Vaccine for leptospira bacteria

Abstract

The present invention relates to a vaccine comprising a non-replicating leptospira immunogen for use in the prophylactic treatment of a pig against infection by leptospira bacteria by injecting a first vaccine into the dermis of the pig.

Description

Technical Field

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

[0002] Pigs are susceptible to many infections and the diseases they cause. Leptospirosis, for example, is an infectious disease in pigs and many other animals, including humans, caused by infection with any of a large group of bacteria in the genus *Leptospira* (“lepto”). Leptospira 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 often studied under a dark-field microscope. They are difficult to culture in laboratory media and grow very slowly (12–26 weeks). Many pathogenic strains can survive for extended periods in slightly alkaline, moist conditions. Most cannot withstand drying and are destroyed by common disinfectants.

[0003] 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 the genus *Leptospira* 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 family Leptospiraceae contains eight pathogenic species, three of which are most important to pigs: *Leptospira questionmark* (…). Leptospira interrogans (Pomona serotype ( pomona ), Jaundice and Hemorrhage Serological Type ( icterohaemorrhagiae ), canine serotype ( canicola ) and Bratislava serotype ( bratislava Leptospira Bois ()), Leptospira Bois ( Leptospira borgpetersenii (Sero serotype ( sejroe ) and Tarasov serotype ( tarassovi )) and Leptospira girdius ( Leptospira kirschneri (Influenza and typhoid serological types ( grippotyphosa The Pomona and Bratislava serotypes exhibit unique adaptability to pigs; other serotypes are also present in other species but can sometimes infect pigs. Leptospira bratislava has been reported to... Leptospira bratislava (This is the most common strain in pigs, but the role of this serotype as a cause of disease is controversial.)

[0004] Control of Leptospira is typically achieved through preventative contact, vaccination, and / or immunization with antibiotics. Preventative contact is difficult to achieve because many species can be carriers of Leptospira. These include infected pigs, rodents (especially mice and rats), and many species of wild animals. Once introduced into a suitable, moist environment, Leptospira typically persists there as a source of infection. Nevertheless, Leptospirosis in closed-environment pig herds 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 with bacteria in breeding herds generally reduces the prevalence of infection and abortion. The bacteria must be adapted to the serotypes of the Leptospira species that cause the disease. Therefore, many vaccines are multivalent.

[0005] Another example of a common swine disease is erysipelas, an infectious disease caused by *Erysipelothrix rubrum*. This is one of the oldest recognized diseases affecting both growing and adult pigs. In intensive pig farming areas, up to 50% of pigs are believed to be colonized by *Erysipelothrix rubrum*. E. rhusiopathiae This organism is typically found in the tonsil tissue. Healthy carriers usually shed it in their feces or nasal / oral secretions, which is a significant source of infection for other pigs. Infection occurs through ingestion of contaminated feed, water, or feces, as well as through skin abrasions. After ingestion, the organism can survive the harsh environment of the stomach and intestines and may remain alive in feces for months. On farms where this organism is prevalent, pigs are naturally exposed to *Erysipelothrix rhusiopathiae* in their early years. Maternally derived antibodies provide passive immunity and suppress clinical disease. Older pigs often tend to develop protective active immunity from exposure to the organism, but this does not necessarily lead to clinical disease. Recovered pigs and pigs with chronic infections can become carriers of *Erysipelothrix rhusiopathiae*. Healthy pigs may also be asymptomatic carriers.

[0006] Vaccination against *Erysipelothrix rhusiopathiae* 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 bacterial vaccines and live attenuated vaccines, delivered by water, provide a longer duration of immunity. The optimal timing for vaccination may vary from farm to farm. When *Erysipelothrix rhusiopathiae* is prevalent in a production environment, vaccination should be administered before an anticipated outbreak. Susceptible pigs can be vaccinated before weaning, at weaning, or a few weeks after weaning. Boars and sows selected for breeding herds should receive a booster shot 3–5 weeks later. Thereafter, breeding stock should be vaccinated twice a year, with follow-up vaccinations. However, vaccination does not always directly achieve 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 can also lead to incomplete immunity and disease outbreaks, but this is a rare event because good cross-protection is believed among the major erysipelas strains that infect pigs.

