter of CMV-IE cytomegalovirus, of human or mouse origin, or possibly of another origin such as rat, pig, guinea pig.

In general, the promoter can be either viral or cellular in origin. As a viral protector other than CMV-IE, the early or late promoter of SV 40 virus or the LTR promoter of Rous Sarcoma virus may be included. It may also be a promoter of the virus from which the gene originates, for example the gene-specific promoter.

The cell promoter may include the promoter of a cytoskeleton gene, such as the promoter of desmine (Polmont et al., Journal of Submicroscopic Cytology and Pathology, 1990, 22, 117-122; and Zhenlin et al., Gene, 1989, 78, 243-254), or the promoter of actin.

When multiple genes are present in the same plasmid, these can be presented in the same transcription unit or in two different units.

The invention also concerns monovalent vaccine formulations containing one or more plasmids coding for one or more genes of one of the aforementioned pathogens, including rhinopneumonia or Lyme disease, equine arteritis, eastern encephalitis, western encephalitis and Venezuelan encephalitis, the genes being described above.

The present invention also concerns a method of vaccination of equidae and particularly horses against infectious diseases, including the administration of an effective dose of a vaccine formula, multivalent or monovalent, as described above.

This method of vaccination includes the administration of one or more doses of the vaccine formula.

The vaccine formulations of the invention may be administered by the various routes of administration proposed in the general framework of polynucleotide vaccination and by known administration techniques, but intramuscular administration is clearly preferable.

Vaccination can also be given intradermally using a liquid jet, preferably multiple jets, and in particular an injector using a injection head with several holes or nozzles, including 5 to 6 holes or nozzles, such as the Pigjet machine manufactured and distributed by Endoscoptic, Laons, France.

The dose volume for such a device should preferably be reduced to between 0.1 and 0.9 ml, in particular between 0.2 and 0.6 ml and preferably between 0.4 and 0.5 ml, the volume being able to be administered in one or more, preferably 2 applications.

The monovalent vaccines mentioned above may be used in particular for the preparation of the multi-valent vaccine of the invention.

Monovalent vaccine formulations may also be used in combination with another type of vaccine (whole live or inactivated, recombinant, subunit) against another disease or as a booster of a vaccine as described below.

The present invention is further intended to use one or more plasmids of the invention for the manufacture of a vaccine for vaccinating primarily vaccinated horses by means of a first conventional (monovalent or multivalent) vaccine of the type described above, selected from the group consisting of whole live vaccine, whole inactivated vaccine, subunit vaccine, recombinant vaccine, the first vaccine being one which contains (i.e. contains or can express) the antigen (s) coded for by the plasmid (s) or antigen (s) providing cross-protection.

Remarkably, the polynucleotide vaccine has a powerful booster effect, resulting in an amplification of the immune response and the establishment of long-lasting immunity.

In general, primary vaccination vaccines will be available from the commercial vaccines available from the various veterinary vaccine manufacturers.

In a preferred embodiment of the method of the invention, an effective dose of the conventional type of vaccine, including inactivated, live, attenuated or recombinant, or a subunit vaccine is first administered to the animal to ensure a primary vaccination and, after a preferential delay of 2-4 weeks, the multi- or monovalent vaccine of the invention is administered.

The invention also concerns a vaccination kit comprising a vaccine formula according to the invention and a primary vaccination vaccine as described above. It also concerns a vaccine formula according to the invention accompanied by a notice indicating the use of this formula as a reminder of a primary vaccination as described above.

The invention also relates to the method of preparation of vaccine formulae, namely the preparation of valences and their mixtures, as shown in this description.

The invention will now be described in more detail using methods of realization of the invention taken with reference to the drawings.

List of figures

The following table shows the results of the analysis of the data collected from the data collected by the Member States in the context of the monitoring of the use of the PAB012 gene:

List of sequences of the SEQ ID No

The following is the list of the substances which are to be classified as 'substances' in Annex I to Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 16 December 2006 on the approximation of the laws of the Member States relating to the labelling of foodstuffs and food ingredients:

Examples Example 1: Virus culture

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Example 2: culture of bacteria:

Tetanus: Bacteria are grown in appropriate environments and under conditions well known to man of the art in such a way as to obtain sufficient bacterial biomass for the extraction of genetic material, which is done using the usual techniques described by Sambrook J. et al. (Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York, 1989). Borrelia burgdorferi strains are grown in appropriate environments and under conditions well known to man of the art. These conditions and environments are particularly described by A. Barbour (J. Biol. Med. 1984. 57. 71-75). The extraction of bacterial DNA was carried out under the conditions described by W. Simpson et al. (Infect. Immun. 1990. 58. 847-853). The usual techniques described by J. Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York. 1989) may also be used.

