Combined vaccine for swine erysipelothrix erysipelas, porcine parvovirus and leptospirosis

Abstract

The present invention relates to a combination of a first vaccine comprising a non-replicating immunogen of porcine erysipelothrix sp. And a non-replicating immunogen of porcine parvovirus and a second vaccine comprising a non-replicating immunogen of leptospira sp. And the first vaccine and the second vaccine are jointly injected into corium layers of a first injection part and a second injection part of the pig at different points to prophylactically treat the pig so as to resist swine erysipelothrix erysipelas infection, porcine parvovirus infection and leptospirosis infection.

Description

Technical Field

[0001] This invention relates to the preventive treatment of pigs through vaccination to resist infection by swine erysipelas, porcine parvovirus, and leptospirosis. Background Technology

[0002] Pigs are susceptible to a variety of infections and diseases caused by these infections. For example, erysipelas is an infectious disease caused by *Erysipelothrix purpureus*, and is one of the oldest known diseases affecting both growing and adult pigs. In intensive pig farming areas, up to 50% of pigs are believed to be colonized by *Erysipelothrix purpureus*. E rhusiopathiae This organism is commonly found in tonsillar tissue. Healthy carriers typically shed it through their feces or nasal / oral secretions, making it a significant source of infection for other pigs. Infection occurs through ingestion of contaminated feed, water, or feces, as well as through skin abrasions. Once ingested, the organism can survive the harsh environment of the stomach and intestines and may remain alive in feces for months. In farms where this organism is prevalent, pigs are naturally exposed to *Erysipelothrix porligus* at a young age. Maternal antibodies provide passive immunity and suppress clinical disease. Older pigs often develop protective active immunity from exposure to the organism, which does not necessarily lead to clinical disease. Recovered pigs and pigs with chronic infections may become carriers of *Erysipelothrix porligus*. Healthy pigs may also be asymptomatic carriers.

[0003] Vaccination against *Erysipelothrix purpureus* is highly effective in controlling disease outbreaks on pig farms and should be encouraged. However, it may be less effective in preventing chronic arthritis. Some farms have stopped vaccination in connection with disease outbreaks. Injectable vaccines and live attenuated vaccines can be delivered by water and provide extended duration of immunity. The optimal timing of vaccination varies from farm to farm. When *Erysipelothrix purpureus* is prevalent in the production environment, vaccination should be administered before the anticipated outbreak. Susceptible pigs can be vaccinated before weaning, at weaning, or several weeks after weaning. Boars and sows selected for breeding herds should receive a booster vaccine 3–5 weeks later. Thereafter, breeding stock should be vaccinated twice a year, with follow-up vaccinations. However, vaccination does not always provide direct protection. Vaccination failure may occur in some herds because management stress can impair the immune system of vaccinated pigs. The use of live vaccines may also lead to clinical disease, particularly chronic erysipelothrix. Antigenic differences between serotypes in vaccines and those prevalent on farms could also lead to incomplete immunity and disease outbreaks, but these are rare events because there is believed to be good cross-protection among the major erysipelas strains that infect pigs.

[0004] In addition, porcine parvovirus infection (PPV, or "parvo") is a common pathogen in pigs. It is a significant cause of infectious infertility. PPV is a potent virus that can multiply normally in the pig's intestines without causing clinical symptoms and is prevalent in pig herds worldwide. PPV is listed as one of the organisms responsible for stillbirth-mummified fetus-embryonic death-infertility (SMEDI) syndrome. In larger herds, it is almost inevitable and an infection that must be addressed and managed, rather than attempted to be eradicated. In smaller herds where PPV-positive pigs have previously been present, they may or may not have died. Most viruses cannot survive long outside the host, but PPV is unusual in that it can persist in the environment for months and is resistant to most disinfectants, which is likely the main reason for its widespread transmission and difficulty in eradication. PPV is most commonly transmitted through the mouth or nose, enters the intestines, multiplies there, and is subsequently transmitted through feces. When pigs are first infected, no clinical signs are present. Boars can also infect sows through semen during mating.

[0005] PPV cannot be eliminated from the herd; therefore, management and prevention of acute cases should be the focus. Routine vaccination of sows and boars before entry into the breeding herd, coupled with annual follow-up vaccinations for all pigs, should be sufficient to ensure herd protection. It is well known that if infected breeding females have been vaccinated against PPV at some point in the past, their immune system is rapidly restimulated upon exposure to PPV (within 5-7 days). This is sufficient to prevent disease and stimulate (semi-)permanent immunity.

