MXPA99005452A - Recombinant equine herpesvirus type 1 (ehv-1) comprising a dysfunctional gene 71 region and use thereof as a vaccine - Google Patents

Abstract

Vaccine formulation comprising EHV-1 gene 71 dysfunctional mutant and uses thereof.

Description

HERPESVIRUS OF EQUINE TYPE 1 (HVE-1) RECOMBINANT THAT UNDERSTANDS A DYSFUNCTIONAL REGION OF GEN 71 AND USE OF IT AS A VACCINE The present invention relates to a viral vaccine containing an attenuated HVE-1 virus comprising a deletion of the gene in the genome thereof, uses thereof and methods for treating diseases related to HVE-1. In particular, the invention relates to a viral vaccine composition for use against equine Herpesvirus type 1 (HVE-1) HVE-1 is a member of the sub-famiha alfaherpesvipnae and is a major viral pathogen of horses. The clinical problems caused for HVE-1 include respiratory disease, abortion and neurological disorders (Bryans JT, and Alien, GP, Kluwer Academic Publishers, Norwell MA 1989) As such, HVE-1 is responsible for significant economic losses within the equine industry The genome of HVEX is a linear double-stranded DNA molecule with a size of approximately 150 kb that can be divided into two components covalently linked to the long and short regions. The long region consists of a single sequence (UL) flanked by a small inverted repeat (IR and TR) The short region comprises a single sequence (Us) flanked by a long inverted repeat (IRS and TRs ^ HVE-1 occurs as pathogenic and nonpathogenic strains) s and recently, the complete DNA sequence of a pathogenic strain, Ab4, has been determined and the sequence has been deposited with the GenBank Bank under accession number M 86664 (Telford, E.A R. and others, Virology 189, pp. 304-316 (1990)). The genome is 150,223 bp in size and contains 81 open reading frames intended to encode pohpeptides. The sizes of its components are UL, 112,870 bp; TR / IRL, 32 bp; Us, 11,861 bp and IRS / TRS, 12,714 bp Interestingly, there are five genes, 1, 2, 67, 71 and 75, which have no homologs in any of the herpesviruses sequenced to date; that is, they are unique to HVE- It is thought that each of the genes 1, 2, 67, 71 and 75 encode a protein, however, the function of individual proteins is not clear. Recently, it has been shown that the gene product 71 of HVE-1 is involved in the adsorption / penetration of the virus and exits the virus from the nuclei of infected cells (Sun Y et al., Juornal of General Virology 77 pages 493-500 (1996)) The prior art does not teach or suggest the use of deletion mutants of gene 71 of HVE-1 comprising a region 71 of the dysfunctional gene in the manufacture and use of vaccines against diseases related to HVE-1 Control by vaccine of infection of HVEX has been a goal sought for long Time Current HVE-1 vaccines include chemically-activated virus vaccines and modified live virus vaccines Inactivated vaccines generally induce a low level of immunity and require additional immunizations and are production expensive The use of such vaccines carries the risk that some infectious viral particles may survive the inactivation process and cause disease after administration to the animal In general, live attenuated virus vaccines are preferred since they evoke a lasting immune response (frequently both humoral and cellular) and are easier to produce Live attenuated HVE-1 vaccines are available, which are based on live HVE-1 virus attenuated by the serial passage of virulent strains in tissue culture. However, the Serial passage of the virulent strains can cause uncontrolled mutations of the viral genome, resulting in a population of heterogeneous virus particles in their virulence and immunization properties. It is also known that said live attenuated HVE-1 virus vaccines can be reversed. virulent diseases resulting in disease to the inoculated animals and the possible dysfunction of OXYGEN TO OTHER ANIMALS The inventors of the present invention have now identified