VACCINE COMPRISING NON-REPLICATING IMMUNOGENS AND PRRS VIRUS, USE THEREOF AND COMBINATION

Abstract

The invention relates to a vaccine comprising, in combination, non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans and live attenuated PRRS virus, and a pharmaceutically acceptable carrier, for use in a method for the prophylactic treatment of a pig against an infection with Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans and PRRS virus, wherein the vaccine is administered in a single dose in relation to treatment against an infection with the PRRS virus.

Description

VACCINE COMPRISING NON-REPLICATING IMMUNOGENS AND PRRS VIRUS, USE THEREOF AND COMBINATION General scope of the invention

[001] The invention generally relates to the field of swine health. Swine are prone to many pathogenic microorganisms. Infection control is commonly achieved through stable management and feeding, treatment with pharmaceuticals such as antiviral drugs and antibiotics, or prophylactic treatment with vaccines. In particular, the invention relates to vaccines against PRRS virus (porcine reproductive and respiratory syndrome), Erysipelothrix rhusiopathiae, porcine parvovirus, and Leptospira interrogans (Lato Sensu), and to a method of protecting an animal against such infections using such vaccines. Background of the invention

[002] The PRRS virus was first reported in 1987 in North America and Central Europe. The PRRS virus is a small, enveloped RNA virus. It contains a single-stranded, positive-sense RNA genome, approximately 15 kilobases in size. The genome contains nine open reading frames. The virus is a member of the genus Arterivirus, family Arteriviridae, order Nidovirales. The two prototype strains of PRRSV are the North American strain VR-2332 and the European strain, Lelystad virus (LV). The European and North American strains of PRRSV cause similar clinical symptoms. In the early 2000s, a highly pathogenic strain of the North American genotype emerged in China. This strain, HP-PRRSV, is more virulent than all other strains and causes significant losses in Asian countries worldwide. Clinical signs include reproductive failure in sows, such as abortions and the birth of stillborn or mummified fetuses, and cyanosis of the ear and vulva.In neonatal piglets, the disease causes respiratory distress, with increased susceptibility to respiratory infections, such as Glasser's disease. Regarding... Petition 870230021160, dated 03 / 13 / 2023, p. 12 / 38 2 / 24 PRRS virus, although inactivated virus vaccines have been described and are commercially available, Modified Live Virus (MLV), comprising the European (type I) or North American (type II) type in live attenuated form, is the main immunological tool for its control. Several vaccines are commercially available in the art. Porcilis® PRRS (available from MSD Animal Health, Boxmeer, Netherlands) is a vaccine comprising live attenuated PRRS virus type I and is registered to reduce infection (viremia) caused by infection with the PRRS virus. Ingelvac PRRS® MLV (available from Boehringer Ingelheim, Ingelheim, Germany) is a vaccine that helps reduce disease caused by the PRRS virus. Fostera® PRRS (available from Zoetis, Florham Park, New Jersey, USA) is also an MLV vaccine and is registered for protection against both the respiratory and reproductive forms of the disease caused by the PRRS virus.Other PRRS vaccines are described, for example, in documents WO2006 / 074986, US 8728487 and WO2014 / 048955.

[003] The infectious disease caused by Erysipelothrix rhusiopathiae (Ery) in pigs is known as erysipelas and is one of the oldest recognized diseases affecting growing and adult swine. Up to 50% of pigs in intensive swine production areas are considered to be colonized by Ery. The organism usually resides in the tonsillar tissue. These typically healthy carriers can shed the organism in their feces or oronasal secretions and are an important source of infection for other pigs. Disease outbreaks can be acute or chronic, and clinically inapparent infections also occur. Acute outbreaks are characterized by sudden and unexpected deaths, episodes of fever, painful joints, and skin lesions ranging from generalized cyanosis to the frequently described diamond-shaped skin lesions (rhomboid urticaria). Chronic erysipelas tends to follow acute outbreaks and is characterized by Petition 870230021160, dated 03 / 13 / 2023, page 13 / 38 3 / 24 joints and increased lameness. A second form of chronic erysipelas is vegetative valvular endocarditis. Pigs with valvular lesions may show few clinical signs; however, when physically exerted, they may show signs of respiratory distress, lethargy, and cyanosis, and suddenly succumb to infection. Severely affected pregnant sows may abort, probably due to fever. Vaccination is very effective in controlling disease outbreaks. Injectable bacterins and other non-replicating immunogens are known to provide long-lasting immunity. Commercially available vaccines comprising non-replicating immunogens of Erysipelothrix rhusiopathiae are Porcilis® ERY+Parvo (MSD Animal Health), FarrowSure® Gold (Zoetis), ErySeng® Parvo (Hipra), and PARVORUVAX® (Merial). The ideal timing for vaccination can vary from farm to farm.Susceptible pigs can be vaccinated before weaning, at weaning, or several weeks after weaning. Male and female pigs selected for addition to the breeding herd should preferably be vaccinated with a booster 3 to 5 weeks later. Subsequently, the breeding stock should be vaccinated twice a year. In general, there is good cross-protection between the main strains of Erysipelothrix rhusiopathiae that infect pigs.

