vaccine against Leptospira

Abstract

The present invention relates to a vaccine containing a non-replicating Leptospira immunogen for use in the prophylactic treatment of pigs against Leptospira infections by injecting a first vaccine into the dermis of the pig.

Description

Technical Field

[0001] The present invention relates to prophylactic treatment of pigs by vaccination against infection with Leptospira bacteria, and optionally prophylactic treatment against infection with Erysipelothrix rhusiopathiae and infection with porcine parvovirus.

Background Art

[0002] Pigs are susceptible to many infectious diseases and disorders caused by such infectious diseases, such as leptospirosis. This is an infectious disease of pigs and many other animals (including humans), and is caused by infection with any one of a large group of Leptospira bacteria ("leptos"). Leptospira is a motile spirochete that is 6 to 12 microns long and 0.1 micron in diameter. They are usually hooked at both ends and can be stained with Giemsa stain, or in tissues, can be stained with silver stain. In the laboratory, they are often studied under a dark field microscope. They are difficult to culture in laboratory media and grow extremely slowly (12 to 26 weeks). Many pathogenic strains can survive for a long time in a moist environment with a weakly alkaline pH. Most are not resistant to drying and are killed by common disinfectants.

[0003] Leptospirosis in pigs is universal, but the details of infection can differ depending on the serotype. Pigs are susceptible to many different serotypes. The classification of Leptospira is mainly based on DNA association with known reference strains, but there is still no universal agreement on the classification of the genus. In the latest classification, the family Leptospiraceae includes eight pathogenic species, three of which are most important for pigs: Leptospira interrogans (serotypes pomona, icterohaemorrhagiae, canicola, and bratislava), Leptospira borgpetersenii (serotypes sejroe and tarassovi), and Leptospira kirschneri (serotype grippotyphosa). Serotypes pomona and bratislava are specifically adapted to pigs, while other serotypes are maintained in other species but occasionally infect pigs. Leptospira bratislava is reported to be the most common strain in pigs, although its role as a disease-causing agent is debated.

[0004] Control of Leptospira infections is typically attempted through exposure prevention, immunization by vaccination, and / or the use of antibiotics. Exposure prevention is difficult to achieve because a wide variety of species can be carriers of Leptospira. These include infected pigs, rodents (especially mice and rats), and many types of wild animals. Once Leptospira are introduced into a suitable moist environment, they often persist there as a source of infection. However, in pig populations raised under restricted conditions, leptospirosis can be effectively controlled (and possibly even eradicated) by using a combination of drug therapy, vaccination, vector control, and providing treated or uncontaminated drinking water sources. Immunization with bacterins, which is widely practiced in breeding populations, usually reduces infection and miscarriage rates. The bacterins must be appropriate for the serotype of the pathogenic Leptospira; therefore, many vaccines are polyvalent.

[0005] Another example of a common disease in pigs is erysipelothrix, an infection caused by Erysipelothrix rhusiopathiae ("ery"). This is one of the oldest recognized diseases affecting growing and adult pigs. It is estimated that up to 50% of pigs in intensive pig-producing areas are infected with E. rhusiopathiae. This microorganism is usually found in the amygdala tissue. Typically, healthy carrier pigs excrete the microorganism in their feces or oral and nasal secretions, and these pigs are a significant source of infection for other pigs. Infection occurs through ingestion of contaminated feed, water, or feces, and through skin abrasions. If ingested, the microorganism can survive through the harsh environment of the gastrointestinal tract and can survive in feces for several months. On farms where this microorganism is endemic, pigs are naturally exposed to E. rhusiopathiae during their youth. Maternal antibodies provide passive immunity and suppress clinical disease. Older pigs tend to develop protective active immunity as a result of exposure to the microorganism, which does not necessarily lead to clinical disease. Recovered and chronically infected pigs can become carriers of Erysipelas rhusiopathiae. Healthy pigs can also be asymptomatic carriers.

