Viral-vector recombinant vaccine against porcine epidemic diarrhoea

Abstract

Described is a recombinant vaccine against porcine epidemic diarrhoea (PED), which comprises a viral vector having an inserted exogenic nucleotide sequence that encodes antigenic sites of PED virus type 2, and a pharmaceutically acceptable vehicle, excipient and / or adjuvant, wherein the vaccine is designed to generate an immune response in pigs without being combined with other vaccines or variants of PED virus type 2.

Description

[0001] RECOMBINANT VACCINE AGAINST EPIDEMIC DIARRHEA IN A VIRAL VECTOR

[0002] FIELD OF INVENTION

[0003] The present invention relates to techniques used in the prevention and control of swine diseases, and more particularly relates to a recombinant viral vector vaccine having inserted an exogenous nucleotide sequence encoding proteins with antigenic activity against the porcine epidemic diarrhea (PED) virus.

[0004] BACKGROUND OF THE INVENTION

[0005] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease of pigs characterized by vomiting, diarrhea, and anorexia in pigs of any age. The disease manifests as an explosive epidemic of diarrhea in nonimmunized weaned piglets (Type I) or in pigs of any age (Type II). After an incubation period of 1 to 3 days, adult piglets or pigs present with similar watery diarrhea with vomiting. While older pigs have a chance of survival, 80 to 100 percent of piglets younger than 10 days old infected with PED virus die within 24 hours of infection. The economic impact of PED virus infection has been substantial worldwide, primarily in Europe, Asia, and North America.

[0006] PED virus (PEDV) is a member of the Coronaviridae family and belongs to the alphacoronavirus genus. These viruses are positive-sense, single-stranded RNA viruses, enveloped, with a helical symmetry nudeocapsid 130 nm in diameter (Pensaert and de Bouck, 1978; Spaan et al., 1988; Kocherhans et al., 2001). Their genomic size ranges from approximately 26 to 32 Kb, relatively large for an RNA virus. Coronaviruses are the largest viruses known to infect humans, other mammals, and birds, and usually cause subclinical respiratory or gastrointestinal illness. Subgenomic mRNAs of PED virus, which are transcribed from the genome, produce viral protein subunits, such as spike glycoprotein S, envelope E, membrane M, nucleoprotein N, and several other proteins of unknown function (Kocherhans et al., 2001; Li et al., 2012).Spike S glycoprotein promotes cell entry and is the primary target of antibodies.

[0007] State-of-the-art techniques, even before vaccines were available, included controlled live virus exposure, or "feedback," to control PED outbreaks. This technique involves exposing an entire pig population to the PED virus through orally administered infectious material, such that the sows develop immunity and transfer it to their piglets. To date, there are no standardized feedback protocols that achieve high and consistent efficacy, and it is also an unsafe practice that can lead to continued reinfections with the PED virus.

[0008] Alternatively, several PED vaccines have been developed, which differ in their genomic sequence, mode of administration, and efficacy, including inactivated viruses, modified live viruses, and recombinant viruses. Modified live vaccines have been attempted for PED control. However, while these can elicit a better immune response than inactivated virus vaccines, they lack the required efficacy (Song et al., 2007; ). In addition, recombinant PED vaccines have been described, as in patent document WO2015153425A1, which describes useful antigenic sequences for a PED vaccine and suggests their use in vectors such as baculoviruses. However, the examples of patent document WO2015153425A1 show that the potency and immunogenicity of the vaccine described in that document can still be improved, as the seroconversion of pigs is only 11% to 60% and mortality is reduced by only 20%.These results are indicative of the low effectiveness of vaccines in controlling the disease.

[0009] For this reason, the feedback technique has not been abandoned despite the availability of vaccines. There are reports that vaccines are more effective when administered in conjunction with the feedback technique. Sato et al. ["Efficacy of genogroup 1-based porcine epidemic diarrhea live vaccine against genogroup 2 field strain in Japan", Virology Journal, 15(1), 28] evaluated the effectiveness of a live vaccine against PED in farms with vaccinated and unvaccinated pigs, comparing the mortality rate of suckling piglets after the onset of PED. To this end, pregnant sows on farms A and B were administered a combined live transmissible gastroenteritis (TGE) / PED vaccine once or twice according to the manufacturer's instructions, while pregnant sows on farm C were not administered the vaccine.Pregnant sows on farms A and C were also subjected to feedback after the onset of the PED outbreak. The results of these experiments showed that, in piglets born to pregnant sows vaccinated 1 and 2 weeks after the onset of PED, there was a greater reduction in mortality rates on farm A, where sows were treated with feedback and also vaccinated with a single dose, compared to farm B, where sows received only two doses of vaccine, and farm C, where pregnant sows only received feedback.