a suitable strain of living HVE-1 mutant virus comprising a dysfunctional region of the HVE-1 genome located within the single short region thereof. Such a mutant can be used in a formulation of live HVE-1 vaccine Specifically, the inventors have found that mutants of HVE-1 are dysfunctional for the production of a protein encoded by gene 71, can be used in a live HVE-1 vaccine formulation. These mutants are shown to be substantially less virulent than wild type HVE-1 virus In addition, it has been found that gene 71 is not essential for the growth of HVE-1 in cell culture (Sun Y and Brown SM, Virology 199 pages 448-452 (1994)) The inventors they have also found that HVE-1 viruses comprising the dysfunctional gene 71 regions of their genome are immunogenic. Said viruses are intended for the use of components in vaccine formulations or therapeutic compositions against HVE-1 infection. Consequently, the present invention refers to HVE-1 viruses that comprise a dysfunctional region located in the protein coding region of gene 71, and in particular, between nucleotides 129.096 and 131 489 of The native genome Statement of the Invention A first aspect of the present invention provides a vaccine formulation comprising a live recombinant HVE-1 virus modified to contain a dysfunctional gene 71 region located within the Us region of the virus genome. and a pharmaceutically acceptable carrier A "dysfunctional gene 71 region" is one that is substantially unable to encode this native pohpeptide or a functional equivalent. Thus, a dysfunctional "gene 71 region" means that the region of gene 71 has been modified by deletion , insertion or substitution (or other change in the DNA sequence such as by rearrangement) so that the region of gene 71 does not express a popeptide of the native 71 HVE-1 gene or a functionally equivalent product thereof. that gene 71 of HVE-1 encodes a polypeptide of 797 amino acids and that the peptide is a 192 kDa glycoprotein linked to O (Sun, Y et al., Jornal of General Virology 75, pp. 3117-3126 (1994)) Therefore, vaccine formulations comprising modified HVE-1 viruses of the invention can include viruses modified in one or more ways via recombinant DNA technology. Types of modifications that can be included are (i) A deletion of the entire 71 gene from the genome of a wild-type HVE-1 virus. For example, a deletion of the nucleotide sequence of the wild-type HVE-1 genome from about 129,096 nucleotides to about 131,489 (n) A deletion of a portion of gene 71 from the genome of a wild-type virus HVE-1 A "portion of gene 71" means a deletion that is sufficient to make any polypeptide encoded by the mutant of deletion of gene 71 and expressed by it is substantially incapable of a physiological activity similar to that of the native pohpeptide produced by wild type HVE-1. The deletion may be between 0% and 100% of the nucleotide sequence located between approximately 129,096 and 131,489 nucleotides and the wild type HVE-1 genome. The deletion can be from about 70% to 100% of the nucleotide sequence of gene 71, or deletion it can be from about 70% to 90% of the nucleotide sequence of the 71 gene, for example, of about 80% of the nucleotide sequence of the 71 gene. (iii) The deletion of all or a portion of the 71 gene, as described in (i) and (ii) above, which will leave a "space" in the HVE-1 genome corresponding to the open reading frame (MLA) of gene 71 or a portion thereof. A suitable gene or genes can be inserted into a "space" such as a marker gene. Suitable marker genes include, but are not restricted to, enzyme marker genes, for example the E. coli lac-Z gene, antibiotic marker genes such as hygromycin, neomycin, and the like. Such marker genes are commonly employed in the art. In general, marker genes, if any, which can be used in a deletion mutant of the gene 71 of the invention, should be such that they do not cause substantial or delayed side effects to a recipient animal.