Example 3: Extraction of viral genomic DNA:

After culture, the overlying and lysed cells are harvested and the entire viral suspension is centrifuged at 1000 g for 10 minutes at +4°C to remove cellular debris. The viral particles are then harvested by ultracentrifugation at 400000 g for 1 hour at +4°C. The coagulation is taken up again in a minimum buffer volume (Tris 10 mM, EDTA 1 mM). This concentrated viral suspension is treated by proteinase K (100 μg/ml final) in the presence of sodium dodecyl sulfate (SDS) (0.5% final volume) for 2 hours at 37°C. The viral DNA is then extracted with a phenol/chloroform mixture, precipitated with 2 volumes of water. After an absolute 20°C, the DNA can be coagulated for 10 minutes at a minimum of 15°C. The coagulation is then carried out by enzymes at a restriction of 10 to 10 000 °C.

Example 4: Isolation of viral genomic RNA

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Example 5: Molecular biology techniques

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The test chemical is used to determine the concentration of the test chemical in the test medium.

Specific oligonucleotides (with restriction sites at their 5' ends to facilitate cloning of amplified fragments) were synthesized in such a way that they completely cover the coding regions of the genes to be amplified (see specific examples).Reverse transcription (RT) and polymerase chain amplification (PCR) were performed using standard techniques (Sambrook J. et al. 1989).Each RT-PCR reaction was done with a pair of specific amplifiers and using the extracted viral genomic RNA as the matrix.The amplified complementary DNA was extracted from phenol/chloroform/alkyl isamylic alcohol (25:24:1) before being digested by restriction enzymes.

The test chemical is used to determine the concentration of the test substance.

The plasmid pVR1012 (Figure 1) was obtained from Vical Inc. San Diego, CA, USA. Its construction was described in J. Hartikka et al. (Human Gene Therapy. 1996. 7.

Example 8 : Construction of the plasmid pAB042 (gene EHV-1 gB)

A PCR reaction was performed with genomic DNA from equine herpesvirus type 1 (EHV-1) (Kentucky strain D) (P. Guo et al. J. Virol. 1990. 64. 2399-2406) and with the following oligonucleotides: - What? to isolate the gene coding for the glycoprotein gB (EHV-1 gB) in the form of a PstI-NotI fragment. After purification, the PCR product of 2981 pb was digested by PstI and NotI to isolate a PstI-NotI fragment of 2959 pb. This fragment was ligated with the vector pVR1012 (Example 7), previously digested with PstI and NotI, to yield the plasmid pAB042 (7841 pb) (Figure No 2).

Example 9: Construction of the plasmid pAB031 (gene EHV-4 gB)

A PCR reaction was performed with genomic DNA from equine herpesvirus type 4 (EHV-4) (strain 1942) (M. Riggio et al. J. Virol. 1989. 63. 1123-1133) and with the following oligonucleotides: - What? to amplify a 2949 pb fragment containing the gene coding for the gB glycoprotein of EHV-4 as a PstI-XbaI fragment. After purification, the PCR product was digested by PstI and Xbal to give a 2931 pb PstI-XbaI fragment. This fragment was ligated with the vector pVR1012 (example 7), which was previously digested with Pstl and Xbal, to give the plasmid pAB031 (7806 pb) (Figure 3).

Example 10: Construction of the plasmid pAB013 (gene EHV-1 gD)

A PCR reaction was performed with genomic DNA from equine herpesvirus type 1 (EHV-1) (Kentucky strain D) (J.C. Audonnet et al. J. Gen. Virol. 1990. 71. 2969-2978) and with the following oligonucleotides: - What? After purification, the 1228 pb PCR product was digested by Pstl and BamHI to isolate a 1211 pb PstI-BamHI fragment. This fragment was ligated with the vector pVR1012 (Example 7), previously digested with PstI and BamHI, to yield the plasmid pAB013 (6070 pb) (Figure No 4).