[004] Porcine parvovirus is ubiquitous in pigs worldwide. Almost all sows are naturally infected before their second gestation, and immunity is lifelong. Consequently, it is a typical disease of first-parity pigs. Sows that are immunologically naive or have high titers of passive antibodies have the highest risk of reproductive disorders caused by the virus. Infection before day 30 of gestation results in early embryonic loss. Fetal infection between 30 and 70 days of gestation can result in fetal death and mummification. Not all fetuses are Petition 870230021160, dated 03 / 13 / 2023, p. 14 / 38 4 out of 24 are infected at the same time, and death at different stages of gestation is typical. Some fetuses survive and are born alive, but persistently infected. Most fetuses infected after 70 days of gestation mount an increased immune response, clear the virus, and are healthy at birth. Litters with dead fetuses of varying sizes, including mummified fetuses, along with stillborn piglets and healthy first-parity sows, are the hallmark of porcine parvovirus. Diagnosis is made by fluorescent antibody tests, virus isolation using the lung tissue of mummified fetuses, or demonstration of pre-colostral antibodies in stillborn piglets. Wild boars transmit the virus through various routes, including semen, for several weeks after acute infection and can introduce the virus into a herd.Effective vaccines comprising non-replicating immunogens are widely available, such as, for example, Porcilis® ERY+Parvo (MSD Animal Health), FarrowSure® Gold (Zoetis), and PARVORUVAX® (Merial).

[005] Leptospira interrogans (Lato Sensu, i.e., all pathogenic leptospira bacteria), especially the Pomona serogroup, is a major cause of reproductive failure in swine (infertility, abortion, stillbirths, and the birth of weak piglets). Although acute leptospirosis occurs in adult swine, most cases are asymptomatic. Pigs infected with Pomona and Australis serogroups, Bratislava serovar, can become chronic renal carriers. Abortion occurs 1 to 4 weeks after infection, and fetuses are autolyzed. Mummification, maceration, stillbirths, and weak piglets are also seen. Diagnosis is based on the demonstration of leptospires in fetal tissues or stomach contents. However, severely autolyzed fetuses may result in poor immunohistochemistry and fluorescent antibody results. PCR testing has better sensitivity and specificity. Vaccination with a bacterin (multivalent) or other immunogene does not Petition 870230021160, dated 03 / 13 / 2023, p. 15 / 38 A 5 / 24 replicate, usually every 6 to 12 months, helps mitigate or even prevent leptospirosis. Field results indicate that Leptospira infection cannot be safely eliminated with antibiotics. Effective vaccines are commercially available, for example, FarrowSure® Gold and Lepto-Eryvac® (Zoetis), and Porcilis® Ery+Parvo+Lepto (MSD Animal Health). Objective of the invention

[006] There is a continuing need for convenient, safe and effective means for managing swine health. In particular, there is a need for a convenient, safe and effective vaccine that can be used for the prophylactic treatment of a pig against infection with Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans and PRRS virus. Infections with these pathogens, in particular, can decrease the reproductive performance of female pigs. Summary of the invention

[007] In order to meet the objective of the invention, a novel vaccine is conceived for the protection of swine against infections with various disease-causing microorganisms, the vaccine comprising, in combination, non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans and live attenuated PRRS virus and a pharmaceutically acceptable vehicle in which the vaccine is administered in a single dose in relation to treatment against an infection with the PRRS virus. This vaccine is very suitable for use in female swine to improve their reproductive performance.

[008] According to the invention, the vaccine is administered in a single dose in relation to treatment against an infection with the PRRS virus. It has been advantageously found that a pig can be successfully vaccinated with the combined vaccine against PRRS virus infection, even after a Petition 870230021160, dated 03 / 13 / 2023, page 16 / 38 6 / 24 single administration of the vaccine. This does not preclude a follow-up vaccination, for example, 6 to 12 months after the first vaccination to renew the level of protection. This follow-up vaccination differs from a booster vaccination in an initial booster vaccination schedule, where protection is believed to be achieved after the booster vaccination. In an initial booster schedule, the two vaccines are typically given 2 to 4 weeks apart.

[009] Although vaccines are known and commercially available to treat Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans, and PRRS virus infections, there is a continuing need for new ways to provide good protection in a safe and convenient manner. A combined vaccine against all these pathogens is commercially available in the USA (Boehringer Ingelheim's ReproCyc® PRRS-PLE). However, this is a two-dose vaccine for all antigens in order to achieve adequate protection (vaccination needs to be repeated within 3 to 4 weeks). Furthermore, even the two-dose administration regimen as described in the art is only successful when animals are positive for the PRRS virus, that is, when it is clear that their immune system is already (pre-)initiated with the virus. The known administration schedule is thus, in fact, a three-dose immunization schedule.Indeed, the PRRS virus is an immune-evasive virus against which adequate protection is not easy. Surprisingly, the applicant found that a single-dose vaccination is sufficient when using a live attenuated PRRS virus in combination with Ery, Paro, and Lepto antigens, even in PRRS-negative animals. This is not understood, but may be the result of positive antigen interference. Furthermore, it is always uncertain whether a contemplated or suggested combination of antigens can... Petition 870230021160, dated 03 / 13 / 2023, page 17 / 38 7 / 24 leading to a safe and effective combination vaccine in any new administration regimen, primarily due to unpredictable interference effects of the antigens. In fact, there is always a level of uncertainty regarding the safety and efficacy of any contemplated combination vaccine in any particular administration regimen.