[0010] On the other hand, Newcastle disease virus (NDV) has been described as a vector that can be used to develop vaccines for mammals, such as in patent documents WO2011059334, US9476033 or US10308913. NDV is a non-segmented negative-strand RNA virus of the Paramyxoviridae family and its natural hosts are birds, so it is antigenically distinct from swine pathogens. Although the virus has been tested as an oncolytic and in various animal diseases, including PRRS as reported in patent document W02012153160A1, in accordance with the results obtained with various vaccines in the state of the art, a recombinant vectored vaccine against PED based on recombinant viruses, such as NDV, is expected to be only partially effective for the treatment or prevention of PED.Furthermore, the most effective way to insert PED genes into a recombinant vaccine to produce an immune response that controls the disease has not been determined, much less in a Newcastle virus vector. Therefore, there is still a need for a PED vaccine that is safe, potent, and highly immunogenic enough to combat this disease on its own, without being used in conjunction with the feedback technique. OBJECTS OF THE INVENTION.

[0011] Taking into account the shortcomings of the prior art, it is an object of the present invention to provide a recombinant viral vector vaccine against porcine epidemic diarrhea (PED) that is immunogenic, even without being combined with other vaccines or versions of PED virus 2.

[0012] It is another object of the present invention to provide the use of a recombinant viral vector vaccine for the control of PED.

[0013] It is a further object of the present invention to provide a viral vector construct with an inserted exogenous nucleotide sequence encoding proteins with antigenic activity against PED virus 2.

[0014] It is also an object of the present invention to provide a viral vector construct with an inserted exogenous nucleotide sequence encoding proteins with antigenic activity against PED virus 2 for use in a vaccine composition for the control of PED, with which it is possible to reduce, after the application of two doses of said vaccine composition in pigs, the number of diarrhea events by at least 50% compared to unvaccinated pigs.

[0015] It is still an object of the present invention to provide a viral vector construct with an inserted exogenous nucleotide sequence encoding proteins with antigenic activity against PED virus 2 for use in a vaccine composition for the control of PED, with which weight gain is achieved in pigs vaccinated with two doses of the vaccine composition, compared to unvaccinated pigs.

[0016] It is still an object of the present invention to provide a viral vector construct with an inserted exogenous nucleotide sequence encoding proteins with antigenic activity against PED virus 2 for use in a vaccine composition for the control of PED, with which after the application of two doses of the vaccine composition to sows before farrowing, the latter transmit to their piglets colostrumed by sows antibodies against the vector and against the PED S protein.

[0017] These and other objects are achieved by a recombinant viral vector PED vaccine according to the present invention.

[0018] BRIEF DESCRIPTION OF THE INVENTION.

[0019] To this end, a recombinant vaccine has been invented comprising a viral vector based on the Newcastle disease virus, which has inserted an exogenous nucleotide sequence from the porcine epidemic diarrhea (PED) virus 2, capable of generating a cellular immune response, and a pharmaceutically acceptable vehicle, adjuvant and / or excipient.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel aspects considered characteristic of the present invention will be set forth with particularity in the appended claims. However, certain embodiments, features, objects, and advantages thereof will be better understood from the detailed description when read in conjunction with the accompanying drawings, in which:

[0022] Figure 1 is a graph showing the temperature measured for each of the groups of pigs in the study.

[0023] Figure 2 shows the histopathology results of the study performed.

[0024] Figure 3 shows the results of the Hemagglutination Inhibition test against the vector virus of group 1 of the study carried out.

[0025] Figure 4 shows the results of the Hemagglutination Inhibition test against the vector virus of group 2 of the study carried out.

[0026] Figure 5 shows the results of the Hemagglutination Inhibition test against the vector virus of group 3 of the study carried out.

[0027] Figure 6 shows the results of the Hemagglutination Inhibition test against the vector virus of all groups in the study.

[0028] Figure 7 shows the results of the PED virus detection test in rectal swabs from group 1 of the study.

[0029] Figure 8 shows the results of the PED virus detection test in rectal swabs from group 2 of the study.

[0030] Figure 9 shows the results of the PED virus detection test in rectal swabs from group 3 of the study.

[0031] Figure 10 shows the results of the PED virus detection test on rectal swabs from study group 4. Figure 11 shows the results of the PED virus detection test on rectal swabs from study group 5.

[0032] Figure 12 shows the antibody titers in serum and colostrum of vaccinated female pigs, determined by ELISA and immunoperoxidase testing.