[0006] Next, leptospirosis is an infectious disease of pigs and many other animals, including humans, caused by infection with any of a large group of Leptospira bacteria (“lepto”). Leptospires are motile spirochetes, 6–12 micrometers long and 0.1 micrometers in diameter. They typically have hooks at both ends and can be stained with Giemsa stain or silver staining in tissues. In the laboratory, they are usually studied under a dark-field microscope. They are difficult to culture in laboratory media and grow very slowly (requiring 12–26 weeks). Many pathogenic strains can survive for extended periods in a slightly alkaline, moist environment. Most cannot withstand drying and can be killed by common disinfectants.

[0007] Although leptospirosis in pigs is widespread, the details of infection can vary between different serotypes. Pigs are susceptible to many different serotypes. The classification of leptospires is primarily based on DNA relevance to known reference strains, but a general consensus on the classification of this genus remains elusive. According to the latest classification, the Leptospiraceae family contains eight pathogenic species, three of which are most important to pigs: *Leptospira questionmark* (…). Leptospira interrogans(Pomona serotype, icteric hemorrhage serotype, canine serotype and Bratislava serotype), Leptospira bovis ( Leptospira borgpetersenii (Sero and Tarasov serotypes) and Leptospira kieri ( Leptospira kirschneri (Influenza / Typhoid Serotypes). Pomona and Bratislava serotypes exhibit unique adaptability to pigs; other serotypes are present in other species but sometimes infect pigs. Leptospira bratislava has been reported to... Leptospira bratislava () is the most common strain in pigs, although the role of this serotype in causing disease remains controversial.

[0008] Leptospirosis control is typically achieved through contact prevention, vaccination, and / or immunization with antibiotics. Contact prevention is challenging because many species can act as carriers of leptospires, including infected pigs, rodents (particularly mice and rats), and various wild animals. Once introduced into a suitable, moist environment, leptospires often persist there as a source of infection. Nevertheless, in closed-environment pig herds, leptospirosis can be effectively controlled (and potentially eradicated) through a combination of medications, vaccination, vector control, and the provision of treated or uncontaminated drinking water. Widespread immunization in breeding herds using vaccines typically reduces infection rates and abortion rates. The vaccine must be matched with pathogenic leptospiral sera; therefore, many vaccines are multivalent.

[0009] PRRS virus was first reported in North America and Central Europe in 1987. PRRS virus is a small enveloped RNA virus. It contains a single-stranded meaningful RNA genome, approximately 15 kilobases in size. This genome contains nine open reading frames. The virus belongs to the genus *Arteriviros*, family *Arteriviridae*, and order *Nestvirales*. The two prototype strains of PRRSV are the North American strain VR-2332 and the European strain Lelistard virus (LV). European and North American PRRSV strains cause similar clinical symptoms. In the early 21st century, a highly pathogenic strain with the North American genotype appeared in China. This strain, HP-PRRSV, is far more virulent than any other strain, causing significant losses in Asian countries worldwide. Subclinical infection is common with any PRRS virus, with clinical signs appearing only occasionally in the herd. Clinical signs include reproductive failure in sows, such as abortion and stillbirth or mummified fetuses, as well as cyanosis of the ears and vulva. In newborn pigs, the disease causes respiratory distress and increases susceptibility to respiratory infections such as Glasser's disease.

[0010] Vaccine compositions targeting all of the aforementioned identified pathogens are well known.

[0011] Purpose of the invention The purpose of this invention is to provide a new method for convenient, safe and effective vaccination against one or more of the swine pathogens mentioned above. Summary of the Invention

