Apathogenic live virus vaccine against porcine reproductive and respiratory syndrome

Abstract

Described is a vaccine against porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus type 1 (PRRSV-1), which comprises an apathogenic live strain of PRRS virus type 2 (PRRSV-2) and a pharmaceutically acceptable vehicle, adjuvant and / or excipient. The vaccine prevents the disease caused by PRRSV-1.

Description

[0001] 241108-AVMX-PS-PRRS

[0002] 1

[0003] Non-pathogenic live virus vaccine against porcine reproductive and respiratory syndrome

[0004] FIELD OF INVENTION

[0005] The present invention relates to techniques used in the prevention and control of Porcine Reproductive and Respiratory Syndrome, and more particularly relates to a non-pathogenic live virus vaccine against Porcine Reproductive and Respiratory Syndrome caused by type 1 and 2 virus strains.

[0006] BACKGROUND OF THE INVENTION

[0007] Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease caused by the PRRS virus (PRRSV), a single-stranded, positive-sense RNA virus currently classified as a member of the order Nidovirales, suborder Arnidovirineae, family Arteriviridae, subfamily Variarterivirinae, genus Betaarterivirus. According to the most recent classification by the International Committee on Taxonomy of Viruses, the previous genotypes (1 and 2) are considered to constitute two distinct species, Betaarterivirus suid 1 and Betaarterivirus suid 2, which are classified into two different subgenera, Eurpobartevirus and Ampobartevirus, whose commonly accepted and recognized conventional names are PRRSV-1 and PRRSV-2.PRRSV-1 (formerly known as genotype 1, type 1, or European) was restricted to Europe, and PRRSV-2 (formerly known as genotype 2, type 2, or North American) to North America, although they are now found worldwide. Furthermore, there is an increasing variety of strains in both species, which is attributed to the high error rate inherent in PRRSV replication and recombination between strains (World Organisation for Animal Health, OIE Terrestrial Manual 2021).

[0008] PRRS virus is transmitted through direct contact with infected animals or with material contaminated by their saliva, urine, semen, mammary secretions, transplacental secretions, and feces. Infected pigs exhibit symptoms such as fever, chills, dyspnea, depression, anorexia, and diarrhea. Reproductively, there is an increase in abortion rates in the later stages of gestation, mummified fetuses, stillbirths, weak births, and repeat breeding. Semen quality in boars also decreases, mortality rates in suckling piglets increase, respiratory problems develop in animals due to the disease itself or in association with bacterial infections, and generally, poor weight gain is observed. Therefore, PRRS is considered one of the diseases with the greatest economic impact on pig farmers worldwide (López-Heydeck et al. Swine Reproductive and Respiratory Syndrome (PRRS). Review).Mexican Journal of Animal Science, 2015; 6(l): 69-89).

[0009] In order to combat the spread of PRRS infection and its effects, modified live vaccines (MLVs) and inactivated or killed vaccines prepared with PRRS virus are currently marketed, with the vaccine presenting greater 241108-AVMX-PS-PRRS

[0010] 2. Effectiveness is reduced when the vaccine virus is more closely related to the field virus. This means that to mitigate the effects of disease caused by PRRSV-1, it is necessary to use a homologous vaccine, that is, one with the antigens corresponding to PRRSV-1, and the same applies to PRRSV-2. MLV vaccines are designed to be administered to young adult sows, 3 to 6 weeks before they enter breeding, and to 3-week-old piglets to help reduce the clinical signs caused by PRRS. These vaccines are not intended for use in herds that have not had prior exposure to the virus, nor in adult boars, as the vaccine virus can be shed and transmitted by contact to unvaccinated pigs or vertically to their offspring.Similarly, recombination between vaccine strains and natural strains has been reported under field conditions, so vaccines have been developed using genetic engineering techniques, which are not yet commercially available (World Organisation for Animal Health, OIE Terrestrial Manual 2021), which increases the complexity of using a homologous strain for vaccination.

[0011] For example, WO2017 / 142798 describes a modified live strain of PRRSV that can be used in immunogenic compositions capable of providing protection against heterologous PRRSV strains. Examples in this document show that vaccines were formulated with PRRSV-2 strains, as well as a vaccine with the PRRSV-1 strain, which were administered intramuscularly to 3-week-old piglets. The piglets were challenged with two PRRSV-2 strains. When the vaccine contained the PRRSV-1 strain, the results were unsatisfactory, as there was no reduction in lung lesions or viral shedding compared to the control; that is, the vaccine did not function adequately against a heterologous challenge strain.

