Combined porcine vaccine

Abstract

To provide further means and a method for immunizing animals against pathogens.SOLUTION: A vaccine contains antigen of Lawsonia intracellularis, and one or a plurality of kinds of antigens of at least one kind of further pathogens selected from a group of porcine circovirus (PCV), mycoplasma hyopneumoniae (M.hyo.) and porcine reproductive and respiratory syndrome virus (PRRSV), where the antigen of Lawsonia intracellularis is viable cell Lawsonia intracellularis.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications and Incorporation by Reference PCT Publication Nos. WO96 / 39629, WO05 / 011731, WO06 / 012949, WO06 / 020730, WO06 / 099561, WO07 / 011993, WO07 / 076520, WO07 / 140244, WO08 / 073464, WO09 / 037262, WO09 / 127684, WO09 / 144088, WO2011 / 054951, WO2015 / 0824 57, WO2015 / 082458, WO2015 / 082465, WO2016 / 124620, WO2016 / 124623, WO2017 / 068126, WO2017 / 162741, WO2018 / 189290, WO2018 / 115435 and WO2019 / 166362, as well as International Application No. PCT / US2020 / 026930, filed April 6, 2020. The aforementioned applications, and all documents cited therein or pending thereon ("application cited documents"), and all documents cited in or referenced in the application cited documents, and all documents cited or referenced herein ("documents cited herein"), and all documents cited in or referenced in the documents cited herein, are hereby incorporated by reference herein and may be utilized in the practice of the present invention, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or referenced in any document incorporated by reference herein. More specifically, all references are incorporated by reference herein to the same extent as if each individual document was specifically and individually indicated to be incorporated herein by reference.

[0002] FIELD OF THEINVENTION Disclosed herein is a combination swine vaccine comprising a Lawsonia intracellularis antigen, a porcine circovirus (PCV) antigen, a Mycoplasma hyopneumoniae (M.hyo.) antigen and a porcine reproductive and respiratory syndrome virus (PRRSV) antigen, methods for its production and uses thereof. [Background technology]

[0003] Lawsonia intracellularis, the causative agent of porcine proliferative enteritis ("PPE"), affects virtually all animals, including humans, rabbits, ferrets, hamsters, foxes, and horses, as well as a variety of other animals such as ostriches and emus, and is a particularly significant cause of losses in pig herds. A consistent feature of PPE is the appearance of non-membrane-bound curved rods in the cytoplasm of enterocytes in affected areas of the intestine. Bacteria associated with PPE have been termed "Campylobacter-like organisms." S. McOrist et al., Vet. Pathol., Vol. 26, 260-64 (1989). This pathogenic bacterium was subsequently identified as a new taxonomic genus and species, commonly referred to as Ileal Commensal (IS) intracellularis. C. Gebhart et al., Int'l. J. of Systemic Bacteriology, Vol. 43, No. 3, 533-38 (1993). These novel bacteria were given the taxonomic name Lawsonia (L.) intracellularis. S. McOrist et al., Int'l. J. of Systemic Bacteriology, Vol. 45, No. 4, 820-25 (1995). These three names are used interchangeably to refer to the same microorganism as further identified and described herein.

[0004] Porcine circoviruses (PCVs) are non-enveloped icosahedral single-stranded DNA (ssDNA) viruses belonging to the genus Circovirus in the family Circoviridae. The genome encodes two major open reading frames (ORFs), ORF1 encoding the replication-related protein (rep) and ORF2 encoding the viral capsid (cap) protein, which determines the antigenic characteristics of the virus. PCV2 shares approximately 80% sequence identity with porcine circovirus type 1 (PCV1). However, in contrast to PCV1, which is generally non-virulent, pigs infected with PCV2 exhibit a syndrome commonly referred to as postweaning multisystemic wasting syndrome (PMWS). PCV3 is genetically distinct from porcine circovirus type 2 (PCV2), specifically, the rep gene shares only 48% amino acid identity with the cap gene, and the cap gene shares only 26% amino acid identity with the rep gene, which is generally non-virulent with the cap gene.

[0005] Mycoplasma hyopneumoniae (M.hyo.) is a small bacterium (400-1200 nm) classified in the family Mycoplasmataceae. M.hyo. is associated with epizootic pneumonia, a porcine respiratory disease commonly seen in growing and finishing pigs. M.hyo. attacks the cilia of epithelial cells in the trachea and lungs, resulting in the cessation of cilia beating (ciliary dyskinesia) and ultimately the collapse of lung segments. M.hyo. is considered a primary pathogen that facilitates the entry of PRRSV and other respiratory pathogens into the lungs. The separate isolates 232, J, and 7448 have had their genomes sequenced (Minion et al., J. Bacteriol. 186:7123-33, 2004; Vasconcelos et al., J. Bacteriol. 187:5568-77, 2005, and Han et al., Genome Announc. 2017 Sep; 5(38): e01012-17). Porcine reproductive and respiratory syndrome (PRRS) is considered by many to be the most important disease currently affecting the swine industry worldwide. PRRS virus (PRRSV) is an enveloped, single-stranded RNA virus classified in the family Arteriviridae. There is a wide variability in the antigenic characteristics of different isolates of PRRSV, limiting the number of effective measures to prevent infection. There are three main vaccine groups available for PRRS: modified live attenuated vaccines (MLV), killed virus vaccines, or recombinant vaccines. The viral envelope proteins of PRRSV are generally categorized into major and minor proteins based on the abundance of the protein in the virion. The major viral envelope proteins are gp5 (ORF 5) and M (ORF 6), which form dimers. The minor envelope proteins are gp2 (ORF2), gp3 (ORF3), gp4 (ORF4), and E (ORF2b), as well as the possibly newly identified viral protein gp5a (ORF 5a). The active antigenic component may include ORF4, ORF5, ORF6, or ORF7 from the PRRSV virus. There is a continuing need for new ways to immunize animals against the above pathogens.

[0006] The technical problem underlying the present invention is therefore the provision of further means and methods for immunizing animals against pathogens. This problem is solved and the above-mentioned needs are addressed by the provision of the embodiments as characterized in the claims and provided herein below. Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides additional means and methods for immunizing animals against pathogens. [Means for solving the problem]

[0008] The present invention relates to a vaccine comprising an antigen of Lawsonia intracellularis and one or more antigens of at least one further pathogen selected from the group of Porcine circovirus (PCV), Mycoplasma hyopneumoniae (M.hyo.) and Porcine reproductive and respiratory syndrome virus (PRRSV), wherein the antigen of Lawsonia intracellularis is a live Lawsonia intracellularis bacterium. Thus, the vaccine may comprise live Lawsonia intracellularis bacteria and antigens of PCV.

[0009] For example, the vaccine may contain the live Lawsonia intracellularis bacteria and the PCV2 ORF2 protein. The vaccine may also include live Lawsonia intracellularis and M. hyo. antigens. For example, the vaccine may contain live Lawsonia intracellularis bacteria and M. hyo. bacterin. The vaccine may also include live Lawsonia intracellularis bacteria and an antigen of PRRSV. For example, the vaccine may also contain live Lawsonia intracellularis bacteria and an attenuated PRRSV virus. The vaccine may also contain live Lawsonia intracellularis bacteria, antigens of PCV and antigens of M. hyo. For example, the vaccine may contain the live Lawsonia intracellularis bacteria, the PCV2 ORF2 protein, and the M. hyo. bacterin. The vaccine may also include a live Lawsonia intracellularis bacterium, an antigen for PCV, and an antigen for PRRSV. For example, the vaccine may contain live Lawsonia intracellularis bacteria, PCV2 ORF2 protein, and an attenuated PRRSV virus. The vaccine may also include live Lawsonia intracellularis bacteria, PRRSV antigens, and M. hyo. antigens. For example, the vaccine may contain live Lawsonia intracellularis bacteria, an attenuated PRRSV virus, and an M. hyo. bacterin. The vaccine may also include live Lawsonia intracellularis, PCV antigens, M. hyo. antigens and PRRS antigens. For example, the vaccine may contain live Lawsonia intracellularis bacteria, PCV2 ORF2 protein, M. hyo. bacterin, and attenuated PRRSV virus.

[0010] Preferably, the vaccine comprises a live Lawsonia intracellularis bacterium and an antigen of PCV. More preferably, the vaccine comprises a live Lawsonia intracellularis bacterium and a PCV2 antigen. More preferably, the vaccine comprises a live Lawsonia intracellularis bacterium and a recombinant polypeptide of PCV2. In a particularly preferred embodiment, the vaccine comprises an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®. The term "live Lawsonia intracellularis" includes "modified live Lawsonia intracellularis" and "attenuated Lawsonia intracellularis".

[0011] The vaccine of the present invention is about 10 3 ~10 9 of bacteria / kg body weight, preferably about 10 5 ~10 7 The vaccine of the present invention may also have a dosage of Lawsonia intracellularis of about 10 bacteria / Kg body weight. 5 ~about 10 7 The Lawsonia intracellularis antigen may have a dosage of 0.01 mg / kg / day or more. The Lawsonia intracellularis antigen may be lyophilized. Preferably, the Lawsonia intracellularis antigen is an antigen contained in Enterisol® Ileitis.

[0012] The PCV antigen of the vaccine of the present invention may be an antigen of PCV1, PCV2 or PCV3. Preferably, the PCV antigen is a PCV2 antigen. The PCV antigen may be a recombinant polypeptide. The recombinant polypeptide may be expressed by a PCV ORF gene. Preferably, the PCV ORF gene is a PCV ORF2 gene. The PCV recombinant polypeptide may be expressed in a baculovirus cell. Preferably, the PCV antigen is an antigen contained in Ingelvac CircoFLEX® or an antigen of PCV contained in 3FLEX®. In the vaccine of the present invention, the antigen of PCV may have a dosage of about 2 μg to about 400 μg. The M.hyo. antigen of the vaccine of the present invention may be a supernatant and / or a bacterin. A detailed description of the supernatant and the bacterin is provided herein below. Preferably, the M.hyo. antigen is an antigen of M.hyo. contained in Ingelvac MycoFLEX® or an antigen of M.hyo. contained in 3FLEX®.

[0013] The PRRSV antigen of the vaccine of the invention can be a live PRRSV virus. The live virus can be a modified and / or attenuated virus. The vaccine of the invention contains approximately 10 1 ~about 10 7 Viral particles, preferably about 10 per dose 3 ~about 10 5 particles, more preferably about 10 per dose 4 ~about 10 5 The vaccine of the present invention may also contain a dosage of about 10 PRRSV antigens per dose. 4 ~about 10 7 The dosage of the PRRSV antigen may be in the form of a viral particle. The PRRSV antigen may be lyophilized. Preferably, the PRRSV antigen is the PRRSV antigen contained in Ingelvac® PRRSV MLV or the PRRSV antigen contained in 3FLEX®. The PCV antigen, M.hyo. antigen, and PRRSV antigen may be antigens contained in 3FLEX®.

[0014] The vaccine of the present invention may be a lyophilized antigen of Lawsonia intracellularis dissolved in 3FLEX®. Thus, the vaccine of the present invention may be a lyophilized live Lawsonia intracellularis dissolved in 3FLEX®. Additionally, the vaccine of the present invention may be Enterisol® Ileitis dissolved in 3FLEX®.

[0015] In one embodiment, the vaccine of the invention may further comprise a pharma- ceutically or veterinarily acceptable carrier. In one embodiment, the vaccine of the invention may further comprise one or more adjuvants. Suitable adjuvants are known in the art and non-limiting examples are described herein. The vaccine of the invention may comprise one or more of the following as adjuvants: polymers of acrylic or methacrylic acid; copolymers of maleic anhydride and alkenyl derivatives; cross-linked polymers of acrylic or methacrylic acid; polymers of acrylic or methacrylic acid cross-linked with polyalkenyl ethers of sugars or polyhydric alcohols; carbomers; acrylic polymers cross-linked with polyhydroxylated compounds having at least three and at most eight hydroxyl groups, where the hydrogen atoms of at least three of said hydroxyl groups may be replaced by unsaturated aliphatic groups having at least two carbon atoms or may be substituted. and wherein the radicals contain 2 to 4 carbon atoms, e.g., vinyl, allyl and other ethylenically unsaturated radicals, which may themselves contain other substituents, e.g., methyl; Carbopol®; Carbopol® 974P; Carbopol® 934P; Carbopol® 971P; Carbopol® 980; Carbopol® 941P; ImpranFLEX®; Aluminum hydroxide; Aluminum phosphate; Saponin; Quil A;QS-21;GPI-0100;Water-in-oil emulsions;Oil-in-water emulsions;Water-in-oil-in-water emulsions;Emulsions based on light liquid paraffin oil or European Pharmacopoeia-type adjuvants;Oils of the isoprene series;Squalane;Squalene oil resulting from the oligomerization of alkenes or isobutene or decene;Esters of acids or of alcohols containing linear alkyl groups;Vegetable oils;Ethyl oleate;Propylene glycol di(caprylic / capric) acid;Glyceryl tri(caprylic / capric) acid;Propylene glycol dioleate;Esters of branched fatty acids or alcohols:Isostearate esters;Non-ionic surfactants;Esters of sorbitan or mannitol or glycol or polyglycerol or propylene glycol or oleic acid or isostearic acid or ricinoleic acid or hydroxystearic acid, optionally ethoxylated, anhydromannitol oleate; polyoxypropylene-polyoxyethylene copolymer blocks, Pluronic products, RIBI adjuvant system; block copolymers; SAF-M; monophosphoryl lipid A; avridine lipid-amine adjuvants; heat-labile enterotoxin from E. coli (recombinant or non-recombinant); cholera toxin; IMS 1314, or muramyl dipeptide; Preferably, the adjuvant is a carbomer. Advantageously, the adjuvant may be ImpranFLEX® and / or Carbopol®.

[0016] The vaccine of the present invention may be in a form for systemic administration. The vaccines of the present invention may be formulated and / or packaged for single dose or one-shot administration. The vaccines of the present invention may be formulated and / or packaged for a multiple dose administration regimen, preferably a two dose administration regimen. The vaccine of the invention may be present in a dosage form, which is delivered from a container containing a larger quantity of the vaccine, and the vaccine dosage form may be delivered from the container, which may contain at least 10, at least 50, at least 100, at least 150, at least 200 or at least 250 doses of the vaccine.

[0017] The invention also includes a vaccine of the invention for use in a method for raising a protective immune response in an animal comprising the step of administering the vaccine to the animal. The invention also encompasses a vaccine of the invention for use in a method for raising a protective immune response in a pig comprising the step of administering said vaccine to said pig.

[0018] The present invention also includes a vaccine of the invention for use in a method for eliciting a protective immune response in an animal against Lawsonia intracellularis and / or PCV and / or M. hyo. and / or PRRSV. In a preferred embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV. In a preferred embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and M. hyo. In a preferred embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PRRS. In a preferred embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV and M. hyo. In a preferred embodiment, the vaccine of the invention is for use in methods for eliciting a protective immune response against Lawsonia intracellularis and PCV and PRRS. In a preferred embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PRRS and M. hyo. In a preferred embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV and M. hyo. and PRRSV.

[0019] The invention also includes a vaccine of the invention for use in a method for raising a protective immune response, wherein the vaccine is administered systemically. The invention also includes a vaccine of the invention for use in a method for raising a protective immune response, wherein the vaccine is administered as a single dose. The invention also includes a vaccine of the invention for use in a method for raising a protective immune response, wherein the vaccine is administered as at least one dose.

[0020] The invention also encompasses a vaccine of the invention for use in a method for raising a protective immune response, wherein an animal is simultaneously / co-treated with one or more antibiotics. The present invention also encompasses a vaccine of the present invention for use in a method for immunizing an animal against clinical disease caused by at least one pathogen in said animal, which vaccine does not cause clinical signs of infection but is capable of inducing an immune response that immunizes the animal against a pathotype of said at least one pathogen.

[0021] The invention also encompasses a vaccine of the invention for use in a method for eliciting a protective immune response, wherein the protective immune response against Lawsonia intracellularis is a protective immune response for reducing intestinal lesions in an animal compared to a non-immunized control animal of the same species. Thus, the vaccine of the invention is a vaccine for use in a method for reducing intestinal lesions in an animal compared to a non-immunized control animal of the same species, the method comprising administering the vaccine to the animal. The intestinal lesions may be ileal lesions. The intestinal lesions and / or ileal lesions may be macroscopic and / or microscopic lesions. The invention also encompasses the vaccine of the invention for use in a method for eliciting a protective immune response, wherein the protective immune response against Lawsonia intracellularis is a protective immune response for reducing fecal shedding in an animal compared to a non-immunized control animal of the same species. Thus, the vaccine of the invention is a vaccine for use in a method for reducing fecal shedding in an animal compared to a non-immunized control animal of the same species, the method comprising administering the vaccine to the animal.

[0022] The invention also encompasses a vaccine of the invention for use in a method for eliciting a protective immune response, wherein the protective immune response against Lawsonia intracellularis is a protective immune response for increasing the average daily weight gain in an animal compared to a non-immunized control animal of the same species. Thus, a vaccine of the invention is a vaccine for use in a method for increasing the average daily weight gain in an animal compared to a non-immunized control animal of the same species, the method comprising the step of administering the vaccine to an animal. The present invention relates to a vaccine of the invention for use in a method for eliciting a protective immune response comprising administering to said patient 8×10 9 The present invention also encompasses a vaccine which is protective against challenge with Lawsonia spp.

[0023] The present invention also includes methods for eliciting an immune or immunological or protective immune or immunological response in an animal against Lawsonia intracellularis, PCV, M. hyo. and PRRSV, the method comprising the step of administering to the animal any of the vaccines disclosed herein. The present invention also encompasses a method of immunizing an animal against clinical disease caused by at least one pathogen in the animal, comprising administering to the animal any one of the vaccines disclosed herein, wherein the vaccine does not cause clinical signs of infection but is capable of inducing an immune response that immunizes the animal against a pathotype of the at least one pathogen. Thus, the present invention encompasses the use of a vaccine of the present invention in the preparation of a composition for inducing a protective immune response against Lawsonia intracellularis and / or PCV and / or M. hyo. and / or PRRSV, and / or the use of a vaccine of the present invention for a method for inducing a protective immune response against Lawsonia intracellularis and / or PCV and / or M. hyo. and / or PRRSV.

[0024] The invention also encompasses a vaccine of the invention for use in a method for raising a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with one or more antibiotics. The present invention further encompasses a vaccine of the present invention for use in a method for eliciting a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with one or more antibiotics, said vaccine comprising live Lawsonia intracellularis bacteria and / or PCV2 ORF2 protein and / or M. hyo. bacterin and / or attenuated PRRSV virus. The present invention further relates to a vaccine of the present invention for use in a method for eliciting a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with one or more antibiotics, said vaccine comprising a live Lawsonia intracellularis bacterium, a PCV2 ORF2 protein, an M. hyo. bacterin and an attenuated PRRSV virus.

[0025] The present invention includes a vaccine of the present invention for use in a method for eliciting a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with one or more antibiotics, said vaccine comprising a live Lawsonia intracellularis bacterium and a PCV2 ORF2 protein. The present invention encompasses a vaccine of the invention for use in a method for eliciting a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with one or more antibiotics, said vaccine comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®. Additionally, the present invention encompasses a vaccine of the present invention for use in a method for eliciting a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with Denagard® (tiamulin) and / or CTC (chlortetracycline), said vaccine comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®.

[0026] It is further noted that the present invention is not intended to encompass within its scope any product, process, or method of making a product, or method of using a product, that does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph), and thus applicants reserve the right to, and hereby disclose the exclusion of, any previously described product, process for making a product, or method of using a product. All rights to expressly disclaim any embodiment that is the subject of any of applicants' issued patents in this line or in any other line or in any previously filed application of any third party are expressly reserved. Nothing herein should be construed as a commitment.

[0027] It is noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like may have the meaning ascribed to them in U.S. patent law, e.g., these terms may mean "includes," "included," "including," and the like, and that terms such as "consisting essentially of" and "consisting essentially of" may have the meaning ascribed to them in U.S. patent law, e.g., these terms take into account elements explicitly recited but exclude elements found in the prior art or which affect a basic or novel characteristic of the invention. These and other embodiments are disclosed or are obvious from, and are encompassed by, the following detailed description. The following detailed description, given by way of example and not intended to limit the invention to only the particular embodiments described, can be best understood in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0028] [Figure 1] Figure 1 shows the mean gross lesion scores of the ileum. Different letters indicate statistical significance (p<0.05) and error bars represent the standard error of the sample mean. [Diagram 2] Figure 2 shows the mean gross lesion length of the ileum. Different letters indicate statistical significance (p<0.05) and error bars represent standard error. [Diagram 3] Figure 3 shows the mean lesion severity in the ileum. Different letters indicate statistical significance (p<0.05) and error bars represent standard error. [Figure 4] Figure 4 shows group mean weight gain (lb) per day. Different letters indicate statistical significance (p<0.05) and error bars represent standard error. [Diagram 5] FIG. 5 shows the percentage of animals with a positive serum ELISA result for Lawsonia. [Figure 6] Figure 6 shows the average amount of L. intracellularis excreted by day, with different letters indicating statistical significance (p<0.05) and error bars representing standard error. [Figure 7] Figure 7 shows the mean microscopic lesion scores measured in the terminal ileum. Different letters indicate statistical significance (p<0.05) and error bars represent standard error. [Figure 8] FIG. 8 shows the mean immunohistochemistry scores for the presence of L. intracellularis antigens in ileal tissue. [Figure 9] FIG. 9 shows the vaccine formulation. [Figure 10]Figure 10 shows the outline of the study. Only the EIIMATB group in the sample received tiamulin / CTC one week before and after vaccination. Both groups (EIIM and EIIMATB) were vaccinated simultaneously, 4 weeks before challenge. All animals were euthanized and necropsied 21 days post infection (dpi). *= blood and fecal collection. Clinical scoring was performed daily from days 0 to 21. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention relates to a vaccine comprising an antigen of Lawsonia intracellularis and one or more antigens of at least one further pathogen selected from the group of Porcine circovirus (PCV), Mycoplasma hyopneumoniae (M.hyo.) and Porcine reproductive and respiratory syndrome virus (PRRSV), wherein the antigen of Lawsonia intracellularis is a live Lawsonia intracellularis bacterium. In one embodiment, the vaccine of the invention comprises a live Lawsonia intracellularis bacterium and an antigen of PCV. In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium and a PCV2 ORF2 protein. In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium and an antigen of M. hyo.