[0010] The Committee for Veterinary Medicinal Products of the European Medicines Agency (EMEA), in its publication Note for guidance: requirements for combined veterinary products (EMEA, 2000, CVMP / IWP / 52 / 97- FINAL), stated (page 2 / 6) that “the development of combined vaccines is not simple. Each combination must be developed and studied individually in terms of quality, safety and efficacy. The committee further indicates that the search for a good combined vaccine typically includes compatibility between the individual components in the combined vaccine, including, for example, preservatives, excipients and stabilizers, inactivating agents and adjuvants. On page 3, top paragraph, it states that In combined vaccines, the presence of more than one component can cause an interaction, leading to a decreased or increased response to individual components, compared to when the specific component(s) is / are administered in isolation.Such interactions are often immunological in nature, but they can also be caused by other factors with less direct effects on the immune system.

[0011] The US Department of Health and Human Services, Food and Drug Administration, Center for Biologies Evaluation and Research, published in April 1997 a guideline, Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies, in which it is stated (page 3, under Compatibility of Components) that Experience has shown that the combination of monovalent vaccines can Petition 870230021160, dated 03 / 13 / 2023, page 18 / 38 8 / 24 result in a new combination that is less safe or effective than desired. Sometimes, the components of inactivated vaccines can adversely affect one or more of the active components, indicating that, in particular, an inactivated vaccine can negatively influence the efficacy of a live vaccine, as occurred when combining a live pertussis vaccine and an inactivated poliovirus vaccine, resulting in a less potent pertussis vaccine. It is indicated that any additional components in the vaccine may complicate the safety and potency of the final product when compared to individual vaccines.

[0012] The World Health Organization (WHO) has published an e-learning course called “Basic Principles of Vaccine Safety”, which in MODULE 2 covers combination vaccines. This module begins with Licensed combination vaccines undergo extensive testing before approval by national authorities to ensure that the products are safe, effective and of acceptable quality. It is also stated that With all combinations, manufacturers must therefore assess the potency of each antigenic component, the effectiveness of the vaccine components when combined to induce immunity, the risk of possible reversion to toxicity and reaction with other vaccine components.

[0013] In short, any combination of particular antigens is not simple and requires experimentation to determine safety and efficacy.

[0014] The present invention, together with the vaccine as such, also relates to a combined vaccine for use in a method for the prophylactic treatment of a pig against infection with Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans and PRRS virus, to a method for constituting such a vaccine and to a method for the prophylactic treatment of a pig against infection with Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira Petition 870230021160, dated 03 / 13 / 2023, p. 19 / 38 9 / 24 interrogans and PRRS virus, comprising administering said vaccine to the animal, in particular, a single dose in relation to the PRRS virus, parenterally, in particular, intramuscularly.

[0015] The invention also relates to a combination of a first vaccine comprising non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvovirus and Leptospira interrogans, and a pharmaceutically acceptable carrier, and a second vaccine comprising lyophilized live attenuated PRRS virus and an instruction that the second vaccine may be mixed with the first vaccine to form the vaccine according to the invention. In practice, the first and second vaccines may be sold as independent and separate vaccines, but with an indication on the label, package leaflet or otherwise, that the two vaccines may be mixed to form a multiple combination vaccine, wherein the vaccine is administered in a single dose in relation to treatment against an infection with the PRRS virus. Definitions

[0016] A vaccine is a pharmaceutical composition that is safe to administer to an animal and is capable of inducing protective immunity in that animal against a pathogenic microorganism (“a pathogen”), that is, to induce successful prophylactic treatment against an infection with the pathogen as defined herein below. A vaccine may be used in conjunction with an adjuvant, that is, a substance or composition that is capable of enhancing the immune response induced by the vaccine.

[0017] A non-replicating immunogene of a pathogen is any substance or compound corresponding to the pathogen, other than the living replicating pathogen as a whole (whether wild-type or attenuated), against which an immune response must be triggered, such that the Petition 870230021160, dated 03 / 13 / 2023, p. 20 / 38 10 / 24 corresponding virulent pathogen or one or more of its virulence factors will be recognized by the host's immune system as a result of this immune response and are, at least in part, neutralized. Typical examples of non-replicating immunogens are inactivated (killed) whole pathogens and whole subunits of these pathogens, such as capsid proteins and surface-expressed proteins, for example, recombinantly expressed proteins. Non-replicating immunogens (e.g., killed whole pathogen, cell lysate, subunit, etc.) evoke an immune response that is primarily humoral (i.e., antibody induction).

