A combination vaccine against Erysipelothrix rhusiopathiae, porcine parvovirus, and Leptospira bacteria.

Abstract

The present invention relates to a combination of a first vaccine containing a non-replicating immunogen of Erysipelothrix rhusiopathiae and a non-replicating immunogen of porcine parvovirus, and a second vaccine containing a non-replicating immunogen of Leptospira bacteria, for use in the prophylactic treatment of pigs against Erysipelothrix rhusiopathiae infection, porcine parvovirus infection, and Leptospira bacteria infection, by injecting the first vaccine and the second vaccine separately into the dermis at a first injection site and a second injection site, respectively, in association with each other.

Description

Technical Field

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

Background Art

[0002] Pigs are prone to many infections and the disorders resulting from such infections. For example, swine erysipelas is a disease caused by an infection by Erysipelothrix rhusiopathiae ("ery") and is one of the oldest recognized diseases affecting growing and adult pigs. It is thought that up to 50% of pigs in intensive pig production areas are colonized by Erysipelothrix rhusiopathiae. This organism is generally present in tonsillar tissue. Typically, healthy carriers excrete this organism in their feces or oral and nasal secretions and are an important source of infection for other pigs. Infection is by ingestion of contaminated feed, water or feces and by skin abrasions. Once ingested, Erysipelothrix rhusiopathiae can survive passage through the harsh environment of the stomach and intestine and can remain viable in feces for several months. On farms where this Erysipelothrix rhusiopathiae is endemic, pigs are naturally exposed to Erysipelothrix rhusiopathiae when young. Maternal antibodies provide passive immunity and suppress clinical disease. Older pigs tend to acquire defensive active immunity as a result of exposure to Erysipelothrix rhusiopathiae, which does not necessarily lead to clinical disease. Recovered and chronically infected pigs can become carriers of Erysipelothrix rhusiopathiae. Healthy pigs may be asymptomatic carriers.

[0003] Vaccination against Erysipelas swine is highly effective and recommended for controlling disease outbreaks in pig farms. However, it may not be as effective in preventing chronic arthritis. Discontinuation of vaccination in some farms has been linked to disease outbreaks. Injectable bacterins and attenuated live vaccines administered via water are available and provide long-lasting immunity. The optimal timing of vaccination may vary from farm to farm. If Erysipelas swine is endemic in the production environment, vaccination should be carried out prior to an expected outbreak. Susceptible pigs can be vaccinated before weaning, at weaning, or a few weeks after weaning. Boars and sows selected for inclusion in breeding stocks should receive a booster vaccination 3–5 weeks later. Subsequently, breeding stocks should be vaccinated twice a year, including follow-up vaccinations. However, vaccination is not always straightforward in obtaining protection. Vaccination failure may occur in some groups due to management stress that impairs the immune system of vaccinated pigs. The use of live vaccines can also cause clinical disease, particularly chronic erysipelas swine. Antigenic differences between serotypes in vaccines and serotypes circulating on farms can also lead to incomplete immunity and disease outbreaks. However, this is a rare occurrence, as good cross-protection is thought to exist among the major strains of Erysipelas swine that infect pigs.

[0004] Porcine parvovirus infection (PPV, or "parvo") is a common pathogen in pigs. It is a significant cause of infectious infertility. PPV is a robust virus that replicates normally in the intestines of pigs without causing clinical signs and is ubiquitous in pig populations worldwide. PPV is one of the organisms listed as a cause of stillbirth, mummification, embryonic death, and infertility (SMEDI) syndrome. In larger populations, its presence is almost certain, and it is an infection that must be managed with coexistence rather than aiming for elimination. In smaller populations, including pigs that were previously PPV-positive, it may or may not have disappeared. While most viruses do not survive long periods outside of a host, PPV is unique in that it can persist in the environment for months and is resistant to most disinfectants, which is the most likely reason why PPV is widespread and difficult to eliminate. PPV is transmitted in most cases either orally or nasally, enters the intestines, replicates there, 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 their semen during mating.