[0007] 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 multiplies normally in the pig's intestines without causing clinical symptoms and is prevalent in pig herds worldwide. PPV is one of the organisms that cause stillbirth-mummified fetus-embryonic death-infertility (SMEDI) syndrome. In larger herds, it is almost certain to be present, making it an infection that must be coexisted with and managed rather than attempted to be eliminated. In smaller herds where pigs previously had PPV-positive pigs, they may have already died or not. Most viruses do not survive long outside the host, and PPV is unusual in that it can persist in the environment for months and is resistant to most disinfectants, most likely due to its widespread presence and difficulty in elimination. PPV is most commonly transmitted orally or through the mouth and nose, entering the intestines where it multiplies and is subsequently transmitted through feces. When pigs are first infected, there are no clinical signs. Boars can also infect sows through semen during mating.

[0008] 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 entering the breeding herd, coupled with annual follow-up vaccinations of all pigs, should be sufficient to ensure herd protection. It is well known that if infected breeding females were vaccinated against PPV at some point in the past, their immune system is rapidly restimulated (within 5-7 days) upon exposure to PPV. This is sufficient to prevent disease and stimulate (semi-)permanent immunity.

[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*. 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 a North American genotype emerged in China. This strain, HP-PRRSV, is more virulent than any other strain and has caused significant losses in Asian countries worldwide. Subclinical infection is common with any PRRS virus, with clinical signs appearing only occasionally in herds. 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 described above. Summary of the Invention

[0012] To achieve the objectives of this invention, it has been discovered that a non-replicating leptospiral immunogen can be successfully used for the prophylactic treatment of pigs against leptospiral bacterial infections by injecting the (first) vaccine into the dermis of pigs. This provides a convenient and safe method for vaccinating pigs. Although leptospiral vaccines can be used in pigs, even with the same antigen, a new application site cannot guarantee the production of a safe and effective vaccine.

[0013] For example, the World Health Organization (WHO) has published an e-learning course entitled "Fundamentals of Vaccine Safety" (available on the World Wide Web at https: / / apps.who.int / iris / handle / 10665 / 340576), which reports on page 53... The route of administration is the way a vaccine (or drug) comes into contact with the body. This is a key factor in successful immunization. The substance must enter the body through the route of administration. The organs are transported to the body parts where they need to take effect. However, achieving this using the body's transport mechanisms is not easy. thing . In this regard, the California Department of Health Services Immunization Service has published guidelines for proper immunization (https: / / www.cdc.gov / vaccines / pubs / pinkbook / vac-admin.html). Regarding the application site, the second full paragraph on page 81, entitled "Route and Site of Vaccination," states that... The recommended route and site for each vaccine are based on clinical trials. Experiment, practical experience and theoretical considerations . There are five routes of vaccination. Deviating from the recommended route may reduce vaccine effectiveness. Or increase local adverse reactions . In general, it is well known that administering a vaccine to a specific site is not a simple matter; experiments are needed to determine whether it is safe and effective, and if so, whether the level is sufficient for commercial use.

[0014] In addition to the vaccines described above, the present invention also relates to a method for the preventive treatment of pigs against infection with Leptospira bacteria, which involves injecting a first vaccine containing a non-replicating Leptospira immunogen into the dermis of the pig.

[0015] definition A vaccine is a pharmaceutical composition that can be safely administered to a test animal and induces protective immunity against a pathogenic microorganism in that animal, i.e., induces successful preventive treatment as defined below.

[0016] Applying a vaccine to the dermis 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 Animal Health), the Pulse 50 MicroDose (pulse needle-free system), or... VaccineOther 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).

[0017] The combination of the first and second vaccines is a set of vaccines in which the first vaccine is distinct from the second vaccine, but the two vaccines are used together for administration to the same subject in a single co-treatment.