[0012] To achieve the objectives of this invention, it has been found that injecting a first vaccine containing non-replicating immunogens of *Erysipelothrix rhusiopathiae* and porcine parvovirus, along with a second vaccine containing a non-replicating immunogen of *Leptospira*, into the dermis of a pig at two separate injection sites (a first and a second injection site) can prevent infection with *Erysipelothrix rhusiopathiae*, porcine parvovirus, and *Leptospira*. At a typical intradermal vaccination dose of less than 1 ml, typically about 0.2 ml, if the first and second injection sites are more than 1.5 to 2 cm apart, the two vaccines will not mix subcutaneously, and the local reactions caused by each vaccine can be distinguished. This means that, according to the EMA guidelines on requirements for combined vaccines and veterinary immunomodulatory products (IVMPs) (July 18, 2013 (EMA / CVMP / IWP / 594618 / 2010)), which defines "separate sites" as "sufficiently far apart to prevent the possibility of product mixing and allow for the differentiation of local reactions of each product," the injection sites can be considered "separate" in the context of this invention. When administering intradermal vaccines, a distance of more than 1.5 cm, preferably more than 2 cm, between the first and second injection sites will result in the same outcome of preventing negative interference with the safety and / or efficacy of co-administering the two vaccines, since the two vaccines do not mix after administration.

[0013] This method provides a convenient way to safely and effectively vaccinate pigs. Although standalone or even combination vaccines can be used on pigs, even with the same antigen, a new application site cannot guarantee a safe and effective vaccine.

[0014] For example, the World Health Organization (WHO) has published an e-learning course titled "Fundamentals of Vaccine Safety" (https: / / apps.who.int / iris / handle / 10665 / 340576). Page 53 of the course states, " The route of application is The pathway of vaccine (or drug) contact with the body. This is a key factor in successful immunization. The substance must be transported from the site of entry to... The parts of the body that need to perform this function. However, achieving this through the body's transport mechanisms is not an easy task. " In response, the California Department of Health Services Immunization Service has published guidelines for proper vaccination (https: / / www.cdc.gov / vaccines / pubs / pinkbook / vac-admin.html). Regarding the application site, the second full paragraph on page 81, titled "Route and Site of Vaccination," states, "..." The recommended route and site for each vaccine are based on clinical trials. The recommended routes of vaccination have been determined through trials, practical experience, and theoretical considerations. There are currently five routes of administration. Deviating from the recommended routes may... Reduced vaccine efficacy or increased local adverse reactions ".

[0015] In conclusion, it is well known that administering vaccines at specific sites is not an easy task and requires experiments to determine whether safety and efficacy can be achieved, and if so, whether the level is sufficient to meet commercial needs.

[0016] In addition to the first and second vaccine combinations described above, the present invention also relates to a component kit comprising a first vaccine containing a non-replicating immunogen of *Erysipelothrix rubrum* and a non-replicating immunogen of porcine parvovirus, and a second vaccine containing a non-replicating immunogen of *Leptospira*, for the prophylactic treatment of pigs against *Erysipelothrix rubrum* infection, porcine parvovirus infection, and *Leptospira* infection by injecting the first and second vaccines together at multiple injection sites into the dermis of the pig at a first and a second injection site.

[0017] The present invention also relates to a method for the preventive treatment of pigs against erysipelas infection, porcine parvovirus infection and leptospirosis infection, which involves injecting a first vaccine containing a non-replicating immunogen of erysipelas and a non-replicating immunogen of porcine parvovirus, and a second vaccine containing a non-replicating immunogen of leptospirosis into the dermis of the pig at multiple sites.

[0018] Finally, the present invention is also characterized by preparing a first vaccine using non-replicating immunogens of *Erysipelothrix rubrum* and porcine parvovirus, and preparing a second vaccine using non-replicating immunogens of *Leptospira*. The combination of these vaccines is used to preventively treat pigs against *Erysipelothrix rubrum* infection, porcine parvovirus infection, and *Leptospira* infection by injecting the first vaccine and the second vaccine together at multiple injection sites into the dermis of the pig at the first and second injection sites.

[0019] definition A vaccine is a pharmaceutical composition that can be safely administered to a test animal and is able to induce protective immunity against a pathogenic microorganism, i.e., successfully induce prophylactic treatment as defined below.

[0020] Vaccines are applied to the dermis.This means that the vaccine is at least partially deposited in the dermis. The World Health Organization, in its paper titled "Intradermal Delivery of Vaccines; A review of the literature and the potential for development for use in low- and middle-income countries," published on August 27, 2009, states that "needle-free" vaccination does not necessarily mean "intradermal" vaccination (see Table 1 on page 3 of the review). A vaccine can only truly be delivered to the dermis when the needle-free device is "configured for intradermal vaccination." Otherwise, it can be delivered subcutaneously or intramuscularly. Several devices are available for intradermal vaccination, such as the IDAL® vaccineer (MSD AnimalHealth), the Pulse 50 MicroDose (pulse needle-free system), or... Vaccine Other devices described in , 2012 Jan 11;30(3):523-38 (see page 525 of “An overview of different devices for liquid and solid formulation administration”, in particular Table 1).