Example 11: Construction of the plasmid pAB032 (gene EHV-4 gD)

A PCR reaction was performed with the genomic DNA of the equine herpesvirus type 4 (EHV-4) (A. Cullinane et al. J. Gen. Virol. 1993. 74. 1959-1964) and with the following oligonucleotides: - What? After purification the 1230 pb PCR product was digested by PstI and BamHI to isolate a 1212 pb PstI-BamHI fragment. This fragment was ligated with the vector pVR1012 (Example 7), previously digested with PstI and BamHI, to yield the plasmid pAB032 (6071 pb) (Figure No 5).

Example 12: Construction of the plasmid pAB043 (Prague strain HA equine influenza gene)

A RT-PCR reaction according to the technique described in Example 6 was performed with genomic RNA from the equine influenza virus (Equine Influenza Virus or EIV) (H7N7 Prague strain) (J. Mc Cauley. - What? to isolate the gene coding for the equine influenza virus glycoprotein HA in the form of a SalI-BamHI fragment. After purification, the RT-PCR product of 1733 pb was digested by SalI and BamHI to isolate a 1720 pb Sall-BamHI fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with SalI and BamHI, to yield the plasmid pAB043 (6588 pb) (Figure No 6).

Example 13: Construction of the plasmid pAB033 (equine influenza HA Suffolk strain gene)

A RT-PCR reaction according to the technique in Example 6 was performed with genomic RNA from the equine influenza virus (EIV) (Suffolk strain) (M. Binns. Sequence access number on Genbank = X68437) prepared according to the technique in Example 4 and with the following oligonucleotides: - What? to isolate the gene coding for the equine influenza virus glycoprotein HA in the form of a Sall-BamHl fragment. After purification, the RT-PCR product of 1729 pb was digested by SalI and BamHI to isolate a 1717 pb Sall-BamHl fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with SalI and BamHI, to yield the plasmid pAB033 (6584 pb) (Figure No 7).

Example 14: Construction of the plasmid pAB099 (equine influenza HA strain Fontainebleau gene)

A RT-PCR reaction according to example 6 was performed with genomic RNA from equine influenza virus (EIV) (Fontainebleau strain), prepared according to example 4, and with the following oligonucleotides: - What? After purification the RT-PCR product of 1724 pb was digested by NotI to isolate a 1710 pb NotI-NotI fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with NotI, to give the pAB099 plasmid (6625 pb) containing the HA (Fontainebleau's equine influenza) gene in the correct orientation to the vector (Figure 9).

Example 15: Construction of the plasmid pAB085 (Equine influenza gene NP strain Prague)

A RT-PCR reaction according to the technique in Example 6 was performed with genomic RNA from the equine influenza virus (EIV) (H7N7 Prague strain) (O. Gorman et al. J. Virol. 1991. 65. 3704-3714) prepared according to the technique in Example 4 and with the following oligonucleotides: - What? to isolate the gene coding for the NP nucleoprotein of the equine influenza virus in the form of a SalI-BamHI fragment. After purification, the 1515 pb RT-PCR product was digested by SalI and BamHI to isolate a 1503 pb SalI-BamHI fragment. This fragment was ligated with the pVR1012 vector (example 7), previously digested with SalI and BamHI, to yield the plasmid pAB085 (6371 pb) (Figure No 10).

Example 16: Construction of the plasmid pAB084 (equine influenza gene NP strain Jillin)

A RT-PCR reaction according to the technique in Example 6 was performed with genomic RNA from the equine influenza virus (EIV) (H3N8 Jillin strain) (O. Gorman et al. J. Virol. 1991. 65. 3704-3714) prepared according to the technique in Example 4 and with the following oligonucleotides: - What? to isolate the gene coding for the NP nucleoprotein of the equine influenza virus in the form of a SalI-BamHI fragment. After purification, the 1515 pb RT-PCR product was digested by SalI and BamHI to isolate a 1503 pb Sall-BamHl fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with Sail and BamHI, to yield the plasmid pAB084 (6371 pb) (Figure No 11).

Example 17: Construction of the plasmid pAB070 (gen subunit C tetanus toxin)

A PCR reaction was performed with Clostridium tetani genomic DNA (Strain CN3911) (N. Fairweather et al. J. Bact. 1986. 165. 21-27), prepared using the technique in Example 2, and with the following oligonucleotides: - What? After purification, the 1377 pb PCR product was digested by Pstl and BamHI to isolate a 1361 pb Pstl-BamHl fragment. This fragment was ligated to the vector pVR1012 (Example 7), previously digested with PstI and BamHI, to yield the plasmid pAB070 (6219 pb) (Figure 12).

