Swine pathogen-containing vaccines for combined, non-mixed use.

Abstract

The present invention relates to a combination of a first vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae, and a second vaccine comprising a live attenuated porcine reproductive and respiratory syndrome (PRRS) virus, for use in prophylactically treating animals against infection with porcine circovirus type 2, Mycoplasma hyopneumoniae, and PRRS virus by combined, unmixed administration of the first and second vaccines to the animal. The present invention also relates to a kit of parts comprising the first and second vaccines, and to methods of using these vaccines to protect animals against such infections. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates generally to the field of swine health. Pigs are susceptible to infection with a number of pathogenic microorganisms. Control of infection is generally achieved by pig farm and feed management, treatment with drugs such as antivirals and antibiotics, or preventative treatment using vaccines. In particular, the present invention relates to vaccines against Porcine Circovirus Type 2 (PCV2 or PCV-2), Mycoplasma hyopneumoniae, Porcine Reproductive and Respiratory Syndrome (PRRS) virus, and optionally Lawsonia intracellularis, as well as methods of using such vaccines to protect animals against such infections. [Background technology]

[0002] PCV-2 is associated with post-weaning multisystemic wasting syndrome (PMWS) seen in young pigs. The disease was first found in Canada in 1991. Its clinical signs and pathology were first published in 1996 and include progressive wasting, dyspnea, tachypnea and sometimes jaundice. Nayar et al. (Can. Vet. J. Volume 38, June 1997) detected porcine circovirus in pigs with clinical signs of PMWS and concluded that PCVs other than the known PCVs recognized as naturally occurring viruses in PK-15 cells may be associated with PMWS. Subsequent publications (Hamel et al., J. Virol., 72(6), 5262-5267, 1998; Meehan et al., J. Gen. Virol., 79, 2171-2179, 1998) confirmed these findings and suggested that the new pathogenic PCV be designated PCV-2, whereas the original PK-15 cell culture isolate (Tischer et al., Nature 295, 64-66, 1982) should be called PCV-1 (Meehan et al., supra). PCV-2 is a small (17 nm-22 nm), icosahedral, non-enveloped virus containing a circular, single-stranded DNA genome. The PCV-2 genome is approximately 1768 bp in length. PCV-2 isolates from different parts of the world appear to be closely related to each other, showing 95-99% nucleotide sequence identity (Fenaux et al., J. Clin. Microbiol., 38(7), 2494-2503, 2000). ORF2 of PCV encodes the capsid protein of the virus. ORF2 of PCV2 encodes a protein of approximately 233 amino acids. The ORF2 genes of all PCV-2 isolates share 91-100% nucleotide sequence identity and 90-100% predicted amino acid sequence identity.

[0003] Mycoplasma hyopneumoniae (Mhyo) is a bacterial species known to cause swine epizootic pneumonia, a highly contagious chronic disease affecting pigs. Mhyo has a small size (400-1200 nm), a small genome (893-920 kilobase pairs (kb)), and lacks a cell wall. Mhyo attach to the cilia of epithelial cells in the lungs of pigs. They cause ciliary beating, clumping, and loss of cilia, ultimately leading to epithelial cell death. This is the cause of the lesions found in the lungs of pigs with swine epizootic pneumonia. This damage prevents normal ciliary clearance and often, secondary infections result. This results in a significant reduction in the weight gain of the animals. The damage in the United States has been estimated so far to be up to $1 billion per year. Swine epizootic pneumonia is endemic worldwide, and Mhyo is found in almost all pig populations. The immune response induced by the presence of Mhyo in pigs is slow and ineffective. Therefore, treatment of the disease is of utmost importance but is limited to antibiotics, which are currently only partially effective as they do not completely eliminate the infection. Vaccines have been shown to reduce the severity of the disease but do not completely prevent the development of the disease in infected pigs.

[0004] 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 with a size of about 15 kilobases. The genome contains nine open reading frames. The virus is a member of the genus Arterivirus in the family Arteriviridae of the order Nidovirales. The two prototype strains of PRRSV are the North American strain VR-2332 and the European strain Lelystad virus (LV). The European and North American PRRSV strains cause similar clinical symptoms. Recently, a highly pathogenic strain of the North American genotype has emerged in China. This strain HP-PRRSV is more virulent than all other strains and has caused great damage in Asian countries. Clinical signs include reproductive disorders in sows, such as abortion and delivery of stillborn or mummified fetuses, and cyanosis of the ears and vulva. In newborn pigs, the disease causes respiratory distress and increases susceptibility to respiratory infections such as Gressey's disease.