[0033] Figure 13 shows the antibody titers in serum, in vaccinated sows and in non-colostrum and colostrum piglets from vaccinated sows, determined by immunoperoxidase test.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] During the development of the present invention, it has been unexpectedly found that a recombinant vaccine against porcine epidemic diarrhea (PED) comprising a viral vector having inserted an exogenous nucleotide sequence that codes for antigenic sites of PED virus 2, and a pharmaceutically acceptable vehicle, adjuvant and / or excipient, generates an immune response in pigs without being combined with other vaccines or versions of PED virus 2, providing adequate protection against PED even without being combined with the "feedback" technique.

[0036] The viral vector used may be active (live) or inactivated (dead), with the term "inactivated" meaning that the recombinant virus that functions as a viral vector and contains the nucleotide sequence encoding the antigenic sites of the PED virus has lost the ability to replicate. Inactivation is achieved by physical or chemical procedures well known in the state of the art, preferably by chemical inactivation with formaldehyde or beta-propiolactone (Office International des Epizooties 2008). Newcastle Disease. OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Office International des Epizooties. France, pp. 576-589). Conversely, an active or live virus is understood to retain its ability to replicate.

[0037] Preferably, the viral vector used is a paramyxovirus, which is selected from Newcastle disease virus (NDV), Sendai virus, avian paramyxovirus (APMV), porcine parainfluenza virus (PPIV), porcine rubulavirus (RVP), Menangle virus, Nipah virus, measles virus, mumps virus, parainfluenza virus and respiratory syncytia virus I (RSV). Preferably, the paramyxovirus is selected from NDV and avian paramyxovirus type 3 (APMV-3). More preferably, the viral vector is NDV, said viral vector preferably being selected from lentogenic or mesogenic strains, such as the LaSota, Bl, QV4, Ulster, Roakin, Komarov strains. Preferably, the recombinant virus is from the LaSota strain.

[0038] As regards the exogenous nucleotide sequence encoding antigenic sites of the PED virus, in the case of the present invention the nucleotide sequence used is preferably selected from a sequence encoding the S spike glycoprotein of the PED virus or a sequence encoding a sequence derived therefrom. The S spike glycoprotein of the PED virus comprises two functional subunits responsible for binding to the host cell receptor (SI subunit) and fusion of the viral and cellular membranes (S2 subunit). In a preferred embodiment of the invention, the exogenous nucleotide sequence encoding antigenic sites of the PED virus is selected from a sequence encoding the SI subunit of the S spike glycoprotein of the PED virus; a sequence encoding the S2 subunit of the S spike glycoprotein of the PED virus;a sequence encoding both the SI and S2 subunits of the spike S glycoprotein of the PED virus; a sequence encoding at least a fragment of the SI or S2 subunits of the spike S glycoprotein of the PED virus; a sequence with at least 80% identity to the sequence encoding the SI subunit of the spike S glycoprotein of the PED virus; a sequence with at least 80% identity to the sequence encoding the S2 subunit of the spike S glycoprotein of the PED virus; a sequence with at least 80% identity to the sequence encoding both the SI and S2 subunits of the spike S glycoprotein of the PED virus;or a sequence with at least 80% identity to a sequence encoding at least a fragment of the SI or S2 subunits of the PED virus spike glycoprotein S. More preferably, the exogenous nucleotide sequence encoding PED antigenic sites is selected from a sequence with at least 80% identity to any of the sequences SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;

[0039] The exogenous nucleotide sequence encoding antigenic sites of the PED vaccine virus of the present invention can be prepared by chemical synthesis of the nucleotide sequence of interest for subsequent insertion into the paramyxovirus viral vector. The insertion of the exogenous nucleotide sequence is performed using standard molecular biology cloning techniques and can be inserted into any of the intergenic regions of the paramyxovirus genome. The infectious clone thus produced is transfected into a cell culture for the generation of the recombinant virus.

[0040] The virus replicates in any system suitable for its growth, such as SPF chicken embryo, or commercial cell lines or those specifically designed to grow viruses, until reaching the concentration of the virus required to achieve the antigenic response, preferably between 10 6 0 and 1O 10 0Chicken embryo infecting dose 50% (DIEP50%) / mL, more preferably between 10 6 0 and 10 8 ° DIEP50% / mL for active virus formulations and between IO 8 0 to 10 9 5 DIEP 50% / mL for inactivated virus vaccines. For virus isolation, the virus is removed from the growth medium and separated from cellular or other components, typically by well-known clarification procedures, such as gradient centrifugation and column chromatography, and may be further purified as desired using well-known procedures, e.g., plaque assays.