[0012] Document WO2021 / 037387 also describes a live, modified strain of PRRSV-1 and its use in a vaccine, which is claimed to be capable of protecting against heterologous PRRSV strains. However, this document does not present experimental data demonstrating that this type of protection is actually achieved, as it only describes how the PRRSV-1 strains to be used in a vaccine formulation were isolated and characterized, without providing data to prove that the ineffectiveness described in document WO2017 / 142798 in protecting against the aforementioned heterologous virus is overcome with these isolates, but in this case to prevent PRRSV-2 disease.

[0013] In fact, in general terms, it has been reported that prevention of PRRSV disease can only be achieved through antigens homologous to the PRRS virus type causing the infection, which in high recombination environments makes establishing effective vaccination schemes to prevent the disease complex and expensive.

[0014] Accordingly, although vaccines exist in the state of the art that have served to mitigate the effects of the disease, a vaccine effective against multiple viruses, that is, one that provides cross-protection against both PRRSV-1 and PRRSV-2 using the same antigen, has not yet been developed. 241108-AVMX-PS-PRRS

[0015] 3

[0016] OBJECTS OF THE INVENTION

[0017] Taking into account the defects of the prior art, it is an object of the present invention to provide a live apathogenic virus vaccine against PRRS that, from a single antigen, can prevent the disease caused by both PRRS-1 and PRRS-2.

[0018] These and other objectives are achieved by means of a non-pathogenic live virus vaccine against Porcine Reproductive and Respiratory Syndrome in accordance with the principles of the present invention.

[0019] BRIEF DESCRIPTION OF THE INVENTION.

[0020] Efforts have been made to resolve the problems encountered in vaccination against various types of PRRSV by obtaining a vaccine that contains a strain of PRRSV-2 that prevents disease against PRRSV-1, making it possible for this vaccine to be used to prevent disease against both types of PRRSV.

[0021] For this purpose, a vaccine against Porcine Reproductive and Respiratory Syndrome (PRRS) caused by a PRRS virus type 1 (PRRS-1) has been invented, which comprises a live, non-pathogenic strain of a PRRS virus type 2 (PRRS-2), and a pharmaceutically acceptable vehicle, adjuvant and / or excipient.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The novel aspects considered characteristic of the present invention will be set forth in detail in the appended claims. However, some embodiments, features, and some objects and advantages thereof will be better understood in the detailed description when read in conjunction with the accompanying drawings, in which:

[0024] Figure 1 shows the distribution of individual rectal temperature readings for the different test groups, where the dotted line indicates a temperature of 40.5°C and the arrow indicates the maximum temperature reached. Figure 1A: group 4, Figure 1B: group 3, Figure 1C: group 2, and Figure 1D: group 1.

[0025] Figure 2 shows the distribution of individual respiratory symptom records according to the scale described in example 3, for each test group. Figure 1A: group 4, Figure 1B: group 3, Figure 1C: group 2, and Figure 1D: group 1.

[0026] Figure 3 shows the evolution of the values ​​obtained from the formula:

[0027] Figure 4 shows the average clinical scores for each day and experimental group.

[0028] Figure 5 shows the average weight gains for the entire challenge phase between day 0 and 28 post-challenge in each group.

[0029] Figure 6 shows the scheme used for the assessment of macroscopic lung lesions. 241108-AVMX-PS-PRRS

[0030] 4

[0031] Figure 7 shows the macroscopic and microscopic evaluation of lung lesions on day 10 post-challenge. Specifically, Figure 7A shows the percentage of lung affected, and Figure 7B shows the severity of the lesions based on histopathological assessment criteria (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0032] Fig. 8 shows the comparison of results of the evaluation of macroscopic (Fig. 8A) and microscopic (Fig. 8B) lesions on days 10 and 28 post-challenge.

[0033] Figure 9 shows the distribution of Ct values ​​for viremia determination in the three challenged experimental groups. Figure 9A: group 3, Figure 9B: group 2, and Figure 9C: group 1. The animals in the unchallenged group tested negative because they had not been inoculated with the Rl-Rosalía challenge virus.

[0034] Figure 10 shows the distribution of Ct values ​​for nasal excretion in the three challenged experimental groups. Figure 10A: group 3, Figure 10B: group 2, and Figure 10C: group 1. The animals in the unchallenged group tested negative because they had not been inoculated with the Rl-Rosalía challenge virus.

[0035] Figure 11 shows the evolution of the antibody response to the PRRS virus.