[0005] Since it is not possible to eliminate PPV from a herd, the focus should be on managing and preventing acute cases. In addition to routine vaccination of heifers and boars before they enter the breeding group, annual follow-up vaccination of all pigs should be sufficient to ensure the herd is protected. If an infected breeding sow has been vaccinated against PPV at some point in the past, it is known that rapid restimulation of the immune system occurs when exposure to PPV occurs (within 5-7 days). This is sufficient to prevent the disease and stimulate (semi-)permanent immunity.

[0006] Next, leptospirosis is a contagious disease affecting pigs and many other animals (including humans), caused by infection with one of the large groups of Leptospira bacteria ("lepto"). Leptospira are motile spirochetes, 6–12 microns long and 0.1 microns in diameter. They are usually hook-shaped at both ends and can be stained by Giemsa staining or silver staining in tissue. In the laboratory, they are often observed under a dark-field microscope. In laboratory media, they are difficult to culture and grow very slowly (12–26 weeks). Many pathogenic strains can survive in the environment for extended periods under humid conditions with a slightly alkaline pH. Most cannot tolerate drying and are destroyed by common disinfectants.

[0007] While leptospirosis in pigs is common, the details of infection by different serotypes can vary. Pigs are susceptible to many different serotypes. The classification of Leptospira is primarily based on DNA association with known reference strains, but a universal agreement on genus classification has yet to be established. Under the latest classification, the Leptospiridae family includes eight pathogenic species, three of which are most important to 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 uniquely adapted to pigs, while the other serotypes are maintained in other species but can also infect pigs. Leptospira bratislava is reported to be the most common strain in pigs, but the role of this serotype in causing disease is debated.

[0008] Control of Leptospira is usually attempted through exposure prevention, immunization by vaccination, and / or the use of antibiotics. Exposure prevention is difficult to achieve because so many species can act as carriers of Leptospira. These include infected pigs, rodents (especially mice and rats), and many types of wildlife. Leptospira often persists in situ as a source of infection when introduced into favorable moist environments. Nevertheless, leptospirosis can be effectively controlled (and possibly even eradicated) from pig populations kept under confinement conditions with treated or uncontaminated drinking water sources, using a combination of drug therapy, vaccination, and vector control. Immunization with bacterins, widely practiced in breeding populations, usually reduces the prevalence of infection and abortion. The bacterin must be appropriate for the serotype of Leptospira causing the disease; therefore, many bacterins are polyvalent.

[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 positive-sense RNA genome approximately 15 kilobases in size. The genome contains nine open reading frames. The virus is a member of the genus Arterivirus, family Arteriviridae, and order Nidovirales. Two prototype strains of PRRSV are the North American strain VR-2332 and the European strain Lelystad virus (LV). Both the European and North American PRRSV strains produce similar clinical symptoms. In the early 2000s, a highly pathogenic strain of the North American genotype emerged in China. This strain, HP-PRRSV, is more virulent than all other strains and has caused significant losses in Asian countries worldwide. Asymptomatic infection is common for all PRRS viruses, and clinical signs occur only sporadically in the population. Clinical signs include reproductive failure in sows, such as miscarriage, stillbirth, or 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 Glasser's disease.

[0010] Vaccine compositions for all of the identified pathogens listed 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 simple, safe, and effective vaccination against one or more of the above-mentioned porcine pathogens. [Means for solving the problem]