[0041] In the live vaccine, the vaccine is either a naturally lentogenic active vaccine virus or one attenuated by procedures known in the state of the art. On the other hand, when the vaccine is inactivated, once the viral concentration required to elicit the antigenic response has been reached, the virus is inactivated. Preferably, inactivation is carried out by physical or chemical procedures well known in the state of the art, preferably by chemical inactivation with aldehyde, beta-propiolactone, or binary ethylene amine (BEI).

[0042] Pharmaceutically acceptable vehicles for the vaccines of the present invention are preferably aqueous solutions or emulsions. More particularly, it is preferred that the vehicle used be a water-in-oil, oil-in-water, or water-in-oil-in-water (WOW) emulsion, preferably a water-in-oil-in-water emulsion. The vaccine can be administered intramuscularly, intranasally, orally, subcutaneously, by spraying, or by nebulization, using the means and methods appropriate for each case. Preferably, the vaccine is administered intramuscularly.

[0043] The PED control vaccine of the present invention allows for a reduction, after administering two doses of said vaccine to pigs, of at least 50% in the number of diarrhea episodes compared to unvaccinated pigs. Furthermore, administering two doses of said vaccine to pigs results in weight gain in vaccinated pigs compared to unvaccinated pigs.

[0044] Likewise, the vaccine of the present invention has been constructed to cause pregnant sows that receive two doses of the vaccine of the present invention before farrowing to transmit antibodies to the vector and to the PED S protein to the colostrum-infused pigs once they are born.

[0045] The present invention will be better understood from the following examples, which are presented solely for illustrative purposes to allow a full understanding of the preferred embodiments of the present invention, without implying that there are no other non-illustrated embodiments that can be put into practice based on the detailed description given above.

[0046] EXAMPLE 1 Generation of the NDV LaSota vector

[0047] To clone the RNA genome of the NDV strain LaSota and thus generate a viral vector in the form of plasmid DNA called pLS40 (SEQ ID NO: 4), total viral RNA from NDV strain LaSota was extracted using the triazole method. From the purified RNA, cDNA (complementary DNA) synthesis of the viral genome was carried out, using the previously purified total RNA as a template. In order to clone all the genes of the NDV genome (15,183 base pairs (bp)), 7 fragments with overlapping ends and cohesive restriction sites were amplified by PCR. Fragment 1 (F1) spans from nucleotide (nt) 1-1755, F2 spans from nt 1-3321, F3 spans from nt 1755-6580, F4 spans from nt 6,151-10,210, F5 spans from nt 7,381-11,351, F6 spans from nt 11,351-14,995 and F7 spans from nt 14,701-15,186.The assembly of the 7 fragments was carried out within a cloning vector called pGEM-pSL11801140 using standard ligation techniques, which allowed the reconstruction of the NDV LaSota genome, which after cloning contains a unique SacII restriction site, between the P and M genes, which is used for cloning any gene of interest in this viral region of the vector.

[0048] EXAMPLE 2

[0049] Cloning of various exogenous nucleotide sequences from the PED virus into the SacII site of the pLS40 vector.

[0050] In order to clone exogenous nucleotide sequences derived from the PED virus spike S glycoprotein, the following version of the PED virus spike S glycoprotein gene called S1S2 PED was assembled In SHico using Vector NTi® software: Sequence of the PED virus spike S glycoprotein (with the SI and S2 subunits) without modifications (SEQ ID NO: 1).

[0051] The above sequence was initially cloned independently into a pUC vector. The pUC insert was then subcloned using standard genetic engineering techniques into the unique SacII restriction site located between the P and M genes of the NDV LaSota genome contained in plasmid pLS40. Plasmid pLS40 also contains all the transcription and translation signal sequences required for the gene to be transcribed and translated to generate the spike S glycoprotein of PED virus. The cloning process resulted in a DNA clone. C (complementary DNA) of NDV called pLS40 S1S2 PED.

[0052] The generated plasmid pLS40 S1S2 PED was characterized by PCR to detect the presence of the PED virus spike S glycoprotein. It was also characterized by restriction enzyme digestion, yielding the expected restriction patterns. The stability and sequence of the PCR product of the PED virus spike S glycoprotein were confirmed by sequencing. EXAMPLE 3

[0053] Generation of the recombinant virus origin.