[0018] Prophylactic treatment against an infection by a pathogen is assistance in preventing or improving an infection with that pathogen or a disorder resulting from that infection, resulting from a post-treatment challenge with a pathogen, in particular, to reduce its burden in the host after that challenge and, optionally, to help prevent or improve one or more clinical manifestations resulting from post-treatment infection with the pathogen.

[0019] A live attenuated pathogen is a viable, replicating form of the pathogen with reduced virulence. The attenuation process takes an infectious pathogen and alters it to become harmless or less virulent, typically by multiple passages of the pathogen through cellular systems or by genetically modifying the pathogen.

[0020] Single-dose administration of a vaccine for use in prophylactic treatment means that, to achieve protective immunity, the vaccination does not need to be reinforced with a second administration of the vaccine. In a two-dose regimen, the first (primary) vaccination is typically reinforced within 6 weeks of the first administration, usually within 3 or even 2 weeks of the first administration, and only after the second (booster) administration is protective immunity achieved, i.e., treatment. Petition 870230021160, dated 03 / 13 / 2023, page 21 / 38 Successful 11 / 24 prophylactic treatment, as defined above, can be achieved.

[0021] A pharmaceutically acceptable vehicle is a biocompatible medium, that is, a medium that after administration does not induce significant adverse reactions in the animal in question, capable of presenting the antigen to the immune system of the host animal after administration of the vaccine. Such a vehicle may be a liquid containing water and / or any other biocompatible solvent, possibly forming an emulsion with one or more hydrophobic liquids, such as an oil. The vehicle, however, may also be a solid, as is commonly used to obtain lyophilized vaccines (based on sugars and / or proteins). Invention modalities

[0022] In one embodiment, the non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvovirus, and Leptospira interrogans are inactivated pathogens of Erysipelothrix rhusiopathiae, porcine parvovirus, and Leptospira interrogans, respectively. For example, by simply killing the pathogens, a simple way of having (all) the immunogens available in a non-replicating form is provided. Although any subunit vaccine may also be suitable for use in the present vaccine, by having the inactivated pathogens available, the relevant immunogens are present per se and in a manner (similar to the manner in which) they are present in the naturally occurring pathogen in the live phase.

[0023] In another embodiment, the Leptospira interrogans pathogen comprises bacteria of the Pomona serogroup, which is the most important pathogen of swine leptospira. Optionally, bacteria from at least one of the Tarassovi, Australis, Grippotyphosa, Icterohaemorrhagiae and Canicola serogroups are also present, wherein the Australis serogroup bacteria, in particular, are bacteria of the Bratislava serovar. Petition 870230021160, dated 03 / 13 / 2023, p. 22 / 38 12 / 24

[0024] As indicated above, also in another embodiment of the vaccine for use in a method for the prophylactic treatment of a pig against infection with Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans and PRRS virus, the non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans are inactivated (i.e., killed) pathogens. In particular, the Leptospira interrogans pathogen comprises bacteria of serogroup Pomona and, optionally, bacteria of at least one of the serogroups Tarassovi, Australis, Grippotyphosa, Icterohaemorrhagiae and Canicola, wherein the bacteria of serogroup Australis, in particular, are bacteria of serovar Bratislava.

[0025] In yet another method, the vaccine is administered parenterally, that is, administered in a part of the body other than the mouth and alimentary canal. The vaccine can, for example, be administered intramuscularly.

[0026] As stated above, the invention also relates to a method for constituting the combination vaccine wherein the method comprises mixing a first composition comprising live attenuated PRRS virus with a second composition comprising immunogens of Erysipelothrix rhusiopathiae, porcine parvovirus and Leptospira interrogans in a pharmaceutically acceptable vehicle. In another embodiment, the first composition comprises lyophilized PRRS virus, for example, in a stabilizer such as Porcilis® PRRS. The mixing preferably takes place no more than 24 hours before the vaccine is administered to the animal, or no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 1 / 2 hour before the start of administration.

[0027] The invention will now be explained in more detail, using the following examples. Examples Experiment 1: the combination of non-living immunogens from Ery, parvo and Petition 870230021160, dated 03 / 13 / 2023, page 23 / 38 13 / 24 lepto

[0028] In a first experiment, the safety and efficacy of the combination of non-replicating immunogens from Erysipelothrix rhusiopathiae, porcine parvovirus, and Leptospira interrogans were evaluated. For such a combination of non-live antigens, in particular, safety is an important aspect. This is due to the relatively high quantity of antigens that is typically used in such a (non-live) vaccine, with the risk of endotoxic shock, particularly when the immunogens comprise inactivated pathogens of gram-negative bacteria (in whose bacteria the cell walls comprise endotoxic lipopolysaccharides).

[0029] The study was designed as a historically controlled field study. It was conducted on a farm with a history of increased abortion rates associated with leptospira infection. High antibody titers against the Pomona serogroup were found in several animals, indicating a recent infection with Leptospira of the Pomona serogroup. The farm was negative for PRRS virus infection.