[0030] In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium and an M. hyo bacterin. In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium and an antigen of PRRSV. In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium and an attenuated PRRSV virus. In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium, an antigen of PCV, and an antigen of M. hyo. In one embodiment, a vaccine of the invention comprises the live Lawsonia intracellularis bacterium, the PCV2 ORF2 protein, and the M. hyo bacterin.

[0031] In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium, an antigen of PCV, and an antigen of PRRSV. In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium, a PCV2 ORF2 protein, and an attenuated PRRSV virus. In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium, an antigen for PRRSV, and an antigen for M. hyo. In one embodiment, the vaccine of the invention comprises a live Lawsonia intracellularis bacterium, an attenuated PRRSV virus, and an M. hyo bacterin. In one embodiment, a vaccine of the invention comprises a live Lawsonia intracellularis bacterium, an antigen for PCV, an antigen for M. hyo and an antigen for PRRSV. In one embodiment, the vaccine of the invention comprises a live Lawsonia intracellularis bacterium, a PCV2 ORF2 protein, an M. hyo bacterin, and an attenuated PRRSV virus.

[0032] It should be noted that, with respect to the components of the vaccines of the invention, the terms "vaccine" and "antigen" may be used interchangeably herein. Thus, "the vaccine comprises a PCV vaccine" may be used interchangeably with, for example, "the vaccine comprises a PCV antigen." With respect to the vaccine of the present invention, the terms "vaccine" and "immunogenic composition" may be used interchangeably herein. The terms "antigen," "immunogen," and "immunogenic component" may be used interchangeably herein. The terms "immune response", "immunological response", "protective immune response" and "protective immunological response" may be used interchangeably herein.

[0033] Furthermore, it should be noted that all disclosures provided herein can be combined. Thus, for example, a specific disclosure provided herein in relation to a PCV vaccine or antigen can be combined with a PRRSV vaccine or antigen, or vice versa, provided that such a transition is considered feasible by those skilled in the art based on the teachings herein. In other words, if a method, technique, route of administration, etc. is disclosed in relation to, for example, PCV, it can not be limited to PCV, but can also be used in relation to, for example, PRRSV. Furthermore, all disclosures provided herein for a particular described vaccine that includes a single antigen can also be applied to the described vaccine that includes more than one antigen. Any disclosure in the context of a method of the invention described herein is applicable to the corresponding uses and vice versa.

[0034] The vaccine of the present invention comprises an antigen of Lawsonia intracellularis. Thus, an immunogenic composition for eliciting a protective immune response in pigs against Lawsonia intracellularis is disclosed.

[0035] As used herein, the term "Lawsonia intracellularis" or "L. intracellularis" refers to the intracellular, curved, Gram-negative bacterium described in detail by C. Gebhart et al., Int'l. J. of Systemic Bacteriology, Vol. 43, No. 3, 533-38 (1993) and S. McOrist et al. Int'l. J. of Systemic Bacteriology, Vol. 45, No. 4, 820-25 (1995), each of which is incorporated herein by reference in its entirety, and deposited with the American Type Culture Collection, Rockville, Md. under ATCC 55672; 12656 and 12657; causative bacteria that may be obtained from pigs or other animals infected with PPE worldwide given the knowledge in the art and the teachings herein; and any variant or mutant of the above bacteria, whether naturally occurring or artificially obtained.

[0036] "Live L. intracellularis" as used herein means that the L. intracellularis bacteria are live bacteria. WO96 / 39629 and WO05 / 011731 describe live or attenuated L. intracellularis strains that are non-pathogenic. However, the vaccine compositions of the invention described herein may contain L. intracellularis bacteria that are inactivated / killed due to the production / formulation steps. As used herein, the term "attenuated strain" refers to any L. intracellularis strain that is prepared according to the culture and passaging techniques known in the art and / or taught herein to achieve reduced virulence, preferably no virulence, while maintaining immunogenic properties when administered to a host animal. As demonstrated below, a variety of different L. intracellularis strains have been cultured and attenuated according to the present teachings to obtain attenuated, immunogenic strains that have vaccine efficacy in pigs and other animals susceptible to L. intracellularis infection.

[0037] A genetically modified virus and / or bacteria, or a modified live virus and / or bacteria, is "attenuated" if it is less virulent than its unmodified parent strain. A strain is "attenuated" if it shows a statistically significant reduction in one or more parameters that determine disease severity. Such parameters can include, but are not limited to, the level of viremia, bacteremia, fever, severity of respiratory distress, severity of reproductive symptoms, or the number or severity of lesions in organs such as the intestine (particularly the ileum) or lungs.

[0038] The attenuated strains for use in the vaccines of the present invention are expected to be useful as immunogens in antimicrobial vaccines for animals, including birds, fish, cattle, pigs, horses, mammals and primates, and humans in general. Such vaccines can be prepared by techniques known to those skilled in the art given the teachings contained herein. Such vaccines will include an immunologically effective amount of the attenuated strain in a pharmaceutically acceptable carrier. The vaccine could be administered in one or more doses. The immunologically effective amount can be determined by means known in the art without undue experimentation given the teachings contained herein. The amount of avirulent bacteria is an amount sufficient to stimulate an immune response in a disease susceptible animal while still remaining avirulent. This will depend on the particular animal, bacteria and disease involved. The recommended dose administered to a susceptible animal is preferably about 10 3 ~10 9 Bacteria / Kg body weight, most preferably about 10 5 ~10 7 Bacteria / Kg body weight. Carriers are known to those skilled in the art and include stabilizers and diluents. Such vaccines may also contain suitable adjuvants. The vaccines of the present invention may be used in combination with other vaccines, for example as a diluent for another freeze-dried vaccine, or may be combined with another vaccine before freeze-drying, or may simply be mixed. In another embodiment, a mixture of two or more liquid vaccines is also contemplated. The vaccine preparation may also be dried, for example by freeze-drying, for storage purposes or for later formulation into a liquid vaccine. Thus, the present invention also includes a method for inducing an immune response in an animal host against virulent wild-type L. intracellularis bacteria for the purpose of protecting the host against such bacteria, comprising administering to the host an immunologically effective amount of a live, modified live, or attenuated bacterium or a bacterium as described herein, preferably administering to the host a vaccine of the present invention.

[0039] As used herein, "large scale cultivation" refers to a cultivation level of L. intracellularis greater than approximately 2.0-3.0 liters, including production at a scale of 100 liters or greater. "Cultivation," as used herein, refers to the process of promoting the growth, reproduction, and / or proliferation of L. intracellularis. When carrying out the method for culturing bacteria described herein, cultured cells may first be contacted with an inoculum containing L. intracellularis bacteria to infect the cells with the bacteria. Numerous cell lines may be used in the practice of the invention, including, but not limited to, IEC-18 (ATCC 1589)--rat intestinal epithelial cells, HEp-2 (ATCC 23)--human epidermoid carcinoma cells, McCoy (ATCC 1696)--mouse (unspecified) cells, MDCK (ATCC 34)--Madin-Darby canine kidney cells, BGMK (Biowhittaker #71-176)--buffalo green monkey kidney cells, and porcine intestinal epithelial cells. Preferred cultured cells are HEp-2, McCoy, or IEC-18 cells. Alternatively, the bacteria may be cultured in a cell-free system, provided that the bacteria are maintained at an appropriate dissolved O2 concentration as taught herein.

[0040] When cultured cells are used, it is preferred, but not necessary, that the cells are in the form of a monolayer before being inoculated. To form a monolayer, the cells can be seeded into conventional flasks. Generally, 25 cm 2 Approximately 1 x 10 per flask 5 ~About 10×10 5 Between 100 and 150 cells are mixed with growth medium and seeded into each flask. The growth medium can be any medium for cell culture that contains a nitrogen source, growth factors required for the selected culture cells, and a carbon source, such as glucose or lactose. A preferred medium is DMEM containing 2-5% fetal bovine serum, although a variety of other commercially available media can be used with good results. Applicants have found that successful cultivation of L. intracellularis is enhanced by keeping the cultured cells in a state of continued growth. Thus, the cultured cell monolayer should be about 20 percent to about 50 percent confluent at the time of inoculation. Preferably, the cells should be about 30 percent to about 40 percent confluent at the time of inoculation, with about 30 percent confluent being most preferred.

[0041] The inoculum can be a pure culture of L. intracellularis obtained, for example, from ATCC deposit 55672, NCTC deposit 12656 or 12657, or obtained from infected pigs or other animals using the isolation and purification methods discussed herein. According to one embodiment, the inoculum for carrying out the invention is an intestinal homogenate prepared by scraping the mucosa of the ileum of a pig or other animal infected with PPE. When preparing the intestinal homogenate, the ileal section selected for culture should show severe lesions with gross thickening of the digestive tract. Due to the fragility of the bacteria, the sample should preferably be stored at -70°C as soon as possible after necropsy. An antibiotic to which L. intracellularis is resistant, such as vancomycin, amphotericin B, or a member of the aminoglycoside group of antibiotics (gentamicin and neomycin, to name a few), is preferably added to the inoculum to suppress bacterial contamination while allowing L. intracellularis growth. Whether the inoculum is a pure culture or an intestinal homogenate, inoculation of cultured cells can be performed by various techniques known in the art, given the teachings herein.

[0042] The bacteria and / or inoculated cell culture are then incubated under reduced dissolved O2 concentrations. At dissolved oxygen concentrations greater than 18%, growth of L. intracellularis is less than optimal, and oxygen concentrations outside this range eventually result in growth cessation. Preferably, the inoculated cell culture is incubated at a dissolved oxygen concentration in the range of about 0% to about 10%. More preferably, the cells are incubated at an oxygen concentration in the range of about 0% to about 8%, with an oxygen concentration of about 0% to about 3.0% being most preferred. An appropriate concentration of carbon dioxide is also important for proper growth of L. intracellularis. Carbon dioxide concentrations above 10% and below 4% result in non-optimal growth, and carbon dioxide concentrations outside this range ultimately result in cessation of growth. Preferably, the carbon dioxide concentration is in the range of about 6% to about 9%, with a carbon dioxide concentration of about 8.8% being most preferred. In addition, the cells are preferably incubated at a hydrogen concentration ranging from about 73% to about 94%. Nitrogen may be used to replace some or all of the hydrogen present. According to certain preferred embodiments, the cells are incubated at about 0-8.0% O2, about 8.8% CO2, and about 83.2% H2.

[0043] The inoculated cells may be incubated in a dual gas incubator or other glass chamber that has suitable oxygen and carbon dioxide concentrations and allows the cells to be suspended during incubation. The chamber should include a means for maintaining the inoculated cells in suspension and a gas monitor and source for providing and maintaining the appropriate gas concentrations. The incubation temperature should be in the range of 30°C to 45°C, more preferably in the range of 36°C to 38°C. Most preferably, the temperature is about 37°C. The equipment required for the culture and attenuation methods of the present invention is readily available to one of skill in the art given the teachings herein. One example of equipment suitable for carrying out the present invention is a dual gas incubator, e.g., Model 480 available from Lab-Line, Melrose Park, Ill., in conjunction with a spinner flask to maintain the cells in suspension. Currently preferred equipment includes fermenters, bioreactors or rotary shakers that contain at least 2 liters of medium, where the cultured cells can be maintained in suspension by sparging with an appropriate concentration of gas or other mechanical agitation means, and where dissolved O2 levels in the medium can be continuously monitored. New Brunswick, Braun and other companies manufacture fermenters and bioreactors suitable for this purpose. By maintaining the inoculated cells in suspension during incubation, maximum growth of the cells, and therefore of L. intracellularis, is achieved by increasing the exposure of the individual cells to the growth medium and to the appropriate mixture of oxygen and carbon dioxide. Cultured cells can be agitated and maintained in suspension by a variety of methods known in the art, including, for example, culture flasks, roller bottles, membrane cultures and spinner flasks. Cells can be kept in suspension during incubation by incubating the cells in a spinner flask inside a dual gas incubator or similar device.The term "spinner flask," as used herein, means a flask or other container that uses paddles, propellers, or other means to agitate the culture and keep the cells contained therein in suspension.

[0044] In a particularly preferred embodiment of the invention, the inoculated cells are incubated until the cells are confluent, then the cells are placed in a spinner flask containing growth medium and incubated in a dual gas incubator while rotating the flask. Preferably, the inoculated cells are scraped into the spinner flask. This can be accomplished by a variety of methods known in the art, for example, by detaching the cells using a cell scraper. Once the cells are introduced into the spinner flask, the paddle of the spinner flask is rotated, typically in the range of about 30 to about 60 rpm, to maintain the infected cells in suspension. A portion of the cultured L. intracellularis is then passaged to fresh medium to increase production of L. intracellularis bacteria. As used herein, the term "passaging" or variations thereof refers to the process of transferring a portion of the cultured L. intracellularis to fresh culture cells for infection of the fresh cells with the bacteria. The term "fresh" as used herein refers to cells that have not yet been infected with L. intracellularis. Preferably, such cells are, on average, approximately one day old or less.

[0045] Passaging of L. intracellularis in suspension culture can be accomplished by removing a portion of the original culture and adding it to a new flask containing fresh cultured cells. If the original culture contains a large number of bacteria per ml, e.g., about 10 per ml, 4With more bacteria, it is preferred to add between about 1-10% (volume to volume) of the culture from the infected flask to the new flask containing fresh cells. This is preferably done when 50-100% of the cells are infected. If less than 50% of the cells are infected, passaging is preferably accomplished by splitting the culture 1:2 into a new flask and upsizing the volume with fresh medium. Either way, in stark contrast to passaging monolayer cultures as in the prior art, no cell lysis and other steps are required.

[0046] Cultured cells were allowed to grow sufficiently, and then TCID 50 At least a portion of the cultured L. intracellularis bacteria is harvested after infection with L. intracellularis at a cell infection rate of greater than about 70% as determined by IFA or other comparable methods. However, if different results are obtained using different techniques to determine cell infection, the results of the IFA method shall be used. The harvesting step can be performed by separating the bacteria from the suspension by various techniques known to those skilled in the art, given the teachings herein. Preferably, the L. intracellularis bacteria are harvested by centrifuging all or a portion of the contents of the suspension to pellet the cultured cells, resuspending the resulting cell pellet, and lysing the infected cells. Typically, at least a portion of the contents is centrifuged at about 3000×g for about 20 minutes to pellet the cells and bacteria. The pellet can then be resuspended, for example, in a sucrose-phosphate-glutamate (SPG) solution and passed through a 25-gauge needle approximately four times to lyse the cells. If further purification is desired, the sample can be centrifuged at about 145×g for about 5 minutes to remove cell nuclei and debris. The supernatant can then be centrifuged at about 3000×g for about 20 minutes, and the resulting pellet can be resuspended in a suitable diluent, for example, SPG containing fetal bovine serum (to prepare harvested bacteria suitable for freezing or for use as an inoculum) or SPG containing growth medium (to prepare harvested bacteria suitable for passaging to fresh cells).

[0047] As previously mentioned, efficient growth of L. intracellularis for large scale production is enhanced by maintaining active growth of the tissue cells. In monolayers, as the culture becomes confluent, the rate of cell division drops significantly. Attempts to grow L. intracellularis in monolayer tissue culture have had limited success and have not been amenable to scale-up. However, the use of suspension cultures significantly aids in maintaining active growth of the cells, allowing for sustained culture expansion and scale-up. Using a fermentor as described above and between about 0-3% dissolved O2, Applicant has successfully grown L. intracellularis in a 10 8 Applicants have also been able to maintain active growth of bacterial cultures for months and expect to be able to do so indefinitely.

[0048] Previously, it was generally believed that cells must be attached to a surface in order to be infected by L. intracellularis. The cell suspension disclosed herein is unique and contradicts this theory. When using McCoy or IEC-18 cells, gelatin, agarose, collagen, acrylamide, or silica beads, such as Cultispher-G porous microcarriers manufactured by HyClone Laboratories, Logan, Utah, can be added with the growth medium. In one embodiment, during the growth of McCoy cells infected with L. intracellularis in cell culture, uninfected McCoy cells can be added to the medium. However, McCoy as well as HEp-2 cells can be used in the culture method of the present invention without the need for microcarriers. This is a particularly advantageous and economical route to large-scale culture.

[0049] For culture maintenance purposes, for HEp-2 cultures, 25-50% of the culture is removed and replaced with fresh medium, preferably at weekly intervals. For cell cultures using microcarriers or beads, 25-50% of the culture is removed and replaced with new microcarriers and fresh medium, preferably once or twice a week. For scale-up purposes, an additional 25-50% of medium, or medium with microcarriers, may be added to the culture. Depending on the rate at which the cultured cells become infected, passage into fresh medium is generally performed about once every 2 weeks to about once every 5 weeks. Assuming that the cultured cells will reach at least 70% within 2 to 3 weeks, passage is preferably performed about once every 3 weeks to about once every 4 weeks.

[0050] Live L. intracellularis antigens for use in the vaccines of the invention can be produced by the production methods outlined above. According to particularly preferred embodiments, after maintaining the infected cells in suspension for an extended period of time (e.g., 6-8 months), at least a portion of the cultured L. intracellularis bacteria are harvested and monitored for possible attenuation. Such monitoring is preferably performed for each host animal or animal model challenge that selects for an attenuated strain. Such attenuated strains are used in vaccines according to the methods taught herein. Attenuated L. intracellularis vaccines according to the invention have shown efficacy against L. intracellularis infection in a variety of animals and are expected to be effective in humans.

[0051] Cultivation in suspension allows for rapid culture expansion, 100-1000 fold increase in yield, and cost reduction. As a result, the abundant supply of L. intracellularis bacteria produced according to the culture methods disclosed herein is easily attenuated for vaccine production purposes. Attenuation is difficult in monolayer cultures due to the low yield of bacteria produced using traditional monolayer growth techniques. In contrast, the disclosed L. intracellularis growth methods greatly increase the ease, speed, and number of bacteria available for this purpose. The more cells and cell divisions that occur, the higher the level of mutations that occur, which are advantageous for vaccine development. Growth in suspension increases the expression of important immunogens that are controlled by environmentally regulated genes and their expression products. The resulting attenuated strain can be cultured in tissue culture monolayers as described in Example 1 of U.S. Patent No. 5,885,823, but is preferably cultured in suspension culture according to the methods disclosed herein. Other attenuation methods can include, for example, chemical attenuation using N-methylnitrosoguanidine, and other attenuation methods known in the art. Whether by multiple passages or by chemical means, attenuated L. intracellularis is produced and selected for vaccine preparation.

[0052] According to one vaccine embodiment of the present disclosure, the antigen is recovered by centrifugation or microfiltration as described above. The antigen is then standardized at a level defined based on the optimal host animal immune response, as determined by dose escalation in the host animal species. According to a particularly preferred vaccine embodiment using the previously described culture methods, the bacteria are serially passaged for derivation and selection of attenuated, avirulent live cultures. The cultures are tested in host animals (preferably after growth in suspension culture for at least 6-8 months or longer) for signs of attenuation. Cultures are harvested and diluted as previously described. Inoculation of pigs with at least 1×10 5 ~1×10 6 Approximately 28 days after vaccination, pigs can be orally vaccinated with 1 × 10 bacteria from a low-passage (approximately 30-45 days old) virulent culture of L. intracellularis. 7 The organism is orally inoculated. Infected animals are necropsied 21 days after challenge and the small intestine is observed for macroscopic as well as microscopic lesions. PCR should also be performed. Approximately 80 percent of control animals will show macroscopic or microscopic lesions and test positive for the presence of L. intracellularis in intestinal mucosal cells using either the PCR or FA test methods. Vaccinated animals will have normal mucosal surfaces as judged by histological observation and will be negative by PCR test.

[0053] Generally, attenuated, immunogenic L. intracellularis strains are produced after continuous culture for at least about 150-250 days, during which the culture is passaged at least about 7 to about 12 times. Using the monitoring and selection methods taught herein, these numbers can be varied to produce other attenuated cultures. A vaccine is then prepared comprising an immunologically effective amount of attenuated L. intracellularis in a pharma- ceutically acceptable carrier. The combined immunogen and carrier can be an aqueous solution, emulsion, or suspension. The immunologically effective amount can be determined by means known in the art without undue experimentation given the teachings contained herein. In general, the amount of immunogen is between 50 and 500 micrograms, preferably 10 micrograms when purified bacteria are used. 7 ~10 9 TCID 50 It will be between.