In one preference, the "space" formed by the deletion of all or a portion of the 71 gene of a wild-type HVE-1 virus is not filled with an insert of the gene, the cut ends of the two pieces of the genome being bound usanao conventional recombinant DNA technology. The skilled person will appreciate that the term 'deletion mutant' encompasses those situations where the space left by partial or omal deletion of the 71 gene can be filled with a gene insert, eg, a marker gene or a nonsense nucleotide sequence (ie say, a sequence capable of originating a protein or polypeptide product) or those situations where the space is not filled by a sequence of heterologous or other nucleotides In such a case, the appropriate free ends of the two pieces of the genome are ligated (iv ) Deletion within the region of gene 71 can comprise a deletion of a small number of nucleotides, for example 1, 2 or more nucleotides. Such deletions can be achieved using recombinant DNA technology. Thus, translational MLA can be altered giving as result in the production of a protein that lacks the physiological function of the native peptide of gene 71 The expert will also appreciate that deletions in the translational MLA of gene 71 can also cause a dysfunctional gene 71 that is not capable of encoding the entire polypeptide, a truncated peptide or even any peptide. Such proteins, if they are produced, generally lack physiological functionality of the protein product. a normal 71 gene MLA (v) Nucleotide insertions can also be made in the region of gene 71 of HVE-1 using recombinant DNA technology that results in dysfunctional gene 71 polypeptides substantially incapable of functional activity. For example, codons of seals can be inserted into the region of gene 71, resulting in the production of non-functional fragments of the polypeptide encoded by the native gene 71 The skilled artisan will appreciate that the nucleotide inserts can be of any length from one or more nucleotides to a number of nucleotides forming, for example, nonsense nucleotide sequences that can have the effect MTA to alter the MLA that results from the lack of production of a polypeptide or in addition, the production of a protein that lacks the physiological function of the native polypeptide of gene 71. The expert will also appreciate that such insertions in translational MLA of gene 71 also they can cause a dysfunctional gene 71 that is unable to encode the entire polypeptide, the truncated peptide or even any peptide. Said proteins, if they are produced, generally lack physiological functionality. Naturally, the skilled artisan will appreciate that deletions of gene 71 and inserts of non-wild type HVE-1 viruses, as described above, are encompassed by the present invention. In a preferred embodiment, a vaccine formulation comprising a mutant virus for suppression of gene 71 of attenuated, recombinant, live immunogenic HVE-1 and a pharmaceutically acceptable carrier is provided. In a second aspect of the invention, a recombinant, live HVE-1 is provided which comprises a dysfunctional gene 71 region to be used as a vaccination agent in a vaccine formulation. Preferably, a mutant deletion virus of the 71 gene is provided. Recombinant attenuated immunogenic HVE-1, live, to be used as a vaccination agent or in a vaccine formulation. Optionally living recombinant HVE-1 may include an inserted gene placed at the site of gene 71 in view of a substantial portion of gene 71 or the entire gene 71. Generally, the vaccine or vaccine formulation is not used in non-pregnant animals because that can cause an abortion. In a third aspect of the invention, the use of a live recombinant HVE-1 virus to produce antibodies or cell-mediated immunity for HVE-1 comprising a dysfunctional gene 71 region located within the Us region of the genome is provided. of the virus for the manufacture of an HVE-1 vaccine for the prophylaxis and / or treatment of HVE-1 infection. Preferably, the use of a mutant virus of suppression of gene 71 of attenuated immunogenic, recombinant HVE-1, alive, is provided for the manufacture of an HVE-1 vaccine for the prophylaxis and / or treatment of infection of HVE-1. More preferably, the use is in horses. In a fourth aspect of the invention, there is provided a method for treating animals which comprises administering to them a vaccine composition comprising a live recombinant HVE-1 virus, modified so as to contain a region of the dysfunctional gene 71 located within the Us region of the virus genome to animals that need it. Preferably, the animals are horses. Still preferably, the method for treating animals comprises administering a vaccine composition comprising a mutant virus deleting gene 71 of immunogenic, attenuated, alive, recombinant HVE-1, to the animals that need it. Naturally, the formulation of The vaccine can be formulated for administration by oral dose (v. g., as an enteric coated tablet), by parenteral injection or otherwise. The invention also provides a process for preparing a live modified HVE-1 virus vaccine, whose process comprises mixing a virus according to the invention with a suitable vehicle or adjuvant. For the