Title:  Polynucleotide vaccine formula in particular against bovine respiratory pathology

United States Patent:  6,818,628

Issued:  November 16, 2004

Inventors:  Audonnet; Jean-Christophe (Lyons, FR); Bouchardon; Annabelle (Lyons, FR); Baudu; Philippe (Craponne, FR); Riviere; Michel (Ecully, FR)

Assignee:  Merial (Lyons, FR)

Appl. No.:  085519

Filed:  February 28, 2002

Abstract

Disclosed and claimed are compositions for inducing in a bovine host an immunological response against bovine respiratory syncytial virus or bovine viral diarrhea virus containing at least one plasmid that contains and expresses in vivo in a bovine host cell nucleic acid molecule(s) having sequence(s) encoding bovine respiratory syncytial virus F protein, or G protein, or F and G proteins; or, at least one plasmid that contains and expresses in vivo in a bovine host cell nucleic acid molecule(s) having sequence(s) encoding bovine viral diarrhea virus E2 protein, or C, E1 and E2 proteins, or E1 and E2 proteins. Methods and kits employing such compositions are also disclosed.

Description of the Invention

The present invention relates to a vaccine formula allowing the vaccination of bovines in particular against respiratory pathology. It also relates to a corresponding method of vaccination.

All bovines are carriers of viruses and bacteria which are potentially pathogenic in widely variable degrees.

Viruses can multiply when the specific immunity is weakened and when there are lesions of the respiratory tract. They are then excreted by the animal and may then contaminate other animals.

Among the viruses which are encountered, there may be mentioned in particular the type 3 parainfluenza virus (PI-3), of moderate inherent pathogenicity, the bovine respiratory syncytial virus (RSV) and the bovine herpesvirus (BHV) also called infectious bovine rhinotracheitis (IBR) virus, of high inherent pathogenicities.

Another virus which is particularly important for its immunodepressant role and its harmful effects on reproduction is the mucosal disease virus or bovine pestivirus (BVDV).

These viruses generally manifest themselves by a primary phase of hyperthermia, flu syndrome and respiratory disorders, with digestive disorders (diarrhoeas) in the case of BVD. This phase may be accompanied by a secondary phase with the onset of bronchopneumonia linked to bacterial, in particular Pasteurella, infections which can lead to death. This phenomenon is exacerbated in particular by the immuno-depression resulting from BVD infection or by the infection of macrophages by PI-3. Other symptoms may further appear, such as abortions with BVD and BHV.

It therefore appears necessary to try to develop an effective prevention against the principal viruses involved in bovine respiratory pathology.

Associations of vaccines against certain viruses responsible for bovine respiratory pathology have already been proposed in the past.

The associations developed so far were prepared from inactivated vaccines or live vaccines and, optionally, mixtures of such vaccines. Their development poses problems of compatibility between valencies and of stability. It is indeed necessary to ensure both the compatibility between the different vaccine valencies, whether from the point of view of the different antigens used or from the point of view of the formulations themselves, especially in the case where both inactivated vaccines and live vaccines are combined. The problem of the conservation of such combined vaccines and also of their safety especially in the presence of an adjuvant also exists. These vaccines are in general quite expensive.

Patent Applications WO-A-90 11092, WO-A-93 19183, WO-A-94 21797 and WO-A-95 20660 have made use of the recently developed technique of polynucleotide vaccines. It is known that these vaccines use a plasmid capable of expressing, in the host's cells, the antigen inserted into the plasmid. All the routes of administration have been proposed (intraperitoneal, intravenous, intramuscular, transcutaneous, intradermal, mucosal and the like). Various vaccination means can also be used, such as DNA deposited at the surface of gold particles and projected so as to penetrate into the animal's skin (Tang et al., Nature 356, 152-154, 1992) and liquid jet injectors which make it possible to transfect at the same time the skin, the muscle, the fatty tissues and the mammary tissues (Furth et al., Analytical Biochemistry, 205, 365-368, 1992).