[0036] Figure 12 shows the ELISPOT test results for IFN-γ after challenge (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001), at days post-challenge (DPD). Fig. 12A: 0 DPD, Fig. 12B: 7 DPD, Fig. 12C: 14 DPD, Fig. 12D: 21 DPD, and Fig. 12E: 28 DPD.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] It has been unexpectedly found that a vaccine comprising a live, non-pathogenic strain of PRRSV-2, and a pharmaceutically acceptable vehicle, adjuvant and / or excipient, is capable of providing protection when used to prevent or control Porcine Reproductive and Respiratory Syndrome caused by at least strains of PRRSV-1, in addition to PRRSV-2.

[0039] In a preferred embodiment, the live, non-pathogenic strain of VPRRS-2 has the sequence SEQ ID NO: 1, known as VPRRS-2 G16X, described in International Application No. PCT / US2013 / 072922, the contents of which are incorporated by reference into this application.

[0040] With regard to pharmaceutically acceptable vehicles for the vaccine of the present invention, these are preferably selected from aqueous solutions or emulsions. More particularly, the vehicle used is selected from a trehalose, phosphate, glutamate, albumin (TPGA) solution, a phosphate-buffered saline (PBS) solution, and combinations thereof.

[0041] To achieve an antigenic response, the vaccine needs to have a virus concentration of at least 10 3 0 cell culture infective dose 50% (CICD50%), preferably between 10 3 0 and 10 4 0 DICC50% / mL, more preferably between 10 3 2 and 10 3 5 DICCso% / mL. 241108-AVMX-PS-PRRS

[0042] 5

[0043] As for the application of the vaccine, it is administered intranasally or intramuscularly, preferably intranasally.

[0044] The vaccine can be administered to pigs of any age, in one or more doses. Preferably, the vaccine is given as a single dose to piglets, particularly between 3 and 21 days of age, although it can also be administered to breeding stock, preferably every 3 to 4 months. In older animals, the intramuscular route is preferred because it is easier to administer.

[0045] Regarding vaccine preparation, techniques already known in the state of the art are used. In general, VPRRS-2 is propagated in a suitable system for its growth, as described in the documents by Calzada-Nova, G. et al. Effect of the host cell line on the vaccine efficacy of an attenuated porcine reproductive and respiratory syndrome virus. Veterinary Immunology and Immunopathology 148 (2012) 116-125; Chen W-Y. et al. 2018. Genotype 2 strains of porcine reproductive and respiratory syndrome virus dysregulate alveolar macrophage cytokine production via the unfolded protein response. Virol 92:e01251-17; Yim-im W, et al. 2021. Comparison of ZMAC and MARC-145 cell lines for improving porcine reproductive and respiratory syndrome virus isolation from clinical samples. 1 Clin Microbiol 59:e01757-20; and Calzada-Nova, G. et al. 2011.North American Porcine Reproductive and Respiratory Syndrome Viruses Inhibit Type I Interferon Production by Plasmacytoid Dendritic Cells. J. Virol. Vol. 85, No. 6, 2703-2713, the contents of which are incorporated by reference into this application. Once the virus concentration required to achieve an antigenic response is reached, the virus is harvested and the vaccine is formulated.

[0046] The present invention will be better understood from the following examples, which are presented for illustrative purposes only to allow a full understanding of the preferred embodiments of the present invention, without implying that there are no other unillustrated embodiments that can be put into practice based on the detailed description above.

[0047] EXAMPLES

[0048] Example 1. Generation of the master seed and the production seed.

[0049] The master seed was produced by propagation in a porcine alveolar macrophage (ZMAC) cell line of a VPRRS-2, strain G16X, at 37°C. After one passage in said cell line, the master seed with a minimum titer of 10 is obtained. 5 5 DICCso% / mL.

[0050] The master seed was propagated in the ZMAC cell line and, after one passage, the production seed was obtained with a minimum titer of 10 5 5 DICC5o% / mL.

[0051] Example 2. Development of a live, non-pathogenic PRRS-2 vaccine.

[0052] From the production seed of Example 1, a vaccine was developed in accordance with the principles of the present invention. 241108-AVMX-PS-PRRS

[0053] 6

[0054] For this purpose, a ZMAC cell line was inoculated with the production seed and incubated at 37° C.

[0055] Once the virus was harvested and purified, the vaccine was formulated with PBS solution and TPGA solution, in such a way that it provided a minimum of 10 3 0 DICC50% / mL, reaching a titer of 10% in this experiment 34 DICC5o% / mL, which is within the parameters that a technician in the field expects when dealing with a biological product.

[0056] Example 3. Evaluation of the cross-effectiveness of the vaccine with VPRRS-2 antigen against the disease caused by highly virulent VPRRS-1.