[0054] L. intracellularis bacteria grown according to the methods of the invention, or components derived from such bacteria, can be used as antigens in ELISA or other immunoassays, such as immunofluorescent antibody tests ("IFAs"), to detect L. intracellularis in serum and other body fluids of animals suspected of infection with the bacteria. A currently preferred immunoassay is an IFA, such as that described in Example 1 of U.S. Patent No. 5,885,823. Alternatively, bacteria grown according to the invention can be used in a Western blot assay.

[0055] WO96 / 39629 and WO05 / 011731 describe the cultivation of Lawsonia intracellularis, attenuated Lawsonia intracellularis and its administration. In an advantageous embodiment, the live Lawsonia intracellularis bacterium is a modified live Lawsonia intracellularis bacterium. In another advantageous embodiment, the live Lawsonia intracellularis bacterium is an attenuated Lawsonia intracellularis bacterium. In an advantageous embodiment, the vaccine of the invention is administered at a concentration of about 10 3 ~109 of bacteria / kg body weight, preferably about 10 5 ~10 7 with a dosage of Lawsonia intracellularis of bacteria / kg body weight. In an advantageous embodiment, the vaccine of the invention comprises about 10 5 ~about 10 7 with a dosage of Lawsonia intracellularis antigen. In an advantageous embodiment, the Lawsonia intracellularis antigen is lyophilized. In an advantageous embodiment, the Lawsonia intracellularis antigen in the vaccine of the invention is an antigen contained in Enterisol® Ileitis. In an advantageous embodiment, the Lawsonia intracellularis vaccine is Enterisol® Ileitis vaccine.

[0056] A preferred method of immunization or vaccination consists in administering the vaccine according to the invention by systemic administration, such as by the intramuscular route. In one aspect, the vaccine of the present invention may comprise an antigen of PCV. Thus, in one aspect of the present invention, an immunogenic composition for eliciting a protective immune response in pigs against PCV is provided. In the context of the present invention, a plasmid construct encoding and expressing a PCV immunogen (antigen) can be used. Furthermore, vaccination methods and DNA vaccines are described herein. In addition, the present invention relates to methods of producing or formulating these vaccines. Inactivated PCV vaccines (see, for example, U.S. Patent No. 6,517,843) are also contemplated.

[0057] According to Meehan 1998, PCV ORF1 and ORF2 encode proteins with predicted molecular weights of 37.7 kD and 27.8 kD, respectively. ORF3 and ORF4 (corresponding to ORF7 and ORF10 in WO9918214, respectively, according to Meehan et al. 1998) encode proteins with predicted molecular weights of 11.9 and 6.5 kD, respectively. The sequences of these ORFs are also available in Genbank AF055392. They can also be incorporated into plasmids and used according to the invention, either alone or in combination, e.g. in combination with ORF1 and / or ORF2 and / or ORF3.

[0058] Other PCV ORFs 1-3 and 5, 6, 8-9, 11-12 disclosed in U.S. Pat. No. 6,391,314 (columns 1-3 and 5, 6, 8-9, 11-12 in WO9918214) can be used in combination or otherwise with each other or with ORFs 1 and 2 defined herein under the conditions described herein. This also includes the use of equivalent sequences within the knowledge given above, in particular the use of ORFs from the various PCVs mentioned herein. The term "equivalent sequence" as used herein may refer to a sequence from a PCV strain having ORF2 and / or ORF1 with homology or identity as further explained below with the corresponding ORF of the Imp 1010 strain. For ORF3 according to Meehan, it can be said that the homology or identity must be, for example, equal to or higher than 80%, in particular higher than 85%, preferably higher than 90% or 95% with ORF3 of the Imp 1010 strain. For ORF4 according to Meehan 1998, it may be equal to or higher than 86%, in particular higher than 90%, preferably higher than 95% with ORF4 of the Imp 1010 strain.

[0059] It is routine to determine the ORFs from the genomic nucleotide sequence, e.g., as disclosed in WO9918214, using standard software such as MacVector™. Also, alignment of the genome with the 1010 strain genome and comparison with the 1010 strain ORFs allows the skilled person to easily determine the ORFs on the genome for another strain (e.g., as disclosed in WO9918214). The use of software to perform alignments is routine for the skilled person and can provide direct access to the equivalent ORFs.

[0060] PCV3 ORF2 and PCV3 genome sequences were obtained from KT869077 (GenBank). Preferably, the PCV antigen of the vaccine of the invention is a PCV1, PCV2 and / or PCV3 antigen. Preferably, the PCV antigen of the vaccine of the invention is a recombinant polypeptide. In a preferred embodiment, the polypeptide of the present disclosure is a recombinant PCV1, PCV2 or PCV3 ORF2 protein, such as a recombinant baculovirus-expressed PCV3 ORF2 protein, or preferably a recombinant baculovirus-expressed PCV2 ORF2 protein. The term "recombinant ORF2 protein" as used herein refers to a protein molecule expressed from a recombinant DNA molecule, such as a polypeptide produced by recombinant DNA techniques. Examples of such techniques may include the case where the DNA encoding the protein to be expressed is inserted into a suitable expression vector, such as a baculovirus expression vector, and also the vector is used to transfect or, in the case of baculovirus expression, infect a host cell to produce the protein or polypeptide encoded by the DNA. Thus, the term "recombinant ORF2 protein" as used herein refers in particular to a protein molecule expressed from a recombinant DNA molecule.

[0061] In other words, the PCV antigen of the vaccine of the present invention is preferably a recombinant polypeptide expressed (encoded) by a PCV ORF gene, preferably the PCV ORF2 gene, most preferably the PCV2 ORF2 gene. The PCV antigen of the vaccine of the present invention is preferably a recombinant polypeptide expressed from (encoded by) a baculovirus cell. The PCV antigen of the vaccine of the present invention is preferably a recombinant polypeptide expressed from (encoded by) a PCV ORF gene, preferably the PCV ORF2 gene, most preferably the PCV2 ORF2 gene, and is expressed in a baculovirus cell. The PCV antigen of the vaccine of the present invention is preferably an antigen of a PCV contained in Ingelvac CircoFLEX® or 3FLEX®.

[0062] According to a particular example, recombinant PCV1, PCV2 or PCV3 ORF2 protein is produced by a method comprising the steps of cloning the PCV1, PCV2 or PCV3 ORF2 gene into a baculovirus transfer vector, using the transfer vector to prepare a recombinant baculovirus containing said gene by homologous recombination in an insect cell, and then expressing the PCV1, PCV2 or PCV3 ORF2 protein in the insect cell during infection with the recombinant baculovirus.

[0063] It is further understood that the term "recombinant PCV protein consisting of a sequence" also relates to any co-translational and / or post-translational modification(s) of the sequence, in particular influenced by the cell in which the polypeptide is expressed. Thus, the term "recombinant PCV ORF2 protein consisting of a sequence", as described herein, also covers a sequence having one or more modifications influenced by the cell in which the polypeptide is expressed, in particular modifications of amino acid residues resulting from protein biosynthesis and / or protein processing, preferably modifications selected from the group consisting of glycosylation, phosphorylation and acetylation. Preferably, the recombinant PCV1, PCV2 or PCV3 ORF2 protein according to the present disclosure is produced or obtainable by a baculovirus expression system, in particular in cultured insect cells.

[0064] As used herein, the term "plasmid" is intended to apply to any DNA transcription unit in the form of a polynucleotide sequence that contains the PCV sequence to be expressed and the elements necessary for its in vivo expression. Supercoiled or otherwise circular plasmid forms are also preferred. Linear forms are also included within the scope of the invention. In the context of the present invention, in particular the plasmids of US Pat. No. 6,943,152 can be used. Each plasmid contains a promoter that can ensure the expression in the host cell of the inserted gene under its control. The promoter is generally a strong eukaryotic promoter, in particular the cytomegalovirus early promoter CMV-IE, which may be of human or mouse origin or of other origins such as rat or guinea pig. More generally, the promoter is either a viral promoter or a cellular promoter. Viral promoters other than CMV-IE can include the SV40 virus early or late promoter or the Rous sarcoma virus LTR promoter. The promoter can also be a promoter from the virus from which the gene originates, for example a promoter specific for that gene. Cellular promoters can include promoters of cytoskeleton genes, such as the desmin promoter, or alternatively the actin promoter. If several genes are present in the same plasmid, they can be provided in the same transcription unit or in two different units.

[0065] The plasmid may also contain other regulatory elements, such as stabilizing sequences of the intron type, preferably intron II of the rabbit beta-globin gene (van Ooyen et al. Science, 1979, 206: 337-344), the signal sequence of the protein encoded by the tissue plasminogen activator gene (tPA; Montgomery et al. Cell. Mol. Biol. 1997, 43: 285-292), and a polyadenylation signal (polyA), in particular the polyA of the bovine growth hormone (bGH) gene (U.S. Pat. No. 5,122,458) or of the rabbit beta-globin gene.

[0066] "Sequence identity", as known in the art, refers to the relationship between two or more polypeptides or between two or more polynucleotide sequences, i.e., the relationship between a reference sequence and a given sequence to be compared to the reference sequence. Sequence identity is determined by comparing a given sequence to a reference sequence after the sequences are optimally aligned to produce a high degree of sequence similarity as determined by the match between strings of such sequences. By such alignment, sequence identity is determined position by position, e.g., a sequence is "identical" at a particular position if the nucleotide or amino acid at that position is identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to obtain the % sequence identity. Sequence identity was determined in Computational Molecular Biology, Lesk, AN, ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York (1993);Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey (1994);Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference.The preferred method for determining sequence identity is designed to produce the maximum match between the sequences tested. Methods for determining sequence identity are codified in publicly available computer programs that determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990)). BLASTX programs are available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights to produce a high level of sequence identity between a given sequence and a reference sequence. By way of illustration, for a polynucleotide sequence having a nucleotide sequence that has at least, for example, 85%, preferably 90%, and even more preferably 95% "sequence identity" to a reference nucleotide sequence, the nucleotide sequence of the given polynucleotide is intended to be identical to the reference sequence, except that the given polynucleotide sequence may contain no more than 15, preferably no more than 10, and even more preferably no more than 5 point mutations per 100 nucleotides of the reference nucleotide sequence.In other words, for a polynucleotide having a nucleotide sequence with at least 85%, preferably 90%, and even more preferably 95% sequence identity to a reference polynucleotide sequence, 15% or less, preferably 10% or less, and even more preferably 5% or less of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or a number of nucleotides may be inserted into the reference sequence, which number is 15% or less, preferably 10% or less, and even more preferably 5% or less of the total nucleotides in the reference sequence. These variations in the reference sequence may be present at the 5' or 3' terminal positions of the reference nucleotide sequence, or may be present anywhere between these terminal positions, either interspersed individually between nucleotides in the reference sequence, or within one or more contiguous groups in the reference sequence. Similarly, for a polynucleotide sequence having a given amino acid sequence with at least, for example, 85%, preferably 90%, and even more preferably 95% sequence identity to a reference amino acid sequence, the given amino acid sequence of the polypeptide is intended to be identical to the reference sequence, except that the given polypeptide sequence may contain no more than 15, preferably no more than 10, and even more preferably no more than 5 changes per 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, and even more preferably 95% sequence identity to a reference amino acid sequence, no more than 15%, preferably no more than 10%, and even more preferably no more than 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids may be inserted into the reference sequence that is no more than 15%, preferably no more than 10%, and even more preferably no more than 5% of the total number of amino acid residues in the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence, or may occur anywhere between these terminal positions, interspersed individually, either between nucleotides in the reference sequence, or within one or more contiguous groups in the reference sequence. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as matches when determining sequence identity.

[0067] "Sequence homology" as used herein refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are aligned and gaps are inserted as necessary. However, in contrast to "sequence identity", conservative amino acid substitutions are not counted as matches when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide with 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or contain a conservative substitution with another amino acid or nucleotide, or a number of amino acids not containing a conservative substitution, not more than 15%, preferably not more than 10%, even more preferably not more than 5% of the total amino acid residues or nucleotides in the reference sequence, can be inserted into the reference sequence. Preferably, the homologous sequence comprises at least a stretch of 50, even more preferably 100, even more preferably 250, even more preferably 500 nucleotides.

[0068] Sequence comparison can be performed over the entire length of the two sequences being compared, or over fragments of the two sequences. Sequence identity can also be performed over a region, e.g., over 20, 50, 100 or more consecutive amino acid residues, but usually the comparison will be performed over the entire length of the two sequences to be compared.

[0069] A "conservative substitution" refers to the replacement of one amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties, including size, hydrophobicity, etc., in a manner that does not significantly alter the overall function.

[0070] In the context of the present invention, PCV1 or PCV2 or PCV3 with mutations, for example, but not limited to, mutations in the capsid protein, may also be used. Despite the differences in the capsid amino acid sequences between PCV2 and Beak and Feather Disease Virus (BFDV), the crystal structures are very similar despite their differences. Advantageously, the mutations in PCV3 are mutations that stabilize virus-like particles (VLPs). Although the PCV3 capsid protein should self-assemble into VLPs, the expression level of the PCV3 protein is significantly lower compared to the PCV2 capsid protein. Specifically, only about 20% of the protein assembles into VLPs, while the remaining 80% of the protein aggregates into an insoluble fraction. Mutations in the PCV3 capsid protein disclosed in International Application No. PCT / US2020 / 026930 may be used in the context of the present invention.

[0071] Suitable assays and techniques for use in the context of the present invention include those used to track or quantify the assembly and disassembly of virus-like particles (VLPs) of porcine circovirus capsid (ORF2) protein, including enzyme-linked immunosorbent assay (ELISA); SDS / PAGE, which may use silver stains or Coomassie stains; Western blots or immunoblots; size-exclusion chromatography (SEC); dynamic light scattering (DLS) or multi-angle light scattering (MALS); transmission electron microscopy (TEM); analytical ultracentrifugation; and fluorescence spectroscopy (FSA) which may be coupled with high performance liquid chromatography (HPLC). Additional suitable techniques may also include agarose gel retardation tests of protein-nucleic acid complexes, immunodiffusion tests, such as single radial immunodiffusion (SRID), nanoparticle tracking analysis (NTA), metabolic labeling, and chemiluminescent enzyme-based assays.Each of these assays is well known in the art and is described, for example, in Fang, Mingli et al. "Detection of the Assembly and Disassembly of PCV2b Virus-Like Particles Using Fluorescence Spectroscopy Analysis" Intervirology vol. 58, 2015, pp. 318-323;Thompson, Christine et al. "Analytical technologies for influenza virus-like particle candidate vaccines: challenges and emerging approaches" Virology Journal vol 10, 2013, p. 141;Steppert, Petra et al. "Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis" Journal of Chromatography A vol. 1487, 2017, pp. 89-99;Yadav, Shalini et al. "A facile quantitative assay for viral particle genesis reveals cooperativity in virion assembly and saturation of an antiviral protein” Virology, vol 429, No. 2, 2012, pp. 155-162; and Zeltins, Andris “Construction and Characterization of Virus-Like Particles: A Review” Molecular Biotechnology vol. 53, 2013, pp. 92-107, each of which references is incorporated herein by reference in its entirety.

[0072] The development of a recombinant baculovirus (BaculoG / PCV3 ORF2 Clone 4B4-2E12 Pre-MSV p8; Lot No. 3624-039) containing the PCV3 ORF2 gene under the control of the baculovirus polyhedrin promoter is described in Example 1 of International Application No. PCT / US2020 / 026930. In some embodiments, the use of such recombinant baculovirus expressed proteins described in Example 1 above in vaccines may include killed and / or inactivated versions of the recombinant virus. Alternatively, recombinant viruses, such as those similar to those shown in Example 1 of International Application No. PCT / US2020 / 026930, may be used as modified live viruses in some vaccines.

[0073] In some embodiments, the amplified PCV ORF2 coding sequence can be subcloned into a baculovirus transfer vector using flanking restriction sites to generate the desired transfer vector. For example, the amplified PCV ORF2 coding sequence can be subcloned into a baculovirus transfer vector using flanking restriction sites to generate the transfer vector. Recombinant baculovirus can be generated by co-transfecting the transfer vector and baculovirus DNA into insect cells. The baculovirus DNA used can include linearized and / or circular baculovirus DNA. For example, in certain embodiments, recombinant baculovirus can be generated by co-transfecting the transfer vector and linearized BaculoGold™ baculovirus DNA into Sf9 (Spodoptera frugiperda) insect cells. The linearized baculovirus DNA may be derived from Autographa californica nuclear polyhedrosis virus (AcNPV) and may contain a lethal deletion at the polyhedrin locus, thus allowing rescue of a viable baculovirus by co-transfection with a transfer vector. The resulting recombinant baculovirus may contain the PCV ORF2 coding sequence under the control of the baculovirus polyhedrin promoter. The recombinant baculovirus may be amplified using Sf9 insect cells and then purified by limiting dilution cloning using Sf9 insect cells. In some embodiments, full-length circular baculovirus DNA, e.g., Bac-to-Bac, may be used. For example, Bac-to-Bac may use transposon-mediated recombination to insert a gene of interest into the polyhedrin locus. Other methods known in the art may also be used. In some embodiments, a method may be selected based on the potential stability of the commercialized method. For example, a baculovirus that results in improved vaccine stability may be selected.

[0074] In some embodiments, after the master cell culture is seeded into the flask, the flask may be incubated at a predetermined temperature for a specific time frame. The culture may then be incubated at 27°C for 4 hours. The flask may then be seeded with a recombinant baculovirus containing the PCV ORF2 gene. For example, a plasmid containing the ORF2 gene may be co-transfected with BaculoGold® (BD Biosciences Pharmingen) baculovirus DNA into Sf+ insect cells (Protein Sciences, Meriden, CT) to generate a recombinant baculovirus containing the ORF2 gene. The recombinant baculovirus containing the ORF2 gene may be plaque purified and the master seed virus (MSV) may be grown using SF+ cell line, aliquoted, and stored at -70°C. The MSV may be unambiguously identified as an ORF2 baculovirus by PCR-RFLP using baculovirus-specific primers. Insect cells infected with ORF2 baculovirus to generate MSV or working seed virus can express ORF2 antigen as detected by polyclonal serum or monoclonal antibody in indirect fluorescent antibody assay. In addition, the identity of ORF2 baculovirus can be confirmed by N-terminal amino acid sequencing. ORF2 baculovirus MSV can also be tested for purity according to 9 CFR 113.27c, 113.28 and 113.55. Each recombinant baculovirus inoculated into spinner flasks can have different multiplicities of infection (MOI).

[0075] After inoculation with baculovirus, the flasks may be incubated at 27±2° C. for 7 days while being agitated at 100 rpm. The flasks may use vented caps to allow airflow. Samples may be taken from each flask every 24 hours for the next 7 days. After collection, each sample may be centrifuged and both the pellet and supernatant separated and then microfiltered through a membrane with a pore size of 0.45-1.0 μm.

[0076] The amount of ORF2 in the resulting samples can then be quantified by ELISA assay. The ELISA assay can be performed with anti-PCV antibody diluted 1:6000 in 0.05M carbonate buffer (pH 9.6). 100 μL of antibody can then be placed into the wells of a microtiter plate, sealed, and incubated overnight at 37° C. The plate is then washed three times with a washing solution that included 0.5 mL of Tween 20 (Sigma, St. Louis, MO), 100 mL of 10×D-PBS (Gibco Invitrogen, Carlsbad, CA), and 899.5 mL of distilled water. Then, 250 μL of blocking solution (5 g of Carnation skim milk powder (Nestle, Glendale, CA) in 10 mL of D-PBS QS, made up to 100 mL with distilled water) is added to each well. The next step is to wash the test plate and then add pre-diluted antigen. Pre-diluted antigen is generated by adding 200 μL of dilution solution (0.5 mL Tween 20 in 999.5 mL D-PBS) to each of the wells of the dilution plate. Samples are then diluted in 1:240 and 1:480 ratios, and 100 μL of each of these diluted samples is added to one of the top wells of the dilution plate (i.e., one top well receives 100 μL of the 1:240 dilution and the other receives 100 μL of the 1:480 dilution). Serial dilutions can then be performed on the remainder of the plate by removing 100 μL from each successive well and transferring it to the next well on the plate. Each well is mixed before the next transfer is made. Washing the test plate involves washing the plate three times with wash buffer. The plate is then sealed and incubated at 37° C. for 1 hour, followed by another 3 washes with wash buffer. The detection antibody used is an antibody against PCV ORF2. The antibody is diluted 1:300 in dilution solution, and then 100 μL of diluted detection antibody is added to the well. The plate is then sealed and incubated at 37° C. for 1 hour, followed by 3 washes with wash buffer.Conjugate diluents are then prepared by adding normal rabbit serum (Jackson Immunoresearch, West Grove, Pa.) to the diluent to a concentration of 1%.

[0077] Conjugated antibody goat anti-mouse (H+1)-HRP (Jackson Immunoresearch) is diluted 1:10,000 in this conjugate diluent. 100 μL of diluted conjugated antibody is then added to each of the wells. The plate is then sealed and incubated for 45 minutes at 37° C., followed by washing three times with wash buffer. 100 μL of substrate (TMB Peroxidase Substrate, Kirkgaard and Perry Laboratories (KPL), Gaithersburg, MD) mixed with an equal volume of Peroxidase Substrate B (KPL) is added to each of the wells. The plate is incubated for 15 minutes at room temperature. 100 μL of IN HCL solution is then added to all wells to stop the reaction. The plate is then loaded onto an ELISA reader.