The polynucleotide vaccines may also use both naked DNAs and DNAs formulated, for example, inside cationic lipid liposomes.

G. J. M. COX has already proposed polynucleotide vaccination against type 1 bovine herpes virus in J. of Virology, Volume 67, No. 9, September 1993, 5664-5667. The authors have also described plasmids integrating the gI (gB), gIII (gC) and gIV (gD) genes.

In Vaccine, Volume 13, No. 4, 415-421, 1995, J. E. CROWE presents a general review of the different methods of vaccination against respiratory syncytial virus and against type 3 parainfluenza virus. This review reexamines all the possibilities offered by the current vaccination techniques and simply suggests that the polynucleotide immunization technique could be useful in the immunization strategy against RSV and PI-3. No plasmid construction or result of vaccination of bovines against these viruses is described in this document.

The invention therefore proposes to provide a multivalent vaccine formula which makes it possible to ensure vaccination against a number of pathogenic viruses involved in particular in bovine respiratory pathology and thus to ensure effective vaccination against this pathology.

Another objective of the invention is to provide such a vaccine formula combining different valencies while exhibiting all the criteria required for mutual compatibility and stability of the valencies.

Another objective of the invention is to provide such a vaccine formula which makes it possible to combine different valencies in the same vehicle.

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

Yet another objective of the invention is to provide such a vaccine formula and a method for vaccinating bovines which makes it possible to obtain a multivalent protection with a high level of efficiency and of long duration, as well as good safety and an absence of residues.

The subject of the present invention is therefore a vaccine formula in particular against bovine respiratory pathology, comprising at least three polynucleotide vaccine valencies each comprising a plasmid integrating, so as to express it in vivo in the host cells, a gene with one bovine respiratory pathogen valency, these valencies being selected from the group consisting of bovine herpesvirus, bovine respiratory syncytial virus, mucosal disease virus and type 3 parainfluenza virus, the plasmids comprising, for each valency, one or more of the genes selected from the group consisting of gB and gD 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, and HN and F for the type 3 parainfluenze virus.

Valency in the present invention is understood to mean at least one antigen providing protection against the virus for the pathogen considered, it being possible for the valency to contain, as subvalency, one or more modified or natural genes from one or more strains of the pathogen considered.

Pathogenic agent gene is understood to mean not only the complete gene but also the various nucleotide sequences, including fragments which retain the capacity to induce a protective response. The notion of a gene covers the nucleotide sequences equivalent to those described precisely in the examples, that is to say the sequences which are different but which encode the same protein. It also covers the nucleotide sequences of other strains of the pathogen considered, which provide cross-protection or a protection specific for a strain or for a strain group. It also covers the nucleotide sequences which have been modified in order to facilitate the in vivo expression by the host animal but encoding the same protein.

Preferably, the vaccine formula according to the invention comprises the four valences.

As regards the BHV valency, use is preferably made of the two genes encoding gB and gD, in different plasmids or in one and the same plasmid. Optionally, but less preferably, either of these genes can be used.

For the RSV valency, use is preferably made of the two G and F genes integrated into two different plasmids or into one and the same plasmid. Optionally, but less preferably, the F gene can be used alone.

For the BVD valency, use will preferably be made of a plasmid integrating the E2 gene. Optionally, but less preferably, a plasmid coding for E1 and E2 together or for the combination consisting of C, E1 and E2 can be used.

For the PI-3 valency, use is preferably made of the combination of the two HN and F genes in two different plasmids or in one and the same plasmid. It is also possible to use only the HN gene.

A preferred vaccine formula according to the invention comprises and ensures the expression of the BHV gB and gD genes, the RSV G and F genes, the BVD E2 genes and PI-3 HN and F genes.

The vaccine formula according to the invention can be provided in a dose volume of between 0.1 and 10 ml and in particular between 1 and 5 ml.

The dose will be generally between 10 ng and 1 mg, preferably between 100 ng and 50 .mu.g and preferably between 1 .mu.g and 250 .mu.g per plasmid type.