[0005] Lawsonia intracellularis causes a proliferative enteropathy, also known as ileitis, which is a common intestinal disease in postweaned pigs worldwide. The characteristic lesion is the proliferation of immature enterocytes in the ileal crypts, which usually contain the causative bacteria within their apical cytoplasm. At necropsy, histological lesions can be confirmed as Lawsonia positive by visualization of 1.5-2.5 μm long Vibrio-like shaped bacteria, especially in the enterocytes, but also frequently in the mesenteric lymph nodes and within macrophages located in the lamina propria between the crypts. Clearance of the bacteria from the enterocytes results in the resolution of the associated proliferative lesions, indicating a direct local effect of the bacteria on the crypts. The presence of Lawsonia intracellularis in these lesions has been demonstrated using PCR, both in animals developing disease and in animals showing only subclinical infection. Clinical cases are usually seen in the grower-finisher period and in some older finishing pigs an acute hemorrhagic form has been recorded.

[0006] Vaccines against the above-identified pathogens are generally known. Conventional vaccines for prophylactically treating animals, especially pigs, against infection with PCV2 can be based on the whole inactivated PCV-2 virus as a (non-replicating) immunogen. It has also been shown in the art that the capsid protein encoded by ORF2 (e.g. when recombinantly expressed) is suitable as a subunit immunogen for porcine circovirus type 2 to be used in a suitable vaccine. This can be understood because this subunit presents the same behavior as the virus itself in the circulation (it forms virus-like particles), differing only essentially in that DNA and nonstructural proteins are not present inside the capsid. Several vaccines against PCV2 are commercially available in the art. Porcilis® PCV (available from MSD Animal Health, Boxmeer, The Netherlands) is a vaccine for protecting pigs against porcine circovirus type 2, used in pigs aged 3 weeks or older. When administered as a two-dose vaccine, the duration of immunity (DOI) is 22 weeks, covering almost the entire fattening period of pigs. Ingelvac CicroFlex® (available from Boehringer Ingelheim, Ingelheim) is a vaccine for protecting pigs against porcine circovirus type 2, used in pigs aged 2 weeks and older. It is registered only as a one-dose vaccine. Circovac® (available from Merial, Lyon, France) is a vaccine for protecting pigs against porcine circovirus type 2, used in pigs aged 3 weeks and older. Suvaxyn® PCV (available from Zoeitis, Capelle a / d IJssel, The Netherlands) is a vaccine for protecting pigs against porcine circovirus type 2, used in pigs aged 3 weeks and older. Other PCV2 vaccines are described, for example, in WO2007 / 028823, WO2007 / 094893 and WO2008 / 076915.

[0007] For Mycoplasma hyopneumoniae, there are numerous commercially available vaccines that are in routine use in the majority of commercial pig farming operations. In general, these vaccines contain non-replicating immunogens such as subunit proteins and / or bacterins (i.e. compositions containing killed bacteria as whole cells, (partial) lysates, homogenates, French presses or combinations thereof, or compositions containing killed bacteria in another form, where the composition is derived from a killed bacterial culture), which are typically injected parenterally. Some examples include RespiSure® (Zoetis), Ingelvac® M.hyo, and MycoFLEX® (Boehringer Ingelheim), Hyoresp® (Merial), Stellamune® Mycoplasma (Elanco Animal Health), Fostera® PCV MH (Zoetis), and M+Pac® and Porcilis® Mhyo (both available from MSD Animal Health).

[0008] For PRRS virus, inactivated virus vaccines have been described and are commercially available, but modified live vaccines (MLVs) containing live attenuated forms of either European (type I) or North American (type II) are the primary immunological means of control. Several vaccines are commercially available in the art. Porcilis® PRRS (available from MSD Animal Health, Boxmeer, The Netherlands) is a vaccine containing live attenuated PRRS virus type I and is registered to reduce infection (viremia) caused by infection with the PRRS virus. Ingelvac PRRS® MLV (available from Boehringer Ingelheim, Ingelheim) is a vaccine that helps reduce disease caused by the PRRS virus, and the vaccine provides cross-protection against different types of strains. Fostera® PRRS (available from Zoeitis, Florham Park, New Jersey, USA) is also an MLV vaccine and is registered to protect against both respiratory and reproductive forms of disease caused by the PRRS virus. Yet another PRRS MLV vaccine is PrimePac PRRS (Type II) available from Merck Animal Health, Madison, NJ, USA. Other PRRS vaccines are described, for example, in WO2006 / 074986, US 8728487 and WO2014 / 048955.