[0030] At the beginning of the study, all breeding sows and replacement gilts on the farm were vaccinated in the neck muscle twice, with a 4-week interval, with a vaccine containing non-replicating immunogens of Erysipelothrix rhusiopathiae (formalin-inactivated bacteria, strain M2), porcine parvovirus (BPL-inactivated viruses, strain 014) and Leptospira interrogans (BPL-inactivated bacteria of serogroups Pomona, Tarassovi, Australis (serovar Bratislava), Grippotyphosa, Icterohaemorrhagiae and Canicola) in an aqueous dl-α-tocopherol acetate-based adjuvant. This vaccine composition is designated Ery / parvo / lepto. The animals were revaccinated with Ery / parvo / lepto in the second week of each subsequent lactation. New replacement gilts were vaccinated using the same schedule. Petition 870230021160, dated 03 / 13 / 2023, page 24 / 38 14 / 24

[0031] The reproductive performance of the pigs in the study was monitored. Farrowing outcomes and relevant reproduction data were collected to determine if vaccination had any effect on the incidence of abortion. The results obtained during the study period were compared with historical data. As parvovirus and Ery also have a significant negative impact on reproduction (parvo can kill fetuses and Ery can cause abortion), a substantial improvement in this respect is a good indication that the vaccine is also effective against these pathogens.

[0032] No adverse events were reported during the study, indicating that the vaccine was safe. Regarding efficacy, the percentage of abortions between the study period and the pre-study period was compared, taking into account the effect of the season. After vaccination, a rapid improvement in abortions was observed, from 12.6% pre-vaccination to 0.5% after the start of vaccination. This is a 96% decrease in the abortion rate. The total number of abortions on the farm decreased from 55 to 6. The trend of having a low number of abortions on the farm remained stable after the start of the vaccination program and in the following season. The frequency of abortions remained low, i.e., 0.6%. The safety and efficacy of the vaccine were confirmed in another study reported in Porcine Health Management in November 2015 (Porcine Health Management 2015 1:16 / DOI: 10.1186 / s40813-015-0011-0). This study focused on viremia, kidney infection, and urinary incontinence.

[0033] It can thus be concluded that the vaccine comprising, in combination, non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvovirus and Leptospira interrogans is safe and effective against all these pathogens. Adding a live PRRS virus to this vaccine, which (inherently) contains a relatively low amount of antigen when compared to the inactivated ery / parvo / lepto pathogens, is not Petition 870230021160, dated 03 / 13 / 2023, page 25 / 38 15 / 24 expected to have a negative effect on the efficacy of ery / parvo / lepto immunogens. However, safety may be compromised and the effect of inactivated immunogens on live PRRS virus is unknown and needs to be evaluated. This was done in another experiment, the results of which are indicated below. Experiment 2: The effect of the EPL combination vaccine on the live PRRS vaccine

[0034] The objective of this study was to evaluate the vaccine potential in six-week-old SPF piglets of a live attenuated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 (Porcilis PRRS) vaccine comprising, in combination, non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvovirus and Leptospira interrogans.

[0035] The study was conducted with forty PRRSV antibody-negative piglets, evenly distributed into 2 groups. Groups 1 and 2 were vaccinated once intramuscularly (IM) with 2 ml of Porcilis PRRS diluted to 4.5 log10 TCID50 per dose (this dose represents a dose slightly above the minimum dose of 4.0 log10 TCID50 of Porcilis® PRRS). Group 1 received the PRRS vaccine diluted in an experimental Ery / Parvo / Lepto vaccine as indicated above in experiment 1. The vaccines were mixed at a temperature of 25°C and left for at least 1 hour before actual administration to the animals. Group 2 received the live attenuated virus diluted in Diluvac Forte, and thus effectively received the normal Porcilis® PRRS vaccine slightly above its minimum dose. This group served as a positive control.

[0036] The following parameters were measured: local and systemic reactions, PRRSV viremia, and PRRSV serology. Thus, it can be Petition 870230021160, dated 03 / 13 / 2023, page 26 / 38 16 / 24 established whether the vaccine strain is capable of replicating normally in the host animal, which is a requirement for inducing an effective immune response. From the day of vaccination until the end of the experiment (28 days post-vaccination, dpv), all piglets were observed daily for clinical signs. Blood samples were collected immediately before vaccination and 2 and 4 weeks post-vaccination. Serum samples obtained at the time points after vaccination were used to determine PRRSV-specific viremia and serological responses.

[0037] Veterinary examination showed that all piglets were healthy on the day of vaccination. No adverse events were found that could be related to the vaccination. One piglet in group 2 was found dead during blood collection in the late afternoon, 14 days after vaccination. Necropsy was performed and the cause of death was likely a hematoma on the neck, due to blood collection.