[0054] The plasmid generated in Example 2 was transformed chemically and then independently propagated in E. cotí for 16 hours under continuous shaking at 37 °C. The clone DNA was purified by standard molecular biology procedures. Then, 10 micrograms (pg) of purified DNA were used in transfection experiments using Lipofectamine in Hep2 and A-549 cells. Forty-eight hours after transfection, recombinant viruses generated from the transfection were recovered from the supernatant and used in viral propagation assays in specific pathogen-free (SPF) embryonated chicken eggs. Amnioallantoic fluid (AAF) was used to characterize the generation of recombinant viruses rescued from E. cotí cell culture by hemagglutination.coH and by RT-PCR, using specific primers to amplify the sequence located between the P and M genes, and to demonstrate the presence of the PED virus spike S glycoprotein sequence cloned in the recovered recombinant viruses. Once the identity was established by RT-PCR, the stability of the insert was determined by sequencing. The recombinant source virus rLS40 S1S2 PED was generated from transfection and propagation assays in embryonated SPF chicken eggs.

[0055] EXAMPLE 4

[0056] Propagation of virus origin rLS40 S1S2 PED to generate Master and Production Seeds.

[0057] Starting from the rLS40 S1S2 PED source virus of Example 3, embryonated SPF chicken eggs were inoculated with the infecting dose previously determined for the recombinant viruses prepared in the previous example. The embryos were incubated at 37°C for a period of 72 hours, with mortality being checked daily. After this period, the live embryos were refrigerated overnight, preferably for 24 hours. The FAA was harvested under aseptic conditions and clarified by centrifugation, thus generating the Master seed. The same propagation process in SPF chicken embryos described above was used to obtain the production seed.

[0058] EXAMPLE 5

[0059] Development of an active vaccine against PED.

[0060] From the production seed obtained in Example 4, 9- to 11-day-old SPF chicken embryos were inoculated with the previously determined dose and incubated for a period of 72 hours at 37°C and 65% relative humidity. After this period, the live embryos were refrigerated for at least 12 hours to obtain the amnioallantoic fluid under aseptic conditions. The amnioallantoic fluid was subsequently clarified by centrifugation at 4,500 rpm for 30 minutes. The active vaccines were prepared for intramuscular and oral administration in aqueous solution. For this purpose, the FAA was mixed with a stabilizing solution (TPGA) such that vaccines were obtained with a concentration that allowed providing a minimum of 10 7 5 DIEP50% per 2.0 mL dose, volume required to administer the vaccine.

[0061] The composition of 1.0 liter of the TPGA stabilizing solution is as follows: 10 g of bovine serum albumin, 75.0 g of trehalose dihydrate, 1.30 g of Sodium Phosphate Dibasic, 0.50 g of Potassium Phosphate Monobasic, 0.90 g of Monosodium Glutamate and 1,000 mL of water.

[0062] EXAMPLE 6

[0063] Study to evaluate the level of safety, immunogenicity and potency produced in pigs by the live virus vaccine rLS40 S1S2 PED

[0064] A study was carried out to evaluate the safety, immunogenicity and potency of the rLS40 S1S2 PED vaccine obtained in Example 5 in SPF pigs at a dose of 10 7 5DIEP 50% / mL of live or active virus was administered with different routes of administration (oral, intramuscular, and a combination thereof) after two doses. Safety was determined by measuring the occurrence or absence of adverse reactions after vaccination. Immunogenicity was assessed by comparing the immune response generated after two doses of the vaccine, as well as the clinical response after challenge with a field isolate of PED, and compared with unvaccinated controls. The study design is shown in Table 1.

[0065] Table 1 where:

[0066] O = Oral,

[0067] IM = Intramuscular,

[0068] X = 1 dose,

[0069] XX = 2 doses, NA = Not Applicable

[0070] At 42 days post-primary vaccination, all pigs in all treatments were humanely euthanized. A strict negative control group and a positive control group (unvaccinated / challenged) were included. A total of 25 age- and weight-matched SPF pigs (3–4 weeks old) were used in the study to evaluate rLS40-SlS2 in the different experimental groups. The animals were randomly allocated according to their weight in isolation cubicles.

[0071] Animals were observed for clinical signs throughout the acclimatization period, the study, and the challenge administration, continuing through necropsy. Each study animal was monitored for clinical signs or abnormal respiration, abnormal attitude, and rectal temperature every morning. For animal welfare reasons, the animals were observed more than once per day.

[0072] The degree of immunogenicity induced by vaccination was evaluated by the production of specific antibodies against the vector. Serological samples were taken on days 0, 7, 14, 21, 28, 35 and 42 days post-first vaccination. Similarly, rectal swabs were taken on days 0, 1, 2, 3, 7, 14 and 21 days post-first vaccination and 1, 2, 3, 7, 14 days post-second vaccination to evaluate the possible presence of the vaccine virus. As well as rectal swabs on days 1, 2 and 3 post-challenge. Clinical signs were evaluated based on the following scale: 0 = Absent, 1 = Mild, 2 = Moderate, 3 = Severe.