[0006] Vaccination against E. rhusiopathiae is highly effective in controlling disease outbreaks in pig farms and should be recommended. However, it may not be effective in preventing chronic arthritis. In some farms, discontinuation of vaccination has been associated with disease outbreaks. Attenuated live vaccines delivered by injection bacterin and drinking water are available and provide long-lasting immunity. The optimal timing for vaccination may vary from farm to farm. If E. rhusiopathiae is endemic in the production environment, vaccination should be carried out before an expected outbreak. Susceptible pigs can be vaccinated before weaning, at weaning, or for several weeks after weaning. Male and female pigs selected for inclusion in breeding groups should be vaccinated with a booster vaccine 3-5 weeks later. Subsequently, breeding stock requires twice-yearly vaccinations and follow-up vaccinations. However, vaccination is not always easy to achieve protective effects. Vaccination may fail in some groups due to management stress that impairs the immune system of vaccinated pigs. The use of live vaccines can also cause clinical diseases (particularly chronic erysipelas). Antigenic differences between serotypes in the vaccine and those circulating on the farm can also lead to incomplete immunity and disease development, although this is rare. This is because good cross-protection is thought to exist among the major strains of E. rhusiopathiae that infect pigs.

[0007] Furthermore, porcine parvovirus infection (PPV, or "parvo") is also a common pathogen in pigs. It is a significant cause of infectious infertility. PPV is a robust virus that replicates normally in the pig intestines without causing clinical symptoms and is ubiquitous in pig populations worldwide. PPV is one of the microorganisms listed as a cause of stillbirths, mummification, embryonic death, and infertility (SMEDI) syndrome. It is almost certainly present in large herds and is an infection that should be managed and coexisted with rather than eliminated. In small herds where there have been pigs that were previously PPV-positive, the virus may or may not have disappeared. Most viruses do not survive outside of a host for long periods. PPV is unusual in that it can survive in the environment for several months and is resistant to most disinfectants. This is the most likely reason why PPV is widespread and difficult to eradicate. PPV is most often transmitted through the mouth or nose, passes through the intestines where it multiplies, and is then excreted in the feces. When pigs are infected for the first time, there are no clinical signs. Male pigs can also infect female pigs through semen during mating.

[0008] PPV cannot be eradicated from herds; therefore, the goal is the management and prevention of acute cases. Routine vaccination of sows and boars before entering the breeding group, in addition to annual follow-up vaccination of all pigs, is sufficient to reliably protect the herd. It is known that if an infected breeding sow has been vaccinated against PPV at some point in the past and is subsequently exposed to PPV, its immune system is rapidly restimulated (within 5-7 days). This is sufficient to prevent the disease and stimulate (semi-)permanent immunity.

[0009] PRRSV was first reported in North America and Central Europe in 1987. PRRSV is a small, enveloped RNA virus. It contains a single-stranded positive-sense RNA genome approximately 15 kilobases in size. Its genome contains nine open reading frames. The virus belongs to the order Nidovirales, family Arteriviridae, and genus Arterivirus. There are two prototype strains of PRRSV: the North American strain (VR-2332) and the European strain (Lelystad virus (LV)). The European and North American PRRSV strains present with 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 pathogenic than all other strains and has caused significant damage in Asian countries. In all types of PRRS virus, asymptomatic infection is common, and clinical signs appear only sporadically within the herd. Clinical signs include reproductive disorders in sows, such as miscarriage, stillbirth, or birth of mummified fetuses, as well as cyanosis of the ears and vulva. In newborn piglets, the disease causes respiratory distress and increases susceptibility to respiratory infections such as Glaser's disease.

[0010] Vaccine compositions for all the pathogens identified above are generally known. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The object of the present invention is to provide a novel method for convenient, safe, and effective vaccination against one or more porcine pathogens mentioned above. [Means for solving the problem]

[0012] To achieve the objectives of this invention, it has been found that non-replicating Leptospira immunogen can be successfully used in the prophylactic treatment of pigs against Leptospira infection by injecting the (first) vaccine into the dermis of pigs. This makes it possible to perform safe and effective vaccination of pigs in a simple manner. Although Leptospira vaccines are available for pigs, it is not a given that a new administration site will result in a safe and effective vaccine, even if the antigen is the same.