preparation of a live attenuated vaccine, normal methodology can be used. The mode of administration of the vaccine of the invention can be by any suitable route that supplies an immunoprotective amount. of the virus of the invention to the subject However, the vaccine is preferably administered parenterally via the intramuscular or deep subcutaneous routes Other modes of administration may also be employed, when desired, such as oral administration or via other parenteral routes, i.e., intradermally, intranasally or intravenously. Generally, the vaccine will usually be presented as a pharmaceutical formulation that includes a vehicle or excipient, for example an injectable vehicle such as saline or pyrogen-free water. The formulation can be prepared by conventional means. The appropriate immunoprotective and non-toxic dose of said vaccine can be easily determined by those skilled in the art, ie the amount Suitable immunoprotective and non-toxic virus contained in the vaccine of this invention may be on the scale of the effective amounts of antigen in all conventional virus vaccines It will be understood, however, that the specific dose level for any particular recipient animal will depend of a a variety of factors including age, general health and sex, the time of administration, the route of administration, the synergistic effects with any other drugs being administered, and seeking the degree of protection. Of course, if necessary, administration can be repeated in suitable ranges The embodiments of the invention will now be illustrated by way of the following Figures and Examples Figure 1: Schematic representation of the arrangement of DNA sequences of HVE-1 and plasmids constructed for gene 71 deletion and substitution Line 1, HVE genome -1 consisting of UL and Us and inverted repeated regions (IRS and TRS) The expanded fragment cloned SamHI / EcoRI of 5 8-kb in pU71 (line 2) Line 3 location and gene direction Line 4, sequence arrangement of the plasmid pD71 of suppression and constructed substitution of gene 71 (line 4) The spaces flanked by solid lines represent regions replaced by acZ (solid squares). Relevant restriction sites: Ba, Ec, EcoRI; Ms, Sg and Bam Hl. Figure 2: Genome structure of the deletion and substitution mutant. Restriction enzyme sites are shown within the region of the genome encompassing gene 71. The genome of wild-type virus is represented by line 1, and the deletion and substitution by line 2. The relevant generated fragments are shown after digestion with Smal. Fragment sizes are given in kb. Relevant restriction sites Ms, Sm, Sg. Figure 3 Virus titers for mice inoculated with Ab4p. Figure 4: Virus titers for mice inoculated with ED 71. Figure 5: Virus titers for mice inoculated with reversed ED 71 Figure 6: Mean virus titers observed in challenged mice previously immunized with RK cell lysate. Figure 7: Mean virus titers observed in confronted mice previously immunized with Ab4p. Figure 8: Mean titers of viruses observed in challenged mice previously immunized with ED 71 Normal methods are as described in "Molecular Cloning A Laboratory Manual", Second Edition, Sambrook J et al., Cold Spring Harbor Laboratory Press 1989 SECTION 1 EXAMPLES Methods Cells v Virus Thirteen baby hamster kidney clones (BHK-21 / C13, Macpherson I &Stoker MG (1962) Virology 16 pp. 147-141) were developed as previously described (Brown et al., 1972 J Gen Virol 18 pages 32 -346) Ab4 strain of HVE-1 was used as the wild-type strain in this study The preparation of the virus material in passage 13 was done by low multiplicity HVE-1 infection in maintained NBL-6 dermal cells of horses in MEM with 1% fetal calf serum The ED71 mutant in which MLA of gene 71 was removed and replaced by the lacZ gene of E coll and Re71 of reversion in which the deletion in ED71 was restored had already been described pr previously (Sun Y and Brown S M (1994) Virology 199 pages 448-452, Sun Y and others (1994) J Gen Virol 75 pages 3117-3126) Purification and Quantification of Vinons The procedure essentially used as described by Szilagyi JFY Cunningham C (1991) J Gen Virol 27 pages 661-668 and Sun et al. (1994) supra Monolayers of BHK-21 / C13 in roller bottles were infected with virus at a multiplicity of infection (mdi) of 0.01 or 5 pfu per cell. At 72 hours after infection (d.i) or 20 hours d.i. the supernatant was recovered and centrifuged at 2500 r.p.m. for 20 minutes to remove debris from the cell. The supernatant virus was pelleted for 2 hours at 12,000 r.p.m. and the pellet was gently resuspended in 1 ml of Eagle medium without phenol red and placed on a gradient of 5 to 15% Ficoll before centrifuging at 12,000 r.p.m. for 2 hours at 4 ° C. The band of virions collected by lateral puncture was diluted and formed into pellets at 21,000 r.p.m. for 2 hours at 4 ° C. The virion pellet was gently resuspended in 200 μl Eagle medium and stored at -70 ° C. Infectivity was determined by titration in BHK-21 / C13 cells. The number of particles was determined by electron microscopy. The specific infectivities (ratio of u.f.p. of particles) of the purified mutant and the wild type virus are presented in Table 1.