[0073] Clinical parameters were monitored daily. Genetic load was assessed at sacrifice in intestinal samples and determined by trq RT-PCR against the challenge virus. Fourteen days after the second application (day 35 post-first vaccination) of the rLS40-SlS2 vaccine, groups 1, 2, 3, and 5 were challenged with an oral inoculum of PED virus, with a genetic load of 2.01 X 10 12 genetic copies / mL, in a volume of 2.0 mL.

[0074] One of the clinical monitoring parameters was rectal body temperature measurement. Figure 1 shows a graph created from the average temperatures per group; the horizontal lines correspond to the mean values ​​of the Strict Negative Control Group, adding and subtracting 1 and 2 standard deviations to determine whether any group presented fever or hypothermia. Only after challenge with the Porcine Epidemic Diarrhea (PED) virus was fever detected in the IM Vaccinated group and the Positive Control group, which lasted approximately 24 hours. This fever could have been due to the stress of the challenge and the PED virus infection.

[0075] In the clinical part, the detection of post-challenge diarrhea events in the different groups was analyzed; the results are shown in Table 2.

[0076] Table 2

[0077] A diarrhea event is considered individual per day (for example, if one piglet in the challenged positive control group presented diarrhea on day 1 post-challenge, the value is 1; if two, the value is 2); the evaluation was conducted for 72 hours post-challenge, and the daily evaluation time ranged from 45 minutes to 1 hour. It can be seen that at least 50% fewer diarrhea events occurred in vaccinated pigs post-challenge than in unvaccinated pigs post-challenge.

[0078] As can be seen in Figure 2, the histopathology results showed that there were no microscopic lesions in group 4 (Strict Negative Control), with group 5 (Positive Control, Unvaccinated / Challenged) being the most affected, followed by group 3 (Orally Vaccinated-IM / Challenged), group 2 (Orally Vaccinated / Challenged), with group 1 (IM Vaccinated / Challenged) appearing with the least presentation of microscopic lesions.

[0079] The Hemagglutination Inhibition test against the vector virus was run on sera obtained during the baseline sampling, and on days 7, 14, 21, 28, and 35 of the experiment. The results for group 1 are shown in Figure 3. The results for group 2 are shown in Figure 4. The results for group 3 are shown in Figure 5. The results for all groups are shown in Figure 6. The above results demonstrate the immunogenicity of the experimental rLS40-SlS2 PED vaccine vector.

[0080] Regarding PEDV detection in rectal swabs, Figures 7 to 11 show the results for groups 1, 2, 3, 4, and 5, respectively. As can be seen, in all challenged groups (groups 1-3 and 5), the highest number of PEDV genetic particles was detected around 36 hours postchallenge. The number of virus particles detected in groups 2 and 3 was similar to the number of particles in the unvaccinated group (group 5), while in group 1, which received two intramuscular doses, there was a significant reduction in the number of virus genetic particles compared to the other challenged groups.

[0081] Table 3 shows the average results of RT-PCRtrq PED in organs from the different groups taken at sacrifice for each study group.

[0082] Table 3

[0083] Although PEDV gene particles were not expected to be found in organs other than the intestines, PEDV particles were identified in other organs, as shown in the table, in all challenged groups (Groups 1 to 3 and 5). While the number of PEDV particles detected in vaccinated groups 1 to 3 was similar in most organs to that in unvaccinated group 5, it is significant that in the lung, there was a very significant reduction in all groups, and particularly in groups 1 and 3, which received at least one dose of vaccine intramuscularly. This same effect was observed in the spleen and lymph node.Regarding the small intestine, because it is the target organ of the PED virus, it is normal to find higher genetic loads corresponding to the challenge virus in this organ than those found in the other organs evaluated. These detections in the intestine confirm that the challenge virus met its target and that the vaccinated groups, depending on the route and dose used, maintained greater or lesser efficacy against the challenge, as demonstrated by the productive differences found between the different study groups.

[0084] For a more accurate analysis of productive performance, the immediate pre-challenge and 5-day post-challenge weights were analyzed for each individual to assess whether vaccination had an effective impact on post-challenge productivity. The results are shown in Table 4.

[0085] Table 4

[0086] A statistical analysis (two-sample t-test assuming unequal variances) determined that there were no statistically significant differences between Group 4 and Groups 1, 2, and 3, with Group 1 performing best. Furthermore, there were significant differences between Group 5 and all other groups, with Group 1 performing best within the vaccinated groups.