[0009] Vaccines for combating Lawsonia intracellularis by inducing active protection are commercially available and described in the art. These vaccines are available under the trade names Enterisol® Ileitis (Boehringer Ingelheim Vetmedica, USA), which is a live attenuated vaccine, and Porcilis® Ileitis (Merck Animal Health, USA), which is a vaccine containing a non-replicating immunogen of Lawsonia intracellularis in the form of a bacterin. Summary of the Invention

[0010] Object of the invention There is a continuing need for simple, safe and effective means for the health management of pigs, in particular for simple, safe and effective vaccines that can be used for the prophylactic treatment of pigs against infection with porcine circovirus type 2, Mycoplasma hyopneumoniae and PRRS virus.

[0011] Overview of the Invention To achieve the object of the present invention, a combination of a first vaccine and a second vaccine is devised in the present invention, where the first vaccine comprises a non-replicating immunogen of Porcine Circovirus Type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae, and the second vaccine comprises a live attenuated Porcine Reproductive and Respiratory Syndrome (PRRS) virus, in which the combination is used to prophylactically treat an animal against infection by Porcine Circovirus Type 2, infection by Mycoplasma hyopneumoniae and infection by PRRS virus by combined, unmixed administration of the first vaccine and the second vaccine to the animal.

[0012] For each of the above pathogens, vaccines are known and commercially available. However, there is no combination of vaccines available for combined non-mixed administration that provides the required efficacy and at the same time is safe for use in animals, especially young animals. As is generally known, not all of the combinations of antigens envisioned or suggested may lead to a safe and effective combination vaccine. Similarly, combined non-mixed use of vaccines, also referred to as co-administration, may cause interactions that lead to safety issues or reduced responses to the individual immunogens present in the vaccine. In the case of live vaccines, reduced replication of the vaccine strain may occur. Interference between different immunogens may result from various immunological events such as antigen competition. Antigen competition refers to the phenomenon in which the immune response to a particular immunogen (also referred to as antigen) may be reduced in the presence of other immunogens compared to when the same immunogen is given alone.

[0013] Evidence of the commonly known problems of vaccine interference, even when administered unmixed, is provided by the European Medicines Agency (EMA) Committee for Medicinal Products for Veterinary Use (CVMP) published on 18 July 2013, entitled "Guideline on requirements for combination vaccines and immunising veterinary medicinal products (IVMP)" (EMA / CVMP / IWP / 594618 / 2010). Section 5.1 states: "The basis for the combination of IVMPs should be the demonstration of acceptable safety and the absence of significant interference between the IVMPs involved. If the safety profile for the combination is less favorable than that established for the individual products, the combination should be justified by an appropriate benefit-risk analysis, in which case the benefits of the combination should clearly outweigh the risk of reduced safety. In such circumstances, the SPCs of the individual products should be modified to reflect the safety profile of the combined use of IVMPs. If any level of interference between the combined products leads to reduced efficacy, the combination of IVMPs will need to be further justified on a case-by-case basis. It should also be noted that changes affecting the manufacture or composition of any of the involved IVMPs will also require a reevaluation of the suitability of the combination."

[0014] Moreover, any combination administration, especially with live PRRS virus, would be expected to have a rather detrimental effect on efficacy against other immunogens, since PRRS virus is generally known to be immunosuppressive. See, e.g., "Suppression of immune responses in pigs by nonstructural protein 1 of porcine reproductive and respiratory syndrome virus" by Zhou Y1, Bai J, Li Y, Wang X, Wang X, Jiang P, Can J Vet Res. 2012 Oct; 76(4): 255-60.

[0015] Overall, it is generally known that combination vaccination against multiple pathogens is not straightforward and requires experimentation to determine safety and efficacy, especially when the combination vaccination includes live PRRS virus.

[0016] It is noted that the present invention also relates to a kit-of-parts comprising a first vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae, and a separate second vaccine comprising a live attenuated PRRS virus, for use in prophylactically treating an animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae and infection with PRRS virus by combined, unmixed administration of the first and second vaccines to the animal.

[0017] The present invention also relates to a method for prophylactically treating an animal against infection with porcine circovirus type 2 (PCV2), infection with Mycoplasma hyopneumoniae and infection with PRRS virus by administering to the animal a first vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae, in combination with a second vaccine comprising a live attenuated PRRS virus.