[0057] In order to determine the effectiveness of the vaccine of the present invention against VPRRS-1 and to demonstrate that it can be equal to or more effective than a commercial live attenuated VPRRS-1 vaccine (Porcilis® PRRS, MSD Animal Health), the effectiveness of the two was compared.

[0058] For this purpose, 68 weaned piglets (Landrace x Duroc), 5 weeks old, from a farm historically free of VPRRS, were used, which were randomly assigned to 4 treatment groups (G1 to G4), as shown in Table 1.

[0059] Table 1. Experimental design

[0060] The VPRRS-2 vaccine of the present invention was administered in a 2 mL dose, via the intranasal (IN) route.

[0061] On the other hand, the commercial vaccine used was kept between 2 and 6°C until the time of use and was reconstituted immediately before vaccination, and was administered in a 2 mL dose by intramuscular (IM) route.

[0062] The animals were challenged at 10 weeks of age (day post-challenge (DPD) 0) with an Rl-Rosalía strain of a VPRRS-1, of sequence SEQ ID NO: 2, with a final concentration of 10 5 5 TCIDso / mL.

[0063] At that same DPD 0, but prior to the challenge, three animals per group were sacrificed to assess the possible presence of lung lesions prior to inoculation with the challenge strain. Additionally, after the challenge, necropsies were performed on half of the animals in each group at 10 DPD, as per 241108-AVMX-PS-PRRS

[0064] 7. Peak phase of the infection. On DPD 28, all remaining pigs were humanely slaughtered and subjected to post-mortem examinations.

[0065] To demonstrate vaccine effectiveness, the presence of fever and clinical signs, weight gain, presence of lung lesions, viremia and nasal viral shedding, humoral immune response, cellular immune response, and the presence of cytokines in the lungs and bronchoalveolar lavage fluid were evaluated. For the various tests performed, blood samples were taken weekly from the day of vaccination until the end of the experiment (day 28). Pigs were weighed weekly during the challenge period. After the challenge and until the end of this period, nasal swab samples were taken on days 0, 4, 7, 10, 14, 21, and 28. On the days necropsies were performed, bronchoalveolar lavage fluid was also collected, and lung samples were frozen at -80°C. Additionally, samples were taken from the apical, middle, and left diaphragmatic lobes of each animal and fixed in formalin.

[0066] Statistical analyses were performed using GraphPad Prism v10.2.3 software. For quantitative variables, comparisons between groups were carried out using the Mann-Whitney U test (for two groups) or the Kruskal-Wallis test (for more than two groups). For qualitative variables, the chi-square test was used, employing exact p-values ​​when necessary.

[0067] On the other hand, during the experiment, one animal died suddenly from bacterial septicemia originating in a heart valve, according to the necropsy performed. Other minor incidents included: one animal in Group 1 received amoxicillin and clavulanic acid to prevent respiratory problems after exhibiting mild respiratory symptoms during the vaccination phase; one animal in Group 2 received amoxicillin and clavulanic acid during the challenge phase due to otitis and was euthanized on day 10 (DPD 10); and one animal in Group 3 received a dose of activated charcoal to control watery feces during the challenge phase.

[0068] Fever and clinical signs

[0069] Body temperature and the development of clinical signs in pigs were monitored from the second day before the challenge until DPD 14, by taking rectal temperature, and assigning a score to the temperatures according to Table 2:

[0070] Table 2. Temperature scoring scale. 241108-AVMX-PS-PRRS

[0071] 8

[0072] Regarding respiratory signs, these were recorded in accordance with Table 3.

[0073] Table 3. Respiratory sign scoring scale.

[0074] For respiratory signs, a proportional aggregate value was calculated as the sum of scores for a group and day group divided by the maximum possible sum of scores in the group, multiplied by 100:

[0075] Finally, the changes in behavior were noted in accordance with what is established in Table 4.

[0076] Table 4. Behavior change scoring scale.

[0077] For each animal, a final score was calculated from the sum of the three parameters mentioned above.

[0078] On the day after the challenge, no respiratory signs were detected, while the recorded rectal temperatures were within normal limits. However, as can be seen in Fig. 1, the rectal temperature of the animals in G3 began to rise from DPD 3 onwards, with temperatures exceeding 40.5°C recorded consecutively until DPD 10. In G2, only rectal temperatures 241108-AVMX-PS-PRRS were observed.

[0079] In the G1 group, temperatures above 40.5°C were recorded between DPD 3 and 7, 3 days less than in the control group (G3). In the G1 group, temperatures above 40.5°C were only recorded between DPD 6 and 8. This is 5 days less than in G3 and 2 days less than in the animals in G2.