Table 1 The Specific Infectivity (particle ratio / u f p) of ED71 and HVE-1 of Virus Wild type Virus Relationship of the particle ratio / u f P * particula / u f p * HVE-1 101 7/1 638/1 ED71 1440/1 2128/1 Re71 103 5/1 1077/1 * Vírus of 4x10 BKH-21 / C13 cells infected with virus at a mdi of 001 pfu per cell and culture at 72 hpi * V? r? ons purified from 4x108 BHK-21 / C13 cells infected with a virus in mdi of 5 pfu / cell and recovered at 20 hpi The suitable deletion mutants of gene 71 of the invention were prepared from According to the teaching of Sun Y and Brown SM (supra) Example 1 In summary, to clone the fragment containing gene 71, equine dermal cells (NBL-6) were infected with the Ab4 strain of HVE-1 (Gibson JS et al., Arch Virol 124 pages 351-366 (1992)) at 0 1 pfu / cells and the progeny vipones were purified by centrifugation in sucrose gradients of 5-55% (w / v) as described by Dumas and others. , J Gen Virol 47 pages 233-235 (1980)) The genomic DNA of Ab4 from HVE-1 was extracted from the vipones purified and digested with a scale of restriction enzymes. for example, the SamHI / EcoR fragment! 5.8 kb (residues 126.517 to 132.305), was cloned into the pUC19 vector so that a plasmid, pU71 containing the 5.8 kb SamHI / EcoRI fragment inserted in the SamHI / EcoRI sites, was constructed (Figure 1). To construct a deletion plasmid, the cloned plasmid was digested by restriction enzymes that were cut at unique sites to remove most of the coding sequence of gene 71. The flanking sequences were re-ligated with complementary synthetic oligonucleotides containing a Single Spel site to allow insertion of the lacZ gene and the stop codon in the upstream frame to prevent the synthesis of a lacZ fusion protein. The lacZ gene in the 4.1-kb Xbal fragment of pFJ3 (Rixon F.J. and McLauchlan J., J. Gen Virol. 71 pages. 2931-2939 (1990)) was inserted into the Spel site. lacZ was in the same orientation as the transcription of the gene. The construct may encode only a very short polypeptide of the remaining N-terminal amino acids of the deleted gene. In this manner, the deletion plasmid pD71 was generated with a deletion of the Mscl site at the SgraAl site (residues 129.211-131.022) in pU71, (Figure 1). Example 2 To generate virus mutants, 1-2 μg of Ab4 DNA of HVE-1 was co-transfected into BHK21 / C13 cells (MacPherson I. and Stoker MG Virology 16 pp. 147-151 (1962)) with amounts pD71 linearized deletion plasmid variables (0.2 to 4 μg, a molar excess of approximately 2 to 20 times) in the presence of carrier-type calf DNA using the calcium phosphate precipitation / DMSO method described by Stow ND, and Wilkie NM, J. Gen. Virol. 33 pages. 447-458 (1976). Cells were incubated at 37 ° C in Eagle medium containing 5% newborn calf serum. When c.p. was disseminated, the virus recovered and was titrated in BHK21 / C13 cells under methylcellulose. Two days after infection, an additional 2 ml of methylcellulose containing 0.7 mg / ml of X-gal was added to each plate. The individual blue plates were isolated for additional plaque purification cycles. A mutant with lacZ: ED71 substitution was isolated with a deletion of 1811 bp of MLA gene 71 of 2393 bp. The suppressed region of the mutant was confirmed by Southern analysis with a probe of the deleted sequence labeled with P32. The structure of the virus mutant was confirmed by Southern analysis and digestion of the restriction enzyme of viral DNA labeled with P32 before the preparation of the virus material. The digestion of the restriction enzyme of the viral DNA labeled with P32 was plotted diagrammatically in Figure 2. The gene 71 is within the 3.8-kb Smal fragment of the wild-type viral DNA. The deletion of gene 71 and the substitution of the lacZ gene resulted in the loss of the 3.8-kb fragment and the generation of a new 6.2-kb fragment. The mutant had the expected genome structure, with no other detectable differences in wild-type viral DNA.