[0021] Combination of first and second vaccines It is a group of vaccines, wherein the first vaccine is distinct from the second vaccine, but wherein the two vaccines are used in combination for administration to the same subject in a single synergistic treatment.

[0022] Non-replicating immunogens of pathogens A non-replicating immunogen is any substance or compound corresponding to a pathogen, not the whole, living, replicating pathogen (whether wild-type or attenuated), that elicits an immune response against the pathogen, thereby allowing the corresponding virulent pathogen or one or more virulence factors to be recognized by the host's immune system and ultimately neutralized, at least partially. Typical examples of non-replicating immunogens are those that kill the whole pathogen (the term includes lysed forms of these pathogens) and their subunits, such as capsid proteins, surface-expressed molecules (e.g., recombinantly expressed proteins or lipopolysaccharides), and excreted molecules, such as toxins.

[0023] Bacterial seedlings It is a suspension of inactivated bacteria, which may be intact cells, partially lysed or completely lysed (e.g., by homogenization, Farchurning or a combination of two or more lysis methods).

[0024] Live detoxificationA pathogen is a live, replicating form of pathogen with reduced virulence. Attenuation is typically achieved through multiple passages of the pathogen in a cellular system or through genetic modification, transforming the pathogen into a harmless or less virulent form.

[0025] Preventive treatment for pathogen infection It can help prevent, improve or cure infection or symptoms caused by a pathogen following a treatment challenge, especially by reducing its burden in the host after such a challenge, or help prevent or improve one or more clinical manifestations caused by a pathogen following a treatment challenge.

[0026] pig Pigs are animals belonging to the family Suidae, order Artiodactyla, family Mammalia, and are commonly called pigs, hogs, or boars. There are currently 18 recognized extant species (or 19 species if domestic and wild pigs are counted separately), classified into 4 to 8 genera. Within this family, the genus Suidae includes the domestic pig (… Sus scrofa domesticus or Sus domesticus ).

[0027] Combined administration of vaccines at multiple sites Simultaneous administration, also known as co-administration, refers to administering these vaccines separately to the target animals, so they are not mixed before administration, but within the time frame where immune interference is expected, usually within 24 hours. Typical examples of co-administration are simultaneous administration at different application sites in the target animals, or at intervals of up to 1-24 hours.

[0028] Simultaneous administration of vaccines This refers to application at exactly the same time or within a time frame of at least one hour. Preferably within time frames of 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 minutes, or even up to one minute, for example, precisely at the same time.

[0029] Single dose of vaccine for preventive treatment This means that in order to achieve protective immunity, a booster vaccination is not required.

[0030] Primary and booster vaccination programs This refers to the use of a second dose of vaccine to boost the initial vaccination in order to achieve protective immunity. Typically, a booster dose is administered within 6 weeks of the initial vaccination, usually within 4 to 5 weeks after the initial vaccination. Sufficient protective immunity is usually achieved after the second (boost) vaccination.

[0031] Further embodiments of the present invention In a first further embodiment of the invention, the first and second injection sites are at most 4 cm apart, preferably at most 3 cm apart, more preferably less than 2.5 cm, for example 2 cm. This method has been found to facilitate vaccination of pigs, particularly by using, for example, the IDAL® 3G Twin device (obtained from MSD AnimalHealth, Boxmeer, The Netherlands; injection site spacing 2.8 cm), which has two containers containing separate vaccines for simultaneous intradermal injection of two vaccines.

[0032] More preferably, the first vaccine and the second vaccine are administered simultaneously at multiple injection sites.

[0033] In another preferred embodiment of the first and second vaccine combination used according to the present invention, the first and second vaccines are administered in a primary and booster vaccination regimen, wherein both vaccines are injected at the primary vaccination and both vaccines are injected at the booster vaccination.