[0013] For example, the World Health Organization (WHO) has published an e-learning course called "Vaccine Safety Basics" (https: / / apps.who.int / iris / handle / 10665 / 340576), which explains on page 53 that "the route of administration is the path by which a vaccine (or drug) comes into contact with the body. This is a crucial element for successful immunity acquisition. The substance must be delivered from the site of introduction to the part of the body where its effect is desired to occur. However, using the body's transport mechanisms for this purpose is by no means straightforward."

[0014] In this regard, the California Department of Health Services' Immunization Branch has published guidelines for proper vaccination (which can be viewed on the World Wide Web at "https: / / www.cdc.gov / vaccines / pubs / pinkbook / vac-admin.html"). Regarding the site of administration, on page 81, in the second paragraph titled "Route and Site for Vaccination," it states that "the recommended routes and sites for each vaccine are based on clinical trials, practical experience, and theoretical considerations. Five routes are used for vaccine administration. Deviation from the recommended route may reduce the effectiveness of the vaccine or increase local adverse reactions."

[0015] Generally speaking, it is known that vaccinating specific sites is not straightforward and requires experimental verification to determine whether safety and efficacy can be achieved, and if so, whether the level is sufficient for commercial use.

[0016] In addition to the vaccine described above, the present invention relates to a method for prophylactically treating pigs against Leptospira infection by injecting a first vaccine containing a non-replicating Leptospira immunogen into the dermis of the pigs.

[0017] definition A vaccine is a pharmaceutical composition that is safe to administer to a target animal and can induce protective immunity against pathogenic microorganisms in that animal, i.e., a pharmaceutical composition that can successfully induce prophylactic treatment as defined below.

[0018] Administering a vaccine into the dermis means that the vaccine will be deposited in the dermis, at least partially. The World Health Organization (WHO), in its August 27, 2009 paper, "Intradermal Vaccine Administration: A Literature Review on the Potential for Development in Low- and Middle-Income Countries," indicated that "needle-free" vaccination does not necessarily mean "intradermal" administration (see Table 1, page 3 of the above review). A vaccine can only be delivered into the dermis if the needle-free device is "configured for intradermal administration." Otherwise, it may be administered subcutaneously or intramuscularly. Several devices are commercially available for intradermal vaccination. For example, the IDAL® Vaccine Vaccine (MSD Animal Health), the Pulse 50 MicroDose (Pulse Needle Free Systems), or other devices described in Vaccine (2012 Jan 11;30(3):523-38) (see Table 1, page 525, "An overview of different devices for liquid and solid formulation administration").

[0019] The combination of the first and second vaccines constitutes a vaccine set, where the first vaccine is distinct from the second vaccine, but the two vaccines are used together to administer to the same target in a single coordinated treatment.

[0020] Non-replicating immunogens of pathogens are any substance or compound corresponding to a pathogen other than the pathogen itself (wild-type or attenuated form), which is alive and capable of replication as a whole, and which induces an immunological response to the pathogen, thereby causing one or more of the corresponding infectious pathogen or its pathogenic factors to be recognized by the host immune system as a result of this immune response and ultimately at least partially neutralized. Typical examples of non-replicating immunogens include dead whole pathogens (this term includes the pathogen in soluble form) and subunits of these pathogens, such as capsid proteins, surface-expressed molecules (e.g., recombinant-expressed proteins or lipopolysaccharides), and secreted molecules (e.g., toxins).

[0021] Bacterins are suspensions of dead bacteria, either whole cells, partially lysated, or completely lysated (e.g., by homogenization, French press, or a combination of two or more lysis methods).

[0022] A living attenuated pathogen is a form of a pathogen with reduced pathogenicity that retains viable replication ability. The attenuation process involves obtaining an infectious pathogen and then modifying it to become harmless or to reduce its pathogenicity, typically by subjecting it to multiple passages in a cell line or by genetic modification.