[0038] The results of the PRRS virus serology are shown in Table 1. The results of the PRRS virus viremia are shown in Table 2. Table 1. PRRSV serological results: percentage of positive animals, 0, 14 and 28 days post-vaccination (dpv). Group 0 DPV 14 DPV 28 DPV 1 0 80 100 2 (positive control) 10 100 100 Table 2 PRRSV viremia results: percentage of positive animals, 14 and 28 days post-vaccination (dpv) Group 14 DPV 28 DPV 1 70 100 2 (positive control) 100 100

[0039] It appeared that the serology, as well as the viremia results for Petition 870230021160, dated 03 / 13 / 2023, p. 27 / 38 17 / 24 The PRRS virus from the combination vaccine was comparable to that of the positive control (Porcilis® PRRS as a standalone vaccine), both at the minimum protective dose and at the highest dose. These data thus indicate that the PRRS virus is capable of surviving incubation in a vaccine comprising non-replicating immunogens in combination of Erysipelothrix rhusiopathiae, porcine parvovirus, and Leptospira interrogans. Based on these data, it is understood that a combined (two-way) vaccine comprising non-replicating immunogens of Erysipelothrix rhusiopathiae and live attenuated PRRS virus will also be safe and effective. For this vaccine, non-replicating immunogens of porcine parvovirus can be added to also provide protection against infection with porcine parvovirus. This three-way vaccine is also understood to be safe and effective based on the current results.All of this offers a range of different vaccines that help in providing the following vaccination schedules for swine, specifically aimed at improving the reproductive performance of these animals: a basic vaccination, for example, an initial booster vaccination, of an animal before its first gestation (typically between 20 and 30 weeks of age) with a combination vaccine comprising non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvovirus and Leptospira interrogans, wherein, if an initial booster schedule is used, either the initial or booster vaccine is combined with live attenuated PRRS virus according to the present invention (the PRRS component of the vaccine in any case being used as a single-dose vaccine). An initial booster vaccination could, for example, occur at 20 and 24 weeks of age.This basic vaccination can then be repeated annually against Erysipelas / Parvovirus / Leptospirosis / Prrs infection with a single dose of the combination vaccine according to the present invention, or with a combination vaccine in which, depending on the circumstances, one or more of the... Petition 870230021160, dated 03 / 13 / 2023, pp. 28 / 38 18 / 24 antigens are not present (e.g., no leptospira antigen if a farm is not expected to be infected with any leptospira bacteria). Regarding PRRS, depending on the PRRS infection pressure on the farm, vaccination may be repeated every 6 months (or even every 3 months) if deemed necessary. Such revaccination at a shorter interval can be done with a combination vaccine comprising near-live attenuated PRRS virus and non-replicating immunogens of Erysipelothrix rhusiopathiae. For parvovirus and leptospira, short-term revaccination is usually considered unnecessary, as leptospira and parvo vaccines can have a duration of immunity of about one year in swine.

[0040] In conclusion, it can be said that the safety as well as the efficacy of the new combination vaccine is sufficient. Additional serological data (in particular, for leptospira bacteria and parvovirus) and / or challenge data (in particular, for PRRS virus and Erysipelothrix rhusiopathiae) can be used to confirm the efficacy of the new combination vaccine for each of the corresponding pathogens. Experiment 3: Serological results of the EPL / PRRS combination vaccine

[0041] The aim of this experiment was to confirm the serological response of the combined mixed use of an Ery+Parvo+Lepto vaccine (Porcilis® Ery+Parvo+Lepto) with a live PRRS vaccine (Porcilis® PRRS) compared to the separate vaccines. Porcilis® PRRS is normally administered once, while the basic Porcilis® Ery+Parvo+Lepto (ELP) vaccination consists of two vaccinations with a 4-week interval. Thus, Porcilis® PRRS can be co-administered during the primary EPL vaccination or during the EPL booster vaccination. Both mixing options were tested in this study. Petition 870230021160, dated 03 / 13 / 2023, pp. 29 / 38 19 / 24

[0042] For this study, sixty-four 20-week-old pigs (seronegative for all vaccine components) were used and distributed into 4 groups of 16 animals each. Group 1 was vaccinated with Porcilis EPL + Porcilis PRRS at 20 weeks of age and with Porcilis EPL at 24 weeks of age. Group 2 was vaccinated with Porcilis EPL at 20 weeks of age and with Porcilis EPL + Porcilis PRRS at 24 weeks of age. Group 3 was vaccinated with Porcilis EPL at 20 weeks of age and 24 weeks of age. Group 4 was vaccinated with Porcilis PRRS at 20 weeks of age. During a period of 20 weeks after vaccination, blood was regularly sampled and sera were tested in the respective serological tests.