[0078] Advantageously, insect cells can be cultured under serum-free conditions to produce PCV ORF2 protein, such as the serum-free insect cells (expresSf+ cell line) of U.S. Patent No. 6,103,526. Other insect cell lines include, but are not limited to, Fall Armyworm (Sf) cell lines, such as Sf21, sf9, expresSf+1 (SF+) from Trichoplusia ni cabbage looper, BTI-TN5B1 (High Five) cells, and BmN cells from Bombyx mori (silkworm), which are widely used in baculovirus research and for recombinant protein production.

[0079] The adjuvants, cell culture supernatants, preservatives, stabilizers, viral vectors, immunomodulators and dosages disclosed in U.S. Pat. Nos. 9,610,345 and 9,669,087 are contemplated, both references being incorporated herein by reference.

[0080] In the context of the present invention, immunogenic preparations and DNA vaccines may also be used that contain at least one plasmid as disclosed herein that encodes and expresses one of the PCV1 or PCV2 or PCV3 immunogens, preferably one of the above-mentioned ORFs, and may additionally contain a veterinarily acceptable vehicle or adjuvant, and may additionally contain a veterinarily acceptable adjuvant. In one embodiment, the adjuvant may include CARBOPOL™ or ImpranFLEX®. In certain embodiments, an immunogenic composition refers to a composition that includes, in a 1 ml dose, i) at least some PCV ORF2 protein, ii) a baculovirus expressing said PCV ORF2 protein, iii) a cell culture, iv) an inactivating agent (e.g., BEI) having a concentration ranging from about 2 to about 8 mM, v) an amount of a neutralizing agent (e.g., sodium thiosulfate) equal to the inactivating agent, and vi) an amount of an adjuvant (e.g., CARBOPOL™ 971 or ImpranFLEX®), and vii) a physiologically acceptable concentration of phosphate.

[0081] Most preferably, the composition provided herein comprises PCV ORF2 protein recovered from the supernatant of in vitro cultured cells, the cells being infected with a recombinant viral vector containing PCV ORF2 DNA and expressing the PCV ORF2 protein, and the cell culture being treated to inactivate the viral vector with about 2 to about 8 mM BEI, preferably about 5 mM BEI, and an equivalent concentration of a neutralizing agent, preferably a sodium thiosulfate solution at a final concentration of about 2 to about 8 mM, preferably about 5 mM. The amount of PCV antigen-encoding DNA used in the vaccine according to the present invention is between about 10 μg and about 2000 μg, preferably between about 50 μg and about 1000 μg. Those skilled in the art will have the faculties necessary to precisely define the effective dose of DNA to be used for each immunization or vaccination protocol. The dose volume may be between 0.5 and 5 ml, preferably between 2 and 3 ml.

[0082] In another embodiment, the invention encompasses a method for eliciting an immune or immunological response or a protective immune or immunological response, inter alia, against Porcine Circovirus (PCV), comprising parenteral or subcutaneous administration to a pig of a single shot, single administration or single dose of (i) at least 2 μg to about 400 μg of PCV ORF2 recombinant protein expressed by a baculovirus system, and (ii) a veterinary acceptable carrier, wherein the veterinary acceptable carrier comprises a solvent, dispersion medium, coating agent, stabilizer, diluent, preservative, antimicrobial agent, antifungal agent, isotonicity agent, adsorption retardant, adjuvant, cell culture supernatant, stabilizer, virus or expression vector, immunomodulatory agent, and / or any combination thereof. In an advantageous embodiment, the PCV vaccine is Ingelvac CircoFLEX® (see, for example, WO2006 / 072065). WO2006 / 072065 and WO2008 / 076915 describe the generation of PCV vaccines, their formulation and their administration.

[0083] In an advantageous embodiment, the vaccine comprises a dosage of about 2 μg to about 400 μg of a PCV antigen. For example, the vaccine comprises a dosage of about 2 μg to about 400 μg of PCV2 ORF2 protein. In another advantageous embodiment, the vaccine comprises a dosage of about 4 μg to about 200 μg of a PCV antigen. For example, the vaccine comprises a dosage of about 4 μg to about 200 μg of PCV2 ORF2 protein. In yet another advantageous embodiment, the vaccine comprises a dosage of about 10 μg to about 100 μg of a PCV antigen. For example, the vaccine comprises a dosage of about 10 μg to about 100 μg of PCV2 ORF2 protein.

[0084] In an advantageous embodiment, the vaccine according to the invention comprises an antigen of Lawsonia intracellularis and one or more antigens of PCV, wherein the antigen of Lawsonia intracellularis is a live Lawsonia intracellularis bacterium, preferably an attenuated Lawsonia intracellularis bacterium or a modified live Lawsonia intracellularis bacterium. In an advantageous embodiment, the vaccine according to the invention comprises an antigen of Lawsonia intracellularis and an antigen of a PCV contained in Ingelvac CircoFLEX® or 3FLEX®, wherein the antigen of Lawsonia intracellularis is a live Lawsonia intracellularis bacterium, preferably an attenuated Lawsonia intracellularis bacterium or a modified live Lawsonia intracellularis bacterium. In an advantageous embodiment, the vaccine according to the invention comprises a 3 ~10 9 of bacteria / kg body weight, preferably about 10 5 ~10 7 The dosage of Lawsonia intracellularis antigen is included in the PCV contained in Ingelvac CircoFLEX® or 3FLEX®, and the Lawsonia intracellularis antigen is a live Lawsonia intracellularis, preferably an attenuated Lawsonia intracellularis or a modified live Lawsonia intracellularis.

[0085] In an advantageous embodiment, the vaccine according to the invention comprises about 10 5 ~about 10 7and comprising an antigen of a PCV contained in Ingelvac CircoFLEX® or 3FLEX®, wherein the Lawsonia intracellularis antigen is a live Lawsonia intracellularis bacterium, preferably an attenuated Lawsonia intracellularis or a modified live Lawsonia intracellularis bacterium. In an advantageous embodiment, the vaccine according to the invention comprises an antigen of Lawsonia intracellularis, as contained in Enterisol® Ileitis, and one or more antigens of PCV. In an advantageous embodiment, the vaccine according to the invention comprises an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and one or more antigens of a PCV, the PCV being PCV1, PCV2 or PCV3. In an advantageous embodiment, the vaccine according to the invention comprises an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and one or more antigens of PCV, the antigen of PCV being a recombinant polypeptide, preferably a recombinant polypeptide expressed in baculovirus cells.

[0086] In an advantageous embodiment, the vaccine according to the invention comprises an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and one or more antigens of PCV, the antigen of PCV being a recombinant polypeptide expressed by a PCV ORF gene, preferably a recombinant polypeptide expressed by a PCV ORF gene expressed in a baculovirus cell. In an advantageous embodiment, the vaccine according to the invention comprises an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and one or more antigens of PCV, the antigen of PCV being a recombinant polypeptide expressed by the PCV ORF2 gene, preferably a recombinant polypeptide expressed by the PCV ORF2 gene expressed in a baculovirus cell. In a very advantageous embodiment, the vaccine according to the invention comprises an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®.

[0087] A preferred method of immunization or vaccination consists in the systemic administration of the vaccine according to the invention and as described immediately above. Systemic administration methods are described herein and include, but are not limited to, intramuscular and intradermal administration. Therefore, a preferred method of immunization or vaccination consists in administering the vaccine according to the invention by the intramuscular route.

[0088] In one aspect, the vaccine of the present invention may comprise an antigen of M.hyo. Thus, in one aspect of the present invention, an immunogenic composition for eliciting a protective immune response in pigs against M.hyo. is provided. Even more preferably, the amount of M.hyo. in each dose has a relative potency (RP) value of at least 1.22, a relative potency of 1.22 meaning that at least 95%, preferably 100%, of mice receiving one-fortieth (1 / 40) such amount of M.hyo. antigen will develop a detectable amount of antibody in a M.hyo.-specific antibody detection assay within 21 days or on the 21st day after treatment. Thus, the 40-fold amount of M.hyo. antigen required to induce a detectable M.hyo.-specific antibody response in at least 95%, preferably 100%, of mice within 21 days or on the 21st day after treatment is sufficient to confer a protective immune response against M.hyo. infection, to reduce the incidence of the infection, and / or to reduce the severity of or prevent clinical signs associated with the infection. In other words, the amount of M.hyo. antigen described above has been proven to be able to overcome any negative interference with PCV antigens when mixed and administered as a combination vaccine. The composition in some preferred forms further includes or comprises an adjuvant. Although a variety of adjuvants are useful in the context of the present invention and can be selected by those skilled in the art, carbomer, more preferably CARBOPOL® (high molecular weight cross-linked polyacrylic acid polymer) or ImpranFLEX®, is particularly preferred. Advantageously, the immunogenic compositions of the invention, when administered in a single dose to pigs, confer a protective immune response against M. hyo. infection, reduce the incidence of said infection, and / or reduce the severity of and / or prevent clinical signs associated with said infection.Such a single dose, when administered to pigs, results in a duration of immunity of at least 100, more preferably at least 110, even more preferably at least 120, even more preferably at least 130, even more preferably at least 140, even more preferably at least 150, even more preferably at least 160, even more preferably at least 170, even more preferably at least 180, and most preferably at least 184 days. In other words, one dose of the immunogenic composition of the present invention results in a reduction in the incidence of infection or a reduction in the severity of clinical signs of infection by M. hyo. in an animal or group of animals for at least 100 days (such as 110, 120, 130, 140, 150, 160, 170, 180 days), most preferably at least 184 days, without booster or subsequent administrations. With regard to antibody detection assays, the skilled artisan will be able to identify and utilize suitable products. ELISA assays and in particular the IDEXX Herdchek M.hyo.Test Kit™ (IDEXX Laboratories, Inc., Westbrook, Me.) are preferred. In particular, the IDEXX Herdchek M.hyo.Test Kit™ (IDEXX Laboratories, Inc., Westbrook, Me.) can be used as a reference assay in accordance with the present invention.

[0089] As used herein, a "protective immune response" refers to a reduction in the incidence of M. hyo. infection or a reduction in the severity of the clinical, pathological or histopathological signs of that infection. A "protective immune response" can be elicited by an immunologically effective amount of an antigen or vaccine.

[0090] The term "M.hyo. antigen" refers to any composition comprising at least one antigen capable of inducing, stimulating or enhancing an immune response against M.hyo. infection when administered to an animal, preferably a pig. Preferably, said M.hyo. antigen is a whole M.hyo. bacterin, preferably in an inactivated form, a live modified or attenuated M.hyo. bacterium, a chimeric virus comprising at least an immunogenic amino acid sequence of M.hyo., or any other polypeptide or component comprising at least an immunogenic amino acid sequence of M.hyo. Preferably, the M.hyo. antigen is an inactivated M.hyo. bacterin. More preferably, the M.hyo. antigen is derived from M.hyo. J strain. Most preferably, the M. hyo. bacterin is an inactivated M. hyo. bacterin contained in the INGELVAC® MYCOFLEX vaccine (Boehringer Ingelheim Vetmedica Inc, St Joseph, Mo., USA) or is INGELVAC® MYCOFLEX. However, M. hyo. antigens that can be used according to the invention can also be selected from any of those contained in the following vaccine compositions: PORCILIS M.HYO, MYCO SILENCER® BPM, MYCO SILENCER® BPME, MYCO SILENCER® ME, MYCO SILENCER® M, MYCO SILENCER® ONCE, MYCO SILENCER® MEH (all from Intervet Inc., Millsboro, Del., USA), STELLAMUNE MYCOPLASMA™ (Pfizer Inc., New York, NY, USA), SUVAXYN MYCOPLASMA™, SUVAXYN M.HYO™, SUVAXYN MH-ONE™ (all from Fort Dodge Animal Health, Overland Park, Kans., USA (Wyeth)). Advantageously, a dose of 2 ml of M. hyo. supernatant and / or bacterin is contemplated.

[0091] Available M.hyo. vaccines are made from killed whole cell mycoplasma preparations (bacterins). Thus, "bacterins" as used herein refers to bacterial whole cell preparations, particularly whole cell preparations of M.hyo., preferably killed whole cell preparations. When a vaccine or antigen is described herein as being a "supernatant", said supernatant may be a soluble fraction / portion of a (killed) whole cell preparation. The present invention contemplates the use of a soluble portion of a M.hyo. whole cell preparation, however, said M.hyo. preparation soluble portion is substantially free of (i) IgG and (ii) immune complexes comprising antigens bound to immunoglobulins (see, e.g., U.S. Pat. No. 10,206,991). In some embodiments, the soluble portion of the M.hyo. preparation comprises at least one M.hyo. protein antigen. In other embodiments, the soluble portion of the M.hyo. preparation comprises two or more M.hyo. protein antigens. In one embodiment, the M.hyo. supernatant fraction contains one or more of the following M.hyo. specific protein antigens: M.hyo. protein of molecular weight approximately 46 kD (p46), M.hyo. protein of molecular weight approximately 64 kD (p64), and M.hyo. protein of molecular weight approximately 97 kD (p97). In another embodiment, the supernatant fraction contains at least the p46, p64, and p97 M.hyo. protein antigens. Alternatively, the M.hyo. protein of approximately 64 kD (p64) may be referred to herein as the p65 surface antigen from M.hyo., as described by Kim et al. (Infect. Immun. 58(8):2637-2643 (1990)) and in U.S. Patent No. 5,788,962. Any M.hyo. strain can be used as the starting antigen for producing the soluble portion of the M.hyo. preparation. Suitable strains of M. hyo. can be obtained from commercial or academic sources, including depositories such as the American Type Culture Collection (ATCC) (Manassas, Va.) and the NRRL Culture Collection (USDA-Agricultural Research Service, Peoria, Ill.).The ATCC has listed six strains for sale alone: ​​M. hyo. ATCC 25095, M. hyo. ATCC 25617, M. hyo. ATCC 25934, M. hyo. ATCC 27714, M. hyo. ATCC 27715, and M. hyo. ATCC 25934D. A preferred M. hyo. strain for use in embodiments of the present invention is identified as strain P-5722-3, ATCC #55052, deposited on May 30, 1990, in accordance with the accessibility rules required by the United States Patent and Trademark Office. Given the widespread nature of the disease, strains can also be obtained by recovering M. hyo from lung secretions or tissues from pigs infected with known strains that cause Mycoplasma pneumonia in pigs.

[0092] The timing of injection is flexible. The compositions described herein can be used from as early as 3 weeks of age until the pigs leave the grower farm for vaccination at least 2 days before exposure to M.hyo. The vaccine according to the invention can be applied by any conventional technique, including intradermal, intratracheal or intravaginal application. The vaccine according to the invention can also be applied by systemic administration. The composition can be preferably administered intramuscularly or intradermally.

[0093] WO2009 / 126356, US Pat. Nos. 8,444,989, 8,852,613 and 8,940,309 describe the production, formulation and administration of M. hyo. bacterin. In an advantageous embodiment, the amount of M. hyo. antigen in each dose has a relative potency (RP) value of at least 1.22. In another advantageous embodiment, the M. hyo. bacterin is at least 5 log 10 ~8log 10 The antigen amount is between 1 and 2. In an advantageous embodiment, the M.hyo. vaccine is Ingelvac MycoFLEX®. Thus, in an advantageous embodiment, the M.hyo. antigen is an M.hyo. antigen contained in Ingelvac MycoFLEX®.

[0094] A preferred method of immunization or vaccination consists in administering the vaccine according to the invention by systemic administration, such as by the intramuscular route. In one embodiment, the vaccine of the present invention comprises an antigen of PRRSV. Thus, in one embodiment of the present invention, an immunogenic composition for eliciting a protective immune response in pigs against PRRSV is provided. The viral envelope proteins of PRRSV are generally categorized into major and minor proteins based on the abundance of the protein in the virion. The major viral envelope proteins are gp5 (ORF 5) and M (ORF 6), which form dimers. The minor envelope proteins are gp2 (ORF2), gp3 (ORF3), gp4 (ORF4) and E (ORF2b), and possibly the newly identified viral protein gp5a (ORF 5a). The active antigenic component may comprise ORF4, ORF5, ORF6 or ORF7 from the PRRSV virus.

[0095] Recombinant PRRSV antigens can be expressed as vectored PRRSV vaccines or compositions that contain one or more recombinant adenovirus vectors carrying and expressing a particular PRRSV antigen, and optionally contain a pharma- ceutically or veterinarily acceptable carrier, adjuvant, diluent or vehicle. Advantageously, the vector is an adenovirus vector, although other vectors, such as baculovirus, are contemplated.

[0096] The PRRSV may be any strain, since the novel inventive compositions and methods disclosed herein are universally applicable to all known and yet to be discovered PRRSV strains. PRRSV viruses exist as two genotypes, called "US" and "EU" types, which share about 50% sequence homology (Dea S et al. (2000). Arch Virol 145:659-88). These two genotypes can also be distinguished by their immunological properties. Most of the sequence information on the various isolates is based on structural proteins, i.e., the envelope protein GP5, which represents only about 4% of the viral genome, and very little is known about the nonstructural proteins (nsp). The isolation of PRRSV and the production of vaccines have been described in numerous publications (WO92 / 21375, WO93 / 06211, WO93 / 03760, WO93 / 07898, WO96 / 36356, EP 0 676 467, EP 0 732 340, EP 0 835 930, U.S. Pat. No. 10,039,821). PRRSV antigens include PRRSV minor proteins (e.g., gp2, gp3, gp4, gp5a, gp5, or E) in any combination, and may also include additional PRRSV major proteins (e.g., gp5 or M). For example, PRRSV antigens could be displayed on the surface of a virus-like particle (VLP). In other embodiments, soluble versions of the antigens could be administered to the host animal (in this case, oligomerization (including trimerization) of the proteins with each other or in addition with components of VSV-G or other viral proteins, or any oligomerization (including trimerization motifs, such as those of bacterial GCN4)). It is further envisioned that the TM / CT domains of type I viral surface glycoproteins serve the same purpose as, and are therefore interchangeable with, the corresponding domains of VSV-G.

[0097] In some embodiments, the PRRSV vaccine is a recombinant vaccine. In this case, the one or more vectors comprise either a nucleotide sequence encoding a PRRSV E antigen, polypeptide, extracellular domain, or variant thereof; or a nucleotide sequence encoding a modified PRRSV gp2, gp3, gp4, gp5a, gp5, or M antigen, polypeptide, extracellular domain, or variant thereof, in which the existing cellular localization sequence of gp2, gp3, gp4, gp5a, gp5, or M antigen is replaced with a cell surface expression determining sequence of a heterologous gene. In some embodiments, the one or more vectors comprise a mixture of two vectors: a first vector expressing a retargeted PRRSV minor protein, and a second vector expressing a retargeted PRRSV major protein.

[0098] In the context of the present invention, methods may be used for the production of live porcine reproductive and respiratory syndrome virus (PRRSV) for use in the production of vaccines and other compositions. In typical production methods, the virus is grown using a cell line that is permissive for PRRSV infection. However, in such common methods, the cell line is grown to confluence or near confluence prior to infection with PRRSV. In an advantageous method, the cell line does not need to be seeded and grown prior to infection with PRRSV, but rather PRRSV and the cell line can be added in parallel to the cell culture process. Thus, the method provides significant benefits of time, cost and material savings when the virus is to be mass-produced on a commercial scale. The term commercial scale refers to a cell culture volume of more than 10 L. For example, commercial scale refers to a production scale ranging from 10 L to 3000 L of live PRRSV. In a more specific embodiment, the volume is 30 L to 300 L.

[0099] The methods described herein may be used to produce any PRRSV strain, including, but not limited to, PRRSV strains deposited under ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402; CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388, or ECACC V93070108. In particularly preferred embodiments, the methods of the invention are used to produce PRRSV strain 94881, deposited in accordance with the provisions of the Budapest Treaty with the European Collection of Cell Cultures (ECACC) under deposit numbers ECACC 11012501 (parental strain) and ECACC 11012502 (high-passage attenuated MSV), respectively, on January 25, 2011, or any progeny or descendant of one of the aforementioned strains. The viruses to be propagated can be any of the above-mentioned viruses in their attenuated form, or the viruses can be genetically modified to include one or more nucleic acids encoding additional antigenic determinants of one or more porcine diseases.

[0100] Those skilled in the art will understand that there are many cell lines that are permissive to infection by PRRSV. An exemplary cell is a porcine alveolar macrophage cell, such as one derived from MARC-145 cells. Other cells that can be infected with PRRSV include MA-104 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO) cells; and African green monkey kidney cells other than MA-104 cells or MARC-145 cells, such as VERO cells, which are transfected. In addition, the cells can be primary cells from porcine animals that are adapted to long-term growth in culture. Particularly suitable host animals are the simian cell line MA-104, Vero cells, or porcine alveolar macrophages. PRRSV grows preferentially in pulmonary macrophages in the alveoli (Wensvoort et al., 1991). A few cell lines, such as CL2621 and other cell lines cloned from the monkey kidney cell line MA-104 (Benfield et al., 1992; Collins et al., 1992; Kim et al., 1993), are also susceptible to viral infection and can be used in the large-scale production methods described herein.

[0101] A current process for the production of PRRSV 94881 MLV is provided in the exemplary method set forth in Example 1 of U.S. Patent No. 9,944,902. Although the procedure is set forth for PRRSV 94881 MLV, one of skill in the art will appreciate that the procedure can be readily used for any PRRSV for which large-scale production is required. Viruses produced by the described production methods can be used to produce PRRSV antigens, particularly MLV PRRSV, for use in vaccines of the invention.