[0034] In another embodiment, at least the first vaccine contains an adjuvant (it is well known that leptospirosis vaccines do not necessarily require adjuvants to achieve high levels of protection), but preferably both the first and second vaccines contain adjuvants, such as oil-in-water adjuvants. Typical adjuvants used in this invention are oil-in-water adjuvants, such as emulsions of mineral oil in water, or emulsions of mineral oil and vitamin E acetate in water. These adjuvants can be stabilized by emulsifiers such as polysorbate.

[0035] In another embodiment of the first and second vaccines used in this invention, the non-replicating immunogen of *Erysipelothrix rubellae* is a *Erysipelothrix rubellae* vaccine. In another embodiment of the first and second vaccines used in this invention, the non-replicating immunogen of porcine parvovirus is inactivated porcine parvovirus. In yet another embodiment of the first and second vaccines used in this invention, the non-replicating immunogen of *Leptospira* is a *Leptospira question mark* vaccine. Preferably, the second vaccine further comprises *Leptospira kieri* and / or *Leptospira savantis* vaccine.

[0036] In another embodiment of the first and second vaccines used in this invention, the non-replicating immunogen of *Erysipelothrix purpureus* is a *Erysipelothrix purpureus* vaccine, the non-replicating immunogen of porcine parvovirus is inactivated porcine parvovirus, and the non-replicating immunogen of *Leptospira* is a *Leptospira question mark* vaccine. Preferably, the second vaccine further comprises *Leptospira kieri* and / or *Leptospira savantis* vaccine.

[0037] Advantageously, it has been found that the combination of the first and second vaccines used according to any one of the preceding claims can also be used in conjunction with a third vaccine injected into pigs, said third vaccine containing live attenuated PRRS virus and achieving safe and effective combined immunization against all four pathogens, thus including protection against PRRS virus infection. Preferably, in prophylactic treatment, pigs receive a primary immunization with the first and second vaccines, and a booster immunization with said first and second vaccines, wherein the third vaccine is injected as a single dose at the time of the booster immunization (i.e., simultaneously with or at least within 24 hours of the booster immunization). In a further embodiment, the third vaccine (i.e., the PRRS vaccine) is mixed with the second vaccine (i.e., the leptospirosis vaccine) for booster vaccination.

[0038] In one embodiment, according to the invention, the combination of a first and a second vaccine is used, both of which are injected using a needle-free device via a jet of the respective vaccine, wherein the jet penetrates the pig's skin to reach the dermis. The third vaccine is also advantageously injected via a jet from a needle-free device.

[0039] The present invention will be described in more detail through the following specific embodiments.

[0040] Example Example 1 is the first trial of the Ery-Parvo-Lepto combined vaccine.

[0041] Example 2 is the second trial of the Ery-Parvo-Lepto combined vaccine.

[0042] Example 3 is the third trial of the Ery-Parvo-Lepto combination vaccine.

[0043] Example 1 Example 1 is the first trial of the Ery-Parvo-Lepto combined vaccine. For this purpose, the existing vaccine Porcilis® EPL (containing Erysipelothrix rhusiopathiae) was used. E. rhusiopathiaeSerotype 2 vaccines, inactivated porcine parvovirus, and various leptospiral vaccines (in this case, Leptospira jaundice hemorrhage serotype, Leptospira bratislava serotype, Leptospira typhus serotype, Leptospira pomona serotype, and Leptospira tarasov serotype) are known to be safe and effective vaccines for pigs, administered intramuscularly to prevent Erythromyces, Parvo, and Leptospira infections. These antigens are all inactivated pathogens. The aim was to observe whether the vaccine could also be administered intradermally while still meeting the pre-set safety and efficacy requirements. The IDAL® 3G Twin device (Twin IDAL) was selected for intradermal delivery. Because the volume of each administration is much smaller than the recommended volume of Porcilis EPL (0.2 ml instead of 2 ml), the antigen content of Porcilis EPL needs to be reduced (to 40% vs. 25%; see below) to accommodate the smaller volume of the aqueous phase of the formulation.

[0044] Various adjuvants were used in the experiments, including the proprietary adjuvant Diluvac Forte (DF; MSD AnimalHealth, Boxmeer, The Netherlands), μDiluvac Forte (μDF; similar to Diluvac but with fewer surfactants and no mineral oil), and an adjuvant containing experimental squalene, vitamin E acetate, and silica (see, for example, WO 2021 / 048338), referred to herein as SVEA. With both Diluvac adjuvants, an antigen concentration of 40% in Porcilis EPL could be formulated. With the squalene-based adjuvant, this was only 25%.