[0012] To achieve the objectives of the present invention, it has been found that a combination of a first vaccine containing a non-replicating immunogen of Erysipelas swine and a non-replicating immunogen of porcine parvovirus, and a second vaccine containing a non-replicating immunogen of Leptospira bacteria, can be used in the prophylactic treatment of pigs against Erysipelas swine, porcine parvovirus, and Leptospira infections by injecting the first and second vaccines separately into the dermis at the first and second injection sites of the pig, respectively. With a typical intradermal vaccine dose of less than 1 ml, typically about 0.2 ml, the two vaccines do not mix subcutaneously when the first and second injection sites are more than 1.5–2 cm apart, and the local reaction of each vaccine can be distinguished from the local reaction of the other vaccine. This is in line with the EMA Guideline on the requirements for combined vaccines and associations of immunological veterinary medicinal products (IVMPs), 18 July 2013 (EMA / CVMP / IWP / 594618 / 2010), which defines “separate sites” as application sites that are sufficiently far apart from each other to prevent the possibility of mixing of the products and to allow the local reactions of each product to be distinct from one another. In the sense of the present invention, injection sites can be considered “separate.” Therefore, in intradermal vaccination, any distance between the first and second injection sites greater than 1.5 cm, preferably greater than 2 cm, results in the same outcome as preventing negative interference with the safety and / or efficacy of the associated vaccination of the two vaccines by preventing mixing of these two vaccines after administration.

[0013] In this way, safe and effective vaccination of pigs can be carried out in a convenient manner. Although separate and combination vaccines are available for pigs, even if the antigen is the same, a new administration site does not necessarily lead to a safe and effective vaccine.

[0014] 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 states on page 53: "The route of administration is the path by which a vaccine (or drug) comes into contact with the body. This is a crucial factor for successful immunization. The substance must be transported from the entry site to the part of the body where its action is to be performed. However, using the body's transport mechanisms for this purpose is not easy."

[0015] In this regard, the California Department of Health Services' Immunization Branch has published guidelines for proper vaccination (https: / / www.cdc.gov / vaccines / pubs / pinkbook / vac-admin.html). Regarding the site of administration, the second complete paragraph on page 81, titled "Routes and Sites for Vaccination," states: "The recommended routes and sites for each vaccine are based on clinical trials, practical experience, and theoretical considerations. There are five routes used to administer vaccines. Deviating from the recommended route may reduce the effectiveness of the vaccine and may increase local adverse reactions."

[0016] Overall, it is generally known that vaccination at specific sites is not straightforward and requires experimentation to determine whether safety and efficacy can be achieved, and if so, whether the level is appropriate for commercial use.

[0017] In addition to the combination of the first and second vaccines for use as described above, the present invention relates to a parts kit comprising a first vaccine containing a non-replicating immunogen for Erysipelas swine and a non-replicating immunogen for porcine parvovirus, and a second vaccine containing a non-replicating immunogen for Leptospirosis, for use in the prophylactic treatment of pigs against Erysipelas swine infection, porcine parvovirus infection and Leptospirosis infection, by injecting the first and second vaccines separately into the dermis in association into a first and second injection site of the pig, respectively.

[0018] The present invention also relates to a method for prophylactically treating pigs against erysipelas swine infection, porcine parvovirus infection, and leptospirosis infection by injecting into the dermis of the pig a first vaccine comprising a non-replicating immunogen of Erysipelas swine and a non-replicating immunogen of porcine parvovirus, and a second vaccine comprising a non-replicating immunogen of Leptospira bacteria, as related separate injections.

[0019] Finally, the present invention is embodied in using a non-replicating immunogen of Erysipelas swine and a non-replicating immunogen of porcine parvovirus to produce a first vaccine, and using a non-replicating immunogen of Leptospira bacteria to produce a second vaccine, and a combination of both vaccines being used to prophylactically treat pigs against Erysipelas swine infection, porcine parvovirus infection, and Leptospira bacteria infection by injecting the first vaccine and the second vaccine separately into the dermis at a first injection site and a second injection site, respectively.

[0020] 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, that is, can induce the success of a prophylactic treatment as defined below herein.

[0021] Intradermal vaccine administration means that the vaccine will be deposited in the dermis, at least partially. The World Health Organization, in an August 27, 2009 paper titled "Intradermal Delivery of Vaccines; A review of the literature and the potential for development for use in low- and middle-income countries," indicates that "needle-free" vaccination does not necessarily mean "intradermal" vaccination (see Table 1 on page 3 of the review). Vaccines can only be delivered to the dermis if the needle-free device is "configured for intradermal vaccination." Otherwise, it may be delivered subcutaneously or intramuscularly. Several devices are commercially available for intradermal vaccination, e.g., the IDAL® vaccine machine (MSD Animal Health), Pulse 50 MicroDose (Pulse Needle Free Systems), or other devices such as those 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").