The test chemical is used to determine the concentration of the active substance in the test chemical.

A PCR reaction was performed with the genomic DNA of Borrelia burgdorferi (Strain B31) (S. Bergstrom et al. Mol. Microbiol. 1989. 3. 479-486), prepared using the technique in Example 2, and with the following oligonucleotides: - What? to isolate the OspA membrane protein-coding gene in the form of a Sall-BamHl fragment. After purification, the 842 pb PCR product was digested by SalI and BamHI to isolate an 829 pb Sall-BamHl fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with Sall and BamHl, to yield the plasmid pAB017 (5698 pb) (Figure No 13).

Example 19: Construction of the plasmid pAB094 (E2 gene of the Eastern encephalitis virus)

A RT-PCR reaction using the technique in Example 6 was performed with Eastern encephalitis virus (EEV) genomic RNA (Strain North America 82V2137) (S. Weaver et al. Virology. 1993. 197. 375-390) prepared using the technique in Example 4 and with the following oligonucleotides: - What? After purification, the 1294 pb RT-PCR product was digested by PstI and BamHI to isolate a 1278 pb PstI-BamHI fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with PstI and BamHI, to yield the plasmid pAB094 (6136 pb) (Figure No 14).

Example 20: Construction of the plasmid pAB093 (gen C of the Eastern encephalitis virus)

A RT-PCR reaction according to the technique in Example 6 was performed with Eastern encephalitis virus (EEV) genomic RNA (Strain North America 82V2137) (S. Weaver et al. Virology. 1993. 197. 375-390) prepared according to the technique in Example 4 and with the following oligonucleotides: - What? to isolate the capsid protein C (EEV C) coding gene in the form of a PstI-BglII fragment. After purification, the 801 pb RT-PCR product was digested by PstI and BglII to isolate a 785 pb PstI-BglII fragment. This fragment was ligated with the vector pVR1012 (Example 7), previously digested with PstI and BglII, to yield the plasmid pAB093·(5650 pb) (Figure No 15).

Example 21: Construction of the plasmid pAB096 (the E2 gene of the western encephalitis virus)

A RT-PCR reaction according to the technique in Example 6 was performed with Western encephalitis virus (WEV) genomic RNA (BSF1703 strain) (C. Hahn et al. Proc. Natl. Acad. Sci. USA. 1988. 85. 5997-6001), prepared according to the technique in Example 4, and with the following oligonucleotides: - What? After purification, the RT-PCR product of 1304 pb was digested by SalI and BamHI to isolate a Sall-BamHl fragment of 1291 pb. This fragment was ligated with the vector pVR1012 (example 7), previously digested with SalI and BamHI, to yield the plasmid pAB096 (6159 pb) (Figure No 16).

Example 22: Construction of the plasmid pAB095 (gen C of the western encephalitis virus)

A RT-PCR reaction according to example 6 was performed with Western encephalitis virus (WEV) genomic RNA (BSF1703 strain) (C. Hahn et al. Proc.Natl.Acad.Sci.USA. 1988. 85. 5997-6001), prepared according to example 4, and with the following oligonucleotides: - What? to isolate the gene coding for the WEV capsid C protein in the form of a Sall-BamHl fragment. After purification, the 809 pb RT-PCR product was digested by SalI and BamHI to isolate a 796 pb SalI-BamHI fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with SalI and BamHI, to yield the plasmid pAB095 (5664 pb) (Figure No 17).

Example 23: Construction of the plasmid pAB098 (gene E2 of the Venezuelan encephalitis virus)

A RT-PCR reaction according to example 6 was performed with genomic RNA from the Venezuelan encephalitis virus (VEV) (Strain P676 (Type IC)) (R. Kinney et al. Virology. 1992. 191. 569-580), prepared according to example 4, and with the following oligonucleotides: - What? After purification, the 1304 pb RT-PCR product was digested by SalI and BamHI to isolate a 1291 pb SalI-BamHI fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with SalI and BamHI, to yield the plasmid pAB098 (6159 pb) (Figure No 18).

Example 24: Construction of the plasmid pAB097 (C gene of the Venezuelan encephalitis virus)

A RT-PCR reaction according to example 6 was performed with genomic RNA from the Venezuelan encephalitis virus (VEV) (Strain P676 (Type IC)) (R. Kinney et al. Virology. 1992. 191. 569-580), pre-packed according to example 4, and with the following oligonucleotides: - What? After purification, the 856 pb RT-PCR product was digested by Pstl and BamHI to isolate an 839 pb PstI-BamHI fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with PstI and BamHI, to yield the plasmid pAB097 (5698 pb) (Figure No 19).