[0102] The viral strains propagated according to the method can be virulent PRRS viruses, attenuated PRRS viruses, or indeed PRRS viruses that have been modified to further confer desirable properties to them. This can be achieved by classical propagation and selection techniques, such as continuous propagation in suitable host cells to expand the attenuated phenotype. Alternatively, the strains can be genetically modified by directed mutation of the nucleic acid sequences of their genomes by suitable genetic engineering techniques. The genome of PRRSV has been completely or partially sequenced (Conzelmann et al., 1993; Meulenberg et al., 1993a, Murthaugh et al., 1995) and encodes six structural proteins in addition to the RNA-dependent RNA polymerase (ORFs 1a and 1b): four envelope glycoproteins designated GP2 (ORF 2), GP3 (ORF 3), GP4 (ORF 4) and GP5 (ORF 5), the non-glycosylated membrane protein M (ORF 6), and the nucleocapsid protein N (ORF 7) (Meulenberg et al. 1995, 1996; van Nieuwstadt et al., 1996). Immunological characterization and nucleotide sequencing of European and American strains of PRRSV have identified subtle antigenic differences among PRRSV strains that are located within the structural viral proteins (Nelson et al., 1993; Wensvoort et al., 1992; Murtaugh et al., 1995).

[0103] In fact, an exemplary virus is the PRRSV 94881 virus. Attenuated strains are grown using the methods described herein, but more easily, the virus is a PRRSV 94881 virus that is made into a chimeric virus, in which the backbone of the PRRSV 948881 virus deposited under ECACC Accession No. 11012502, or indeed the parent strain deposited under ECACC Accession No. 11012501, is modified to replace the endogenous sequences for one or more of ORF 1a, ORF 1b, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, or ORF 7 with the corresponding ORF of a different PRRS virus strain. For example, the different PRRS virus strains can be different European strains, such as the Lelystad virus strain (Lelystad Agent (CDI-NL-2.91), or other strains, such as those having accession numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM Accession No. I-1140, CNCM Accession No. I-1387, CNCM Accession No. I-1388, ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402; CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388, or ECACC V93070108, or may in fact be a US-type strain, e.g., North American-type PRRS virus, pT7P129A; ATCC deposit VR-2332, ATCC deposit VR-2368; ATCC VR-2495; ATCC VR 2385, ATCC VR 2386, ATCC VR 2429, ATCC VR 2474, and ATCC VR 2402.

[0104] Recombinant techniques for preparing modified sequences are well known to those skilled in the art and usually involve the construction of a full-length complementary DNA copy (infectious clone) of the viral genome, which can then be modified by DNA recombination and engineering methods (such as site-directed mutagenesis). In this way, for example, antigenic sites or enzymatic properties of viral proteins can be modified. Infectious clones of PRRS viruses of European and North American genotypes have been reported in the literature and can be propagated using the methods of the invention.

[0105] Preferably, the vaccine according to the invention comprises a modified live PRRSV comprising one or more of these strains living in a suitable carrier, although killed vaccines (KV) can also be prepared using inactivated virus. MLVs are typically administered at a concentration of 10 1 ~10 7 Viral particles, preferably 10 per dose 3 ~10 5 particles, more preferably 10 per dose 4 ~10 5 Particles (4.0 to 5.0 log 10 TCID 50 The vaccine of the present invention is formulated to allow administration of approximately 10 4 ~about 10 7 The viral particles may have a dosage of PRRSV antigen. KV may have a dosage of about 10 per dose. 3 ~about 10 10 The vaccine may be formulated based on the pre-inactivation titer of viral particles. The vaccine may include a pharma- ceutically acceptable carrier, such as saline. The vaccine may or may not include an adjuvant. An example of a suitable adjuvant is alpha-tocopherol acetate, available under the trade name Diluvac Forte®. Alternatively, for example, an aluminum-based adjuvant may be used.

[0106] Pigs can be infected with PRRSV by the oronasal route. The virus in the lungs is taken up by alveolar macrophages in the lungs, and PRRSV viral replication in these cells is completed within 9 hours. PRRSV migrates from the lungs to the pulmonary lymph nodes within 12 hours, and to the peripheral lymph nodes, bone marrow, and spleen within 3 days. At these sites, only a small number of cells stain positive for viral antigens. The virus is present in the blood for at least 21 days, and often longer. After 7 days, antibodies to PRRSV are found in the blood. The combined presence of virus and antibodies in PRRS-infected pigs indicates that viral infection can persist for long periods, albeit at low levels, despite the presence of antibodies. For at least 7 weeks, the alveolar cell population in the lungs is different from normal SPF lungs.

[0107] The vaccine may be provided in the form of a lyophilized preparation of live virus, which will be reconstituted with a solvent to obtain a solution for injection. Thus, after the recovery step of the described method, the viruses may be combined and lyophilized. The solvent may be, for example, water, saline or buffer, or an adjuvanted solvent. The solvent may contain an adjuvant, for example alpha-tocopherol acetate. The reconstituted vaccine may then be injected into the pig, for example as an intramuscular or intradermal injection in the neck. For intramuscular injections, a volume of 2 ml may be applied, for intramuscular injections typically 0.2 ml. Thus, in a further aspect, the present invention relates to a vaccine product comprising a lyophilized composition of virus and a solvent for reconstitution in separate containers, which may further comprise a pamphlet or label containing instructions for use. Vaccines prepared from viruses produced by the above method may contain one or more of the above mentioned strains, but may also contain components that are active against PRRS or against other porcine viral or bacterial diseases, such as Lawsonia intracellularis, PCV and / or M.hyo. Thus, the present invention further relates to the vaccines described, characterized in that they have at least one additional antigenic activity against a porcine disease other than PRRS. In addition, the vaccine may contain certain pharmacologic or veterinary acceptable adjuvants. One such adjuvant is alpha-tocopherol. Thus, the new vaccine compositions, in particular PRRS virus vaccines, including PRRSV 94881, may be further improved by the addition of adjuvants. Such improvements include preparing the vaccine in combination with an adjuvant that enhances the efficacy of the vaccine, such that administration of the combination of adjuvant and vaccine results in a better clinical response / outcome compared to administration of the vaccine alone. For example, the vaccine composition of the present invention may include a PRRSV 94881 virus vaccine and an adjuvant selected from MCP-1, Haemophilus sonmus fraction, Carbopol®, and combinations thereof. In some embodiments, the virus vaccine includes a PRRSV 94881 virus vaccine, which may be a recombinant subunit vaccine or alternatively may be an attenuated live virus vaccine. An exemplary live vaccine that exists is Ingelvac® PRRS MLV, and PRRSV 94881 may be formulated in a manner similar to Ingelvac® PRRS MLV.

[0108] In addition to the above, the vaccine composition may contain other components, provided they do not interfere with the adjuvant properties of MCP-1, Haemophilus somnus fraction, Carbopol® or the base viral vaccine. Such other components include, for example, binders, colorants, drying agents, preservatives, humectants, stabilizers, excipients, adhesives, plasticizers, tackifiers, thickeners, patch materials, ointment bases, keratin removers, basic substances, absorption enhancers, fatty acids, fatty acid esters, higher alcohols, surfactants, water, and buffers. Preferred other components include buffers, ointment bases, fatty acids, preservatives, basic substances, or surfactants.

[0109] The content or amount of the adjuvant used in the present invention may vary and may be determined, for example, by considering the characteristics and dosage form of the PRRS virus vaccine used. The vaccine composition of the present invention can be formulated by any method known in the field of formulation, for example, into a liquid preparation, a suspension, an ointment, a powder, a lotion, a W / O emulsion, an O / W emulsion, an emulsion, a cream, a cataplasm, a patch and a gel, and preferably, the vaccine composition of the present invention is used as a medicament. Therefore, according to another aspect of the present invention, a pharmaceutical composition comprising the above vaccine composition is provided. The vaccine composition according to the present invention can significantly induce antibody production when administered transdermally. Therefore, in another preferred embodiment, the vaccine composition of the present invention can be provided as a transdermal preparation.

[0110] When the adjuvant and the PRRS virus vaccine are administered to an organism, the clinical outcome of the animal is improved. Those skilled in the art can appropriately determine the effective amount of the adjuvant and the immunologically effective amount of the PRRS virus vaccine by considering, for example, the type and characteristics of the antigenic substance, the species, age, weight, severity of the disease, type of disease, number of administrations and administration method of the organism, and further using the amount of antibodies produced against the antigenic substance in the organism as an indicator. The PRRS virus vaccine, adjuvant or combination thereof may be administered to an organism by any suitable method selected depending, for example, on the patient's condition and disease characteristics. Examples of such methods include intraperitoneal administration, transdermal administration (e.g., subcutaneous injection, intramuscular injection, intradermal injection, and patch application), intranasal administration, oral administration, mucosal administration (e.g., rectal administration, intravaginal administration, and corneal administration). Of these, intramuscular administration is preferred.

[0111] An exemplary therapeutic dose of PRRSV MLV is about two milliliters (2 mL). Those skilled in the art will appreciate that dosages may vary based on the breed, size and other physical factors of the individual subject, as well as the specific PRRSV MLV formulation and route of administration. Preferably, the PRRSV MLV is administered in a single dose, although additional doses may be useful. Again, those skilled in the art will appreciate through the present invention that dosage and frequency of administration will be influenced by the age and health of the subject pig, as well as other considerations common to the industry, and the specific condition under which the PRRSV MLV is administered.

[0112] In certain other embodiments, the vaccine may be a multivalent vaccine comprising two or more PRRS viruses, wherein at least one of the PRRS viruses is the attenuated 94881 virus deposited under ECACC Accession No. 11012502. Other PRRS viruses may be PRRSV strains deposited under accession numbers Lelystad virus strain (Lelystad Agent (CDI-NL-2.91) or other strains, e.g., accession numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM accession number I-1140, CNCM accession number I-1387, CNCM accession number I-1388, ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402; CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388, or ECACC V93070108, or may in fact be a US-type strain, e.g., North American-type PRRS virus, pT7P129A; ATCC deposit VR-2332, ATCC deposit VR-2368; ATCC VR-2495; ATCC VR 2385, ATCC VR 2386, ATCC VR 2429, ATCC VR 2474, and ATCC VR 2402.

[0113] Vaccines based on the PRRS virus can be used to vaccinate both piglets and sows. In one aspect of the present invention, a particular dose administration regimen is selected based on the age of the pig and the antigen selected for administration. This allows pigs of all ages to be administered the most effective dose based on our discovery that PRRSV infection (both from wild type exposure and vaccination) is cleared much more quickly in older animals. Thus, while vaccination of older animals is preferred in some respects, vaccination of younger pigs, including those aged 3 weeks and younger, helps induce active immunity and is also highly beneficial. Animal age can be an important factor in controlling PRRS and can be a factor that influences vaccination and the development of an effective immune response. Therefore, age, disease management, animal husbandry management, natural and active immunity are important and need to be considered in the control strategy.

[0114] The PRRSV vaccine can be administered in any conventional manner, and in some preferred methods, administration is intranasal administration.Preferably, the administered PRRSV vaccine provides the advantage of treating PRRSV infection after a single administration, or of reducing the severity or incidence of infection, similar to Ingelvac PRRS®.However, it should be understood that if other antigens or mixed or multivalent vaccines are selected, they can be administered in their conventional manner, which may include one or more booster vaccinations after the initial administration.Those skilled in the art will be able to determine the appropriate dosage level based on the PRRSV vaccine selected and the age range of the animal to which the antigen will be administered. In an advantageous embodiment, the PRRSV vaccine is Ingelvac PRRS® MLV. Thus, in an advantageous embodiment, the PRRSV antigen is a PRRSV antigen contained in Ingelvac® MLV. A preferred method of immunization or vaccination consists in administering the vaccine according to the invention by systemic administration, such as by the intramuscular route.

[0115] In a particularly advantageous embodiment, the PCV antigen, the M. hyo. antigen and the PRRSV antigen are the PCV antigen, the M. hyo. antigen and the PRRSV antigen contained in 3FLEX®.

[0116] In a particularly advantageous embodiment, the antigen of L. intracellularis is lyophilized and dissolved in the 3FLEX® vaccine. In another particularly advantageous embodiment, the antigen of L. intracellularis contained in Enterisol® Ileitis is dissolved in the 3FLEX® vaccine. In an even more particularly advantageous embodiment, the antigen of L. intracellularis contained in Enterisol® Ileitis is dissolved in Ingelvac CircoFLEX®. The volume of the vaccine may be 2 ml.

[0117] The present invention further contemplates a vaccine that may further include one or more antimicrobial agents, including but not limited to tiamulin and / or chlortetracycline. In this case, the dosage of tiamulin may be about 35g / ton or about 35ppm, and the dosage of chlortetracycline may be about 400g / ton or about 400ppm. The combination vaccine of the present invention is advantageously administered intramuscularly, although oral administration is also contemplated.

[0118] The present invention also encompasses combinations with other microorganisms causing disease in pigs. Preferably, the other microorganism causing disease in pigs is Actinobacillus pleuropneumonia; adenovirus; alphavirus, e.g., Eastern equine encephalomyelitis virus; Bordetella bronchiseptica; Brachyspira spp., preferably B. hyodysenteriae; B. piosicoli; Brucella suis, preferably biovars 1, 2 and 3; Classical swine fever virus; Clostridium spp. spp., preferably Cl. difficile, Cl. perfringens types A, B and C, Cl. novyi, Cl. septicum, Cl. tetani; coronaviruses, preferably porcine respiratory coronavirus; Eerythrozoonosis suis; Erysipelothrix rhusiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtypes 1, 7 and 14; hemagglutinating encephalomyelitis virus; Japanese encephalitis virus; Leptospira spp., preferably Leptospira australis, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagicae, and Leptospira interrogans, Leptospira pomona, Leptospira tarassovi; Mycobacterium spp., preferably M. avium, M. intracellulare, and M. bovis; Pasteurella multocida multocida; porcine cytomegalovirus; porcine parvovirus; pseudorabies virus; rotavirus; Salmonella spp., preferably S. thyphimurium and S. choleraesuis; Staphylococcus spp., preferably Streptococcus spp., preferably Strep. suis; swine herpes virus; swine influenza virus; swinepox virus; vesicular stomatitis virus; swine vesicular rash virus; Leptospira Hardjo and / or Mycoplasma hyosynoviae.

[0119] The immunogenic preparation of the present invention can also be combined with at least one conventional vaccine (live attenuated, inactivated or subunit) or recombinant vaccine (viral vector) against at least one different or identical swine pathogen. The present invention provides in particular a combination with a conventional vaccine (live attenuated, inactivated or otherwise subunit) containing an adjuvant. For the inactivated or subunit vaccines, in particular those containing alumina gel alone or mixed with saponin as adjuvant, or those formulated in the form of an oil-in-water emulsion.

[0120] In addition, the composition may include one or more veterinarily acceptable carriers. As used herein, "veterinarily acceptable carriers" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonicity agents, and adsorption retardants, etc. In a preferred embodiment, the immunogenic composition includes PCV3 ORF2 protein or PCV2 ORF2, as provided herein, preferably provided at the concentrations described above, mixed with an adjuvant, preferably CARBOPOL®, and saline.

[0121] It will be understood by those skilled in the art that the compositions used herein may include physiologically acceptable sterile solutions for injection. For example, isotonic aqueous solutions, such as saline or corresponding plasma protein solutions, are readily available for preparing ready-to-use solutions for parenteral injection or infusion. In addition, the immunogenic and vaccine compositions of the present disclosure may include diluents, isotonic agents, stabilizers, or adjuvants. Diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers may include albumin and alkali salts of ethylenediaminetetraacetic acid, among others.

[0122] "Adjuvants" as used herein may include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech Inc., Cambridge, Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions. The emulsions may be based in particular on light liquid paraffin oil (European Pharmacopoeia type); isoprene-based oils such as squalane or squalene oils resulting from the oligomerization of alkenes, in particular isobutene or decene; esters of acids or of alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di(caprylic / capric), glyceryl tri(caprylic / capric) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifier is preferably a non-ionic surfactant, in particular esters of sorbitan, esters of mannitol (e.g., anhydrous mannitol oleate), esters of glycols, esters of polyglycerols, esters of propylene glycol, and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which may be ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular Pluronic products, in particular L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, DES). John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

[0123] For example, it is possible to use the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of the same book.

[0124] Further examples of adjuvants are compounds selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are polymers of acrylic or methacrylic acid that are crosslinked, in particular with polyalkenyl ethers of sugars or polyhydric alcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). The skilled person may also refer to US Pat. No. 2,909,462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least three, and preferably at most eight, hydroxyl groups, in which the hydrogen atoms of at least three of the hydroxyl groups are replaced by unsaturated aliphatic groups having at least two carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated groups may themselves contain other substituents, such as methyl. Particularly suitable are the products sold under the name CARBOPOL® (BF Goodrich, Ohio, USA). They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among them, CARBOPOL® 974P, 941P, 934P and 971P can be mentioned. The use of CARBOPOL®, especially CARBOPOL® 971P, is most preferred, preferably in an amount of 500 μg to about 5 mg per dose, even more preferably in an amount of about 750 μg to about 2.5 mg per dose, most preferably in an amount of about 1 mg per dose. In particular, the dose of the final composition may contain CARBOPOL® or CARBOPOL® 971 in the range of about 750 μg to about 2.5 mg of CARBOPOL®. For example, in some embodiments, a dose of the final composition may contain 1 mg of CARBOPOL® 971.

[0125] Additional suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), block copolymers (CytRx, Atlanta, Ga.), SAF-M (Chiron, Emeryville, Calif.), monophosphoryl lipid A, avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or non-recombinant), cholera toxin, IMS 1314, or muramyl dipeptide, among others. In other words, the vaccines of the present invention may comprise one or more adjuvants, non-limiting examples of which are provided throughout this specification.

[0126] Furthermore, the vaccine of the invention may comprise one or more of the following as adjuvants: polymers of acrylic or methacrylic acid; copolymers of maleic anhydride and alkenyl derivatives; cross-linked polymers of acrylic or methacrylic acid; polymers of acrylic or methacrylic acid cross-linked with polyalkenyl ethers of sugars or polyalcohols; carbomers; acrylic polymers cross-linked with polyhydroxylated compounds having at least three and at most eight hydroxyl groups, the hydrogen atoms of at least three of said hydroxyl groups being optionally replaced by unsaturated aliphatic groups having at least two carbon atoms, or substituted, the groups containing 2 to 4 carbon atoms, e.g., vinyl, allyl and other ethylenically unsaturated groups, the unsaturated groups themselves may contain other substituents, e.g., methyl; acrylic polymers; Carbopol®; Carbopol® 974P; Carbopol® 934P; Carbopol® 971P; Carbopol® 980; Carbopol® 941P; ImpranFLEX®; Aluminum hydroxide; Aluminum phosphate; Saponin; Quil A;QS-21;GPI-0100;Water-in-oil emulsions;Oil-in-water emulsions;Water-in-oil-in-water emulsions;Emulsions based on light liquid paraffin oil or European Pharmacopoeia-type adjuvants;Oils of the isoprene series;Squalane;Squalene oil resulting from the oligomerization of alkenes or isobutene or decene;Esters of acids or of alcohols containing linear alkyl groups;Vegetable oils;Ethyl oleate;Propylene glycol dicaprylate / caprate;Glyceryl tricaprylate / caprate;Propylene glycol dioleate;Esters of branched fatty acids or alcohols:Isostearate;Non-ionic surfactants;Esters of sorbitan or of mannitol or of glycol or of polyglycerol or of propylene glycol or of oleic acid or isostearic acid or of ricinoleic acid or of hydroxystearic acid, optionally ethoxylated, anhydrous mannitol oleate;Polyoxypropylene-polyoxyethylene copolymer block, Pluronic products, RIBI adjuvant system; block copolymers; SAF-M; monophosphoryl lipid A; avridine lipid-amine adjuvant; heat-labile enterotoxin from Escherichia coli (recombinant or non-recombinant); cholera toxin; IMS 1314, or muramyl dipeptide.

[0127] In a preferred and advantageous embodiment, the vaccine of the invention comprises one or more carbomers. In a preferred and advantageous embodiment, the vaccine of the invention comprises Carbopol® and / or ImpranFLEX®. Specific examples of Carbopol® are provided herein. In a further embodiment, the vaccine of the invention may comprise a pharma- ceutically or veterinarily acceptable carrier.

[0128] Preferably, the adjuvant is added in an amount of about 100 μg to 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 100 μg to 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 μg to 5 mg per dose. Even more preferably, the adjuvant is added in an amount of about 750 μg to 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose. In addition, the composition may include one or more pharma- ceutically acceptable carriers. As used herein, "pharma-ceutically acceptable carriers" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, and adsorption retardants, etc.

[0129] According to a further embodiment, the immunogenic composition further comprises a pharma- ceutically acceptable salt, preferably a phosphate salt, in a physiologically acceptable concentration.Preferably, the pH of said immunogenic composition is adjusted to physiological pH, meaning between about 6.5 and 7.5.

[0130] Dosing regimens can be used to improve the economics of pig farming management. For example, an immunogenic composition, such as a vaccine, can be administered to sows and / or piglets as part of an effort to protect the sows, piglets, or both. It is further asserted that the vaccine of the present invention is capable of protecting fertilized gilts and sows when challenged with Lawsonia intracellularis, PCV, M. hyo. or PRRSV during all, two or at least one trimester of gestational age 114 days. It is further asserted that the vaccine of the present invention is capable of significantly reducing the incidence of mummification, stillbirth and fetuses in vaccinated gilts and sows when challenged with Lawsonia intracellularis, PCV, M. hyo. or PRRSV during all or two or at least one trimester of a gestational period of 114 days.