[0022] The combination of the first and second vaccines constitutes a set of vaccines; the first vaccine is individually different from the second vaccine, but the two vaccines are used together to administer to the same target in a coordinated treatment.

[0023] A non-replicating immunogen of a pathogen is any substance or compound corresponding to the pathogen, other than a living replicating pathogen as a whole (either in its wild-type or attenuated form), which induces an immunological response against the pathogen, such that one or more of the corresponding pathogenic pathogens or their pathogenic factors are recognized by the host immune system as a result of this immune response and are ultimately at least partially neutralized. Typical examples of non-replicating immunogens are killed whole pathogens (this term includes these pathogens in their lysed form) and subunits of these pathogens, such as capsid proteins, surface-expressed molecules (e.g., recombinant expressed proteins or lipopolysaccharides) and secreted molecules such as toxins.

[0024] A bacterin is a suspension of killed bacteria that has been partially or completely lysed (e.g., by homogenization, French press, or a combination of two or more lysis methods) as whole cells.

[0025] An attenuated pathogen is a viable replicable form of a pathogen with reduced pathogenicity. The process of attenuation takes an infectious pathogen and typically alters it, either by multiple passages of the pathogen through a cell line or by genetically modifying the pathogen, such that it becomes harmless or less toxic.

[0026] A prophylactic treatment against infection by a pathogen is to prevent, ameliorate, or assist in curing an infection by that pathogen, or a disorder resulting from that infection, which occurs upon challenge after treatment with the pathogenic pathogen, thereby, in particular, reducing the burden in the host after such a challenge or preventing or ameliorating one or more clinical symptoms resulting from a post-treatment infection by the pathogen.

[0027] Pigs are animals belonging to the family Suidae, a family of even-toed (artiodactyl) mammals, commonly referred to as pigs, hogs, or boars. 18 extant species are currently recognized (or 19 if domestic pigs and wild boars are counted separately), and they are classified into 4 to 8 genera. Among this family, the genus Sus includes the domestic pig, Sus scrofa domesticus or Sus domesticus.

[0028] Separate administrations of vaccines, also called co-administrations, are the administrations of these vaccines separately to the target animal, and thus not mixed before administration, but are done within a time frame where immunological interference is expected to occur, typically within 24 hours. A typical example of related use is co-administration at separate application sites in the target animal, or separated temporally by up to 1 to 24 hours.

[0029] Co-administration of vaccines means administering them precisely simultaneously, 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 within a time frame of 1 minute, for example, administering them precisely simultaneously.

[0030] Single-dose administration of a vaccine for use in prophylactic treatment means that for the purpose of achieving protective immunity, it is not necessary to boost vaccination with a second administration of that vaccine.

[0031] Prime and boost vaccination schemes mean that for the purpose of achieving protective immunity, the first vaccination is boosted by a second administration of that vaccine. Typically, the prime vaccination is boosted within 6 weeks, generally within 5 weeks, and further within 4 weeks from this first vaccination, and typically sufficient protective immunity is obtained after the second (boost) vaccination.

Mode for Carrying Out the Invention

[0032] In a first further embodiment of the present invention, the first and second injection sites are separated from each other by a maximum of 4 cm, preferably a maximum of 3 cm, and more preferably less than 2.5 cm, for example, 2 cm. Thus, it has been found that pigs can be more conveniently vaccinated by simultaneously administering the associated intradermal injections of two vaccines, particularly using an IDAL® 3G twin device (available from MSD Animal Health (Boxmeer, The Netherlands)) which has, for example, two barrels and holds separate vaccines; with an inter-injection site distance of 2.8 cm.

[0033] It is even more preferable that the associated separate injections of the first vaccine and the second vaccine be administered simultaneously.