[0080] Regarding clinical signs (Fig. 2), respiratory symptoms were evident in group G3 from day 8 (DDD 8) through the rest of the challenge phase. Affected animals exhibited respiratory signs including coughing and labored breathing, with or without a clear alteration of normal respiratory mechanics. In contrast, the vaccinated groups G1 and G2 showed less severe signs, mainly nasal discharge, although more severe signs were occasionally observed in group G1 from day 10 post-challenge onwards.

[0081] The calculation of the proportional aggregate values ​​for the respiratory symptom observations (Fig. 3) showed a clear reduction in symptomatology for G2 and a smaller reduction for G1, since in the latter there was an increase in respiratory symptomatology from DPD 13 onwards that could correspond to bacterial complications.

[0082] When the proportion of animals with a respiratory score greater than 1 at any time was considered, the results were 0% (0 / 14) for G4, 76.9% (9 / 13) for G3, 28.6% (4 / 14) for G2, and 35.7% for G1, with no significant differences observed between G1 and G2. The p-values ​​for the comparison between G3 and G1 were p=0.120 and p=0.056 for the comparison between G2 and G3.

[0083] Regarding the observed behavioral changes, only lethargy was noted and only one animal exceeded a score of 1 on one occasion (data not shown).

[0084] Based on the average values ​​of the overall clinical scores (Fig. 4), a reduction in severity was observed in the vaccinated animals (G1 and G2). Daily examination of the clinical scores (data not shown) revealed differences favoring G1 compared to G3 (p<0.05) on days 4-6, 9-10, and 12, while on days 11, 13, and 14 the differences were not significant, which could be due to the presence of an animal with moderate or severe respiratory signs. In the case of G2, the differences compared to G3 were significant on day 4 and between days 7 and 13. The scores of G1 and G2 were not different on most days.

[0085] Weight gain

[0086] The animals were weighed upon arrival at the facility, on the day of the challenge, and subsequently weekly until the end of the experiment. Weight gains were calculated individually by subtracting the initial and final values ​​for each period.

[0087] Analysis of weight gain during the challenge period revealed significant differences between the groups. As shown in Fig. 5, G4 gained an average of 27.7 kg (989.3 g / day), while G1 gained only 15.79 kg (563.92 g / day) (p<0.05). The vaccinated groups gained 21.79 kg (778.21 g / day) and 20.29 kg (724.46 g / day) for G2 and G1, respectively. Therefore, weight gain in the vaccinated animals was similar for both groups.

[0088] Determination of macroscopic and microscopic lung lesions 241108-AVMX-PS-PRRS

[0089] 10

[0090] In groups DPD 0 (n=3 per group), 10 (n=7 per group), and 28 (n=7 per group), the designated animals were euthanized by anesthetic overdose. Macroscopic lesions were assessed visually, and dorsal and ventral photographs of each lung (not shown) were taken and used to compare the scores.

[0091] Lung injuries were assessed both macroscopically (percentage of lung affected) and microscopically (severity of injuries).

[0092] The scoring scheme used to assess macroscopic lesions is shown in Fig. 6. The total lung volume was considered to be 100, and each lobe (dorsal and ventral) was assigned a value based on its proportional contribution to the total lung volume. Thus, in the dorsal view, the apical and middle lobes were assigned a value of 5, and the diaphragmatic lobes a value of 15 (5+5+5+5+15+15 = 50 points for the dorsal view). In the ventral view, the apical and middle lobes were again assigned a value of 5, the diaphragmatic lobes a value of 12.5, and the accessory lobe a value of 5 (5+5+5+5+12.5+12.5+5 = 50 points for the ventral view). A score was assigned to each lobe based on the area of ​​pneumonia. Thus, for example, if in the dorsal view there was a score of 2.5 for the apical lobe, this indicated that pneumonic lesion was observed in 50% of that lobe.

[0093] Additionally, the observed lesions were recorded (lack of collapse, edema, multifocal pattern, fibrin, hemorrhages, etc.).

[0094] For the evaluation of microscopic lesions, samples were taken from the apical, middle, and left diaphragmatic lobes of each lung and fixed in 10% neutral formalin. Once paraffin embedding and the corresponding sections were made, the samples were stained with hematoxylin and eosin for microscopic evaluation.

[0095] In the histopathological evaluation, the degree of interstitial pneumonia (0-4), bronchopneumonia (0-4), and the presence or absence of necrotizing proliferative pneumonia, observed in previous tests with the challenge strain used in this experiment, were assessed. The presence of this lesion was considered in the assessment by adding one point to the degree of interstitial pneumonia. For this assessment, the mean of the scores for each sample was reported.