Use will preferably be made of naked plasmids simply placed in the vaccination vehicle which will be in general physiological saline (0.9% NaCl), ultrapure water, TE buffer and the like. All the polynucleotide vaccine forms described in the prior art can of course be used.

Each plasmid comprises a promoter capable of ensuring the expression of the gene inserted, under its control, into the host cells. This will be in general a strong eukaryotic promoter and in particular a cytomega-lovirus early CMV-IE promoter of human or murine origin, or optionally of another origin such as rats, pigs and guinea pigs.

More generally, the promoter may be either 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 from the virus from which the gene is derived, for example the gene's own promoter.

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 plasmid, these may be presented in the same transcription unit or in two different units.

The combination of the different vaccine valencies according to the invention may be preferably achieved by mixing the polynucleotide plasmids expressing the antigen(s) of each valency, but it is also possible to envisage causing antigens of several valencies to be expressed by the same plasmid.

The subject of the invention is also monovalent vaccine formulae comprising one or more plasmids encoding one or more genes from one of the viruses selected from the group consisting of BRSV, BVD and PI-3, the genes being those described above. Besides their monovalent character, these formulae may possess the characteristics stated above as regards the choice of the genes, their combinations, the composition of the plasmids, the dose volumes, the doses and the like.

The monovalent vaccine formulae may be used (i) for the preparation of a polyvalent vaccine formula as described above, (ii) individually against the actual pathology, (iii) combined with a vaccine of another type (live or inactivated whole, recombinant, subunit) against another pathology, or (iv) as booster for a vaccine as described below.

The subject of the present invention is in fact also the use of one or more plasmids according to the invention for the manufacture of a vaccine intended to vaccinate bovines first vaccinated by means of a first conventional vaccine of the type in the prior art, in particular, selected from the group consisting of a live whole vaccine, an inactivated whole vaccine, a subunit vaccine, a recombinant vaccine, this first vaccine having, that is to say containing or capable of expressing, the antigen(s) encoded by the plasmid(s) or antigen(s) providing cross-protection.

Remarkably, the polynucleotide vaccine has a potent booster effect which results in an amplification of the immune response and the acquisition of a long-lasting immunity.

In general, the first-vaccination vaccines can be selected from commercial vaccines available from various veterinary vaccine producers.

The subject of the invention is also a vaccination kit grouping together a first-vaccination vaccine as described above and a vaccine formula according to the invention for the booster. It also relates to a vaccine formula according to the invention accompanied by a leaflet indicating the use of this formula as a booster for a first vaccination as described above.

The subject of the present invention is also a method for vaccinating bovines against respiratory pathology, comprising the administration of the effective vaccine formula as described above. This vaccination method comprises the administration of one or more doses of the vaccine formula, it being possible for these doses to be administered in succession over a short period of time and/or in succession at widely spaced intervals.

The vaccine formulae according to the invention can be administered, in the context of this method of vaccination, by the different routes of administration proposed in the prior art for polynucleotide vaccination and by means of known techniques of administration.

The subject of the invention is also the method of vaccination consisting in making a first vaccination as described above and a booster with a vaccine formula according to the invention.

In a preferred embodiment of the process according to the invention, there is administered in a first instance, to the animal, an effective dose of the vaccine of the conventional, especially inactivated, live, attenuated or recombinant, type, or alternatively a subunit vaccine, so as to provide a first vaccination, and, within a period preferably of 2 to 6 weeks, the polyvalent or monovalent vaccine according to the invention is administered.

The invention also relates to the method of preparing the vaccine formulae, namely the preparation of the valencies and mixtures thereof, as evident from this description.

Claim 1 of 11 Claims

What is claimed is:

1. An immunogenic composition for inducing an immunological response against bovine parainfluenza virus type 3 consisting essentially of one or more plasmids that contain and express in vivo in a bovine host cell nucleic acid molecule(s) having sequence(s) encoding type 3 bovine parainfluenza virus hemaglutinin/neuraminidase (HN) protein, or fusion (F) protein, or HN and F proteins.

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