[0018] The present invention also relates to a vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae, which is used to prophylactically treat an animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae and infection with PRRS virus by administering the vaccine to the animal in combination with another vaccine comprising a live attenuated porcine reproductive and respiratory syndrome (PRRS) virus. Similarly, the present invention relates to a vaccine comprising a live attenuated porcine reproductive and respiratory syndrome (PRRS) virus, which is used to prophylactically treat an animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae and infection with PRRS virus by administering the vaccine to the animal in combination with another vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae. Each of the individual embodiments described below in the "Embodiments of the Invention" section may be combined with these two vaccines for use in accordance with the present invention.

[0019] It is noted that in vaccines, the immunogen (also called antigen) is typically combined with a pharma- ceutically acceptable carrier. The carrier is a biocompatible medium, i.e., a medium that does not induce significant adverse reactions in the target animal after administration, and that can present the immunogen to the immune system of the host animal after administration of the vaccine, such as a liquid containing water and / or any other biocompatible solvent, or a solid carrier, such as a solid carrier that is commonly used to obtain freeze-dried vaccines (based on sugars and / or proteins), and can optionally contain an immunostimulatory substance (adjuvant) that, upon administration to an animal, induces an immune response to treat the animal against infection by a wild-type microorganism (i.e., helps prevent, ameliorate or cure such infection or the disorder resulting therefrom). Optionally, other substances, such as stabilizers, viscosity modifiers or other ingredients, are added depending on the intended use or desired properties of the vaccine.

[0020] definition A vaccine is a pharmaceutical composition that may be safely administered to a subject animal, which is capable of inducing protective immunity against pathogenic microorganisms in the subject animal, ie, successfully effecting prophylactic treatment as described below.

[0021] A non-replicating immunogen of a pathogen is any substance or compound corresponding to a pathogen other than a live replicating pathogen in its entirety (in an attenuated wild type form) that elicits an immune response against the corresponding pathogen, where the corresponding virulent pathogen or one or more of its virulence factors are recognized and ultimately at least partially neutralized by the host immune system as a result of this immune response. Typical examples of non-replicating immunogens are killed whole pathogens (this term includes cytolytic forms of these pathogens), as well as subunits of these pathogens, such as capsid proteins, surface expressed proteins (e.g. recombinantly expressed proteins or lipopolysaccharides) and excretion molecules, such as toxins.

[0022] A live attenuated pathogen is a viable, replicable form of a pathogen that has reduced virulence. The method of attenuation involves taking an infectious pathogen and altering it so that it is harmless or attenuated, typically by multiple passaging of the pathogen through cell lines or by genetic modification of the pathogen.

[0023] Prophylactic treatment against infection by a pathogen is intended to help prevent, ameliorate or cure infection by or damage resulting from that infection (resulting from post-treatment challenge with a pathogenic pathogen), in particular to reduce the burden in the host following such challenge, and, optionally, to help prevent or ameliorate one or more clinical symptoms resulting from post-treatment infection with the pathogen.

[0024] Combined, unmixed administration of vaccines, also referred to as concomitant administration, is the administration of these vaccines to a target animal separately, without mixing prior to administration, within a time frame during which immunological interference may occur, typically within a 24-hour period. Examples of combined, unmixed use include simultaneous administration at separate application sites on the target animal, and administration at the same or separate application sites at different times, typically 1-6 hours apart.

[0025] Simultaneous administration of vaccines means administration at exactly the same time or within a time frame of at least one 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 even within a time frame of one minute.

[0026] A single (single dose) administration of the vaccine used for prophylactic treatment means that vaccination does not need to be boosted with a second dose of vaccine to obtain protective immunity. In a two-dose regimen, the first (prime) vaccination is typically boosted (boosted) within 6 weeks of the first dose, generally within 3 weeks or even 2 weeks of the first dose, and it is only after the second (boost) dose that protective immunity, i.e., the successful prophylactic treatment described above, can be achieved.

[0027] EMBODIMENTS OF THE PRESENTINVENTION In one embodiment of the combination of the first and second vaccines for use in the present invention, the combined non-mixed administration is carried out simultaneously, which has the advantage that the animals only need to be handled once to apply both vaccines.