[0096] As mentioned above, the initial assessments were performed at the time of the challenge to determine any potential vaccination-induced lesions. The data obtained indicated some level of macroscopic injury in the vaccinated groups G1 and G2, with signs of edema and a lack of partial collapse, a pattern suggestive of possible interstitial pneumonia, although of low severity and difficult to assess macroscopically. Subpleural hemorrhages or petechiae were observed in some animals, likely due to the agony, as no significant lesions were seen on microscopic examination.

[0097] Lung lesions were re-examined at DPD 10, revealing the development of severe lesions in the challenged animals. Macroscopic and microscopic assessment at 10 DPD showed a wide range of lesion development. 241108-AVMX-PS-PRRS

[0098] 11 in the different groups, although significant differences favoring G1 compared to the control G3 were observed in relation to the percentage of lung affected (Fig. 7). The percentage of lung affected in the animals of G3 ranged from 17.5% to 70.5%, for G2 this variation was from 0.5% to 48%, and for G1 it ranged from 5% to 21%. The two vaccinated groups were similar to each other. Setting 20% ​​as an arbitrary cutoff point to indicate the severity of pneumonia, in G1 and G2, the percentage of animals above this value was 14.3% (1 / 7; 95% CI: 0.8-58.0%), while this proportion was 8.3% in the animals of G3.

[0099] On the other hand, the histopathological examination showed lesions compatible with PRRS, but of an uncommon severity and variety, reinforcing that it is a highly virulent challenge strain.

[0100] Figure 8 shows a comparison of the results at 10 and 28 days post-dwell (DPD). As can be seen, at 28 DPD macroscopic lesions were still visible in the lungs of the challenged animals, with lung involvement exceeding 20% ​​in all three challenged groups. In contrast, the intensity of the microscopic lesions appeared to decrease, and no animal scored higher than 4. Furthermore, there were no significant differences between the challenged animal groups (G1-G3).

[0101] Determination of viremia and nasal excretion of the virus

[0102] Blood samples were obtained by puncture of the caudal or jugular vein using silicone blood collection tubes (BD Vacutainer). These samples were allowed to clot at room temperature, after which they were stored at 4°C for at least 30 minutes to promote clot retraction. Subsequently, they were centrifuged at 300 x g for 10 minutes at 4°C. The resulting serum was aliquoted and stored at -80°C until use. A commercial kit (LSI VETMAX PRRS EUNA 2.0, Thermo Fisher) was used to determine the presence of viruses in the serum samples.

[0103] Nasal samples were obtained by deep insertion of swabs specifically designed for virus detection (Virocult®) into both nostrils of pigs. Once obtained, the samples were manually resuspended in transport medium. The tubes containing the swabs and medium were vigorously vortexed to ensure effective resuspension of the collected material. The sample was then centrifuged at 500 x g for 5 minutes at 4°C, and the supernatant was stored in aliquots at -80°C.

[0104] RNA extraction from the samples was performed using the MagMax Core Nucleic Acid Purification kit (Thermofisher). The obtained nucleic acids were immediately used for VPRRS detection using the LSI VETMAX PRRS EUNA 2.0 kit (Thermofisher) according to the manufacturer's instructions. This kit detects both VPRRS-1 and VPRRS-2.

[0105] To determine the development of viremia and the pattern of nasal virus shedding, blood samples were taken from animals in the different groups from the day of challenge until the end of the experiment. The results showed that all challenged animals except one had developed viremia by DPD 4, which persisted for several weeks, with viremic animals found in all groups until the end of the experiment (Fig. 9). 241108-AVMX-PS-PRRS

[0106] 12

[0107] Viremia associated with vaccination was detected in G1, in which VPRRS-2 was detected, and G2, in which VPRRS-1 was detected.

[0108] The possible existence of differences between groups with respect to the proportion of positive animals was also evaluated, according to Table 5.

[0109] Table 5. Proportion of animals positive in serum RT-qPCR for PRRS virus.

[0110] As can be seen, in the vaccinated groups G1 and G2 there was no clear decrease in the proportion of positive animals until DPD 28, although these differences did not reach statistical significance.

[0111] Regarding nasal viral shedding, it was observed in at least one animal in all inoculated groups throughout the observation period (Fig. 10). In this case, significant differences were observed in the mean viral loads estimated from the Ct values ​​on DPD 7, with lower loads in both vaccinated groups compared to the controls. On DPD 0, viral shedding was observed in 3 / 17 animals in G1 and in 5 / 17 animals in G2.