[0018] A non-replicating immunogen of a pathogen is any substance or compound, not the whole, living replicating pathogen (whether wild-type or attenuated), that elicits an immune response against the pathogen, thereby enabling the host's immune system to recognize the corresponding virulent pathogen or one or more virulence factors and ultimately neutralize it, 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.

[0019] A vaccine is a suspension of inactivated bacteria, which may be whole-cell, partially lysed, or completely lysed (e.g., by homogenization, Farchurning, or a combination of two or more lysis methods).

[0020] A live attenuated pathogen is a live, replicating form of pathogen with reduced virulence. The attenuation process requires an infectious pathogen to be altered to make it harmless or less virulent, usually through multiple passages of the pathogen through cellular systems or through genetic modification of the pathogen.

[0021] Prophylactic treatment for pathogen infection can help prevent, improve or cure infection or symptoms caused by pathogen infection following treatment of a pathogen, particularly by reducing the burden of the pathogen in the host after such a challenge, or by helping to prevent or improve one or more clinical manifestations caused by infection following treatment of a pathogen.

[0022] Pigs are members of the family Suidae (order Artiodactylus), commonly known as pigs, hogs, or boars. There are currently 18 recognized extant species (or 19 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 ).

[0023] Combined administration of vaccines, also known as simultaneous administration, refers to administering these vaccines separately to the target animal so that they are not mixed before administration, but within the time frame where immune interference is expected, usually within 24 hours. Typical examples of combined use are simultaneous administration to different application sites in the target animal, or at intervals of up to 1-24 hours.

[0024] Simultaneous administration of vaccines refers to administration at exactly the same time or within a time frame of at least 1 hour, preferably within a time frame 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 1 minute, such as at precisely the same time.

[0025] A single dose of a vaccine used for preventive treatment means that, in order to achieve protective immunity, a booster dose of the vaccine is not required.

[0026] The primary and booster vaccination regimen refers to the use of a second dose of vaccine to reinforce the first vaccination in order to achieve protective immunity. Typically, a booster dose is administered within 6 weeks of the primary vaccination, usually 5 weeks or even 4 weeks after the primary vaccination, and after the second (booster) vaccination, sufficient protective immunity is usually achieved. Detailed Implementation

[0027] In a first further embodiment of the vaccine according to the invention (i.e., the "first" vaccine), the non-replicating Leptospira immunogen is Leptospira question mark. Preferably, the vaccine further comprises Leptospira kiribonucleic acid and / or Leptospira sandrosi ( Leptospira santarosai ) bacterial seedlings.

[0028] In another embodiment, the vaccine comprises an adjuvant. Although it is well known that Leptospira vaccines do not necessarily require adjuvants to achieve a high level of protection. Adjuvants are, for example, 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 emulsion-type adjuvants can be stabilized with emulsifiers such as polysorbate.

[0029] In one embodiment, the first vaccine according to the invention is injected using a needle-free device via a jet of the vaccine, wherein the jet penetrates the pig's skin and reaches the dermis.

[0030] In another embodiment, a first vaccine and a second vaccine are used in combination for the prophylactic treatment of pigs against infection by Leptospira bacteria, Erysipelas rubrum bacteria, and porcine parvovirus. The second vaccine comprises a non-replicating immunogen of Erysipelas rubrum bacteria and a non-replicating immunogen of porcine parvovirus. The first and second vaccines are administered by combined injection into the dermis of the pig at first and second injection sites, respectively. In cases where the typical intradermal vaccination volume is less than 1 mL, typically about 0.2 mL, if the first and second injection sites are more than 1.5-2 cm apart, the two vaccines will not mix under the skin, and the local reaction of each vaccine can be distinguished from the other. This means that, consistent with the EMA guidance on requirements of the Institute of Veterinary Medicines and Immunization (IVMP) (July 18, 2013, EMA / CVMP / IWP / 594618 / 2010), which defines "separate sites" as application sites "sufficiently far apart to prevent the possibility of product mixing and to allow for differentiation of local reactions of each product," the injection sites can be considered "separate" in the sense of this embodiment of the invention. In intradermal vaccination, since the two vaccines do not mix after administration, any distance between the first and second injection sites exceeding 1.5 cm, preferably exceeding 2 cm, will result in the same outcome of preventing negative interference with the safety and / or efficacy of the combined vaccine.