[0034] 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 given in a primary and booster immunization scheme, where both vaccines are injected at the time of primary vaccination and both vaccines are injected at the time of booster immunization.

[0035] In another embodiment, at least the first vaccine contains an adjuvant (as is generally known, leptospirosis vaccines do not necessarily require an adjuvant to achieve a high level of protection), but preferably both the first and second vaccines contain an adjuvant, such as an oil-in-water adjuvant. Typical adjuvants for use 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.

[0036] In yet another embodiment of the first and second vaccines for use according to the present invention, the non-replicating immunogen for Erysipelas swine is Erysipelas swine bacterin. In yet another embodiment of the first and second vaccines for use according to the present invention, the non-replicating immunogen for porcine parvovirus is inactivated porcine parvovirus. In yet another embodiment of the first and second vaccines for use according to the present invention, the non-replicating immunogen for Leptospira bacteria is Leptospira interrogans bacterin. Preferably, the second vaccine further comprises Leptospira kirschneri bacterin and / or Leptospira santarosai bacterin.

[0037] In yet another embodiment of the first and second vaccines for use according to the present invention, the non-replicating immunogen for Erysipelas swine is Erysipelas swine bacterin, the non-replicating immunogen for porcine parvovirus is inactivated porcine parvovirus, and the non-replicating immunogen for Leptospira bacteria is Leptospira interrogans bacterin. Preferably, the second vaccine further comprises Leptospira kirschneri bacterin and / or Leptospira santarosai bacterin.

[0038] Advantageously, a combination of the first and second vaccines for use according to any of the prior claims may also be used in combination with a third vaccine injected into the pig, the third vaccine comprising attenuated live PRRS virus, and it has been found that this results in a safe and effective combined vaccine against all four pathogens and thus includes protection against infection by the PRRS virus. Preferably, in prophylactic treatment, the pig receives an initial vaccination with the first and second vaccines and a booster immunization with the first and second vaccines, and in the booster immunization (i.e., concurrently with or at least within 24 hours of this booster immunization), the third vaccine is injected as a single-dose vaccine. In further embodiments, the third vaccine (i.e., the PRRS vaccine) is mixed with the second vaccine (i.e., the leptospirosis vaccine) for booster immunization.

[0039] In one embodiment, in a combination of a first vaccine and a second vaccine for use according to the present invention, both the first and second vaccines are injected by a jet stream of each vaccine using a needleless device, where the jet stream penetrates the pig's skin and reaches the dermis. The third vaccine is advantageously also injected by a jet stream using a needleless device.

[0040] The present invention will be explained in more detail using the following specific examples.

[0041] [Examples] Example 1 is the first trial of the Ery-Parvo-Lepto combination vaccine.

[0042] Example 2 is a second trial of the Ery-Parvo-Lepto combination vaccine.

[0043] Example 3 is the third trial of the Ery-Parvo-Lepto combination vaccine.

[0044] [Example 1] Example 1 is the first trial of the Ery-Parvo-Lepto combination vaccine. For this purpose, the existing vaccine Porcilis® EPL was used (containing E. rhusiopathiae, serotype 2 bacterin, inactivated porcine parvovirus, and various leptospirabacterins, namely, in this case L. interrogans serogroup Icterohaemorrhagiae, L. interrogans serogroup Australis serovar Bratislava, L. kirschneri serogroup Grippotyphosa, L. interrogans serogroup Pomona serovar Pomona, and L. santarosai serogroup Tarassovi). This is known to be a safe and effective vaccine for intramuscular vaccination in pigs against ery, parvo, and lepto infections. All antigens are inactivated pathogens. The aim was to confirm whether the vaccine could also be applied intradermally and still meet the specified safety and efficacy requirements. The device selected for intradermal delivery was the IDAL® 3G twin device (Twin IDAL). Since the volume per dose is substantially smaller than that recommended for Porcilis EPL, i.e., 0.2 ml instead of 2 ml, it was necessary to reduce the amount of Porcilis EPL antigen to accommodate the smaller volume of the aqueous phase of the formulation (40 and 25%; see below).