[0028] In another embodiment of this combination of vaccines, the second vaccine is administered intradermally to the animal. Such administration of at least the PRRS vaccine has been shown to be safe and effective, while at the same time less stressful for the animal compared to intramuscular administration. With regard to intradermal administration (also referred to as intradermal administration), such administration is often performed using a needle-free vaccination device, such as the IDAL® Vaccinator (available from MSD Animal Health, Boxmeer, The Netherlands), but "intradermal" administration in itself should not be equivalent to "needle-free" administration. The World Health Organization, in its August 27, 2009 paper entitled "Intradermal Delivery of Vaccines; A review of the literature and the potential for development for use in low- and middle-income countries," has indeed clearly indicated that "needle-free" vaccination does not necessarily mean "intradermal" vaccination (see Table 1 on page 3 of the review). Only if the needle-free device is "configured for intradermal vaccination," the vaccine is actually delivered (at least partially) intradermally. Alternatively, the vaccine may be delivered in its entirety subcutaneously or intramuscularly.

[0029] In yet another embodiment of the combination of the first and second vaccines, the first vaccine comprises a non-replicating immunogen of Lawsonia intracellularis. In this embodiment, the novel combination of vaccines can provide protection against four major swine pathogens by using only one vaccination protocol. In one embodiment, the immunogen of Lawsonia intracellularis is combined with PCV2 and Mycoplasma hyopneumoniae within 24 hours prior to administration, preferably within 6 hours prior to administration. Combining the antigens immediately prior to administration allows greater freedom in the choice of excipients. Because, as is known for many pharmaceutical compositions, and even for combination vaccines comprising PCV2 ORF2 antigens known per se (e.g. Porcilis® PCV M Hyo available from MSD Animal Health), long-term stability may still not be easily achieved, at least for any and all pharma- ceutical acceptable carrier compositions.In another embodiment, the Lawsonia intracellularis immunogen is added to the vaccine in the form of a composition comprising lyophilized killed whole cells of Lawsonia intracellularis.

[0030] In yet another embodiment of the novel combination of the first and second vaccines, the first and second vaccines are administered in a single dose. It has been found that a single dose provides effective vaccination against all pathogens. This provides a very simple and economical method for protecting animals against these pathogens.

[0031] In another embodiment, the non-replicating immunogen is a recombinant expression protein encoded by the ORF2 gene of PCV2, for example, expressed by baculovirus, as known in the art.This recombinant protein has been found to be suitable for application in the present invention.In particular, the ORF2 protein can be expressed in a baculovirus expression system, for example, as described in WO2007 / 028823, WO2007 / 094893 or WO2008 / 076915.

[0032] In yet another embodiment, the non-replicating Mycoplasma hyopneumoniae immunogen comprises killed whole Mycoplasma hyopneumoniae. Such Mhyo antigens are relatively easy to produce and have a good track record of efficacy in routine pig farming practices.

[0033] The present invention will now be described in more detail with the aid of the following examples.

[0034] Working Example Study 1: Safety and protection against PRRSv challenge with unmixed use of Porcilis® PCV MHyo in combination with Lawsonia immunogens reconstituted therein and Porcilis® PRRS This study was conducted to evaluate the safety and efficacy of Porcilis® PRRS in combination with lyophilized killed whole cells of Lawsonia intracellularis (also referred to as "Lawsonia lyophilisate") reconstituted (reconstituted) in Porcilis® PCV M Hyo. The study was conducted using 60 PRRSV antibody-negative piglets equally distributed into 5 groups of 12 piglets per group. Four groups were vaccinated at 3 weeks of age with 2 mL of Porcilis® PRRS intramuscularly (IM; groups 1 and 3) or 0.2 mL of Porcilis® PRRS intradermally (ID; groups 2 and 4). Piglets in groups 1 and 2 were vaccinated with Lawsonia lyophilisate reconstituted in Porcilis® PCV M Hyo (2 mL; IM) within minutes of administration of the PRRS vaccine. Piglets in group 5 (also 12 piglets) were not vaccinated and served as non-vaccinated challenge controls. Four weeks after vaccination, all piglets were challenged by the intranasal (IN) route with a heterologous virulent PRRSV type 1 strain (isolate 2).

[0035] After challenge, the following parameters were measured: clinical signs, rectal temperature, and PRRSV viremia and serology. No clinical signs associated with PRRSV challenge were observed in any of the groups. Rectal temperatures were measured for 12 days, starting from 1 day before challenge to the 10th day after challenge. No significant differences were observed between any of the vaccinated groups. Rectal temperatures in all PRRS vaccinated groups were lower compared to the control group at some time points after challenge. No statistically significant differences were observed between IM or ID vaccinated groups, or between groups treated with Porcilis® PRRS alone or Porcilis PRRS combined with Lawsonia lyophilisate reconstituted in Porcilis® PCV M Hyo.