[0023] Prophylactic treatment for pathogenic infection aims to promote the prevention, mitigation, or cure of infection or disease resulting from a challenge by the pathogen after treatment, particularly to reduce the pathogenic burden on the host after such challenge, or to promote the prevention or mitigation of one or more clinical symptoms resulting from infection by the pathogen after treatment.

[0024] Pigs belong to the family Suidae, which is a family of even-toed ungulate mammals, and are commonly referred to as pigs, hogs, or boars. There are currently 18 recognized extant species (or 19 if domestic pigs and wild boars are counted separately), classified into 4 to 8 genera. Among these genera, the genus Sus includes domestic pigs ("Sus scrofa domesticus" or "Sus domesticus").

[0025] Separate administrations of related vaccines (also referred to as co-administrations) are administrations to the subject animal of these vaccines separately (thus, without mixing the vaccines before administration), but within a time frame in which immunological interference is expected to occur (typically, within 24 hours). Typical examples of related uses are co-administration at separate administration sites of the subject animal or administration with an interval of up to 1 to 24 hours.

[0026] Co-administration of vaccines is administration at exactly the same time or within a time frame of at least 1 hour, preferably within 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 within 1 minute (e.g., exactly the same time).

[0027] Single-dose administration of a vaccine for use in prophylactic treatment means that the vaccination does not require a booster vaccination by a second administration of the vaccine in order to acquire protective immunity.

[0028] Prime and booster vaccination schemes mean that, in order to acquire protective immunity, a second vaccine is administered after the first vaccination to boost. Typically, the prime vaccination is boosted within 6 weeks, generally within 5 weeks or within 4 weeks, from the first vaccination, and it is common to obtain sufficient protective immunity after the second (booster) vaccination.

Modes for Carrying Out the Invention

[0029] In a first further embodiment of the vaccine according to the present invention (i.e., the "first" vaccine), the non-replicating Leptospira immunogen is a Leptospira interrogans bacterin. Preferably, the vaccine further comprises a Leptospira kirschneri bacterin and / or a Leptospira santarosai bacterin.

[0030] In another embodiment, the vaccine includes an adjuvant, despite the fact that it is generally known that leptospirosis vaccines do not necessarily require an adjuvant to obtain a high level of protection. The adjuvant is, for example, an oil-in-water adjuvant. Typical adjuvants used in the present invention are oil-in-water adjuvants, such as an emulsion of mineral oil in water or an emulsion of mineral oil and vitamin E acetate in water. These adjuvants can be stabilized with an emulsifier such as a polysorbate.

[0031] In one embodiment, the first vaccine used according to the present invention is injected by a jet stream of vaccine using a needleless device, the jet stream penetrating the skin of the pig and reaching the dermis.

[0032] In yet another embodiment, the first vaccine is intended for use in combination with the second vaccine in the prophylactic treatment of pigs from infections caused by Leptospira, Erysipelothrix rhusiopathiae, and porcine parvovirus, by injecting the first vaccine and the second vaccine into the dermis of the pig at the first and second injection sites, respectively, by associated separate injections, wherein the second vaccine contains a non-replicating immunogen of Erysipelothrix rhusiopathiae and a non-replicating immunogen of porcine parvovirus. A typical intradermal vaccine dose is less than 1 mL (typically about 0.2 mL), and if the first and second injection sites are separated by 1.5–2 cm or more, the two vaccines do not mix subcutaneously, and the local reactions of each vaccine can be distinguished from each other. This means that the injection sites can be considered "separate sites" in the sense of this embodiment of the present invention, in accordance with the EMA Guideline dated 18 July 2013 (EMA / CVMP / IWP / 594618 / 2010) on requirements for combinations of concomitant vaccines and immunological veterinary drugs (IVMPs) [wherein the Guideline defines "separate sites" as administration sites that are "sufficiently far apart from each other in order to prevent the possibility of mixing of the products and to enable the local reactions to each product to be distinguished from each other"]. Accordingly, in the case of intradermal vaccination, any distance greater than 1.5 cm (preferably greater than 2 cm) between the first and second injection sites will result in no mixing of the two vaccines after administration, thus preventing negative interference with the safety and / or efficacy of the associated vaccination of those two vaccines.