Example 3 The growth characteristics of the mutant in tissue culture were also investigated. Monolayers of BHK21 / C13 cells were separately infected at a multiplicity of infection (m.d.i.) of 5 u.f.p. / cells and 0.01 u.f.p. / cells with wild type virus and ED71 mutant deletion and substitution. The culture was recovered and the virus was released by sound treatment at intervals for a period of 72 hours. The virus titers were measured by plaque analysis and the growth patterns were compared with those of the wild type virus. The plate morphology of the mutants was obviously not different from the wild type virus plates. The mutant ED71 grew very slowly and the yield was finally reduced approximately 5-fold compared to that of the wild-type virus. Final results were observed at a high multiplicity (data not shown), although the reduction in the yield of mutant ED71 was lower than that of low multiplicity. To determine if the mutant was sensitive to temperature or if it had a phenotype on the host scale they were developed at a m.d.i. high of 5 u.f.p. / cell in BHK21 / C13 cells at different temperatures (31 °, 37 ° C and 38.5 ° C) and at 37 ° C in NBL-6, Vero, HFL and 3T6 cells. The cultures were recovered 24 hours after infection and the progeny virus was triturated in BHK21 / C13 cells. The mutant of ED71 at 38 ° C was developed 10 times less well than at 31 ° C and purchased with the wild-type virus at 38 5 ° C (data not shown). The slightly damaged growth of the ED71 mutant was evident in the NBL-6, Vero, HFL and 3T6 cells as well as in BHK21 / C13. Therefore, it was concluded that gene 71 is not essential for the growth of HVE-1 in cell culture. Example 4: Infection Experiments: Mortality v Clinical Signs Materials and Methods Virus strains Wild-type and mutant viruses were developed in RK cells at the Department of Clinical Veterinary Medicine, Cambridge and BHK cells at the Institute of Virology, Glasgow. The wild type strain for primary infection experiments was the Ab4p strain of HVE-1. The virus used to confront the previously immunized mice was the Ab4 strain of HVE-1. Mice Model Female Balb / c mice 3-4 weeks old (Bantin and Kingman, UK) were obtained. The mice were inoculated intranasally under isoflurane / oxygen anesthesia. Cultivation of Teiidos The monolayers of RK cells were cultured in Essential Medium Eagle Minimum (EMEM) with Earle Salts with 10% newborn calf serum Virus titration Tissue samples obtained from three mice per group were homogenized using a Ultraturrax motorized homogenizer Samples were then treated with sound in a water bath cooled in ice and centrifuged at low speed to separate cellular debris Ten-fold serial dilutions of the supernatant were made and 100 μl of each dilution was inoculated into a confluent monolayer of RK cells, in duplicate. the virus absorbed the cell layer for 45 minutes before all the samples were covered with medium containing 4% fetal calf serum and 2% carboxymethyl cellulose. The plates were incubated for 37 ° C for about 3 days and then washed in saline solution regulated with sterile phosphate before fixing them and dyeing them with violet crystal in 20% ethanol Protocol Expe On days 1, 3 and 5 after infection, the three groups of mice were killed with 0 15 ml of pentobarbitone sodium solution (Sagatal, Rhéne Mepeux), the tissues were removed, placed in 1 ml of medium of virus isolation, were frozen at -70 ° C and then titrated for virus growth Tissue samples were taken from the lung, turbinates, olfactory bulb and tuberculin ganglia Clinical signs were monitored in a separate group of mice from day 0 to day 8 after infection Blood samples were taken on days 8, 16, 23 and 30 after infection for immunological tests A group of surviving animals was then confronted with a dose of 5x10 6 pfu / mouse of the H4-H4 strain of Ab4. The tissue samples were taken as before and the signs were monitored. Clinical mortality and clinical results are shown in Table 2. The virus titration results are shown in Figures 3 to 8 and Tables 4 (a) -4 (d) inclusive. Mortality and Clinical Signs TABLE 2 the infection.