[0031] In a further embodiment of this combined vaccination, the first and second injection sites are at most 4 cm apart, preferably at most 3 cm apart, and even more preferably less than 2.5 cm, such as 2 cm. Studies have found that this method makes it easier to vaccinate pigs, particularly by using devices such as the IDAL® 3G Twin device (available from MSDAnimal Health, Boxmeer, The Netherlands; the distance between application sites is 2.8 cm), which has two containers containing separate vaccines for simultaneous intradermal injection of two vaccines.

[0032] A further preferred approach is to administer the first and second vaccines simultaneously at multiple injection sites.

[0033] In another preferred embodiment of the first vaccine combined with the second vaccine according to the present invention, the first and second vaccines are administered in a primary and booster vaccination schedule, wherein both vaccines are injected during the primary vaccination and both vaccines are injected during the booster vaccination.

[0034] In yet another embodiment, the non-replicating immunogen of erysipelas of pig is erysipelas of pig vaccine, and the non-replicating immunogen of porcine parvovirus is inactivated porcine parvovirus.

[0035] Advantageously, it has been found that the first vaccine used in conjunction with the second vaccine according to any of the preceding claims can also be used in combination with a third vaccine injected into pigs, the third vaccine comprising live attenuated PRRS virus, and achieving safe and effective combined vaccination against all four pathogens, thereby including protection against PRRS virus infection. Preferably, in prophylactic treatment, pigs receive a primary immunization using the first and second vaccines, and a booster immunization using the first and second vaccines, wherein the third vaccine is administered 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 first vaccine (i.e., the Leptospira vaccine) for the booster immunization.

[0036] The invention will now be explained in more detail using the following specific embodiments.

[0037] Example Example 1 is the first trial of the Ery-Parvo-Lepto combined vaccine.

[0038] Example 2 is the second trial of the Ery-Parvo-Lepto combined vaccine.

[0039] Example 3 is the third trial of the Ery-Parvo-Lepto combination vaccine.

[0040] Example 1 Example 1 is the first trial of the Ery-Parvo-Lepto combined vaccine. For this purpose, the existing vaccine Porcilis® EPL (containing serotype 2 of *Erysipelothrix rubella*, inactivated porcine parvovirus, and various *Leptospira* serotypes, in this case, *Leptospira question mark* serogroup jaundice hemorrhage serotype, *Leptospira question mark* serogroup Bratislava serogroup, *Leptospira kieri* serogroup influenza typhoid serogroup, *Leptospira question mark* serogroup Pomona serogroup Pomona, and *Leptospira stanislavis* serogroup Tarasov serogroup) was used, known to be a safe and effective vaccine for pigs, preventing *ery*, *parvo*, and *lepto* infections via intramuscular vaccination. These antigens are all inactivated pathogens. The aim of this example was to observe whether the vaccine could also be administered intradermally while still meeting the expected safety and efficacy requirements. The device selected for intradermal delivery was the IDAL® 3G Twin device (Twin IDAL). Since the volume of each application is much smaller than the recommended Porcilis EPL (i.e., 0.2 ml instead of 2 ml), the amount of antigen in Porcilis EPL needs to be reduced (to 40% and 25%, see below) to accommodate the smaller volume of the aqueous phase of the formulation.

[0041] Various adjuvants were used in the tests, including the patented adjuvant Diluvac Forte (DF; MSD AnimalHealth, Boxmeer, The Netherlands), µDiluvac forte (µDF; same as 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), denoted here as SVEA. With both Diluvac adjuvants, a 40% antigen concentration in PorcilisEPL could be formulated. With the squalene-based adjuvant, this was only 25%.

[0042] 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 regimen), and Porcilis PRRS was administered separately (as a single vaccine) during the booster vaccination period. Group 2 also received two ID vaccinations (in the primary-booster regimen), but this time with EPL-ID-40%-µDF, and Porcilis PRRS was administered separately during the booster vaccination period. Group 3 received two ID vaccinations with EPL-ID-25%-SVEA, and Porcilis PRRS was 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.