[0033] In a further embodiment of this combination vaccine, the first and second injection sites are separated by a maximum of 4 cm, preferably a maximum of 3 cm, and more preferably less than 2.5 cm (e.g., 2 cm). This method has been found to allow for more convenient vaccination of pigs, particularly by using the IDAL® 3G Twin device (available from "MSD Animal Health, Boxmeer, The Netherlands"; the distance between the administration sites is 2.8 cm) (which has two barrels and holds separate vaccines) for simultaneous intradermal injection of two vaccines.

[0034] Even more preferable is that the associated separate injections of the first and second vaccines are administered simultaneously.

[0035] In another preferred embodiment of the combination of the first and second vaccines for use according to the present invention, the first and second vaccines are administered in a prime and booster vaccination scheme, where both vaccines are injected at the time of prime vaccination and both vaccines are injected at the time of booster vaccination.

[0036] In yet another embodiment, the non-replicating immunogen of Erysipelothrix rhusiopathiae is Erysipelothrix rhusiopathiae bacterin, and the non-replicating immunogen of porcine parvovirus is inactivated porcine parvovirus.

[0037] Advantageously, a combination of the first and second vaccines for use according to any of the preceding claims can also be used in combination with a third vaccine injected into the pig, wherein the third vaccine contains live attenuated PRRS virus, and it has been found that safe and effective combination vaccination against all four pathogens is achieved, including protection against infection by the PRRS virus. Preferably, in prophylactic treatment, the pig receives a prime vaccination with the first and second vaccines, and a booster vaccination with the first and second vaccines, wherein the third vaccine is injected as a single-dose vaccine at the time of the booster vaccination (i.e., simultaneously with or at least within 24 hours of the booster vaccination). In a further embodiment, the third vaccine (i.e., the PRRS vaccine) is mixed with the first vaccine (i.e., the leptospirosis vaccine) in the booster vaccination.

[0038] The present invention will be described in more detail using the following specific examples. [Examples]

[0039] Examples Example 1 is the first trial of the Ery-Parvo-Lepto combination vaccine.

[0040] Example 2 is the second trial of the Ery-Parvo-Lepto combination vaccine.

[0041] Example 3 is the third trial of the Ery-Parvo-Lepto combination vaccine.

[0042] Example 1 Example 1 is the first trial of the Ery-Parvo-Lepto combination vaccine. For this, an existing vaccine, Porcilis® EPL (which contains E. rhusiopathiae serotype 2 bacterin, inactivated porcine parvovirus, and various leptospirabacterins (in this case, L. interrogans serogroup Icterohaemorrhagiae, L. interrogans serogroup Australis serotype Bratislava, L. kirschneri serogroup Grippotyphosa, L. interrogans serogroup Pomona serotype Pomona, and finally, L. santarosai serogroup Tarassovi) was used. This vaccine is known to be a safe and effective vaccine for use in pigs by intramuscular vaccination against ery, parvo, and lepto infections. Its antigens are all inactivated pathogens. The objective was to determine whether this vaccine could be administered intradermally and whether it met the predetermined safety and efficacy requirements. The device chosen for intradermal delivery was the IDAL® 3G Twin device (Twin IDAL). Since the volume per dose was substantially less than the recommended amount for Porcilis EPL (i.e., 0.2 mL instead of 2 mL), it was necessary to reduce the amount of Porcilis EPL antigen to fit into the smaller volume of the aqueous phase of the formulation (by 40% and 25%, details below). The study used a variety of adjuvants, namely the proprietary adjuvants Diluvac Forte (DF; MSD Animal Health, Boxmeer, The Netherlands), μDiluvac Forte (μDF; similar to Diluvac but with fewer surfactants and no mineral oil), and an experimental adjuvant containing squalane, vitamin E acetate, and silica (see WO2021 / 048338) (referred to herein as SVEA). The two Diluvac adjuvants were able to formulate 40% of the antigen present in Porcilis EPL. This was only 25% for the squalene-based adjuvant.