Example 5: Immunology - ELISA The protocol of Tewari D., et al. (1994) Journal of Gen. Virol, 75 pages. 1735-1741. The results are shown in Table 3.

TABLE 3 Acute Phase ELISA After Confrontation TABLE 4 (a) Day +1 After confrontation TABLE 4 (b) Day +3 After Confrontation TABLE 4 (c) Day +5 After confrontation TABLE 4 (d) Day +8 After Confrontation SECTION 2 OF EXAMPLES 1. Experimental Details An analysis was made in ponies in ponies using 3 animals per group and two groups, one vaccinated and the other not (control group). The animals of the analysis were selected on the basis that they did not have or have low neutralizing HVE-1 and complement fixation antibodies (FC) of HVE-1. The experimental groups were kept in separate rooms in isolation with filtered air in and out. Foals, 7, 15 and 20 were each vaccinated intranasally with 6.0 Iog10 of TCID50 of HVE-1 deleted from gene 71 (ED71), in 2.0 ml of MEM (Gibco) containing neomycin (100μg / ml), 2% serum of fetal calf irradiated with? (SBF) (Tissue Culture Services), giving 1.0 ml in each pot. After vaccination both the vaccinated test ponies (internal numbers 7, 15 and 20) and the control foals (internal numbers 5, 8 and 16) were tested for replication of the virus in the upper respiratory tract by taking nasal samples daily during 2 weeks. The six animals are bled at intervals and their serum was tested for neutralizing FC antibodies of HVE-1 (Table 7). The intranasal challenge infection with the wild type strain AB-4 was carried out 51 days after the vaccine when each of the groups of foals were given 6.0 log 1 or TCID50 of AB-4 in 2.0 ml of MEM medium supplemented with 2% SBF. After the confrontation, the procedures carried out were the same as those after vaccination, ie the evaluation of the growth of the virus in the respiratory tract (Table 5). TABLE 5 Experimental groups and procedures 2. Results 2.1 Replication of the ED71 virus in the respiratory tract Nasal swab virus was isolated in supplemented MEM medium as described above, after normal procedures. The results of the isolation of the nasal swab virus daily after intranasal vaccination are given in Table 6. The ED71 virus at low titration (most below 3.0 log? 0 of TCIDS0 / ml) were isolated from 2 of 3 foals vaccinated on days 2 and 3 of colt 7, and days 1 to 5 of colt 15 HVE-1 was not recovered from control colts of daily nasal swab samples for 15 days 2.2 Serological responses after vaccination virus neutralizing antibody (NV) sera and complement fixers (FC). The results of NV tests carried out according to the method of Thompson G.R, and other Equine Vet. Journal Vol 8 pp. 58-65, for after vaccination and confrontation are given in Table 7 and for the FC test performed according to the Thompson method and others supra (vaccination only) are given in Table 8 NV test against two different strains of HVE-1 namely ED71 virus (AB-4 parent strain) and M8 no significant differences were recorded in the titers In the vaccinated group the three foals had detectable antibodies NV positive in intranasal vaccination All three animals responded with significant antibody response (elevation> 4 times), numbers 7 and 20 in week four and number 15 in week two. There was no elevation of NV antibodies in the control animals until after the confrontation. For the FC test against HVE-1, two (15 and 20) of three foals showed a significant elevation (> 4 times of elevation) in week two after vaccination; Colt No. 7 had high activity in the vaccination (Table 8). The control animals (5, 8 and 16) did not show significant changes in FC antibody titers. Maintaining the results of virus isolation, there was no seroconversion in control animals indicating absence of a field infection or recrudescence of HVE-1. 3. Findings of the Confrontation 3.1 Replication of confrontation virus in the upper respiratory tract. The results of isolation of the nasal swab virus are given in Table 9. The viruses with low titration (2.0 logio of TCIDso / ml) isolated from only one (No. 7) of the three foals on two occasions (day 1 and 2). This was a notorious contrast for the control foals (5, 8 and 16) from which the virus was recovered for 3 (No. 5) to 5 to 6 days (Nos. 8 and 16) in quite superior titers. 3.2 Viremia due to confrontation viruses Isolation of HVE-1 from leukocyte confrontation is given in Table 10 There was no confrontation virus detected in foals vaccinated with ED71. In contrast, the three control foals became viremic, giving a peak between 12 to 200 infected leukocytes / 2x107 cells. TABLE 6 Replication of the vaccine virus on the respiratory tract 4x200μl of the lowest dilution (10"1) of the nasal swab titration.