[0045] The study used a variety of different adjuvants, namely the proprietary adjuvant Diluvac Forte (DF; MSD Animal Health, Boxmeer, The Netherlands), μDiluvac Forte (μDF; the same as Diluvac but with fewer surfactants and no mineral oil), and an adjuvant referred to here as SVEA, containing experimental squalane, vitamin E acetate, and silica (see, in particular, International Publication No. 2021 / 048338). With the two Diluvac adjuvants, 40% of the antigen present in Porcilis EPL could be formulated. With the squalene-based adjuvant, this was only 25%.

[0046] For the experiment, seronegative, healthy 12-week-old pigs for PRRS and Ery were used. The pigs were divided into four groups. The vaccinated group consisted of 10 pigs, and the control group consisted of 5 pigs. Group 1 was vaccinated intradermally (ID) twice (primary-booster immunization scheme) with EPL-ID-40%-DF, with Porcilis PRRS administered unmixed (as a separate vaccine) during the booster immunization. Group 2 was also vaccinated twice (primary-booster immunization scheme) with ID, but in this group, it was vaccinated with EPL-ID-40%-μDF, with Porcilis PRRS administered unmixed during the booster immunization. Group 3 was vaccinated twice with ID using EPL-ID-25%-SVEA, with Porcilis PRRS administered unmixed during the booster immunization. Group 4 remained unvaccinated. All vaccinations were administered intradermally (0.2 ml) in the neck (first dose on the right side, booster dose on the left side) using an IDAL device, with a 4-week interval between vaccinations.

[0047] After vaccination, groups 1, 2, and 3 were observed for local injection site reactions.

[0048] Three weeks after the last vaccination, groups 1 and 4 were intradermally challenged (0.1 ml) with challenge strains of Erysipelas swine serotype 1 and serotype 2 in the right and left sides of the chest, respectively. The pigs were monitored for clinical signs and rectal temperature for 10 days, starting two days prior to the challenge. Blood was collected for serological testing of Ery and Parvo on each day of vaccination and on the day of the challenge.

[0049] Group 1 showed an Ery antibody response, while the control group remained seronegative. None of the pigs in the test groups induced an HI response to Parvo. The results are shown in Table 1 below. [Table 1]

[0050] None of the formulations were sufficiently safe (in this study, the mean / maximum local reaction was set at 5 / 7 cm), and the formulation tested for Ery efficacy also showed insufficient efficacy. Furthermore, this formulation did not induce a Parvo serological reaction. Based on these results, it can be concluded that EPL is not feasible as a vaccine for intradermal vaccination because neither formulation meets the safety and efficacy requirements.

[0051] [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 the previous trial. However, instead of formulating all antigens into a single monovaccine, they were divided into two separate vaccines, namely the EP vaccine and the L vaccine. These vaccines were administered simultaneously as separate vaccines into the dermis of pigs using an IDAL device.

[0052] For the experiment, three different adjuvants were used: the proprietary Emunade® and X-Solve® adjuvants (both oil-in-water adjuvants are available from MSD Animal Health (Boxmeer, The Netherlands)) and the SVEA adjuvant identified above.

[0053] Twenty healthy 18-week-old pigs that had not been vaccinated with any of the vaccine components were used. The pigs were divided into four groups of five animals each. Groups 1, 2, and 3 were vaccinated twice intradermally at 4-week intervals in the EP-ID and L-ID (opposite side of the neck) 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) (using the IDAL device). The adjuvant Emunade was used in Group 1, X-Solve in Group 2, and SVEA in Group 3. Group 4 was vaccinated intramuscularly with Porcilis EPL as a reference vaccine.

[0054] 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, Pomona, and Thalassovi antigens—were determined.