[0043] Following vaccination, the local injection site reactions were observed in groups 1, 2, and 3. Three weeks after the last vaccination, groups 1 and 4 underwent intradermal challenge (0.1 ml), with challenge strains of *Erysipelothrix rhusiopathiae* serotype 1 and serotype 2 administered to the right and left sides of the chest, respectively. Clinical signs and rectal temperature were monitored in animals for 10 days, starting two days prior to challenge. Blood samples were collected on each vaccination and challenge day for Ery and Parvo serological testing.

[0044] Group 1 showed an Ery antibody response, while the control group remained serum negative. No HI response against Parvo was induced in the experimental groups. The results are shown in Table 1 below.

[0045] Table 1. Post-vaccination and post-challenge data for EPL-ID

[0046] No formulation was sufficiently safe (avg / max local response was 5 / 7 cm for this test set), and the formulation tested for efficacy against Ery was also insufficiently effective. Furthermore, the formulation did not induce a serological response in Parvo. These results lead to the conclusion that EPL is not feasible as a vaccine for intradermal administration due to its failure to meet safety and efficacy requirements.

[0047] Example 2 Example 2 is the 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 divided into two separate vaccines, namely EP and L vaccines. These vaccines were simultaneously administered as separate vaccines into the dermis of pigs using the IDAL device.

[0048] 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.

[0049] 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). Emunade was used as an adjuvant in Group 1, X-Solve in Group 2, and SVEA in Group 3. Group 4 received an intramuscular vaccination with Porcilis EPL as a control.

[0050] 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.

[0051] The results are summarized in Tables 2 and 3 below (responses are expressed in relative units when no absolute units are given). Note that due to field infection, all animals had high titers, making it impossible to determine parvo titers induced by vaccination.

[0052] Table 2. Post-vaccination and post-challenge data for EP-ID.

[0053] Table 3. Post-vaccination and post-challenge data for L-ID

[0054] For each adjuvant, site reactions were found to be acceptable, therefore vaccination was considered safe. However, in terms of safety and efficacy, oil-in-water adjuvants yielded better results, with X-solve achieving the best results. The latter is an oil dispersion in water and, unlike Emunade, does not contain aluminum hydroxide.

[0055] Ery antibody responses were comparable across all formulations, at levels equal to or higher than those of Porcilis EPL. Parvo vaccine titers typically ranged from 2 to 10 log2, while titers could be >10 log2 after field infection. It is clear from the results that field infection occurred during the study period, therefore no conclusions can be drawn regarding vaccine titers. Lepto antibody responses induced by the different formulations (Ictero, Pomona, and Tarassovi) tended to be lower than those induced by 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).

[0056] 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 administered a live attenuated PRRS vaccine concurrent with a booster EPL primiparous immunization.

[0057] 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 (left side) with the L-ID 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 (therefore, approximately 3% compared to the antigen content of Porcilis EPL). Group 4 was unvaccinated and served as a negative control. All vaccinations 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 was collected on each vaccination day and challenge day for Parvo and PRRS serological testing.

[0058] 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).

[0059] Table 4. Post-vaccination and post-challenge data for EP-ID and L-ID.

[0060] 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 obtained with the commercially available Porcilis PRRS product, and parvo HI titers were consistent with those obtained using the commercially available Porcilis EPL product.

[0061] In summary, the combined, non-mixed use of the individually safe and effective vaccines EP-ID and L-ID, even when used in conjunction with a live attenuated PRRS vaccine, results in safe vaccination and effective protection against infections caused by swine erysipelas, porcine parvovirus, various Leptospira bacteria, and PRRS virus.

[0062] 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. The vaccinations administered to these groups were the same as those described in Example 3.

[0063] At 11 weeks of age, pigs were transported to the challenge site and challenged intravenously 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 specified time points until the end of the study, 2 weeks post-challenge. Serum blood samples were collected on each vaccination day, challenge day, and 2 weeks post-challenge for antibody titer determination. Heparinized blood samples were collected before challenge, 24 and 48 hours post-challenge, and at 2, 3, 4, 7, and 10 days post-challenge for re-isolation of the challenge strain.