[0036] Vaccination of piglets with Porcilis® PRRS and vaccination of piglets with Porcilis® PRRS in combination with vaccination with Lawsonia lyophilisate reconstituted in Porcilis® PCV M Hyo showed a significant reduction in PRRSV viremia compared to non-vaccinated controls (Figure 1). No significant differences were found between the use of Porcilis® PRRS alone and the unmixed combination administered by IM or ID routes.

[0037] All vaccinated groups showed anti-PRRSV antibody titers on the day of challenge, 4 weeks after vaccination, but the control group did not. Four weeks after challenge, all groups showed anti-PRRSV antibody titers, with the antibody titers in the vaccinated groups being higher than on the day of challenge. No statistically significant differences were found between the use of Porcilis® PRRS alone and the use of the unmixed combination administered by IM or ID route.

[0038] In conclusion, concomitant administration of Lawsonia lyophilisate reconstituted in Porcilis® PCV M Hyo did not adversely affect Porcilis® PRRS vaccination, either by IM or ID route. It was unexpected that the combined use of the Lawsonia-PCV-Mhyo triple vaccine did not have any adverse effect on the efficacy of the PRRS vaccine (see Study 5).

[0039] Study 2: PCV2 Efficacy Study of Porcilis® PCV MHyo in Combination with Lawsonia Immunogen Reconstituted Therein and Porcilis® PRRS Unmixed Use A total of 45 piglets with low to moderate maternal PCV2 antibody titers (<5.5 log2), no PCV2 viral load, and no PRRSV antibody titers were assigned to three treatment groups with 15 piglets per group. The piglets were vaccinated once at 3 weeks of age. Piglets in groups 1 and 2 were vaccinated with Lawsonia FD dissolved in Porcilis PCV M Hyo, both as a single dose (intramuscular route; IM), and piglets in group 1 were also vaccinated with Porcilis PRRS at approximately the same time (by both intramuscular and intradermal routes; IM and ID). Animals in group 3 were left unvaccinated (control).

[0040] At 5 weeks of age (2 weeks after vaccination), animals were challenged with an intranasally administered wild-type PCV2 challenge virus. Twenty-one days after challenge, all animals were necropsied and samples were taken from inguinal lymph nodes, tonsils, and lungs for detection of PCV2.

[0041] Serum samples were collected at the time of vaccination, 1 day before challenge, and at weeks 2 and 3 after challenge. Samples were tested for PCV2 antibodies and viral nucleic acid. Feces and nasal swabs collected 1 day before challenge, 2 weeks after challenge, and 3 weeks after challenge were tested for viral nucleic acid.

[0042] Serological tests showed that both vaccinated groups produced comparable levels (6 log2) of anti-PCV2 antibodies upon challenge, while antibodies in the control group were significantly reduced. After challenge, antibodies in the vaccinated group rose to about 13 log2 at 3 weeks after challenge. In the control group, the level at that time point was 5 log2. After challenge, it was possible to detect viral nucleic acid in serum, nasal and fecal swabs in control animals. The levels were substantially lower in vaccinated animals. In fecal samples, animals vaccinated simultaneously with both vaccines did not show detectable levels of viral nucleic acid. This suggests that this group was best protected against PCV2 challenge. This surprising finding of possible improved protection was confirmed by qPCR measurements of the tissues, as can be seen in Figure 2 (left to right, respectively, represent the PCV2 copies found in tonsils, lungs and inguinal lymph nodes). The lowest viral loads were consistently seen in animals receiving both vaccines (left column) and were even lower in animals receiving the commercial PCV vaccine, Porcilis® PCV M Hyo (middle column), which were significantly lower than the controls (right column).

[0043] Study 3: Efficacy of lyophilized Lawsonia reconstituted in Porcilis® PCV M Hyo combined with Porcilis® PRRS in pigs against Lawsonia challenge For this study, 75 3-week-old piglets divided into three groups were used. Group 1 was vaccinated with lyophilized Lawsonia antigen reconstituted in Porcilis® PCV M Hyo (2 ml IM) administered simultaneously (within 1 h) with Porcilis® PRRS (2 ml IM + 0.2 ml ID), group 2 was vaccinated with lyophilized Lawsonia antigen reconstituted in Porcilis PCV M Hyo (2 ml IM), and group 3 remained as an unvaccinated control. At 7 weeks of age (4 weeks after vaccination), all pigs were orally challenged with homogenized Lawsonia -infected intestinal mucosa. After challenge, pigs were observed daily for clinical signs. Serum (for serological tests) and fecal (for qPCR) samples were collected at regular intervals before and after challenge. Three weeks after challenge, pigs were euthanized and postmortem examinations were performed. The intestine was macroscopically inspected for Lawsonia intracellularis infection, and ileal samples were collected for qPCR and (immuno)histological scoring.