[0112] The proportion of excreting animals at different time points after the challenge is shown in Table 6, and the differences between groups were not significant. It is important to note that at the end of the experiment, only one animal per group tested positive via nasal swab.

[0113] Table 6. Proportion of animals positive in the nasal swab RT-qPCR for PRRS virus. 241108-AVMX-PS-PRRS

[0114] 13

[0115] Humoral immune response

[0116] The development of antibodies against PRRSV was determined using the IDEXX PRRS X3 Ab kit, which detects antibodies produced against the PRRSV nucleoprotein. This kit recognizes antibodies produced against both PRRSV-1 and PRRSV-2.

[0117] Antibody development following vaccination or challenge was examined using the PRRS X3 Ab Idexx ELISA. In both G1 and G2, seroconversion occurred primarily on day post-vaccination (DPV) 14 (Fig. 11), with similar mean S / P values ​​achieved in both groups (2.05±0.49 vs. 1.89±0.86, respectively, not significant). Subsequently, on the day of challenge, higher antibody levels were observed in G2 compared to G1 (2.84±0.48 vs. 2.37±0.4 for G2 and G1, respectively, pc0.00l).

[0118] Following the challenge, antibody levels in group G1 did not increase, remaining stable until the end of the experiment (mean sensitivity / percentage difference 2.07 ± 0.43 at 28 days post-diastolic (DPD)). In contrast, in group G2, antibody levels continued to increase after the challenge, reaching a maximum of 3.03 ± 0.53 at 7 DPD, a value significantly higher (pC0.00) than that of the animals in group G1 (2.2 ± 0.56). However, this increase in antibody levels in G2 did not reach statistical significance compared to the values ​​achieved by the same group on the day of the challenge. For the animals in group G3, the seroconversion pattern was similar, with the majority of seroconversion occurring on DPD 14. The maximum antibody levels in this group were reached on the last day of the trial (mean sensitivity / percentage difference: 2.55 ± 0.15).

[0119] Cellular immune response

[0120] The development of virus-specific interferon-gamma (IPF-γ)-producing cells was determined using the ELISPOT technique. Blood samples were collected in heparinized tubes (BD Vacutainer) to prevent clotting. The collected blood samples were mixed in equal parts with Hanks saline solution and then deposited onto a sucrose gradient column (Histopaque 1077, Merck). The columns were centrifuged at 550 x g for 45 minutes, after which the peripheral blood mononuclear cell (PBMC) layer was separated. The PBMCs were resuspended in RPMI 1640 medium supplemented with sodium pyruvate, non-essential amino acids, antibiotics, and 10% fetal bovine serum, and stored at 4°C until use (less than 24 hours).In parallel, PVDF background plates (MultiScreen® HTS IP, Merck) were coated with the IFN-γ P2G10 capture antibody (BD Pharmingen) at a concentration of 10 pg / ml in PBS. Prior to coating, the plates were pretreated with 35% ethanol for 30 seconds. After incubating the plates overnight at 4°C, PBMCs were added at a rate of 2.5 x 10⁻⁵. 5 cells / well and were stimulated with the vaccine strain used in G1, in G2 or the challenge strain, to an infection multiplicity of 0.1, depending on the groups and whether it was 241108-AVMX-PS-PRRS

[0121] 14. Immunization or challenge phase. Viral stimulation was performed in triplicate wells. In parallel, unstimulated wells (culture medium) and wells in which cells (50,000 cells / well) were stimulated with Concanavalin A (10 pg / ml) were added. Viability and cell concentration were determined by counting in a Neubauer chamber and staining with trypan blue. After 20 hours of incubation, the cells were removed, the plates were washed with PBS, and the biotinylated detection antibody P2C11 (BD Pharmingen) was added at a concentration of 0.75 pg / ml. After 2 hours of incubation, the plates were washed again, and streptavidin-peroxidase (Sigma, 0.67 pg / ml) was added, followed by incubation for 1 hour at room temperature. Next, non-plaque-bound streptavidin was removed by washing with PBS, and the AEC substrate (BD Pharmingen) was added. Spot development was determined by stereoscopic examination.Virus-specific CPIFN-γ counts were obtained by subtracting the mean number of spots per well in the control wells from the mean number of spots in the unstimulated wells. Wells in which concanavalin A had been added were used as positive controls indicating cell viability and functionality. Results were expressed as the frequency of virus-specific CPIFN-γ per million PBMCs.