[0131] The dosing regimen may include vaccinating young sows (i.e., 5 months of age or younger) with at least one dose of the immunogenic composition described herein prior to breeding. A dose of the immunogenic composition described herein may be administered intramuscularly as a 1 mL dose prior to breeding. In some embodiments, one or more doses of the vaccine may be administered to the sow. For example, a primary vaccine may be administered, followed by a booster vaccine 21 days later and prior to breeding. In some embodiments, the sow may be bred within 14-21 days after the booster vaccination. This time frame allows the sow to mount an immune response. Utilizing such a dosing regimen may reduce and / or inhibit the number of mummifications at farrowing.

[0132] Furthermore, the use of a dosing regimen comprising administering an immunogenic composition comprising Lawsonia intracellularis, PCV, M.hyo. or PRRSV may reduce, reduce and / or suppress lymphadenopathy, lymphocyte depletion and / or polynuclear / giant histiocytes in pigs infected with Lawsonia intracellularis, PCV, M.hyo. or PRRSV. Advantageously, the dose is about 2 ml. Methods of immunization are also described that allow inducing an immune response in pigs to circovirus. In particular, methods of vaccination are described that are effective in pigs. These immunization and vaccination methods comprise the administration of one of the above preparations or one of the monovalent or polyvalent vaccines. These methods of immunization and vaccination comprise the administration of one or more successive doses of these preparations or vaccines. The preparations and vaccines in connection with this immunization or vaccination method can be administered by the various administration routes proposed in the prior art for polynucleotide vaccination, in particular by the intramuscular and intradermal routes, as well as by known administration techniques, in particular by injection with a syringe having a needle, by liquid jet (Furth et al. Analytical Bioch., 1992, 205: 365-368) or by projection of gold particles coated with DNA (Tang et al. Nature, 1992, 356: 152-154).

[0133] This method allows administration not only to adult pigs, but also to young pigs and to pregnant sows, the latter in particular allowing active immunity (maternal antibodies) to be conferred to the newborn. Preferably, the sows are inoculated before breeding and / or before conception and / or during pregnancy. Advantageously, at least one inoculation is performed before conception, followed by inoculations during pregnancy, for example at about mid-pregnancy (at about 6-8 weeks of pregnancy) and / or at late pregnancy (at about 11-13 weeks of pregnancy). Thus, an advantageous regime is an inoculation before conception and a booster inoculation during pregnancy. There may then be revaccinations before each conception and / or during pregnancy at about mid-pregnancy and / or at late pregnancy. Preferably, the revaccinations are during pregnancy. In a further aspect, the present invention relates to the use of the vaccines of the present invention as described herein.Furthermore, the present invention relates to methods comprising the use of the vaccines of the present invention as described herein. Thus, in one embodiment the vaccine of the invention is for use in a method for raising a protective immune response in an animal comprising the step of administering said vaccine to the animal.

[0134] Thus, in one embodiment the vaccine of the invention is for use in a method for raising a protective immune response in a pig comprising the step of administering said vaccine to the pig. In an advantageous embodiment the vaccine of the invention is for use in a method for raising a protective immune response in an animal, wherein the vaccine is administered systemically, preferably intramuscularly or intradermally. In an advantageous embodiment, the vaccine of the invention is for use in a method for raising a protective immune response in an animal, in which the vaccine is administered in one dose or at least one dose.

[0135] The present invention is a vaccine for use in a method for eliciting a protective immune response in an animal against Lawsonia intracellularis and / or PCV and / or M. hyo. and / or PRRSV. In an advantageous embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and M. hyo. In an advantageous embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PRRS. In an advantageous embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV and M. hyo.

[0136] In an advantageous embodiment, the vaccine of the invention is a vaccine for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV and PRRS. In an advantageous embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PRRS and M. hyo. In an advantageous embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV and M. hyo. and PRRSV. In an advantageous embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV.

[0137] In an advantageous embodiment, the vaccine of the present invention is a vaccine for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV, comprising a live Lawsonia intracellularis bacterium, preferably an attenuated Lawsonia intracellularis or a modified live Lawsonia intracellularis bacterium, and an antigen of PCV. In an advantageous embodiment, the vaccine of the present invention is a vaccine for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV, comprising a live Lawsonia intracellularis bacterium, preferably an attenuated Lawsonia intracellularis or a modified live Lawsonia intracellularis bacterium, and a recombinant polypeptide of PCV.

[0138] In an advantageous embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV, comprising a live Lawsonia intracellularis bacterium, preferably an attenuated Lawsonia intracellularis or a modified live Lawsonia intracellularis bacterium, and a recombinant polypeptide of PCV expressed by the PVC ORF2 gene. In an advantageous embodiment, the vaccine of the present invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV, and comprises an antigen of Lawsonia intracellularis contained in Enterisol (registered trademark) Ileitis and an antigen of PCV.

[0139] In an advantageous embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV, comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®. In an advantageous embodiment, the vaccine of the present invention is a vaccine for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV, comprising an antigen of Lawsonia intracellularis and an antigen of PCV, which is administered systemically, preferably intramuscularly or intradermally. In an advantageous embodiment, the vaccine of the invention is a vaccine for use in a method for eliciting a protective immune response against Lawsonia intracellularis and PCV, comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®, which is administered systemically, preferably intramuscularly or intradermally.

[0140] In one embodiment, the vaccine of the present invention is for use in a method for immunizing an animal against clinical disease caused by at least one pathogen in the animal, which does not cause clinical signs of infection but is capable of inducing an immune response that immunizes the animal against a pathotype of said at least one pathogen. In an advantageous embodiment, the vaccine of the present invention is for use in a method for immunizing an animal against clinical disease caused by Lawsonia intracellularis and PCV in an animal, which does not cause clinical signs of infection but is capable of inducing an immune response that immunizes the animal against a pathotype of the pathogen. In one embodiment, the vaccine of the present invention is for use in a method for eliciting a protective immune response against Lawsonia intracellularis, which is a protective immune response that reduces intestinal pathology in an animal compared to a non-immunized control animal of the same species.

[0141] Thus, in an advantageous embodiment, the vaccine of the present invention is a vaccine for use in a method for raising a protective immune response against Lawsonia intracellularis, which protective immune response reduces intestinal pathology in an animal compared to a non-immunized control animal of the same species, said vaccine comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®. The intestinal lesions may be ileal lesions. The intestinal and / or ileal lesions may be macroscopic and / or microscopic lesions.

[0142] In one embodiment, the vaccine of the present invention is for use in a method for eliciting a protective immune response, wherein the protective immune response against Lawsonia intracellularis reduces fecal shedding in an animal compared to a non-immunized control animal of the same species. In an advantageous embodiment, the vaccine of the invention is a vaccine for use in a method for raising a protective immune response against Lawsonia intracellularis, which is a protective immune response to reduce fecal shedding in an animal compared to a non-immunized control animal of the same species, said vaccine comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®.

[0143] In one embodiment, the vaccine of the present invention is for use in a method for eliciting a protective immune response, wherein the protective immune response against Lawsonia intracellularis increases the average daily weight gain of an animal compared to a non-immunized control group of animals of the same species. In an advantageous embodiment, the vaccine of the invention is a vaccine for use in a method for eliciting a protective immune response against Lawsonia intracellularis, which protective immune response increases the average daily weight gain of an animal compared to a non-immunized control group of animals of the same species, said vaccine comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®.

[0144] The present invention further includes a method for eliciting a protective immune response in an animal against Lawsonia intracellularis and / or PCV and / or M. hyo. and / or PRRSV, the method comprising the step of administering a vaccine of the present invention to the animal. The present invention also includes a method for eliciting a protective immune response in an animal against Lawsonia intracellularis and PCV, the method comprising the step of administering to the animal a vaccine of the present invention. The present invention also includes a method for eliciting a protective immune response in a pig against Lawsonia intracellularis and PCV, the method comprising the step of administering to the pig a vaccine of the present invention.

[0145] The present invention also encompasses a method of immunizing an animal against clinical disease caused by at least one pathogen in the animal, comprising the step of administering to the animal a vaccine of the present invention, wherein the vaccine does not cause clinical signs of infection but is capable of inducing an immune response that immunizes the animal against a pathotype of the at least one pathogen. The present invention also encompasses a method for immunizing an animal against clinical disease caused by Lawsonia intracellularis and PCV in an animal, comprising the step of administering to the animal a vaccine of the present invention, wherein the vaccine does not cause clinical signs of infection but is capable of inducing an immune response that immunizes the animal against a pathotype of the pathogen. The present invention also encompasses a method for immunizing pigs against clinical disease caused by Lawsonia intracellularis and PCV in pigs, comprising the step of administering to the animal a vaccine of the present invention, which does not cause clinical signs of infection but is capable of inducing an immune response that immunizes the pig against a pathotype of the pathogen. The present invention further encompasses the use of a vaccine of the present invention in the preparation of a composition for inducing a protective immune response against Lawsonia intracellularis and / or PCV and / or M. hyo. and / or PRRSV.

[0146] An advantageous embodiment is the use of a vaccine according to the invention in the preparation of a composition for inducing a protective immune response against Lawsonia intracellularis and PCV. An advantageous embodiment is the use of a vaccine according to the invention in the preparation of a composition for inducing a protective immune response against Lawsonia intracellularis and PCV and M. hyo. and PRRS. The present invention further encompasses the use of the vaccine of the present invention for a method for inducing a protective immune response against Lawsonia intracellularis and / or PCV and / or M. hyo. and / or PRRSV. In an advantageous embodiment, the use of the vaccine of the invention is for a method of inducing a protective immune response against Lawsonia intracellularis and PCV. In an advantageous embodiment, the use of the vaccine of the invention is for a method of inducing a protective immune response against Lawsonia intracellularis and PCV and M. hyo. and PRRS.

[0147] The vaccine of the present invention may be administered as a single dose, ie, a one-shot administration. Thus, in one embodiment, the vaccine of the invention may be formulated and / or packaged for single dose or one-shot administration. In one embodiment, the vaccine of the invention may be formulated and / or packaged for a multiple dose regimen, preferably a two dose regimen. In one embodiment, the vaccine of the invention is present as a dosage form, said dosage form being delivered from a container containing a larger quantity of said vaccine, said dosage of said vaccine being deliverable from said container, which may contain at least 10, at least 50, at least 100, at least 150, at least 200 or at least 250 doses of said vaccine.

[0148] Normally, porcine proliferative enteritis (PPE), a disease caused by Lawsonia intracellularis, can be controlled using a commercially available live vaccine (Enterisol® Ileitis) administered orally in the drinking water or by drench. It is required that animals vaccinated with Enterisol® Ileitis are not administered any antibiotics active against Lawsonia intracellularis 3 days prior to, on the day of, and 3 days after vaccination. The present inventors have surprisingly and unexpectedly found that a live Lawsonia intracellularis vaccine, when administered intramuscularly, is effective despite simultaneous / concomitant antibiotic treatment of animals. It is therefore expected that the vaccines of the present invention can be administered to an animal despite concurrent / concomitant antibiotic treatment of the animal. When the vaccines of the present invention are administered despite concurrent / concomitant antibiotic treatment of the animal, the route of administration is preferably systemic. Thus, the present invention also encompasses a vaccine for use in a method for eliciting a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with one or more antibiotics.

[0149] Furthermore, the present invention also encompasses a vaccine for use in a method for eliciting a protective immune response in a pig, said method comprising administering said vaccine to a pig, wherein the pig is simultaneously / concomitantly treated with one or more antibiotics. The phrase "simultaneously / concomitantly treated with one or more antibiotics" as used herein means that the animal / pig received antibiotic treatment (i.e. one or more antibiotics were administered to the animal / pig) 3 days prior to vaccination, 2 days prior to vaccination, and / or 1 day prior to vaccination. The phrase may also mean that the animal / pig received antibiotic treatment and vaccination on the same day. The phrase may also mean that the animal / pig will receive antibiotic treatment 1 day, 2 days and / or 3 days after vaccination.

[0150] The term "antibiotic" is well known in the art and is used herein in its broadest sense. The term "antibiotic" as used herein may refer to a compound that exerts a harmful effect on bacteria. Non-limiting examples of antibiotics include beta-lactamases (e.g., penicillin VK, penicillin G, amoxicillin trihydrate), nitroimidazoles, macrolides (e.g., tylosin tartrate, erythromycin, azithromycin, and clarithromycin), tetracyclines, glycopeptides (e.g., vancomycin), pleuromutilins, and fluoroquinolones.

[0151] Preferably, the antibiotics Denagard® (tiamulin) or CTC (chlortetracycline) or a combination thereof are used as antibiotics. Preferably, the antibiotics are administered at dosages of 35 g / ton Denagard® (tiamulin) and 400 g / ton CTC (chlortetracycline) for a total of two weeks. Thus, the present invention encompasses a vaccine of the present invention for use in a method for eliciting a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with one or more antibiotics, said vaccine comprising an antigen of Lawsonia intracellularis and an antigen of PCV.

[0152] In an advantageous embodiment, the vaccine of the invention is for use in a method for eliciting a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with one or more antibiotics, said vaccine comprising a live Lawsonia intracellularis bacterium and an antigen of PCV, and which is administered systemically, preferably intramuscularly. The invention further includes a vaccine for use in a method for raising a protective immune response in an animal, the method comprising administering the vaccine to an animal, the animal being simultaneously / co-treated with one or more antibiotics, the vaccine comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis, and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®. The invention further includes a vaccine for use in a method for raising a protective immune response in an animal, the method comprising administering the vaccine to an animal, the animal being simultaneously / co-treated with Denagard® (tiamulin) and / or CTC (chlortetracycline), the vaccine comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis, and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®.

[0153] In an advantageous embodiment, the vaccine of the present invention includes a vaccine for use in a method for eliciting a protective immune response in an animal, said method comprising the step of administering said vaccine to an animal, said animal being simultaneously / co-treated with Denagard® (tiamulin) and / or CTC (chlortetracycline), said vaccine comprising an antigen of Lawsonia intracellularis contained in Enterisol® Ileitis, and an antigen of PCV contained in Ingelvac CircoFLEX® or 3FLEX®, and administered systemically, preferably intramuscularly. Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

[0154] Certain features of the present invention will now be described by numbered items. 1. A vaccine suitable for use as a swine vaccine, comprising an immunogenic Lawsonia intracellularis component, an immunogenic porcine circovirus (PCV) component, an immunogenic Mycoplasma hyopneumoniae (M.hyo.) component, and an immunogenic porcine reproductive and respiratory syndrome virus (PRRSV) component. 2. The vaccine of item 1, comprising one or more adjuvants. 3. The adjuvant or adjuvants are selected from the group consisting of polymers of acrylic or methacrylic acid; copolymers of maleic anhydride and alkenyl derivatives; crosslinked polymers of acrylic or methacrylic acid; polymers of acrylic or methacrylic acid crosslinked with polyalkenyl ethers of sugars or polyhydric alcohols; carbomers; acrylic polymers crosslinked with polyhydroxylated compounds having at least three and at most eight hydroxyl groups, in which the hydrogen atoms of at least three of the hydroxyl groups may be or have been replaced by unsaturated aliphatic groups having at least two carbon atoms, said groups containing from 2 to 4 carbon atoms, e.g. vinyl, allyl and other ethylenically unsaturated groups, which unsaturated groups may themselves contain other substituents, e.g. methyl; Carbopol; Carbopol 974P; Carbopol 934P; Carbopol 971P; aluminum hydroxide; aluminum phosphate; saponin; Quil A;QS-21;GPI-0100;Water-in-oil emulsions;Oil-in-water emulsions;Water-in-oil-in-water emulsions;Emulsions based on light liquid paraffin oil or European Pharmacopoeia-type adjuvants;Oils of the isoprene series;Squalane;Squalene oil resulting from the oligomerization of alkenes or isobutene or decene;Esters of acids or of alcohols containing linear alkyl groups;Vegetable oils;Ethyl oleate;Propylene glycol dicaprylate / caprate;Glyceryl tricaprylate / caprate;Propylene glycol dioleate;Esters of branched fatty acids or alcohols:Iso Stearic acid esters; non-ionic surfactants; esters of sorbitan or of mannitol or of glycols or of polyglycerols or of propylene glycol or of oleic acid or of isostearic acid or of ricinoleic acid or of hydroxystearic acid, optionally ethoxylated, anhydromannitol oleate; polyoxypropylene-polyoxyethylene copolymer blocks, Pluronic products, RIBI adjuvant system; block copolymers; SAF-M; monophosphoryl lipid A; avridine lipid-amine adjuvants;The vaccine of item 2, comprising one or more of the following: heat-labile enterotoxin from Escherichia coli (recombinant or non-recombinant); cholera toxin; IMS 1314; or muramyl dipeptide;

[0155] 4. The vaccine of any one of the preceding items, wherein the immunogenic Lawsonia intracellularis component is a Lawsonia intracellularis vaccine. 5. The vaccine of any one of the preceding items, wherein the immunogenic Lawsonia intracellularis component is a live vaccine. 6. The vaccine of any one of the preceding items, wherein the Lawsonia intracellularis component is an attenuated vaccine. 7. Lawsonia intracellularis ingredients are about 10 3 ~10 9 Bacteria / kg body weight or approximately 10 5 ~10 7 The vaccine of any one of the preceding items comprising a dose of bacteria / Kg body weight.

[0156] 8. Lawsonia intracellularis component is 1 x 10 5 ~1×10 7 The vaccine of any one of the preceding items comprising a dose of 9. The vaccine of any one of the preceding items, wherein the Lawsonia intracellularis component is lyophilized. 10. The vaccine of any one of the preceding items, wherein the Lawsonia intracellularis vaccine further comprises an adjuvant. 11. The vaccine of the preceding item, wherein the adjuvant is ImpranFLEX®.

[0157] 12. The vaccine of any one of the preceding items, wherein the Lawsonia intracellularis component is Enterisol® Ileitis vaccine. 13. The vaccine of any one of the preceding items, wherein the immunogenic porcine circovirus (PCV) component is a porcine circovirus (PCV) vaccine. 14. The vaccine of any one of the preceding items, wherein the PCV is PCV1.

[0158] 15. The vaccine of any one of the preceding items, wherein the PCV is PCV2. 16. The vaccine of any one of the preceding items, wherein the PCV is PCV3. 17. The vaccine of any one of the preceding items, wherein the PCV is PCV2 and PCV3. 18. The vaccine of any one of the preceding items, wherein the PCV component is a recombinant PCV component. 19. The vaccine of any one of the preceding items, wherein the recombinant PCV component is, comprises, or is expressed by a PCV ORF gene, e.g., a protein expressed by a PCV ORF gene.

[0159] 20. The vaccine of any one of the preceding items, wherein the recombinant PCV component is, comprises, or is expressed by a PCV ORF gene, e.g., a protein expressed by a PCVPCV ORF gene, and the PCV ORF gene encodes the PCV ORF2 gene. 21. The vaccine of any one of the preceding items, wherein the recombinant PCV component is, comprises, or is expressed by a PCV ORF gene, e.g., a protein expressed by a PCV ORF gene, wherein the PCV ORF gene encodes a PCV ORF2 gene, and wherein the PCV ORF2 gene is a PCV2 ORF2 gene. 22. The vaccine of any one of the preceding items, wherein the PCV component is or comprises a recombinant PCV ORF2 protein. 23. The vaccine of any one of the preceding items, wherein the PCV component is or comprises recombinant PCV ORF2 protein, and the vaccine comprises a dosage of about 2 μg to about 400 μg of recombinant PCV ORF2 protein. 24. The vaccine of any one of the preceding items, wherein the PCV component is or comprises DNA.

[0160] 25. The vaccine of any one of the preceding items, wherein the PCV component is or comprises DNA, and the DNA is present in an amount between about 10 μg and about 2000 μg, preferably between about 50 μg and about 1000 μg. 26. The vaccine of any one of the preceding items, wherein the PCV component is or has been expressed in baculovirus cells. 27. The vaccine of any one of the preceding items, wherein the PCV component further comprises an adjuvant. 28. The vaccine of any one of the preceding items, wherein the vaccine or one of the vaccine components comprises an adjuvant, and the adjuvant is CARBOPOL™. 29. The vaccine of any one of the preceding items, wherein the PCV component is Ingelvac CircoFLEX®.

[0161] 30. The vaccine according to any one of the preceding items, wherein the immunogenic Mycoplasma hyopneumoniae (M. hyo) component is a Mycoplasma hyopneumoniae (M. hyo) vaccine. 31. The vaccine of any one of the preceding items, wherein the M.hyo. component is supernatant and / or bacterin. 32. The vaccine of the preceding paragraph wherein the M.hyo. component is a bacterin. 33. The vaccine of any one of the preceding items, wherein the dose of M.hyo. component is about 2 ml of supernatant and / or bacterin. 34. The vaccine of any one of the preceding items, wherein the M.hyo. component is Ingelvac MycoFLEX®.

[0162] 35. The vaccine according to any one of the preceding items, wherein the immunogenic porcine reproductive and respiratory syndrome virus (PRRSV) component is a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. 36. The vaccine of any one of the preceding items, wherein the PRRSV component is a live vaccine. 37. The vaccine of the preceding item, wherein the PRRSV component is an attenuated vaccine. 38. The vaccine of any one of the preceding two items, wherein the PRRSV component is a modified live vaccine. 39. PRRSV components are approximately 10 per dose. 1 ~about 10 7 Viral particles, preferably about 10 per dose 3 ~about 10 5 particles, more preferably about 10 per dose 4 ~about 10 5 The vaccine of any one of the preceding items comprising a dose of the particles.