[0087] Based on the information gathered in the described study, it is determined that vaccination using the rLS40-SlS2 vaccine is harmless, produces a moderate immunogenicity towards the vector used and when applied intramuscularly in two doses, with a schedule of 0 and 21 days, significantly reduces the clinical picture, macroscopic and microscopic lesions produced when challenged with the PED virus; in addition to being the group with the best productive performance (weight gain 5 days post-challenge), without having statistical differences against the strict negative control (group 4), which is surprising since better effects were expected with the vaccine administered orally.

[0088] EXAMPLE 7

[0089] Antibody production in sows against the S alichoprotein of PED virus due to the use of the rLS40 S1S2 PED vaccine

[0090] A dose of 10 was administered 7 5DIEP50% / mL of the live or active rLS40 S1S2 PED vaccine obtained in Example 5 was administered intramuscularly to 5 sows that were virologically and serologically negative against the PED virus, with two applications at 5 and 3 weeks pre-partum, before each farrowing. Serum samples were taken 14 days before farrowing, as well as colostrum samples immediately after each farrowing, at 2, 3, 4, 5 and 6 farrowings. IgG antibody titers were measured by ELISA using a commercial kit (ID Screen® PEDV Indirect), while the immunoperoxidase test also measured IgG antibody titers as well as IgA antibodies produced against the S glycoprotein of the PED virus. The results are shown in Figure 12.

[0091] As can be seen, the S / P values ​​for serum and colostrum antibodies are negative for the PED ELISA test because it does not detect antibodies against the Spike protein of the PED virus, which are produced in response to the active rLS40 S1S2 PED vaccine. The antibodies detected by the ELISA test are against Nudeocapsid or Matrix, antigens that are not present in the rLS40 S1S2 PED vaccine.

[0092] As regards the IgG and IgA antibodies measured by the immunoperoxidase test, in serum and colostrum, similar titres were reached as also observed in Figure 12. With these results, it can be said that the production of IgG and IgA antibodies is consistent throughout the applications of the vaccine of the present invention.

[0093] EXAMPLE 8

[0094] Antibody production in sows and piglets against the S glycoprotein of PED virus due to the use of the rLS40 S1S2 PED vaccine

[0095] A dose of 10 was administered 7 5 DIEP50% / mL of the live or active rLS40 S1S2 PED vaccine obtained in example 5, administered intramuscularly to 20 sows, with two applications at 5 and 3 weeks pre-partum.

[0096] Serum samples were taken from each sow 14 days before farrowing. Serum samples were also taken from 20 piglets colostrum fed by the vaccinated sows on day three postpartum, and from 20 non-colostrum fed piglets immediately after farrowing. From these samples, IgG antibody titers produced against the S glycoprotein of the PED virus, as well as against the NDV vector, were measured (Figure 13).

[0097] As can be seen, sows vaccinated with two pre-partum doses produced antibodies to both the recombinant vaccine vector and the PED S protein, with similar titers. Similarly, antibodies to the vector and the PED S protein were observed in colostrum-fed piglets, although the latter were lower. Finally, as expected, piglets without colostrum did not have antibodies against the NDV vector or the PED S glycoprotein, because these antibodies produced by the vaccinated sow were not absorbed by the piglets as they were not colostrum-fed.

[0098] Based on the foregoing, piglets will exhibit protection against PED provided they are adequately colostrum fed by sows that have received two doses of the vaccine of the present invention prior to farrowing. Although specific embodiments of the invention have been illustrated and described, it should be emphasized that numerous modifications are possible, such as the virus used as a viral vector and the exogenous viral sequence employed. Therefore, the present invention should not be considered as restricted except as required by the prior art and the appended claims.

Claims

CLAIMS 1. A recombinant vaccine against porcine epidemic diarrhea (PED) characterized in that it comprises a viral vector that has inserted an exogenous nucleotide sequence that codes for antigenic sites of the PED virus 2, and a pharmaceutically acceptable vehicle, adjuvant and / or excipient, wherein said vaccine is adapted to generate an immune response in pigs without being combined with other vaccines or versions of the PED virus 2.

2. The recombinant vaccine according to claim 1, further characterized in that the viral vector is live or active, or inactivated.

3. The recombinant vaccine according to claim 1, further characterized in that the viral vector is a paramyxovirus.

4. The recombinant vaccine according to claim 3, further characterized in that the paramyxovirus is selected from Newcastle disease virus (NDV), Sendai virus, avian paramyxovirus (APMV), porcine parainfluenza virus (PPIV), porcine rubulavirus (RVP), Menangle virus, Nipah virus, measles virus, mumps virus, parainfluenza virus and respiratory syncytial virus (RSV).