Example 25: Construction of the plasmid pAB041 (rabies virus G gene)

A RT-PCR reaction according to example 6 was performed with the genomic RNA of the rabies virus (ERA strain) (A. Anilionis et al. Nature. 1981. 294. 275-278), prepared according to example 4, and with the following oligonucleotides: - What? After purification, the RT-PCR product was digested by Pstl and BamHI to yield a 1578 pb PstI-BamHI fragment. This fragment was ligated with the vector pVR1012 (example 7), previously digested with PstI and BamHI, to yield the plasmid pAB041 (6437 pb) (Figure No 20).

Example 26: Preparation and purification of plasmids

In particular, the alkaline lysis technique followed by two successive ultracentrifugations on industrial cesium chloride gradient in the presence of ethidium bromide as described in J. Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York. 1989). Reference may also be made to the PCT applications WO 95/21250 and WO 96/02658 for PCT which describe methods for producing ultra-high resistivity plasmids. To obtain resistivity plasmids for the manufacture of vaccines, for example, the pH of these plasmids (HCl) is 10 mL (Tm) to 17 mL (Tm) depending on whether the solutions are purified in water.

Example 27: Manufacture of associated vaccines

The various plasmids required to make a matched vaccine are mixed from their concentrated solutions (Example 16). The mixtures are made in such a way that the final concentration of each plasmid corresponds to the effective dose of each plasmid. The solutions used to adjust the final concentration of the vaccine can be either a 0.9% NACI solution or a PBS buffer. Special formulations such as liposomes, cationic lipids, can also be used for the manufacture of vaccines.

Example 28: Vaccination of horses

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Claims (14)

1. Equine vaccine characterised by the inclusion of a plasmid that integrates, in order to express it in vivo in cells, the genes coding for the encephalitis virus C and E2 antigens.
2. The vaccine claimed 1, characterised by the plasmid incorporating the gene coding for the encephalitis virus E2 antigen in a way that it is expressed in vivo in cells.
3. The vaccine according to any of claims 1 to 2, characterised by the encephalitis virus being selected from the group consisting of Eastern EEV encephalitis virus, Western WEV encephalitis virus and Venezuelan VEV encephalitis virus.
4. The vaccine as claimed 3, characterised by the inclusion of a plasmid incorporating in vivo expression in cells of a gene coding for the E2 antigen of one or more encephalitis viruses selected from the group consisting of Eastern encephalitis virus, Western encephalitis virus and Venezuelan encephalitis virus.
5. The vaccine according to any of claims 1 to 4, characterised by the combination of genes coding for several antigens is achieved by the mixture of plasmids expressing a single antigen.
6. The vaccine according to any of claims 1 to 4, characterised by the combination of genes coding for multiple antigens is achieved by inserting multiple genes into the same plasmid.
7. The vaccine as claimed 6, characterised by the presence of genes in the same transcription unit or in two different units.
8. The vaccine according to any of claims 1 to 7, characterised by the inclusion of the early CMV-IE cytomegalovirus promoter in the plasmid.
9. The vaccine according to any of claims 1 to 7, characterised by the inclusion of a promoter selected from the group of early SV40 virus promoter, late SV40 virus promoter, LTR Rous Sarcoma virus promoter, desmine promoter and actin promoter.
10. The vaccine according to any of claims 1 to 9, characterised as comprising 10 ng to 1 mg per plasmid type.
11. The vaccine as claimed 10, characterised by comprising 100 ng to 500 μg per plasmid type.
12. The vaccine as claimed 11, characterised by comprising 1 μg to 250 μg per plasmid type.
13. Use of one or more plasmids as described in any of claims 1 to 12 for the manufacture of a vaccine for vaccinating primogen vaccinated equidae by means of a first vaccine selected from the group consisting of a live whole vaccine, inactivated whole vaccine, subunit vaccine, recombinant vaccine, this first vaccine having the antigen (s) providing cross-protection.
14. Equine vaccination kit comprising a vaccine according to any of claims 1 to 12 and a primary vaccination vaccine selected from the group consisting of whole live vaccine, whole inactivated vaccine, subunit vaccine, recombinant vaccine, this primary vaccination vaccine has the antigen (s) providing cross-protection.