[0163] 40. The vaccine of any one of the preceding items, wherein the PRRSV component is lyophilized. 41. The vaccine of the preceding item, wherein the lyophilized PRRSV component is or has been reconstituted with 2 ml of solvent for administration. 42. The vaccine of any one of the preceding items, wherein the PRRSV component is Ingelvac® PRRS MLV. 43. The vaccine of any one of the preceding items, wherein the PCV component, the M.hyo. component and the PRRSV component are, or are derived from, the 3FLEX(registered trademark) vaccine. 44. The vaccine of any one of the preceding items, wherein the PCV component, the M. hyo. component and the PRRSV component are or are derived from the 3FLEX(registered trademark) vaccine, and the Lawsonia intracellularis component is lyophilized and dissolved in the 3FLEX(registered trademark) vaccine.

[0164] 45. The vaccine of any one of the preceding two items, wherein the Lawsonia intracellularis component is Enterisol(R) Ileitis. 46. ​​The vaccine of any one of the preceding items, wherein the volume of the vaccine is from about 0.5 ml to about 4 ml. 47. The vaccine of any one of the preceding items, wherein the volume of the vaccine is about 2 ml. 48. The vaccine of any one of the preceding items, further comprising a pharma- ceutically or veterinarily acceptable carrier. 49. The vaccine of any one of the preceding items, further comprising an adjuvant.

[0165] 50. The vaccine of the preceding item, wherein the adjuvant is ImpranFLEX®. 51. The vaccine of any one of the preceding items in a form for oral administration. 52. The vaccine of any one of the preceding items in a form for oral administration via drinking water or oral drench. 53. The vaccine of any one of the preceding items in a form for intramuscular administration. 54. The vaccine of any one of the preceding items, formulated and / or packaged for single dose or one-shot administration.

[0166] 55. The vaccine of any one of the preceding items, formulated and / or packaged for a multiwell plate regimen. 56. The vaccine of any one of the preceding items, formulated and / or packaged for a two-dose regimen. 57. The vaccine of any one of the preceding items, present as a dosage form, said dosage form being delivered from a container containing a larger amount of vaccine, said vaccine dosage form being deliverable from said container. 58. The vaccine of any one of the preceding items, present in a dosage form, said dosage form being delivered from a container containing a larger amount of said vaccine, said vaccine dosage form being deliverable from said container, said container containing at least 10 doses of said composition. 59. The vaccine of any one of the preceding items, present in a dosage form, said dosage form being delivered from a container containing a larger amount of said vaccine, said vaccine dosage form being deliverable from said container, said container containing at least 50 doses of said composition.

[0167] 60. The vaccine of any one of the preceding items, present in a dosage form, said dosage form being delivered from a container containing a larger amount of said vaccine, said vaccine dosage form being deliverable from said container, said container containing at least 100 doses of said composition. 61. The vaccine of any one of the preceding items, present in a dosage form, said dosage form being delivered from a container containing a larger amount of said vaccine, said vaccine dosage form being deliverable from said container, said container containing at least 150 doses of said composition. 62. The vaccine of any one of the preceding items, present as a dosage form, said dosage form being deliverable from a container containing a larger amount of said vaccine, said vaccine dosage form being deliverable from said container, said container containing at least 200 doses of said composition. 63. The vaccine of any one of the preceding items, present in a dosage form, said dosage form being delivered from a container containing a larger amount of said vaccine, said vaccine dosage form being deliverable from said container, said container containing at least 250 doses of said composition. 64. A vaccine according to any one of the preceding items for use in eliciting an immune or immunological response or a protective immune or immunological response in an animal.

[0168] 65. The vaccine of any one of the preceding items for use in eliciting an immune or immunological or protective immune or immunological response in an animal, wherein the animal is a porcine animal. 66. A vaccine of any one of the preceding two items for use as described in any one of the preceding two items, wherein the use is for eliciting an immune or immunological response or a protective immune or immunological response in an animal against Lawsonia intracellularis, PCV, M. hyo. and PRRSV. 67. The vaccine of any one of the preceding three items for use according to any one of the preceding items, which is administered orally. 68. The vaccine of any one of the preceding items for use according to any one of the preceding four items, administered orally via drinking water or oral drench. 69. The vaccine of any one of the preceding items for use according to any one of the preceding five items, administered intramuscularly.

[0169] 70. The vaccine of any one of the preceding six items for use according to any one of the preceding items, administered as one dose. 71. The vaccine of any one of the preceding items for use according to any one of the preceding seven items, administered as one dose, said one dose eliciting an immune or immunological or protective immune or immunological response in an animal against Lawsonia intracellularis, PCV, M. hyo. and PRRSV. 72. The vaccine of any one of the preceding items for use according to any one of the preceding 8 items, administered as at least one dose. 73. A vaccine of any one of the preceding items for use according to any one of the preceding nine items, administered as at least one dose, said one dose eliciting an immune or immunological response or a protective immune or immunological response in an animal against Lawsonia intracellularis, PCV, M. hyo. and PRRSV. 74. The vaccine of any one of the preceding items for use according to any one of the preceding 10 items, wherein one dose is administered to a porcine animal.

[0170] 75. The vaccine of any one of the preceding items for use according to any one of the preceding 11 items, wherein only one dose is administered to a porcine animal. 76. The vaccine of any one of the preceding items for use according to any one of the preceding 12 items, wherein at least one dose is administered to a porcine animal. 77. A method for eliciting an immune or immunological or protective immune or immunological response in an animal against Lawsonia intracellularis, PCV, M. hyo. and PRRSV, the method comprising the step of administering to the animal a vaccine of any one of the preceding items. 78. A method for immunizing an animal against clinical disease caused by at least one pathogen in said animal, comprising administering to the animal a vaccine of any one of the preceding items, wherein the immunological composition is capable of inducing an immune response that does not cause clinical signs of infection but immunizes the animal against a pathotype of said at least one pathogen. 79. Use of a vaccine of any one of the preceding items for use in the preparation of a composition for eliciting an immunological or immune response or a protective immunity or a protective immunological response against Lawsonia intracellularis, PCV, M.hyo. and PRRSV, or for use in a method for inducing an immunological or immune response or a protective immunity or a protective immunological response against Lawsonia intracellularis, PCV, M.hyo. and PRRSV. * * *

[0171] Although the preferred embodiments of the present invention have been described in detail, it should be understood that the invention defined by the above paragraphs is not limited to the specific details set forth in the above description, since many obvious variations thereof are possible without departing from the spirit or scope of the present invention. The present invention is further described in the following examples, which are offered for illustrative purposes and are not intended to limit the invention in any respect. EXAMPLES

[0172] Example 1 Efficacy of Enterisol® Administered Intramuscularly and Efficacy Study Design with the Presence of Antimicrobial Agents the purpose: The primary objective of this example is to evaluate the efficacy of Enterisol® Ileitis in combination with 3FLEX® vaccine when injected intramuscularly in pigs challenged with gastrointestinal homogenate containing virulent Lawsonia intracellularis, the causative agent of PPE. A secondary objective is to evaluate the efficacy of this vaccine combination when administered to pigs treated with antimicrobial combinations.

[0173] basis: PPE can be controlled using a commercial live vaccine (Enterisol® Ileitis) administered orally in drench or drinking water. It is required that animals vaccinated with Enterisol® Ileitis are not administered any antibiotics effective against Lawsonia 3 days before, on the day of and 3 days after vaccination. Although there is a trend to reduce antibiotic use, this is still a major hurdle to overcome in many farms. The use of a different application route may result in vaccine efficacy in the presence of antibiotic treatment, which would encourage vaccine use. At the same time, there is a desire in the industry to reduce the number of injections that pigs receive, and a better understanding of the efficacy of Enterisol® Ileitis when injected in combination with the 3FLEX® vaccine will provide insight into the feasibility of developing a combination vaccine against Lawsonia, PCV2, M.hyo. and PRRSV. In this study, we will investigate vaccination of pigs by the intramuscular route with Enterisol® in combination with 3FLEX®, followed by a 3-week post-challenge with gastrointestinal homogenate containing virulent Lawsonia intracellularis.

[0174] General design description: One hundred and twelve weaned pigs, 21 days old (+ / - 2 days), were obtained from a source with a known lack of history of ileitis. In addition, pigs used in this study will be sero-negative to Lawsonia intracellularis as well as fecal qPCR negative. Pigs will be randomly assigned to four groups (three groups with 24 animals in one group and one group with 16 animals in one group) and blocked by weight, litter, and sex. Treatment groups are positive challenge control (PC); Enterisol® Ileitis administered IM with 3® FLEX vaccine (EIIM), and Enterisol® Ileitis administered IM with 3® FLEX vaccine in combination with an antimicrobial agent (EIIMATB). A negative control group of 16 pigs will not be challenged and will be compared to the PC group to evaluate Lawsonia challenge alone. This negative control group will be euthanized along with the other treatment groups at the end of the study. Treatment group EIIMATB is the only group to receive antimicrobials, and this group receives the labelled dosage of 35 g / ton Denagard® (tiamulin) and 400 g / ton CTC (chlortetracycline) for a total of two weeks. Antimicrobial treatment begins on (D-7) after a five-day adaptation period to allow the pigs to recover from the lack of feed intake caused by weaning. After one week of Denagard® CTC on the feed, the EIIMATB group receives an IM vaccination with a 2 ml dose of Enterisol® Ileitis in combination with the 3FLEX vaccine. The lyophilized form of Enterisol® Ileitis is rehydrated to obtain one 2 ml dose of Enterisol® Ileitis for every 2 ml dose of 3FLEX using the 3FLEX vaccine. This means that the resulting 2 ml dose of the experimental "4FLEX" vaccine contains the appropriate amount of all four antigens. All treatment groups are vaccinated at the same time (DO). Archived samples of vaccine remaining after vaccination will be stored at -80° C. After vaccination, animals will be observed for lesions at the injection site and any other adverse reactions. Observations will be recorded.Positive and negative challenge control groups are vaccinated with 3FLEX only and do not receive Lawsonia antigen. After a 28 day (4 week) period to develop immunity, 10 organisms per pig are administered. 8~9 All animals will be challenged orally with a mucosal homogenate containing L. intracellularis, containing a target amount of 1000 mg / kg of L. intracellularis. The mucosal homogenate will be sequenced by next generation sequencing to investigate its total content and by quantitative PCR to quantify L. intracellularis. The gastrointestinal homogenate material should be free of other pathogens, including Salmonella, PRRSV and Brachyspira species. All pigs will be weighed at the time of challenge to allow for the determination of weight gain before and after challenge. After challenge, all animals will be evaluated daily for fecal changes, changes in body condition and altered behavior until the end of the study.

[0175] The study will be terminated 21 days after challenge, at which time all animals will be euthanized and weighed again. At necropsy, macroscopic lesions in all segments of the intestine will be measured and assessed, and terminal ileum samples as well as any additional affected tissues will be taken up in formalin to measure microscopic lesions.

[0176] Microscopic lesions are estimated by immunohistochemistry (IHC) and by hematoxylin and eosin (H&E) staining, and proliferative lesions are measured. Blood and fecal samples are collected from all animals at the time of vaccination, challenge, and then weekly until necropsy for Lawsonia intracellularis serology and qPCR. Fecal samples are collected by finger insertion with gloves changed between animals, or by fecal loop. If fecal samples are collected by fecal loop, they should not be reused, a different fecal loop should be used for each animal. All blood and fecal samples should be aliquoted and only one aliquot should be submitted for Lawsonia serology and fecal PCR. All sample tubes are labeled with the date of collection, study date, and pig ID number.

[0177] Experimental unit: each individual pig. Rationale for number of replicates: Power calculation: Assuming an incidence rate in the challenge control group of at least 75%, 21 animals per treatment group is expected to provide approximately 80% power to detect a 40 percentage point difference between treatment and control for a two-sided test using a=0.05. A total of 24 animals per treatment group is used to allow for possible side effects and to allow for the acceptance of a power level slightly higher than 80%. Method for randomization: Animals are blocked by weight, sex and litter and a random number generator is used to assign pigs to treatments. Level and description of blinding: Patients will be blinded to treatment for the assessment of lesions, fecal shedding and serology. Blinding will also be performed for statistical analyses.

[0178] Diagnostic details and requirements: After vaccination and challenge, blood and fecal samples are collected weekly from all study animals. This equates to approximately 560 blood and fecal samples (112 animals x 5 samplings). All samples are submitted for diagnosis. At least one aliquot of each sample is stored at -80°C until the end of the study. Production stage: The nursery stage of production. Sex of the animals: barrows and gilts are distributed as equally as possible between the treatment groups. Inclusion / exclusion and post-inclusion removal criteria: Inclusion criteria at study start: commercial production herds of pigs with normal health status on D(-7). Exclusion criteria at study start: pigs that were clinically sick or stunted on D(-7). Exclusion criteria during the study: If welfare or disease concerns arise, the Principal Investigator and on-site veterinarian and / or designer will evaluate and decide on the best course of action, which may include euthanasia.

[0179] Example 2 Efficacy of Enterisol® Administered Intramuscularly and with the Presence of Antimicrobial Agents, Final Study Report This study evaluated the efficacy of Enterisol® Ileitis administered intramuscularly and in combination with Ingelvac® 3FLEX vaccine in protecting against Lawsonia intracellularis, the causative agent of porcine proliferative enteritis (PPE). Potential interference of antimicrobial agents with vaccine efficacy was also evaluated. PPE was successfully reproduced in this study by measuring the characteristic macroscopic lesions, microscopic lesions, fecal shedding of L. intracellularis, seroconversion to L. intracellularis, clinical signs, and impact on production performance. The combination of Enterisol® Ileitis and 3FLEX® for intramuscular administration, "4FLEX", resulted in a meaningful and significant reduction in the severity of macroscopic lesions, the severity of microscopic lesions, clinical diarrhea scores, and fecal shedding of L. intracellularis. An increase in the average daily weight gain was also observed in this treatment group compared to the unvaccinated control. These results indicate that the intramuscular route and the combination of Enterisol® Ileitis and 3FLEX® vaccines are a suitable and effective option for the prevention of PPE. The vaccine combination was also observed to be safe, with no adverse events or injection site reactions observed.

[0180] When antimicrobial agents were administered during vaccination, changes were observed in some of the parameters evaluated in this study. A reduction in macroscopic and microscopic lesions was observed in the group treated with antimicrobial agents (EIIMATB) compared to the unvaccinated challenged controls, but these levels did not reach statistical significance and were numerically elevated compared to the same treatment without antimicrobial agents (EIIM). However, the vaccinated group that also received antimicrobial agents (EIIMATB) was the vaccinated group that had the highest weight gain after challenge, which was significantly increased compared to the unvaccinated controls. A significant reduction in the incidence of changes in clinical diarrhea scores was also observed. These results indicate that antimicrobial agents may potentially interfere with the efficacy of the vaccine, but a significant level of protection was still conferred when vaccinated in the presence of the tested antimicrobial agents.

[0181] The primary objective of this study was to evaluate the efficacy of the intramuscular route of administration of Enterisol® Ileitis in combination with 3FLEX® vaccine in protecting pigs against Lawsonia intracellularis, the causative agent of porcine proliferative enteritis (PPE). A secondary objective was to evaluate the efficacy of this vaccine combination when administered to pigs treated with antimicrobial combinations. Vaccine efficacy was determined by reduction in gross and microscopic intestinal lesions. Other variables of interest were also assessed, including growth potential, clinical signs and fecal shedding.

[0182] PPE can be controlled using a commercial live vaccine (Enterisol® Ileitis) administered orally in drench or drinking water. It is required that animals vaccinated with Enterisol® Ileitis are not administered any antibiotics active against Lawsonia 3 days prior to, on the day of and 3 days after vaccination. Although there is a trend to reduce antibiotic use, this remains a major hurdle to overcome on many farms. The use of a different route of application may result in vaccine efficacy in the presence of antibiotic treatment and would encourage vaccine use. At the same time, there is a desire in the industry to reduce the number of injections that pigs receive, and a better understanding of the efficacy of Enterisol® Ileitis when injected in combination with the 3FLEX® vaccine will provide insight into the feasibility of developing combination vaccines against Lawsonia, PCV2, Mycoplasma hyopneumoniae and PRRSV. This study investigated vaccination of pigs by the intramuscular route with Enterisol® in combination with 3FLEX®, followed by challenge 4 weeks later with gastrointestinal homogenate containing virulent Lawsonia intracellularis.

[0183] Design considerations: Pigs aged 21 days (+ / - 2 days) were obtained from a source with a known lack of history of ileitis. Pigs were randomly assigned to 4 groups (3 groups with 24 animals in each group, 1 group with 16 animals). Treatment groups were positive challenge control (PC); Enterisol® Ileitis administered IM with 3® FLEX vaccine (EIIM), and Enterisol® Ileitis administered IM with 3® FLEX vaccine in combination with antimicrobial (EIIMATB). A negative control (NC) group of 16 pigs was not challenged and served the purpose of assessing the severity of challenge in the PC group. Treatment group EIIMATB was the only group that received antimicrobial and was given the labelled dosage of 35 g / ton Denagard® (tiamulin) and 400 g / ton CTC (chlortetracycline) for a total of 2 weeks. Antimicrobial treatment began (D-7) after a 13-day adaptation period to allow pigs to recover from the lack of feed intake due to weaning. After one week of Denagard® CTC on the diet, the EIIMATB group received an IM vaccination with a 2 ml dose of Enterisol® Ileitis in combination with the 3® FLEX vaccine. All treatment groups were vaccinated at the same time (D0). After a 28-day (4-week) period to generate immunity, an oral challenge with mucosal homogenate containing Lawsonia intracellularis was administered to all animals. The study was terminated 21 days after challenge, at which time all animals were euthanized, lesions were scored, and samples were taken. Further design details are in the table below.

[0184] [Table 1]

[0185] [Table 2]

[0186] Treatment by group: The treatment groups were positive challenge control (PC); Enterisol Ileitis administered IM with Ingelvac 3FLEX vaccine (EIIM), and Enterisol Ileitis administered IM with Ingelvac 3FLEX vaccine in combination with antimicrobial (EIIMATB). A negative control (NC) group of 16 pigs was not challenged and served the purpose of evaluating the severity of the challenge in the PC group. Treatment group EIIMATB was the only group that received antimicrobial, and this group received the labelled dosage of 35 g / ton Denagard (tiamulin) and 400 g / ton CTC (chlortetracycline) for a total of two weeks. After one week of Denagard CTC in the feed, the EIIMATB group was vaccinated IM with a 2 ml dose of Enterisol Ileitis in combination with Ingelvac 3FLEX vaccine. The lyophilized form of Enterisol® Ileitis was rehydrated using the Ingelvac® 3FLEX vaccine to give one 2 mL dose of Enterisol® Ileitis for every 2 mL dose of Ingelvac® 3FLEX, meaning that the resulting 2 mL dose of the experimental "4FLEX" vaccine contained the appropriate amounts of all four antigens.

[0187] Treatment Medication: Vaccines were administered to groups 1-5 on DO by the routes shown in the table below. IM injections were administered on the right side of the neck, midway between the base of the ear and the tip of the shoulder, using an appropriately sized sterile needle and syringe.

[0188] [Table 3]

[0189] [Table 4] Animal inclusion / exclusion criteria: Prior to the start of the study, the health of each animal was assessed and animal health forms were completed. Only animals in normal health that were qPCR and seronegative for Lawsonia were included in the study. If any welfare or disease concerns arose during the study, the principal investigator and on-site veterinarian and / or designer determined the best course of action, which may include euthanasia, and recorded these events.

[0190] Experimental unit: Pig was the experimental unit. Randomization: Randomization of pigs to housing and treatments was performed by statistical services provided by GBI using SAS statistical software. Available pigs, litter information, sex, age, weight, and housing arrangements were provided to GBI statisticians prior to the start of the study. Animals were blocked by weight, sex, and litter, and a random number generator was used to assign pigs to treatments.

[0191] Blinding criteria: Personnel involved in scoring the lesions and performing the laboratory assays were blinded to the allocation of pigs to groups throughout the study. All laboratory personnel involved in collecting data for this study completed a signature record for documentation purposes.

[0192] Veterinary care and concomitant procedures: Upon arrival at the study facility, no medications were administered prior to consent from the supervisor or designer. Animals were under veterinary care upon arrival at the facility until the end of the study. Any animals presenting with trauma or illness unrelated to challenge administration were to receive appropriate veterinary care. Documentation provided by the investigator included a description of clinical signs observed, the outcomes of any diagnostic tests, and the outcomes of any treatments administered. All treatments were to be documented in the Biological and Pharmaceutical Treatment Record, and these included antimicrobials administered to the EIIMATB group. Euthanized or expired animals were to be necropsied and samples taken as necessary to establish a diagnosis, which was then entered into the Veterinary Reporting Record. Any unexpectedly ill or moribund animals were to be handled appropriately by the on-site investigator, on-site veterinarian in collaboration with the supervisor and PI to determine the best course of action to alleviate pain / distress from either treatment or euthanasia.

[0193] General Observations: Beginning upon arrival and continuing until D28, all animals were observed daily for general health and observations were recorded in the General Health Observation Record. Injection Reactions: Pigs were monitored for injection site reactions by observation of the injection site for 4-8 hours after vaccination and for any adverse events. Pigs were re-evaluated on days 1, 2, and 3 after vaccination. Any animals with injection site abnormalities were to be monitored daily until resolution of lesions. Abnormalities were noted and described in terms of size (cm), redness, swelling, heat, and pain. Injection site observations were recorded in the Injection Site Observation Record.