[0043] The test was valid if the animals were seronegative at the start of the study and if at least 10 animals in each control group remained seronegative for the omitted component of the vaccinations, i.e., group 4 negative for Ery, parvo and Leptospira and group 3 negative for PRRS. These criteria were met. Overall results by antigen are described below. PRRS serology (using the X3 PRRS test, IDEXX)

[0044] At the beginning of the study, most animals were seronegative. Responses after vaccinations associated with mixed use (groups 1 and 2) were at least as good as for Porcilis PRRS alone (group 4), while group 3 of Porcilis Ery+parvo+Lepto alone remained at a low level. Therefore, it can be concluded that mixed use associated with 20 weeks or 24 weeks of age had no negative effect on the PRRS component of Porcilis PRRS. Ery serology (using CIVTEST suis SE / MR, Hipra)

[0045] At the beginning of the study, most animals were seronegative. Responses after vaccinations associated with mixed use (groups 1 and 2) were at least Petition 870230021160, dated 03 / 13 / 2023, pp. 30 / 38 20 / 24 less as good as for Porcilis Ery+Parvo+Lepto alone (group 3), while group 4 of Porcilis PRRS alone remained at a low level. Therefore, it can be concluded that mixed use associated with 20 weeks or 24 weeks of age had no negative effect on the Ery component of Porcilis Ery+Parvo+Lepto. Parvovirus serology (using an at-home HI test and the INgezim PPV, Ingenasa test)

[0046] Although the HI test is frequently used to detect post-infection Parvo antibodies, it is less suitable for post-vaccination serological studies because after vaccination, many non-responders are found. Therefore, this antigen was tested in the HI test as well as in a more sensitive commercial ELISA.

[0047] At the beginning of the study, most animals were seronegative in both tests. Responses after vaccinations associated with mixed use (groups 1 and 2) were at least as good as for Porcilis Ery+Parvo+Lepto alone (group 3), while group 4 of Porcilis PRRS alone remained at a low level. Therefore, it can be concluded that mixed use associated with 20 weeks or 24 weeks of age had no negative effect on the Parvo component of Porcilis Ery+Parvo+Lepto. Lepto serology (using internal serogroup-specific ELISA)

[0048] The MAT (microagglutination) test is generally used to detect post-infection Leptospira antibodies. However, this test primarily measures IgM antibodies, which are short-lived and predominantly induced after primary vaccination, while IgG antibodies are mainly induced after a booster vaccination. Furthermore, some serotypes hardly respond to the MAT test, which also makes this test less suitable for serological studies. As IgG Petition 870230021160, dated 03 / 13 / 2023, pages 31 / 38 21 / 24 was implicated in protection, and since the MAT test is less suitable, serotype-specific ELISA antibodies were developed and validated internally. These serotype-specific inhibition ELISA tests were used to measure serotype-specific IgG responses in this study.

[0049] At the beginning of the study, all animals were seronegative. Responses after the vaccines combined with mixed use (groups 1 and 2) were at least as good as for Porcilis Ery+Parvo+Lepto alone (group 3), while group 4 with Porcilis PRRS alone remained at a low level. Therefore, it can be concluded that mixed use combined with 20 weeks or 24 weeks of age had no negative effect on any of the components of Porcilis Ery+Parvo+Lepto. Furthermore, the decline in antibody titer prolongation produced similar profiles, suggesting that the duration of immunity is also not influenced.

[0050] In conclusion, antibody responses after the combined mixed use of Porcilis PRRS and Porcilis Ery+Parvo+Lepto were at least as high as after vaccination with the vaccine alone, and therefore it can be concluded that vaccine intake and immunity levels are not negatively influenced by the combined mixed use. Similarly, the decline in antibody titers over time showed similar profiles indicating that the duration of immunity for all vaccine components is also not negatively influenced by the combination. Experiment 4: Efficacy of minimum dose PRRS in a combination vaccine with Ery+Parvo+Lepto

[0051] The objective of this experiment was to evaluate the efficacy of a live attenuated PRRSV Type 2 vaccine (Prime Pac® PRRS) reconstituted with a minimum dose of 4.0 log10 TCID50 / animal in an EPL vaccine.

[0052] Sixty-six 5-week-old piglets seronegative for Petition 870230021160, dated 03 / 13 / 2023, pp. 32 / 38 22 / 24 PRRSV were included in this study. Piglets were vaccinated with a minimum dose of Prime Pac® PRRS reconstituted in an EPL formulation (group 1; one hour waiting at 25°C between reconstitution and vaccination) or with Diluvac Forte (group 2), intramuscularly on the right side of the neck. Piglets in group 3 were vaccinated intramuscularly on the right side of the neck with 2 ml of the same EPL formulation and served as PRRSV challenge controls. Four weeks after vaccination, piglets were challenged with a dose of 5.0 lg 10⁻⁶ TCID₅₀ of a virulent strain of PRRSV Type 2, Nebraska-1, via intranasal (IN) injection, 1 ml per nostril. Blood samples from the piglets were collected on the day of vaccination, the day of the challenge, and 5, 7, 10, 14, and 28 days after the challenge. Rectal temperatures were measured 1 day before the challenge, immediately before the challenge, 4 hours after the challenge, and then daily from 1 to 10 days after the challenge.Body weight was measured the day before the challenge, and 9 and 25 days after the challenge. Ten days after the challenge, 11 pigs per group were euthanized and observed for lung lesions. Twenty-eight days after the challenge, the remaining piglets were euthanized. Body temperature and body weight