[0064] At the start of the study, all pigs were seronegative against Leptospira pomona serotype and PRRS virus. Following vaccination, group 1 showed similar or higher antibody responses to Ery, Parvo, and Lepto compared to group 2, indicating that Porcilis PRRS had no negative impact on EP-ID and L-ID intake.

[0065] EP-ID and L-ID vaccination induced small, transient local reactions at the vaccination site (maximum diameters of 4 and 3 cm, respectively). All reactions disappeared within 2 weeks post-vaccination. Vaccination did not induce any clinical abnormalities.

[0066] The results after the challenge are summarized in Table 5 below.

[0067] Table 5 shows the post-challenge data for EP-ID and L-ID.

[0068] The results showed that vaccination with EP-ID in combination with L-ID and PRRS in pigs was safe. Furthermore, all three vaccine groups, including those used in combination with live attenuated PRRS vaccine and those containing 25% of the normal antigen, induced complete protection against Leptospira pormona serogroup.

Claims

1. A first vaccine comprising a non-replicating Leptospira genus immunogen, used for prophylactic treatment of pigs against Leptospira genus infection by injecting the first vaccine into the dermis of the pig.

2. The first vaccine used according to claim 1, wherein the non-replicating Leptospira immunogen is a Leptospira question mark vaccine.

3. The first vaccine used according to claim 2, characterized in that... The first vaccine also contains Leptospira kiuri and / or Leptospira sandrosi.

4. The first vaccine used according to any one of the preceding claims, characterized in that... The vaccine contains an adjuvant.

5. The first vaccine used according to claim 4, characterized in that... The adjuvant is an oil-in-water adjuvant.

6. The first vaccine used according to any one of the preceding claims, characterized in that... The first vaccine is injected using a needle-free device via a jet of the vaccine, wherein the jet penetrates the pig's skin.

7. The first vaccine used according to any one of the preceding claims, characterized in that... The first and second vaccines are used in combination for the prophylactic treatment of pigs against infection by Leptospira bacteria, Erysipelothrix rhusiopathiae, and porcine parvovirus. The second vaccine contains a non-replicating immunogen of Erysipelothrix rhusiopathiae and a non-replicating immunogen of porcine parvovirus. The first and second vaccines are injected into the dermis of the pig at two injection sites, respectively.

8. The first vaccine used in combination with the second vaccine according to claim 7, characterized in that... The first and second injection sites are at most 4 cm apart, preferably at most 3 cm apart.

9. The first vaccine used in combination with the second vaccine according to claim 7 or claim 8, characterized in that... The first vaccine and the second vaccine were administered simultaneously at multiple injection sites.

10. The first vaccine used in combination with the second vaccine according to any one of claims 7 to 9, characterized in that... The first and second vaccines are administered in a primary and booster vaccination regimen, wherein both vaccines are administered at the time of the primary vaccination and at the time of the booster vaccination.

11. The first vaccine used in combination with the second vaccine according to any one of claims 7 to 10, characterized in that... The non-replicating immunogen of swine erysipelas is a swine erysipelas vaccine, and the non-replicating immunogen of porcine parvovirus is inactivated porcine parvovirus.

12. The first vaccine used in combination with the second vaccine according to any one of claims 7 to 11, characterized in that... The pigs were injected with a third vaccine, which contained live attenuated PRRS virus.

13. The first vaccine used in combination with the second vaccine according to claim 12, characterized in that... In the preventive treatment, the pigs receive a primary immunization with the first and second vaccines, and a booster immunization with the first and second vaccines, wherein the third vaccine is administered as a single dose during the booster immunization.

14. The first vaccine used in combination with the second vaccine according to claim 13, characterized in that... The third vaccine is mixed with the first vaccine.

15. A method for the preventive treatment of pigs against infection by Leptospira bacteria, comprising injecting a first vaccine containing a non-replicating Leptospira immunogen into the dermis of the pig.

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