[0122] Vaccination induced detectable levels of IFN-γ producing cells from the third week post-vaccination, although at low levels. On the day of challenge, groups G1 and G2 showed significant but low responses to the homologous antigen (vaccination strain), while the unvaccinated groups were non-reactive to the virus. Regarding the response to the challenge virus (Fig. 12), the true indicator of protection, it should be noted that, on the day of challenge, the responses of different groups were low and below what can be considered specific. However, at 7 days post-dose (DPD), a clear increase in IFN-γ producing cells was observed in the vaccinated groups G1 and G2, suggesting the generation of reactive memory cells as a result of vaccination. At that time, G1 showed superiority over G2 (p<0.05), although these differences between vaccinated groups disappeared in the following days.The specific response to the challenge virus in the G3 group began to be detected on DPV 14.

[0123] Based on the results obtained, it can be concluded that both vaccines evaluated provided protection, producing a significant improvement in clinical and productive parameters and alleviating lung lesions, although with poorer performance in controlling viremia and lung lesions. However, it is worth noting that the vaccine of the present invention was the most effective at controlling fever, with a significant reduction in respiratory signs.Furthermore, as shown by the ELISPOT results, the vaccine of the present invention was able to induce priming against the Rl-Rosalía challenge strain, even though the vaccine is formulated with a PRRSV-2 strain and the challenge model was extremely virulent. Therefore, it can be said that a remarkable effect is achieved, particularly with regard to the development of lung lesions, and the clinical and productive improvement when using the vaccine of the present invention for the control of PRRS caused by a type 1 virus (PRRSV-1). 241108-AVMX-PS-PRRS.

[0124] 15

[0125] In accordance with the foregoing, it will be observed that the live, non-pathogenic PRRS virus vaccine has been designed for use in the prevention of disease caused by PRRSV-1 and PRRSV-2, and it will be evident to any person skilled in the art that the vaccine embodiments of the present invention, as described above and illustrated in the accompanying drawings, are merely illustrative and not limiting to the present invention, since numerous substantial changes in their details are possible without departing from the scope of the invention. For example, it is possible to use different strains for the preparation of the vaccine, or different vehicles.

[0126] Therefore, the present invention shall not be considered restricted except as required by prior art and within the scope of the appended claims.

Claims

241108-AVMX-PS-PRRS 16 CLAIMS 1. A vaccine against Porcine Reproductive and Respiratory Syndrome (PRRS) caused by at least one PRRS type 1 virus (PRRS-1), characterized in that it comprises a live, non-pathogenic strain of a PRRS type 2 virus (PRRS-2), and a pharmaceutically acceptable vehicle, adjuvant and / or excipient.

2. The vaccine according to claim 1, further characterized in that the live apathogenic strain of VPRRS-2 has the sequence SEQ ID NO:

1.

3. The vaccine according to claim 1, further characterized in that the pharmaceutically acceptable vehicle is selected from aqueous solutions or emulsions.

4. The vaccine according to claim 3, further characterized in that the pharmaceutically acceptable vehicle is selected from a trehalose, phosphate, glutamate, albumin (TPGA) solution, a phosphate buffer saline (PBS) solution, and combinations thereof.

5. The vaccine according to claim 1, further characterized in that it has a concentration of VPRRS-2 of at least 10 3 0 cell culture infective dose 50% (CICC50%).

6. The vaccine according to claim 5, further characterized in that the concentration of the VPRRS-2 virus is between 10 3 0 and 10 4 0 DICC5o% / mL.

7. The vaccine according to claim 6, further characterized in that the concentration of VPRRS-2 is between 10 3 2 and 10 3 5 DICC5o% / mL.

8. The vaccine according to claim 1, further characterized in that it is adapted for intranasal or intramuscular administration.

9. The vaccine according to claim 8, further characterized in that it is adapted for intranasal administration.

10. The vaccine according to claim 1, further characterized in that it also prevents disease caused by PRRS virus type 2.

11. A live, non-pathogenic strain of a PRRS type 2 virus (PRRS-2) for use in a vaccine for the prevention or control of PRRS caused by at least one PRRS-1 virus.

12. The live, non-pathogenic strain of VPRRS-2 according to claim 11, further characterized in that it has the sequence SEQ ID NO:

1.

13. The live, non-pathogenic strain of VPRRS-2 according to claim 11, further characterized in that the strain is used in a vaccine according to any one of claims 3 to 10.

14. The live, non-pathogenic strain of VPRRS-2 according to claim 11, for use in the vaccine for the prevention or control of PRRS caused by VPRRS-1 and / or VPRRS-2 types. 241108-AVMX-PS-PRRS 17 15. Use of a live, non-pathogenic strain of a PRRS-2 virus for the preparation of a vaccine useful for the prevention or control of PRRS caused by a PRRS-1 virus in accordance with any of claims 1 to 10.

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