[0053] Regarding body temperature, in each of the vaccine groups the temperature was lower than in control group 3. It was also found that there is no difference between the mixed-use group 1 and the group with only PRRS Prime Pac® (group 2). Regarding body weight, vaccinated animals grew faster than unvaccinated animals in group 3. It was also found that animals in the mixed-use group 1 grew at a similar rate to animals in the group with only Prime Pac® PRRS (group 2). Lung injury scores

[0054] Half of the animals, a total of 11 animals per group, were euthanized 10 days after the challenge to observe their lungs for Petition 870230021160, dated 03 / 13 / 2023, pages 33 / 38 23 / 24 PRRS-related pneumonia. Table 3 shows the estimated average percentage of lung affected by visible pneumonia by group. From the table, it can be concluded that, in general, the PRRSV challenge strain did not cause, on average, almost any lung lesions. However, 10 days after vaccination, vaccinated animals showed fewer lung lesions than controls. Overall, there was not much difference between the mixed-use group and the Prime Pac® PRRS-only group. Table 3 Mean lung injury scores by group, 10 days post-expected Group 10 dpc PrimePac + EPL 0 PrimePac 0.2 Control 0.85 Serological response

[0055] On the day of vaccination, all animals were seronegative for PRRSV, demonstrating that PRRSV-negative animals were used for this study. The unvaccinated control group animals remained seronegative until the day of the challenge (i.e., 4 weeks after vaccination). In contrast, on the day of the challenge, an antibody response was measured for the vaccinated groups, indicating that the animals in both the mixed-use group 1 and the Prime Pac® PRRS-only group (group 2) were successfully initiated through PRRS vaccination. At later times after the challenge, i.e., 10 and 28 dpc, an increase in the height of the antibody level was observed, probably caused by the challenge infection. PRRS viremia

[0056] The average viral load per group is shown in Table 4. The table clearly shows that the vaccinated animals (groups 1 and 2) have, on average, a reduced viral load in their serum compared to the control animals (group 3). There is little difference in the height of viremia between each of the Petition 870230021160, dated 03 / 13 / 2023, pages 34 / 38 24 / 24 groups vaccinated. Table 4 PRRS dpc titration, 10g TCID50 / ml Group 0 5 7 10 14 28 1 0 0.6 0.5 0.7 0.6 0 2 0 0.1 0.2 0.2 0.3 0 3 0 2.0 1.8 2.2 2.1 0

[0057] In conclusion, it was established that even at the lowest dose (i.e., the lower limit according to the label) the commercial Prime Pac® PRRS vaccine reconstituted in an EPL formulation adequately seroconverted the animals, and protection against pathogenic PRRSV was obtained.

Claims

CLAIMS 1. A vaccine, characterized in that it comprises, in combination, inactivated non-replicating immunogens of the M2 strain of Erysipelothrix rhusiopathiae, the 014 strain of porcine parvovirus, the Pomona, Tarassovi, Australis (serovar Bratislava), Grippotyphosa, Icterohaemorrhagiae and Canicola serogroups of Leptospira interrogans and live attenuated PRRS virus, and a pharmaceutically acceptable carrier, wherein the vaccine is for use in a method for the prophylactic treatment of a PRRS virus seronegative pig against infection with Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans and PRRS virus, and wherein the vaccine is formulated to be administered in a single dose in relation to treatment against infection with PRRS virus.

2. Vaccine according to claim 1, characterized in that the vaccine is administered parenterally.

3. Vaccine according to claim 2 or 3, characterized in that the vaccine is administered intramuscularly.

4. Use of a combination of inactivated non-replicating immunogens of the M2 strain of Erysipelothrix rhusiopathiae, the 014 strain of porcine parvovirus, the Pomona, Tarassovi, Australis (serovar Bratislava), Grippotyphosa, Icterohaemorrhagiae and Canicola serogroups of Leptospira interrogans and live attenuated PRRS virus, and a pharmaceutically acceptable carrier, characterized in that it is for manufacturing a vaccine for the prophylactic treatment of a PRRS virus seronegative pig against infection with Erysipelothrix rhusiopathiae, porcine parvovirus, Leptospira interrogans and PRRS virus, wherein the vaccine is formulated to be administered in a single dose in relation to treatment against infection with the PRRS virus.

5. Combination, characterized by the fact that it is a first vaccine Petition 870260049857, dated 05 / 25 / 2026, page 13 / 14 2 / 2 comprising inactivated non-replicating immunogens of the M2 strain of Erysipelothrix rhusiopathiae, the 014 strain of porcine parvovirus and the Pomona, Tarassovi, Australis (serovar Bratislava), Grippotyphosa, Icterohaemorrhagiae and Canicola serogroups of Leptospira interrogans, and a pharmaceutically acceptable carrier, and a second vaccine comprising lyophilized live attenuated PRRS virus, and wherein the second vaccine is mixed with the first vaccine, wherein the vaccine is administered in a single dose in relation to treatment against an infection with the PRRS virus.