[0045] In this experiment, healthy 12-week-old pigs that were PRRS and Ery seronegative were used. The pigs were divided into four groups. The vaccine group consisted of 10 animals, and the control group consisted of 5 animals. Group 1 received two intradermal (ID) vaccinations with EPL-ID-40%-DF (in the primary-booster immunization regimen) and Porcilis PRRS administered separately (as a single vaccine) during the booster vaccination period. Group 2 also received two ID vaccinations (in the primary-booster immunization regimen), but this time with EPL-ID-40%-μDF and Porcilis PRRS administered separately during the booster vaccination period. Group 3 received two ID vaccinations with EPL-ID-25%-SVEA and Porcilis PRRS administered separately during the booster vaccination period. Group 4 was unvaccinated. All vaccinations were administered intradermally (0.2 ml) in the neck (right side for primary vaccination, left side for booster vaccination) using an IDAL device, with a vaccination interval of 4 weeks.

[0046] Following vaccination, the local injection site response was observed in groups 1, 2, and 3. Three weeks after the last vaccination, groups 1 and 4 underwent intradermal challenge (0.1 ml), with each group receiving challenge strains of *Erysipelothrix rhusiopathiae* serotype 1 and serotype 2 on the right and left sides of the chest. Clinical signs and rectal temperature were monitored in animals for 10 days, starting two days prior to challenge. Blood samples were collected on the day of each vaccination and challenge for serological testing of *Ery* and *Parvo*.

[0047] Group 1 showed an Ery antibody response, while the control group remained serum negative throughout. No HI response against Parvo was induced in any of the experimental groups. The results are shown in Table 1 below.

[0048] Table 1 Data after EPL-ID vaccination and challenge

[0049] None of the formulations were sufficiently safe (average / max local response was 5 / 7 cm for this test set), and the formulation tested for Ery efficacy was also insufficiently effective. Furthermore, the formulation failed to induce a Parvo serological response. These results lead to the conclusion that EPL is not feasible as an intradermal vaccine due to its failure to meet safety and efficacy requirements.

[0050] Example 2 Example 2 is a second trial of the Ery-Parvo-Lepto combined vaccine. In this trial, the ery, parvo, and lepto antigens were the same as in previous trials. However, instead of formulating all antigens in a single vaccine, they were separated into two separate vaccines: an EP vaccine and a L vaccine. These vaccines were simultaneously administered as separate vaccines to the dermis of pigs using the IDAL device.

[0051] For these experiments, three different adjuvants were used: the patented Emunade® and X-Solve® adjuvants (both of which are available from MSD Animal Health, Boxmeer, The Netherlands), and the aforementioned identified SVEA adjuvant.

[0052] Twenty healthy 18-week-old pigs that had not received any vaccine components were used. The pigs were divided into four groups of five animals each. Groups 1, 2, and 3 were vaccinated twice intradermally with EP-ID and L-ID (on the opposite side of the neck) at four-week intervals (using an IDAL device), using either a vaccine containing 50% of the antigen content compared to Porcilis EPL (Group 3) or a vaccine containing 12.5% ​​of the antigen content compared to Porcilis EPL (Groups 1 and 2). Group 1 received the adjuvant Emunade, Group 2 received the adjuvant X-Solve, and Group 3 received SVEA. Group 4 received an intramuscular vaccination with Porcilis EPL as a control.

[0053] Following vaccination, local injection site reactions were observed in all groups, and serological responses to Ery, Parvo, and three of the five Lepto antigens (i.e., Ictero, Pomona, and Tarassovi antigens) were measured.

[0054] The results are summarized in Tables 2 and 3 below (responses are expressed in relative units when absolute units are not provided). Note that due to field infection, all animals had high titers, making it impossible to determine parvo titers induced by vaccination.

[0055] Table 2. Data after EP-ID vaccination and challenge.

[0056] Table 3. Data after L-ID vaccination and challenge.

[0057] For each adjuvant, site reactions were found to be acceptable, therefore vaccination was considered safe. However, in terms of both safety and efficacy, oil-in-water adjuvants yielded better results, with the best results achieved using X-solve adjuvant. The latter is an oil dispersion in water, and unlike Emunade, it does not contain aluminum hydroxide.