[0044] On the day of vaccination, most pigs were seronegative to Lawsonia, while others had low to moderate antibody titers. After vaccination, antibody titers in groups 1 and 2 showed a similar increase in antibody titers, while the controls showed a slight decrease and remained at low levels. On the day of vaccination, the pigs showed low to moderate maternally derived PCV antibody titers. After vaccination, PCV antibody titers in groups 1 and 2 showed a similar increase, while the controls showed a decrease in maternal antibodies. On the day of vaccination, the pigs were seronegative for Mhyo and PRRSv. After vaccination, groups 1 and 2 showed similar antibody responses to Mhyo, while the control group 3 remained seronegative until the end of the study. Group 1 responded to PRRS vaccination, while groups 2 and 3 remained seronegative until challenge.

[0045] The results regarding the different parameters after Lawsonia challenge are summarized in Table 1 below (log 10 PCR data in pg DNA / μl). [Table 1]

[0046] From the results it can be concluded that Lawsonia lyophilized antigen dissolved in Porcilis® PCV M Hyo administered together with Porcilis® PRRS (group 1) induced significant protection against Lawsonia infection 4 weeks after vaccination. This was demonstrated by a significant reduction in shedding, infection (qPCR ileal mucosa), gross ileal lesion score and microscopic ileal lesion score. No adverse effect of Porcilis® PRRS on the efficacy of Lawsonia was observed. On the contrary, the combined use group tended to be better with regard to said parameters.

[0047] Study 4: Efficacy of combined unmixed use of Porcilis® PCV M Hyo, Lawsonia lyophilisate reconstituted therein, and Porcilis® PRRS in 3-week-old SPF piglets against Mycoplasma hyopneumoniae challenge infection 4 weeks after vaccination One hundred pigs from a 3-week-old herd free of M. hyopneumoniae and PRRS virus were used in this study in groups of 25 pigs each. One group was vaccinated IM with Porcilis® PCV M Hyo + Lawsonia lyophilisate and vaccinated with Porcilis® PRRS (IM+ID; see above), one group was vaccinated with Porcilis® PCV M Hyo and vaccinated approximately simultaneously (within 1 hour) with Porcilis® PRRS (IM+ID), one group was vaccinated with Porcilis® PCV M Hyo, and the fourth group was unvaccinated and served as a challenge control. Four weeks after vaccination, all animals were infected with a virulent M. hyopneumoniae strain and 3 weeks later, all animals were examined post-mortem for lung lesions.

[0048] All animals were seronegative to Mycoplasma hyopneumoniae at the time of vaccination, and non-vaccinated animals remained seronegative until challenge. Vaccinated groups showed similar serological responses to Mycoplasma hyopneumoniae 4 weeks after vaccination and 3 weeks after challenge.

[0049] A significant reduction in lesion scores was observed for all vaccinated groups compared to non-vaccinated animals. Porcilis® PCV M Hyo + Lawsonia Lyophilisate vaccinated with Porcilis® PRRS showed a 90% reduction and Porcilis® PCV M Hyo vaccinated with Porcilis® PRRS showed a 95% reduction. Porcilis® PCV M Hyo induced a 100% reduction in median Mycoplasma hyopneumoniae lesions. Differences between vaccinated groups were not significant.

[0050] It can be concluded that the unmixed combination of Porcilis® PCV M Hyo, alone or in combination with lyophilized Lawsonia antigen, and Porcilis® PRRS is effective in reducing Mycoplasma hyopneumoniae-induced pulmonary lesions.