[0055] The results are summarized in Tables 2 and 3 below (responses are in relative units unless absolute units are indicated). Note that parvo antibody titers due to vaccination could not be determined because the animals had been infected in the field, and all animals showed high antibody titers. [Table 2]

[0056] [Table 3]

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

[0058] Ery antibody responses were comparable across all formulations, at or above the same level as Porcilis EPL. Parvovirus vaccine titers typically ranged from 2 to 10log2, although titers exceeding 10log2 could be observed after field infection. The results clearly indicate that field infection occurred during the study, and therefore no conclusions can be drawn regarding vaccine antibody titers. Lepto antibody responses induced by different formulations (Ictero, Pomona, and Tarassovy) 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 antibody titer typically corresponds to effective protection).

[0059] [Example 3] Example 3 is a third trial of the Ery-Parvo-Lepto combination vaccine, aiming to establish the Parvo response of the second group of combination vaccines based on the oil-in-water adjuvant X-Solve, i.e., the best vaccine from Example 2. Furthermore, the response to PRRS was evaluated when the patient was vaccinated with an attenuated live PRRS vaccine as a booster immunization during the initial EPL vaccination.

[0060] Thirty-five healthy 12-week-old pigs weighing over 20 kg, negative for PRRS, and seronegative for Ery and Parvo were used. The pigs were divided into four groups (10 pigs in each vaccine group and 5 in the control group). Group 1 received two ID vaccinations at 4-week intervals with the EP-ID vaccine from Group 2 of Example 2 (right) in association with the L-ID vaccine from Group 2 of Example 2 (left), in an unmixed use. In addition, during booster immunization, Porcilis PRRS was administered intradermally using Twin IDAL in association with L-ID (left). Group 2 was vaccinated in the same manner as Group 1, except for the associated use with Porcilis PRRS. Group 3 was vaccinated identically to Group 2, except that the EP-ID and L-ID vaccines contained only 25% of the antigen content (and therefore approximately 3% compared to the antigen content of Porcilis EPL). Group 4 was left unvaccinated as a negative control. All vaccines were administered intradermally (0.2 ml) in the neck using IDAL or Twin IDAL. After vaccination, groups 1 and 2 were observed for local injection site reactions. Blood was collected for Parvo and PRRS serological testing on each day of vaccination and on the challenge day.

[0061] 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 (local reactions are shown in correspondence with Tables 2 and 3). [Table 4]

[0062] Site reactions were acceptable. Serological results for Parvo and PRRS (established 52 days post-vaccination) were acceptable and considered to meet the efficacy requirements. Post-vaccination PRRS antibody titers were comparable to those obtained with the commercially available Porcilis PRRS, and parvo HI antibody titers were consistent with those obtained with the commercially available Porcilis EPL.

[0063] In conclusion, the relevant non-combined use of the separate vaccines EP-ID and L-ID, even when administered with the attenuated live PRRS vaccine, results in safe vaccination and is effective in protection against infection by Erysipelas swine, porcine parvovirus, various Leptospira bacteria, and PRRS virus.

[0064] [Example 4] Example 4 is the fourth trial of the Ery-Parvo-Lepto combination vaccine aimed at establishing protection against L-pomona challenge. Essentially, this experiment used the same experimental setup as Example 3, except that 40 six-week-old piglets were divided into four groups. The vaccination of these groups was identical to that described in Example 3.

[0065] At 11 weeks of age, pigs were transported to a challenge facility and intravenously challenged with Leptospira interrogans serogroup Pomona one week later. For two weeks post-challenge, pigs were observed daily for clinical signs and / or behavioral and appetite abnormalities. Rectal temperature was measured at regular intervals until the end of the two-week post-challenge period. Serum blood was sampled on each vaccination day, the day of challenge, and two weeks after challenge, and used to determine antibody titers. Heparinized blood was sampled immediately before challenge, 24 and 28 hours after challenge, and 2, 3, 4, 7, and 10 days after challenge, and used for re-isolation of the challenge strain.

[0066] At the start of the study, all pigs were seronegative for Leptospira serotypes Pomona and PRRS virus. After vaccination, antibody responses in group 1 against Ery, Parvo, and Lepto were comparable to or higher than in group 2, demonstrating no negative effect of Porcilis PRRS on EP-ID and L-ID colonization.