[0058] Ery antibody responses were comparable across all formulations, at the same or higher levels compared to Porcilis EPL. Parvo vaccine titers typically ranged from 2 to 10 log2, while after field infection, titers could be >10 log2. As the results clearly show, field infection occurred during this study, making it impossible to draw conclusions regarding vaccine titers. Lepto antibody responses induced by different formulations (jaundice-hemorrhagic serotype, Pomona serotype, and Tarasov serotype) tended to be lower compared to Porcilis EPL, but were still considered effective (based on the known use of Porcilis EPL, any level of positive lepto titer generally equates to effective protection).

[0059] Example 3 Example 3 is the third trial of the Ery-Parvo-Lepto combination vaccine, designed to establish a parvo response to the best vaccine of Example 2 (i.e., the group 2 combination vaccine based on the oil-in-water adjuvant X-Solve). Additionally, the response to PRRS was evaluated when a live attenuated PRRS vaccine was administered during a booster EPL primaries.

[0060] Thirty-five healthy 12-week-old pigs weighing at least 20 kg, PRRS-negative, and Ery and Parvo serologically negative were used. The pigs were divided into four groups (n=10 per vaccine group and 5 per control group). Group 1 received the EP-ID vaccine (right side) from Group 2 of Example 2 twice (4 weeks apart), administered in combination with the L-ID vaccine (left side) from Group 2 of Example 2, without mixing. Additionally, during a booster vaccination, Porcilis PRRS was administered intradermally with the L-ID (left side) using Twin IDAL. Group 2 was vaccinated in the same manner as Group 1, except for the combined use of Porcilis PRRS. Group 3 received the same vaccination as Group 2, except that the EP-ID and L-ID vaccines contained only 25% of the antigen (thus approximately 3% compared to the antigen content of Porcilis EPL). Group 4 was unvaccinated and served as a negative control. All vaccines were administered intradermally in the neck using IDAL or Twin IDAL (0.2 ml). Local injection site reactions were observed in Groups 1 and 2 after vaccination. Blood samples were collected on the day of each vaccination and on the day of the challenge for serological testing of Parvo and PRRS.

[0061] At the start of the study, all pigs were seronegative against Ery, Parvo, Lepto, and PRRS. The results are shown in Table 4 below (local responses correspond to Tables 2 and 3).

[0062] Table 4. Post-EP-ID and L-ID vaccination data and post-challenge data.

[0063] Site reactions were acceptable. Serological results for Parvo and PRRS (established 52 days post-vaccination) were acceptable and considered to meet efficacy requirements. Post-vaccination PRRS titers were comparable to those available with the commercial product Porcilis PRRS, and parvo HI titers were consistent with those available with the commercial product Porcilis EPL.

[0064] In summary, the combined, non-mixed use of the individual EP-ID and L-ID vaccines, even when used in conjunction with a live attenuated PRRS vaccine, results in safe vaccination and effective protection against infections by Erysipelothrix rhusiopathiae, porcine parvovirus, various leptospirosis strains, and PRRS virus.

[0065] Example 4 Example 4 is the fourth trial of the Ery-Parvo-Lepto combined vaccine, designed to establish protection against L-pomona challenge. Essentially, the same experimental setup as in Example 3 was used, except that 40 six-week-old piglets were divided into four groups. These groups received the same vaccination as described in Example 3.

[0066] At 11 weeks of age, pigs were transported to the challenge site and intravenously challenged one week later with the Pomona serotype of Leptospira question mark. Clinical signs and / or behavioral and appetite abnormalities were observed daily for 2 weeks post-challenge. Rectal temperature was measured at scheduled time points until the end of the study, 2 weeks post-challenge. Serum blood was collected on the day of each vaccination, on the day of challenge, and 2 weeks post-challenge for antibody titer determination. Heparinized blood was collected before challenge, 24 and 28 hours post-challenge, and at 2, 3, 4, 7, and 10 days post-challenge for re-isolation of the challenge strain.

[0067] At the start of the study, all pigs were seronegative against Leptospira serogroup Pomona and PRRS virus. Following vaccination, Group 1 showed antibody responses to Ery, Parvo, and Lepto equal to or higher than Group 2, indicating that Porcilis PRRS had no negative impact on EP-ID and L-ID intake.