[0043] The experiment used healthy 12-week-old pigs that were seronegative for PRRS and Ery. The pigs were divided into four groups. Each vaccine group consisted of 10 pigs, and the control group consisted of 5 pigs. Group 1 was vaccinated twice intradermally (ID) with EPL-ID-40%-DF (prime-booster scheme), and Porcilis PRRS was administered separately (as a separate vaccine) at the time of booster vaccination; Group 2 was also vaccinated twice with ID (prime-booster scheme), but in this group, EPL-ID-40%-μDF and Porcilis PRRS were administered separately without mixing at the time of booster vaccination. Group 3 was vaccinated twice with EPL-ID-25%-SVEA with ID, and Porcilis PRRS was administered separately at the time of booster vaccination. Group 4 was not vaccinated. All vaccines were administered intradermally (0.2 mL) to the neck (right side for the first dose, left side for the booster) using an IDAL device at 4-week intervals.

[0044] After vaccination, groups 1, 2, and 3 were observed for local injection site reactions. Three weeks after the final vaccination, groups 1 and 4 received intradermal challenge doses (0.1 mL) of serotype 1 and serotype 2 challenge strains of Erysipelas rhusiopathiae, respectively, to the right and left sides of their respective chests. Clinical signs and rectal temperature were monitored in the animals for 10 days, starting two days before the challenge. Blood samples were collected for serological testing of Ery and Parvo on each vaccination day and the challenge day.

[0045] Group 1 showed an Ery antibody response, while the control group remained seronegative. None of the tested pigs induced an HI response to Parvo. The results are shown in Table 1 below. [Table 1]

[0046] None of the above formulations demonstrated sufficient safety (in this study, the mean / maximum local reaction was set at 5 / 7 cm), and the formulation tested for efficacy against Ery also did not demonstrate sufficient efficacy. Furthermore, this formulation did not induce a serological response to Parvo. From these results, it can be concluded that EPL is not feasible as an intradermal vaccine because it does not meet the safety and efficacy requirements.

[0047] Example 2 Example 2 is the second trial of the Ery-Parvo-Lepto combination vaccine. In this trial, the ery, parvo, and lepto antigens were the same as in the previous trial. However, instead of combining all antigens into a single vaccine, they were divided into two separate vaccines (i.e., EP vaccine and L vaccine). These vaccines were administered simultaneously as separate vaccines to the dermis of pigs using an IDAL device.

[0048] The experiment used three different adjuvants: the proprietary Emunade® adjuvant and X-Solve® adjuvant (both oil-in-water adjuvants available from MSD Animal Health, Boxmeer, The Netherlands), and the SVEA adjuvant identified above.

[0049] Twenty healthy 18-week-old pigs that had not been vaccinated against any of the vaccine components were used. These pigs were divided into four groups of five pigs each. Groups 1, 2, and 3 were intradermally vaccinated twice with EP-ID and L-ID (opposite side of the neck) at 4-week intervals (using an IDAL device). The vaccines used were either a vaccine containing 50% of the antigen compared to Porcilis EPL (group 3) or a vaccine containing 12.5% ​​of the antigen compared to Porcilis EPL (groups 1 and 2). As adjuvants, Emunade was used in group 1, X-Solve in group 2, and SVEA in group 3. Group 4 was intramuscularly vaccinated with Porcilis EPL as a control vaccine.

[0050] After vaccination, all groups were observed for local injection site reactions, and serological responses to Ery antigen, Parvo antigen, and three of the five Lepto antigens (Ictero antigen, Pomona antigen, and Tarassovi antigen) were measured.

[0051] The results are summarized in Tables 2 and 3 below (unless absolute units are given, responses are in relative units). However, because the animals were infected in the field (all animals showed high titers), the parvo titer after vaccination could not be measured. [Table 2]

[0052] [Table 3]

[0053] For each adjuvant, the site reaction was within acceptable limits, and therefore, vaccination was deemed safe. However, better results were obtained with oil-in-water adjuvants, and the best results in terms of both safety and efficacy were obtained with the X-solve adjuvant. The latter is an oil dispersion in water and, unlike Emunade, does not contain aluminum hydroxide.