[0067] 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.

[0068] The results after the challenge are summarized in Table 5 below. [Table 5]

[0069] The results indicate that vaccination with EP-ID in unmixed use associated with L-ID and PRRS is safe in pigs. Furthermore, all three vaccine groups, including unmixed use associated with attenuated live PRRS vaccine and vaccines containing 25% of the normal dose of antigen, induced complete protection against Leptospira interrogans serogroup Pomona.

Claims

1. A combination of a first vaccine comprising a non-replicating immunogen of Erysipelothrix rhusiopathiae and a non-replicating immunogen of porcine parvovirus and a second vaccine comprising a non-replicating immunogen of Leptospira bacteria, for use in the prophylactic treatment of pigs against Erysipelothrix rhusiopathiae infection, porcine parvovirus infection and Leptospira bacteria infection, by injecting the first vaccine and the second vaccine separately into the dermis of the first and second injection sites of the pig, respectively.

2. The combination of a first vaccine and a second vaccine for use according to claim 1, 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.

3. A combination of a first vaccine and a second vaccine for use according to claim 1 or 2, characterized in that the associated separate injections of the first vaccine and the second vaccine are administered simultaneously.

4. A combination of a first vaccine and a second vaccine for use according to any one of claims 1 to 3, characterized in that the first and second vaccines are administered in a primary and booster immunization scheme, both vaccines are injected at the time of the primary immunization, and both vaccines are injected at the time of the booster immunization.

5. A combination of a first vaccine and a second vaccine for use according to any one of claims 1 to 4, characterized in that at least the first vaccine comprises an adjuvant.

6. A combination of a first vaccine and a second vaccine for use according to any one of claims 1 to 5, characterized in that both the first vaccine and the second vaccine contain an adjuvant.

7. The combination of the first vaccine and the second vaccine for use according to claim 5 or 6, characterized in that the adjuvant is an oil-in-water adjuvant.

8. A combination of a first vaccine and a second vaccine for use according to any one of claims 1 to 7, wherein the non-replicating immunogen of Erysipelas swine is Erysipelas swine bacterin, the non-replicating immunogen of porcine parvovirus is inactivated porcine parvovirus, and the non-replicating immunogen of Leptospira bacteria is Leptospira interrogans bacterin.

9. A combination of the first vaccine and the second vaccine for use according to claim 8, further comprising a Leptospira kirschneri bacterin and / or a Leptospira santarosai bacterin.

10. A combination of a first vaccine and a second vaccine for use according to any one of claims 1 to 9, characterized in that a third vaccine is injected into the pig, and the third vaccine contains an attenuated live PRRS virus.

11. The combination of the first vaccine and the second vaccine for use according to claim 10, characterized in that, in the prophylactic treatment, the pig receives an initial vaccination with the first vaccine and the second vaccine, and a booster immunization vaccination with the first vaccine and the second vaccine, and in the booster immunization vaccination, the third vaccine is injected as a single-dose vaccine.

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

13. A combination of a first vaccine and a second vaccine for use according to any one of claims 1 to 12, characterized in that the first vaccine and the second vaccine are injected by a jet stream of each vaccine using a needleless device, the jet stream penetrating the skin of the pig.

14. A parts kit comprising a first vaccine containing a non-replicating immunogen of Erysipelas swine and a non-replicating immunogen of porcine parvovirus, and a second vaccine containing a non-replicating immunogen of Leptospira bacteria, for use in prophylactic treatment of pigs against Erysipelas swine infection, porcine parvovirus infection and Leptospira bacteria infection by injecting the first vaccine and the second vaccine separately into the dermis of a first injection site and a second injection site, respectively, of the pig.

15. A method for prophylactically treating pigs against erysipelas swine infection, porcine parvovirus infection, and leptospirosis infection by injecting a first vaccine containing a non-replicating immunogen of Erysipelas swine and a non-replicating immunogen of porcine parvovirus, and a second vaccine containing a non-replicating immunogen of Leptospira bacteria, separately and in association into the dermis of the pigs.

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