[0068] EP-ID and L-ID vaccination induced small, transient local reactions at the vaccination site (maximum diameter 4 cm and 3 cm, respectively). All reactions disappeared within 2 weeks post-vaccination. Vaccination did not induce any clinical abnormalities.

[0069] The results after the challenge are summarized in Table 5 below.

[0070] Table 5. Post-EP-ID and L-ID vaccination data and post-challenge data.

[0071] The results showed that vaccination with EP-ID in combination with L-ID and PRRS in pigs without mixing was safe. Furthermore, all three vaccine groups, including those with live attenuated PRRS vaccine and those with 25% normal antigen, induced complete protection against the Pomona serotype of Leptospira question mark serogroup.

Claims

1. A combination of a first vaccine comprising a non-replicating immunogen of *Erysipelothrix purpureus* and a non-replicating immunogen of porcine parvovirus, and a second vaccine comprising a non-replicating immunogen of *Leptospira*, for the prophylactic treatment of pigs against *Erysipelothrix purpureus* infection, porcine parvovirus infection, and *Leptospira* infection by injecting the first vaccine and the second vaccine together at multiple injection sites into the dermis of a first injection site and a second injection site.

2. The combination of the first and second vaccines according to claim 1, characterized in that... The first and second injection sites are at most 4 cm apart, preferably at most 3 cm apart.

3. The combination of the first and second vaccines used according to any one of the preceding claims, characterized in that... The first vaccine and the second vaccine were administered simultaneously at multiple injection sites.

4. The combination of the first and second vaccines used according to any one of the preceding claims, characterized in that... The first and second vaccines are administered using a primary and booster vaccination regimen, wherein both vaccines are administered at the primary immunization and both vaccines are administered at the booster vaccination.

5. The combination of the first and second vaccines used according to any one of the preceding claims, characterized in that... At least the first vaccine contains an adjuvant.

6. The combination of the first and second vaccines used according to any one of the preceding claims, characterized in that... Both the first and second vaccines contain adjuvants.

7. The combination of the first and second vaccines used according to any one of claims 5 or 6, characterized in that... The adjuvant is an oil-in-water adjuvant.

8. The combination of the first and second vaccines used according to any one of the preceding claims, characterized in that... The non-replicating immunogen of *Erysipelothrix rhusiopathiae* is *Erysipelothrix rhusiopathiae* vaccine, the non-replicating immunogen of porcine parvovirus is inactivated porcine parvovirus, and the non-replicating immunogen of *Leptospira* is *Leptospira question mark* vaccine.

9. The combination of the first and second vaccines according to claim 8, characterized in that... The second vaccine also contains Leptospira kiuri and / or Leptospira sandrosi.

10. The combination of the first and second vaccines used according to any one of the preceding claims, characterized in that... The third vaccine, which contains live attenuated PRRS virus, is injected into the pigs.

11. The combination of the first and second vaccines used according to claim 10, characterized in that... In the preventive treatment, the pigs receive a primary immunization with the first and second vaccines, as well as a booster immunization with the first and second vaccines, wherein the third vaccine is administered as a single-dose vaccine during the booster immunization.

12. The combination of the first and second vaccines used according to claim 11, characterized in that... The third vaccine is mixed with the second vaccine.

13. The combination of the first and second vaccines used according to any one of the preceding claims, characterized in that... The first and second vaccines are injected using a needle-free device via a jet of the respective vaccine, wherein the jet penetrates the pig's skin.

14. A component kit comprising a first vaccine containing a non-replicating immunogen of *Erysipelothrix purpureus* and a non-replicating immunogen of porcine parvovirus, and a second vaccine containing a non-replicating immunogen of *Leptospira*, for prophylactic treatment of pigs against *Erysipelothrix purpureus* infection, porcine parvovirus infection, and *Leptospira* infection by injecting the first vaccine and the second vaccine together at multiple injection sites into the dermis of a pig at a first injection site and a second injection site.

15. A method for the prophylactic treatment of pigs against infection by *Erysipelothrix rubrum*, porcine parvovirus, and *Leptospirosis*, comprising injecting a first vaccine containing a non-replicating immunogen of *Erysipelothrix rubrum* and a non-replicating immunogen of porcine parvovirus, and a second vaccine containing a non-replicating immunogen of *Leptospirosis*, into the dermis of the pigs at multiple sites.

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