[0054] Ery antibody responses were comparable across all formulations and were at or above the level of Porcilis EPL. Parvo vaccine titers are typically in the range of 2–10log2, but titers exceeding 10log2 may be observed after field infection. From the above results, it is clear that field infection occurred during the study period, and therefore, it is not possible to draw conclusions regarding vaccine titers. Lepto antibody responses (Ictero, Pomona, and Tarassovi) induced by different formulations tended to be lower compared to Porcilis EPL, but were still considered effective (as is known from the use of Porcilis EPL, any level of positive Lepto titer is usually equivalent to effective protection).

[0055] Example 3 Example 3 is the third trial of the Ery-Parvo-Lepto combination vaccine, and its purpose is to establish the Parvo response of the best-performing vaccine in Example 2 (i.e., the combination vaccine in Group 2) based on the oil-in-water adjuvant X-Solve. Furthermore, the response to PRRS was also evaluated when the attenuated live PRRS vaccine was administered after boosting with the EPL Prime vaccine.

[0056] Thirty-five healthy 12-week-old piglets weighing over 20 kg, negative for PRRS, and seronegative for Ery and Parvo were used. These piglets were divided into four groups (10 piglets in each vaccine group and 5 in the control group). Group 1 received two doses (4 weeks apart) of the ID vaccine (right) from Group 2 of Example 2, unmixed with the L-ID vaccine (left) from Group 2 of Example 2. Furthermore, for booster vaccination, Porcilis PRRS was administered intradermally (left) using Twin IDAL, unmixed with L-ID. Group 2 was vaccinated in the same manner as Group 1, except for the relevant use of Porcilis PRRS. Group 3 was vaccinated in the same manner as Group 2, but the EP-ID and L-ID vaccines had an antigen content of only 25% (and therefore approximately 3% compared to the antigen content of Porcilis EPL). Group 4 was not vaccinated as a negative control. All vaccinations were administered intradermally (0.2 mL) to the neck using IDAL or Twin IDAL. After vaccination, groups 1 and 2 were observed for local injection site reactions. Blood samples were collected for serological testing of Parvo and PRRS on each vaccination day and challenge vaccination day.

[0057] At the start of the study, all pigs were serologically negative for Ery, Parvo, Lepto, and PRRS. The results are shown in Table 4 below (the local reactions shown correspond to Tables 2 and 3). [Table 4]

[0058] The site reaction was within acceptable limits. Serological tests for Parvo and PRRS (established 52 days after vaccination) were within acceptable limits and were deemed to meet the efficacy requirements. Post-vaccination PRRS titers were equivalent to those obtained with the commercially available Porcilis PRRS, and Parvo HI titers were consistent with those obtained with the commercially available Porcilis EPL.

[0059] In conclusion, the relevant non-combined use of the safe and effective separate vaccines EP-ID and L-ID, even when used in combination with the attenuated live PRRS vaccine, results in safe vaccination and is effective in protecting against infections caused by Erysipelothrix rhusiopathiae, porcine parvovirus, various Leptospira species, and PRRS virus.

[0060] Example 4 Example 4 is the fourth trial of the Ery-Parvo-Lepto combination vaccine, with the objective of establishing protection against L-pomona challenge. The experimental setup was essentially the same as in Example 3, but this experiment used 40 six-week-old piglets, divided into four groups. The vaccination of these groups was identical to that described in Example 3.

[0061] At 11 weeks of age, the pigs were transferred to a challenge vaccination facility, and one week later, they were intravenously challenged with Leptospira interrogans serotype Pomona. For two weeks post-challenge, the pigs were observed for clinical signs and / or behavioral and appetite abnormalities. Rectal temperature was measured regularly until the end of the study, two weeks post-challenge. Serum blood was collected on each vaccination day, the challenge vaccination day, and two weeks after challenge vaccination and used to measure antibody titers. To re-isolate the challenge strain, heparinized blood was collected immediately before challenge vaccination, 24 and 28 hours after challenge vaccination, and on days 2, 3, 4, 7, and 10 after challenge vaccination.