5. The recombinant vaccine according to claim 4, further characterized in that the paramyxovirus is selected from Newcastle disease virus (NDV) and avian paramyxovirus type 3 (APMV-3).

6. The recombinant vaccine according to claim 5, further characterized in that the paramyxovirus is the Newcastle disease virus (NDV).

7. The recombinant vaccine according to claim 6, further characterized in that the Newcastle disease virus (NDV) is selected from LaSota, Bl, QV4, Ulster, Roakin, and Komarov strains.

8. The recombinant vaccine according to claim 7, further characterized in that the Newcastle disease virus (NDV) is the LaSota strain.

9. The recombinant vaccine according to claim 1, further characterized in that the exogenous nucleotide sequence is selected from a sequence encoding the spike glycoprotein S of the PED virus or a sequence encoding a sequence derived therefrom.

10. The recombinant vaccine according to claim 9, further characterized in that the exogenous nucleotide sequence is selected from a sequence encoding the SI subunit of the spike glycoprotein S of the PED virus; a sequence encoding the S2 subunit of the spike glycoprotein S of the PED virus; a sequence encoding the two subunits SI and S2 of the spike glycoprotein S of the PED virus; a sequence encoding at least a fragment of the SI or S2 subunits of the spike glycoprotein S of the PED virus; a sequence with an identity of at least 80% with the sequence encoding the SI subunit of the spike glycoprotein S of the PED virus; a sequence with an identity of at least 80% with the sequence encoding the S2 subunit of the spike glycoprotein S of the PED virus; a sequence with an identity of at least 80% identity with the sequence encoding both the SI and S2 subunits of the S spike glycoprotein of the PED virus; and a sequence with at least 80% identity with a sequence encoding at least a fragment of the SI or S2 subunits of the S spike glycoprotein of the PED virus.

11. The recombinant vaccine according to claim 10, further characterized in that the exogenous nucleotide sequence is selected from a sequence with an identity of at least 80% with any of the sequences SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

12. The recombinant vaccine according to claim 1, further characterized in that the concentration of the virus required to achieve the antigenic response is between 10 6 ° and 1O 10 0 DIEP50% / mL.

13. The recombinant vaccine according to claim 12, further characterized in that the concentration of the virus is between 106 0 and 10 8 ° DIEP50% / mL when the virus is alive or active.

14. The recombinant vaccine according to claim 12, further characterized in that the concentration of the virus is between 10 8 0 to 10 9 5 DIEP50% / mL when the virus is inactivated.

15. The recombinant vaccine according to claim 1, further characterized in that the pharmaceutically acceptable carrier is aqueous solutions or emulsions.

16. The recombinant vaccine according to claim 15, further characterized in that the pharmaceutically acceptable vehicle is selected from a water-oil, oil-water or water-oil-water emulsion.

17. The recombinant vaccine according to claim 16, further characterized in that the pharmaceutically acceptable vehicle is a water-oil-water emulsion.

18. The recombinant vaccine according to claim 1, further characterized in that it is adapted to be administered by intramuscular route, intranasal route, oral route, subcutaneous route, spray, or nebulization.

19. The recombinant vaccine according to claim 18, further characterized in that it is adapted to be administered intramuscularly.

20. A recombinant paramyxovirus viral vector comprising an exogenous nucleotide sequence encoding antigenic sites of PED virus 2 for use in a vaccine composition for the control of PED that does not require combination with other PED vaccines or viruses to generate an effective immune response in pigs.

21. A recombinant viral vector for use according to claim 20, further characterized in that after the application of two doses of the vaccine composition in pigs, the number of diarrhea events is reduced by at least 50% compared to unvaccinated pigs.

22. A recombinant viral vector for use according to claim 20, further characterized in that after the application of two doses of the vaccine composition in pigs, there is a weight gain in vaccinated pigs that are infected with PED virus 2.

23. A recombinant viral vector for use according to claim 20, further characterized in that after the application of two doses of the vaccine composition to pregnant sows before farrowing, they transmit to their colostrum piglets antibodies against the vector and against the PED S protein.

24. Use of a paramyxovirus viral vector that has an exogenous nucleotide sequence inserted that codes for antigenic sites of PED virus 2, for the production of a recombinant vaccine for the control of PED, wherein said recombinant vaccine is adapted to generate an immune response in pigs without being combined with other vaccines or versions of PED virus 2.

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