[0051] Study 5: Effect of use of inactivated PCV vaccine on the efficacy of live PRRS vaccine In this experiment, the effect of mixing a first vaccine (CircoFLEX, Boehringer Ingelheim) containing a non-replicating immunogen of porcine circovirus type 2 with a second vaccine (Ingelvac PRRS, Boehringer Ingelheim) containing a live attenuated porcine reproductive and respiratory syndrome virus was investigated. Since the survival of live PRRS virus is the key parameter of critical importance when mixing these two vaccines, the survival of PRRS virus after 1, 2 and 4 hours of incubation was examined. On day -1, 10 MA104 cells were inoculated with 100 mL of 10% porcine circovirus type 2 vaccine.5 The cells were seeded at 1000 cells / ml and placed in the wells at 225 μl per well. The cells were maintained at 37° C. in air with a carbon dioxide content of 5%. On day 0, Ingelvac PRRS was pre-diluted in PBS to a titer of 5.3 log10 TCID50 / ml. This mixture was diluted in PBS (control) or with CircoFLEX (two different batches) to a final titer of 5.0 log10 TCID50 / ml. The final mixture was incubated at room temperature for 1, 2 and 4 hours to mimic the actual situation at steady state when inoculating a freshly mixed combination vaccine. After this, 25 μl of each of the incubated mixtures was incubated in MA104 cells at 37° C., 5% carbon dioxide for 7 days to allow the PRRS virus to grow. On day 7, the PRRS virus was measured. The data are shown in Table 2 below. [Table 2]

[0052] As can be seen from the table, PRRS virus loses viability upon short incubation at room temperature with PCV vaccine, and vaccine efficacy is adversely affected by a reduction in vaccine titer. [Brief description of the drawings]

[0053] [Figure 1] 1 shows the change in viral infectivity after PRRSV challenge. [Diagram 2] PCV2 copy numbers found in tonsils, lungs and inguinal lymph nodes after viral challenge are shown.

Claims

1. A method for prophylactically treating a non-human animal against infection with porcine circovirus type 2 (PCV2), infection with Mycoplasma hyopneumoniae, and infection with PRRS virus by combined, non-mixed administration of a first vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae, and a second vaccine comprising a live attenuated PRRS virus, wherein the method is characterized in that the second vaccine is administered intradermally to the non-human animal and the combined, non-mixed administration is performed simultaneously.

2. 2. The method of prophylactically treating a non-human animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae and infection with PRRS virus according to claim 1, wherein the first vaccine comprises a non-replicating immunogen of Lawsonia intracellularis.

3. 3. A method for prophylactically treating a non-human animal against infection with porcine circovirus type 2, Mycoplasma hyopneumoniae and PRRS virus as described in claim 2, wherein a Lawsonia intracellularis immunogen is added to the first vaccine within 24 hours prior to administration of the vaccine.

4. 4. The method of prophylactically treating a non-human animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae and infection with PRRS virus as claimed in claim 3, wherein the Lawsonia intracellularis immunogen comprises lyophilized killed whole cells of Lawsonia intracellularis.

5. The method for prophylactically treating a non-human animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae, and infection with PRRS virus according to any one of claims 1 to 4, wherein the first vaccine and the second vaccine are administered in a single dose.

6. A method for prophylactically treating a non-human animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae, and infection with PRRS virus according to any one of claims 1 to 5, wherein the non-replicating immunogen of PCV2 is a recombinantly expressed protein encoded by the ORF2 gene of PCV2.

7. The method for prophylactically treating a non-human animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae and infection with PRRS virus according to claim 6, wherein the non-replicating immunogen of PCV2 is a baculovirus-expressed protein of PCV2.

8. The method of prophylactically treating a non-human animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae, and infection with PRRS virus according to any one of claims 1 to 7, wherein the non-replicating immunogen of Mycoplasma hyopneumoniae comprises whole killed Mycoplasma hyopneumoniae.

9. A kit comprising a first vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae, and independently a second vaccine comprising a live attenuated PRRS virus, for use in a method for prophylactically treating a non-human animal against infection with porcine circovirus type 2 (PCV2), infection with Mycoplasma hyopneumoniae, and infection with PRRS virus according to any one of claims 1 to 8.

10. A vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae, which is used to prophylactically treat a non-human animal against infection with porcine circovirus type 2, infection with Mycoplasma hyopneumoniae and infection with PRRS virus by combined, unmixed administration of the vaccine to the animal with another vaccine comprising a live attenuated porcine reproductive and respiratory syndrome (PRRS) virus, characterized in that the separate vaccine is administered intradermally to the animal and the combined, unmixed administration is performed simultaneously.

11. 1. A vaccine comprising a live attenuated porcine reproductive and respiratory syndrome (PRRS) virus for use in prophylactically treating a non-human animal against infection with porcine circovirus type 2 (PCV2), Mycoplasma hyopneumoniae, and PRRS virus by combined, unmixed administration of the vaccine to the animal with another vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae, wherein the vaccine is administered intradermally to the animal at the same time as the combined, unmixed administration.

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