[0062] At the start of the study, all pigs were seronegative for Leptospira serotypes Pomona and PRRS virus. After vaccination, the antibody response in group 1 against Ery, Parvo, and Lepto was equivalent to or better than that in group 2, indicating that Porcilis PRRS does not adversely affect the intake of EP-ID and L-ID.

[0063] Vaccination with EP-ID and L-ID induced small, transient local reactions at the vaccine administration site (maximum diameters of 4 cm and 3 cm, respectively). All reactions resolved within two weeks post-vaccination. Vaccination did not induce any clinical abnormalities. [Table 5]

[0064] The above results indicate that EP-ID vaccination in pigs is safe in unmixed use with L-ID and PRRS. Furthermore, all three vaccine groups, including unmixed use with attenuated live PRRS vaccine and vaccines containing the usual 25% antigen, demonstrated complete protection against Leptospira interrogans serotype Pomona.

Claims

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

2. A first vaccine for use according to claim 1, wherein the non-replicating Leptospira immunogen is Leptospira interrogans bacterin.

3. A first vaccine for use in accordance with claim 2, characterized in that the first vaccine further comprises a Leptospira kirschneri bacterin and / or a Leptospira santarosai bacterin.

4. A first vaccine for use in accordance with any of the preceding claims, characterized in that the vaccine contains an adjuvant.

5. A first vaccine for use according to claim 4, characterized in that the adjuvant is an oil-in-water adjuvant.

6. A first vaccine for use according to any of the preceding claims, characterized in that the first vaccine is injected by a jet stream of the vaccine using a needleless device, the jet stream penetrating the skin of a pig.

7. The first vaccine is for use in combination with the second vaccine in the prophylactic treatment of a pig for infections caused by Leptospira, Erysipelothrix rhusiopathiae, and porcine parvovirus, wherein the first vaccine and the second vaccine are injected into the dermis of the pig at a first injection site and a second injection site by associated separate injections, the first vaccine comprising a non-replicating immunogen of Erysipelothrix rhusiopathiae and a non-replicating immunogen of porcine parvovirus, the first vaccine for use according to any of the preceding claims.

8. A first vaccine, in combination with a second vaccine, for use according to claim 7, characterized in that the first injection site and the second injection site are at most 4 cm apart from each other, preferably at most 3 cm apart from each other.

9. A first vaccine combined with a second vaccine for use according to claim 7 or 8, characterized in that the associated separate injections of the first vaccine and the second vaccine are administered simultaneously.

10. The first vaccine and the second vaccine are administered in a prime and booster vaccination scheme, wherein both vaccines are injected at the time of prime vaccination and both vaccines are injected at the time of booster vaccination, and the first vaccine is used in combination with the second vaccine, according to any one of claims 7 to 9.

11. A first vaccine, in combination with a second vaccine for use according to any one of claims 7 to 10, characterized in that the non-replicating immunogen of Erysipelothrix rhusiopathiae is Erysipelothrix rhusiopathiae bacterin, and the non-replicating immunogen of porcine parvovirus is inactivated porcine parvovirus.

12. A first vaccine, in combination with a second vaccine, for use according to any one of claims 7 to 11, characterized in that a third vaccine is injected into a pig, wherein the third vaccine contains a live, attenuated PRRS virus.

13. A first vaccine combined with a second vaccine for use according to claim 12, characterized in that, in prophylactic treatment, the pig receives a prime vaccination with a first vaccine and a second vaccine, and a booster vaccination with a first vaccine and a second vaccine, wherein a third vaccine is injected as a single-dose vaccine at the time of booster vaccination.

14. A first vaccine combined with a second vaccine for use according to claim 13, characterized in that the third vaccine is mixed with the first vaccine.

15. A method for prophylactically treating a pig against an infection caused by Leptospira bacteria, comprising injecting a first vaccine containing a non-replicating Leptospira immunogen into the dermis of the pig.

Images