TABLE 7 Neutralization antibody (NV) responses of the virus NV tests were performed against virus ED71 (figures on the left side) and M8 of HVE-1 (Figures on the right side). The titrations denote the reciprocal of dilution of completely neutralizing serum. From 200 (ED71) to 316 (M8) TCID50 of HVE-1 Vac denotes vaccination at week 0 Confrontation at week +7.

TABLE 8 Complement fixation antibody (FC) responses to HVE-1 vac denotes vaccination in week 0.

TABLE 9 Replication of the confrontation virus in the upper respiratory tract TABLE 10 Leukocyte viremia after confrontation of intranasal HVE-1 Potro group Number of infected leukocytes infected with ED71 virus at 2 × 10 7 cells after confrontation No. 11 13 TEST (ED71) 15 20 CONTROL 12 2.5 200 1.3 16 20 2.5 2.5 The coating cell layer of 10 ml of citrated blood was separated on a wash pad washed in phosphate-buffered saline and titrated in monolayers of equine dermal cells ( Animal Health Trust, Newmarket, Suffolk) using 8 monolayers / dilution and 3 ten-fold dilutions (MEM, 10% SBF irradiated with and supplemented with neomycin). There is no HVE-1 isolated after two passages in series at 37 ° C.

Claims (1)

1. CLAIMS 1 Vaccine formulation comprising a live recombinant HVE-1 virus modified to contain a dysfunctional gene 71 region located within the Us region of the virus genome and a pharmaceutically acceptable carrier 2 A vaccine formulation according to with claim 1, which comprises a mutant virus deleting gene 71 of immunogenic, attenuated, recombinant HVE-1, alive and a pharmaceutically acceptable carrier 3 A vaccine formulation according to claim 1 or claim 2, wherein the region of the dysfunctional gene 71 of the recombinant HVE-1 virus comprises a deletion of at least one nucleotide between nucleotide 129.096 and nucleotide 131.489 of a genome of wild type HVE-1 A vaccine formulation according to any of claims 1 to 3, wherein the recombinant HVE-1 comprises a marker gene 5 A living recombinant HVE-1, comprising a region of the gene 71 di Functional to be used as a vaccination agent 6 A mutant virus for suppressing the 7a gene of immunogenic, attenuated, recombinant, live, to be used as a vaccination agent 7 The use of a mutant virus for suppression of gene 71 of HVE- 1 recombinant live, in the manufacture of an HVE-1 vaccine for the prophylaxis and / or therapy of infection of HVE-1 8. A method for treating an animal that comprises administering to an animal a vaccine composition comprising a recombinant HVE-1 virus, alive, modified so as to contain a dysfunctional gene 71 region located within the Us region of the virus genome . 9. A method according to claim 8, wherein the animal is a horse. A method according to claim 8 or claim 9, wherein the vaccine composition comprises a mutant, immunogenic, attenuated, live, recombinant HVE-1 gene 71 deletion virus.