[0194] Clinical Observations: All pigs were observed daily from D28 to D49 for clinical signs associated with L. intracellularis challenge. Findings were recorded in the Clinical Observations Record using the observational scores shown in the table below.

[0195] [Table 5] body weight: Each pig was weighed (lb) as indicated on the event date and the results were recorded on a Body Weight Record. The average daily weight gain for all test animals was calculated for group comparisons. autopsy: On D49, all remaining pigs were euthanized according to site procedures. The ileum, jejunum, cecum, and colon were examined for gross lesions and scored on the Off Test Necropsy Record according to the scoring scheme in the table below.

[0196] [Table 6]

[0197] [Table 7]

[0198] Blood Sample Collection: Blood collection was recorded in a Sample Collection Record. Venous whole blood was collected into serum separator tubes (SST) according to the date of event. Venous whole blood was collected from each pig via the anterior vena cava by the investigator or designer using a sterile 18-20g x 1-1.5" Vacutainer® needle, Vacutainer® needle holder, and 9 or 13 ml SST tube. Each sample was labeled with the animal ID number, study number, collection date, study date, and sample type. The SST was left at room temperature to clot and then centrifuged at approximately 2000 x g for 5-10 minutes. Upon completion of centrifugation, aliquots were made for testing and long-term storage at -80°C. Aliquots of serum stored at -80°C were shipped to the diagnostic laboratory (ISU-VDL) for Lawsonia ELISA testing on dry ice to standardize handling of all samples.

[0199] Fecal sample collection: Fecal samples were collected by digital insertion into the rectum according to the date of event for testing at the ISU-VDL for Lawsonia shedding by qPCR. Fecal collection was recorded in a Sample Collection Record. Fecal samples were collected in tubes labeled with the study number, collection date, study date, and pig ID number. All samples were aliquoted into two tubes and stored at -80°C. One aliquot was shipped in an insulated container with dry ice to keep the samples frozen during shipping and to ensure that all samples would be processed equally at the ISU-VDL. Samples collected were recorded in a Sample Collection Record. Evaluation of microscopic lesions was performed by hematoxylin and eosin (H&E) and Lawsonia immunocytochemistry (IHC). Testing procedures and results will be included in the final report. Raw data was recorded using the Histology and Immunohistochemistry Record. Ileal samples were taken and fixed in formalin for microscopic histological examination with scoring by established methods using the Histology and Immunohistochemistry Record.

[0200] Statistical analysis: Statistical tests followed those used in peer-reviewed publications with similar data and allowed pairwise comparisons. Quantitative data were evaluated with chi-square tests. Significance was determined when p<0.05 and trend when 0.05≦p<0.10. General Observations / Adverse Events: Three adverse events occurred during the study. None were related to vaccination, but one event was related to Lawsonia intracellularis challenge. These events are listed in the table below.

[0201] [Table 8] Injection Reactions: None of the vaccinated animals developed any redness, swelling, fever or pain during the three days they were evaluated after vaccination. No differences in injection site reactions were noted in the groups receiving Ingelvac® 3FLEX vaccine IM alone or when mixed and injected in combination with Enterisol® Ileitis.

[0202] Clinical Observations: Clinical diarrhea scores were zero at the time of challenge in all animals and began to change in the various groups at 6 days post-infection (dpi). The incidence of diarrhea score changes was significantly (p<0.0001) higher in the PC group compared to the NC group (see table below), indicating that L. intracellularis challenge resulted in characteristic clinical signs. All groups treated with Enterisol® Ileitis did not have a significant reduction in the incidence of clinical diarrhea score changes compared to the non-vaccinated PC group (see table below). Behavior was not significantly altered after challenge, with only one altered behavioral event observed among 501 evaluations in the PC group. Body condition scores were found to change due to challenge. All treatment groups experienced significantly more body condition changes compared to the negative control group (see table below). The EIIM treatment group was the group with the lowest incidence of body condition changes and tended to have reduced body condition (p=0.054) compared to the non-vaccinated PC group.

[0203] [Table 9] Gross Lesion Score: The macroscopic lesion score in the ileum, the preferential colonization site of L. intracellularis, was highest in the PC group with a mean score of 1.55 (Figure 1). This score was significantly higher than the non-challenged control group (NC), thus validating the validity of the challenge model (Figure 1, p<0.05). Among the vaccinated groups, the EIIM group had the lowest lesion score with a mean score of 0.67, which was significantly (p<0.05) lower than the non-vaccinated PC group (Figure 1). The EIIMATB group developed similar levels of lesions that were less severe than the PC group but did not reach statistical significance. The length of the macroscopic lesion was also measured and this length followed a similar pattern as the lesion score. The EIIM group had a mean ileal lesion length that was approximately half the length of the PC group (p=0.097) (Figure 2). A severity score was assigned to the lesion length by multiplying it by the lesion score. The mean lesion severity scores can be found in Figure 3. Severity scores followed a similar trend as those of lesion scores and lesion length. The EIIM group had a mean severity score of 15.63, while the PC group had a mean score of 40.68 (p=0.053; FIG. 3). The vaccinated group also led to a reduction in lesions in other parts of the intestinal tract. As there was no significant difference in lesions between the PC and NC groups, this was not investigated further. This was likely due to the lower number of animals with lesions outside the ileum.

[0204] body weight: Average daily weight gain (ADG) was very similar between treatment groups during the pre-challenge study period, with no significant differences found between groups. During the post-challenge period, all challenged groups had a significant reduction in ADG compared to the non-challenged NC group (Figure 4). The EIIMATB group was the best performing vaccinated group during the post-challenge period, with an ADG of 1.71 lb / day, which was significantly greater than the non-vaccinated challenged PC group, which had an ADG of 1.43 (P<0.05). The EIIM group had the second highest post-challenge ADG, with an ADG of 1.65, which was also significantly greater than the PC group (p<0.05); (Figure 4).

[0205] Lawsonia Serum ELISA: The number of animals with serum antibodies against Lawsonia measured by ELISA at ISU-VDL is shown in Figure 5. All animals were seronegative at the time of vaccination on study day 0 (28 days before challenge, -28 dpi). Only two animals were seropositive at the time of challenge (study day 28), one animal in the EIIM group and one animal in the EIIMATB group. This indicates that IM vaccination with Enterisol® Ileitis in combination with the Ingelvac® 3FLEX vaccine results in a very significant seroconversion. As expected, most animals seroconverted and were positive at 21 dpi, again confirming the validity of the Lawsonia challenge according to the expectations of this challenge model. The PC group had the highest number of animals with anti-Lawsonia antibodies at 21 pdi, with 95% of pigs being positive. At 14 dpi, the EIIM and PC groups had 67% and 65% seropositive pigs, respectively.

[0206] Lawsonia fecal qPCR: All animals were fecal qPCR negative for Lawsonia at the time of vaccination on study day 0 (28 days prior to challenge, -28 dpi). At the time of challenge, two animals in the EIIMATB group had detectable Lawsonia levels in their feces by qPCR. One of the animals in the PC group had a Ct value of 34.1 or 252 / organism per gram of feces at the time of challenge. Lawsonia shedding peaked at 14 dpi, at which point the PC group had an average of 6.88 log seroprevalence per gram of feces. 10 At this point, the EIIM groups had a log 10 This resulted in a significant (p<0.05) reduction in fecal shedding compared to the PC group, which had a shedding level of 5.96 microorganisms. At 21 dpi, the EIIM group shed the least Lawsonia among the challenged animals, with a log 4.28 per gram of feces. 10The microbial count averaged 3.20, which is an average of 4.64 log per gram of feces. 10 The EIIMATB group also reduced shedding compared to PC, although to a lesser extent, with a log 1000 / g feces shedding rate of 1000 / g feces. 10 The average microbial count was 4.05 (Figure 6).

[0207] Microscopic lesions and Lawsonia IHC: Microscopic lesions were measured by hematoxylin and eosin (H&E) staining of terminal ileum taken at necropsy. The group that developed the most severe lesions was the PC group with a mean lesion score of 2.05, while animals in the NC group did not develop lesions, again validating the validity of this infection model (Figure 7). The vaccinated group that developed the least severe lesions was the EIIM group, whose mean score was 1.29, which was significantly reduced from the PC group (Figure 7, p<0.05). The EIIMATB group had a higher but similar mean lesion score compared to the EIIM group, with a mean score of 1.57. Terminal ileum tissue taken at necropsy and fixed in formalin was also subjected to immunohistochemistry (IHC) staining for L. intracellularis antigens. Similar to HE staining, the group with the highest score was the PC group, with a mean score of 2.23, which was significantly (p<0.05) increased compared to the NC group, again demonstrating the validity of disease infection and reproduction in this study (Figure 8). Again, similar to the HE score, the EIIM group was the vaccinated group with the lowest sensitivity score, with a score of 1.63, which was significantly reduced compared to the PC group (p<0.05).

[0208] Consideration: This study investigated the efficacy of a lyophilized version of Enterisol Ileitis when combined with a 2 ml dose of 3FLEX vaccine administered by the intramuscular route. In addition, the interference of tiamulin and chlortetracycline when offered in the feed during vaccination was evaluated.

[0209] Porcine proliferative enteritis (PPE) caused by L. intracellularis was successfully reproduced in this study by measuring the characteristic macroscopic lesions, microscopic lesions, fecal shedding of L. intracellularis, seroconversion to L. intracellularis, clinical signs, and impact on production performance. The combination of Enterisol® Ileitis and 3FLEX® for intramuscular administration, "4FLEX", resulted in a meaningful and significant reduction in the severity of macroscopic lesions, the severity of microscopic lesions, clinical diarrhea scores, and fecal shedding of L. intracellularis. A non-significant numerical increase in the average daily weight gain was also observed in this treatment group (EIIM) compared to the unvaccinated control (PC). These results indicate that the intramuscular route and the combination of Enterisol® Ileitis and 3FLEX® vaccine are a suitable and effective option for the prevention of PPE. The vaccine combination was also observed to be safe, with no adverse events or injection site reactions observed.

[0210] When antimicrobial agents were administered during vaccination, changes were observed in the parameters evaluated in this study. A reduction in macroscopic and microscopic lesions was observed in the EIIMATB group compared to the non-vaccinated challenged controls, but these levels did not reach statistical significance and were numerically elevated compared to the same treatment without antimicrobial agents (EIIM). However, the EIIMATB group was the vaccinated group with the greatest numerical weight gain after challenge (Figure 4) and there was a significant reduction in the incidence of changes in clinical scores for diarrhea (Table of clinical score evaluation between treatment groups). These results indicate that although antimicrobial agents can interfere with the efficacy of the vaccine, vaccination in the presence of the tested antimicrobial agents that are active against Lawsonia still conferred a certain level of protection.

[0211] Nogueira MG et al. (Immunological responses to vaccination following experimental Lawsonia intracellularis virulent challenge in pigs. Vet Microbiol. 2013) also found that intramuscular administration of Enterisol® Ileitis led to reduced microscopic lesions and reduced fecal shedding of L. intracellularis compared to unvaccinated and challenged animals. In that study, the live L. intracellularis antigen present in Enterisol® Ileitis was administered alone without any adjuvant. The combination of Enterisol® Ileitis and 3FLEX® could improve the immune response to the vaccine, possibly due to the inclusion of the ImpranFLEX® adjuvant.

[0212] This study demonstrates that intramuscular administration of Enterisol® Ileitis in combination with Ingelvac 3FLEX® provides significant protection against L. intracellularis. The potential impact of antimicrobials administered during vaccination is unclear and warrants further investigation. However, intramuscular vaccination in combination with antimicrobial administration provided a meaningful level of protection in several relevant parameters of the disease.

[0213] Example 3 Efficacy of Enterisol® Administered Intramuscularly and with the Presence of Feed-Grade Antimicrobials Introduction: Enterisol® Ileitis vaccine, administered via drinking water or by oral drench, is a highly effective and successful commercial product licensed for use in healthy postweaned pigs for the prevention of porcine proliferative enteritis caused by Lawsonia intracellularis. There are no data regarding the efficacy of this product, administered by intramuscular injection, when combined with 3FLEX® and when administered with concomitant antimicrobial therapy.

[0214] The objectives of this example are: 1. to evaluate the efficacy of the Enterisol® Ileitis vaccine when administered by intramuscular injection in combination with 3FLEX® vaccination, and 2. to evaluate the efficacy of this vaccine combination when administered to pigs receiving an in-feed antimicrobial combination treatment. FIG. 9 shows the vaccine formulation. Figure 10 provides an outline of the study. Primary parameters measured include macroscopic lesion score, microscopic lesion score and fecal output. Secondary parameters measured include mean daily gain, clinical score and seroconversion.

[0215] Materials and Methods: Weaned pigs aged 17-21 days were obtained and randomized into three treatment groups with 24 animals each. The treatment groups were: unvaccinated positive challenge control (PC); Enterisol® Ileitis administered IM with 3®FLEX vaccine (EIIM); and Enterisol® Ileitis administered IM with 3®FLEX vaccine in combination with an antimicrobial agent (EIIMATB). Vaccine preparation was performed by reconstituting a lyophilized form of Enterisol® Ileitis with 3FLEX® vaccine. This resulted in a final 2 ml dose containing modified live Lawsonia intracellularis antigen together with PCV2, M. hyo. and PRRSV MLV vaccine fractions. To investigate whether in-feed antimicrobial treatments would inhibit the efficacy of vaccination with live modified Enterisol® Ileitis vaccine, the EIIMATB group received in-feed tiamulin (35 ppm) and chlortetracycline (400 ppm) starting 1 week before vaccination and continuing until 1 week after vaccination. All animals were challenged with gut homogenate containing virulent Lawsonia intracellularis 28 days after vaccination and necropsied 21 days later. Clinical signs, average daily weight gain (ADG), fecal output, and macroscopic and microscopic lesions were evaluated at necropsy.

[0216] result: Both vaccination treatment groups led to a significant reduction in clinical scores of diarrhea (P<0.05). Both vaccinated groups also led to an increase in ADG after challenge. The challenge control group had a post-challenge ADG of 1.43 lbs, while the EIIM and EIIMATB groups had ADGs of 1.65 and 1.71 lbs, respectively (P<0.05). Macroscopic lesion scores were reduced in both vaccinated groups with values ​​of 0.67 and 1.04 compared to the unvaccinated group, which had a mean score of 1.55. Similarly, microscopic lesions were also reduced by vaccination with a mean score of 2.05 in the PC group compared to a mean of 1.29 and 1.57 in the EIIM and EIIMATB groups, respectively. This IM vaccine combination was found to be safe and no adverse reactions were noted. Conclusion: The two injected vaccine groups compared to the unvaccinated group following Lawsonia challenge improved ADG, resulted in reduced macroscopic and microscopic lesion scores, reduced Lawsonia shedding, and reduced clinical signs. No evidence of antimicrobial interference was observed with the combination of Enterisol® Ileitis and 3FLEX® intramuscular vaccine. This study reveals a new tool for pig producers that warrants further investigation.

[0217] Example 4 Efficacy of porcine circovirus type 2a (PCV2a) ORF2 VLP vaccine combined with Lawsonia ALC vaccine administered intramuscularly The objective of this study was to demonstrate the efficacy of porcine circovirus type 2a (PCV2a) ORF2 VLP vaccine in combination with Lawsonia ALC (avirulent live culture) vaccine (Enterisol® Ileitis, lyophilized) administered intramuscularly in 3-week-old pigs against porcine circovirus type 2a challenge 4 weeks later.

[0218] Pigs are randomized upon enrollment in the study. During the vaccination phase, pigs are housed with their littermates. See table below, day 0 pigs are 21±3 days at the time of vaccination. PCV2a ORF2 VLP vaccine (Ingelvac CircoFLEX®) is combined with L. intracellularis ALC (non-toxic live culture; Enterisol® Ileitis, lyophilized), and the Enterisol Ileitis lyophilisate is reconstituted in Ingelvac CircoFLEX (Group 1). Group 2 (non-toxic live culture of Lawsonia, monovalent) contains non-toxic Lawsonia, as well as saline and the adjuvant Carbopol, since Carbopol and saline are present in Ingelvac CircoFLEX®, and therefore Carbopol and saline are also present in Groups 1 and 3. Group 3 (PCV2 VLP, monovalent) contains PCV2a ORF2 VLP. The NTX group consists of 6 pigs that serve as non-treated controls. The pigs are observed for any adverse reactions to inoculation, including injection site reactions and anaphylaxis. Lawsonia ALC (Avirulent Live Culture; Enterisol® Ileitis, freeze-dried form) and PCV2a ORF2 VLP (Ingelvac CircoFLEX®) are well-known proprietary veterinary vaccines. However, WO2006 / 072065 and WO2008 / 076915 describe the generation of PCV vaccines, their formulation and their administration. WO96 / 39629 and WO05 / 011731 describe the cultivation of Lawsonia intracellularis, attenuated Lawsonia intracellularis and their administration.

[0219] Pigs are mixed prior to challenge (D27). The NTX group is necropsied on D20 to ensure that pigs have not been exposed to wild-type PCV2 during the vaccination phase. On day 28, virulent PCV2a (4.77 log 10 TCID 50Pigs are challenged with 100 mg / 2 mL of fecal pelleted broth (100 mg / 2 mL dose) and assigned a clinical score based on fecal consistency, body condition and behavior beginning on day 27 and continuing throughout the remainder of the study.

[0220] autopsy 22 days after challenge, animals from groups 1-3 are euthanized and necropsied. Lymph nodes and ileum are evaluated, scored, and harvested for histopathology and immunohistochemistry.

[0221] [Table 10] A general examination of all organs and injection site areas for abnormalities will be completed during necropsy. Samples of tonsils, tracheobronchial lymph nodes (TBLN), mesenteric lymph nodes (MLN), external iliac lymph nodes (ILN), and ileum will be collected and fixed in 10% neutral buffered formalin. Samples will be processed according to standard procedures and evaluated by hematoxylin and eosin staining (H&E) for histopathology and by immunohistochemistry (IHC) for PCV2 antigen. Tissues will be scored according to the scoring system below.

[0222] [Table 11]

[0223] Lymphocyte depletion Pigs are considered positive if one or more of the four lymphoid tissue samples (tonsil, TBLN, MLN, ILN) or ileum were histologically positive for lymph node depletion (score >0).

[0224] Lymphocyte colonization Pigs are considered positive if one or more of the four lymphoid tissue samples (tonsil, TBLN, MLN, ILN) or ileum were positive for PCV2 lymphocyte colonization by IHC (score >0).

[0225] Tissue results (histopathology and immunohistochemistry) The results presented in the table below represent every tissue that had a positive score (score of 1, 2, 3) as listed in the Tissue Scoring Table. The percentages indicate the number of pigs with a positive score out of the total number of pigs in the herd.

[0226] [Table 12]

[0227] The results in group 2 show that the challenge was successful, since animals vaccinated only with avirulent live Lawsonia vaccine show a high incidence of infection with PCV2. Furthermore, the results show that the PCV2 vaccine is effective in combination with avirulent live Lawsonia vaccine, since group 1 does not show any clinical signs of PCV2 infection. Group 1 (abortogenic live culture of Lawsonia + PCV2 VLP; bivalent vaccine) behaves similarly to group 3 (PCV2 VLP, monovalent vaccine), which was vaccinated only with PCV2 vaccine. Thus, no interference is observed when the PCV2 vaccine is combined with avirulent live Lawsonia vaccine.

Claims

1. 1. A vaccine for use in a method for raising a protective immune response in an animal, the vaccine comprising an antigen of Lawsonia intracellularis and one or more antigens of Mycoplasma hyopneumoniae (M. hyo), and one or more adjuvants, the method comprising administering the vaccine to the animal, the vaccine being administered intramuscularly or intradermally, and the Lawsonia intracellularis antigen being live Lawsonia intracellularis.

2. The vaccine of claim 1, wherein the M. hyo. antigen is a bacterin.

3. A vaccine as described in claim 1 or 2, wherein the live Lawsonia intracellularis bacteria are modified live Lawsonia intracellularis bacteria, or the live Lawsonia intracellularis bacteria are attenuated Lawsonia intracellularis bacteria.

4. A vaccine described in any one of claims 1 to 3, wherein the animal is a pig.

5. The vaccine of any one of claims 1 to 4, wherein the method is for eliciting a protective immune response in an animal against Lawsonia intracellularis and / or M. hyo.

6. A vaccine according to any one of claims 1 to 5, administered intramuscularly and / or as a single dose.

7. A vaccine described in any one of claims 1 to 6, wherein the animal is treated / combinedly treated with one or more antibiotics simultaneously.

8. A vaccine described in any one of claims 1 to 7, wherein the method is for immunizing an animal against clinical disease caused by at least one pathogen in the animal, and the vaccine does not cause clinical signs of infection but is capable of inducing an immune response that immunizes the animal against a pathogenic form of the at least one pathogen.

9. A vaccine described in any one of claims 1 to 8, wherein the protective immune response against Lawsonia intracellularis is a protective immune response that reduces intestinal lesions in animals compared to non-immunized control animals of the same species.

10. The vaccine of claim 9, wherein the intestinal lesion is an ileal lesion, or the intestinal lesion is a macroscopic lesion and / or a microscopic lesion.

11. A vaccine described in any one of claims 1 to 10, wherein the protective immune response against Lawsonia intracellularis is a protective immune response that reduces fecal shedding in the animal compared to a non-immunized control group of animals of the same species, or wherein the protective immune response against Lawsonia intracellularis is a protective immune response that increases the average daily weight gain in the animal compared to a non-immunized control group of animals of the same species.

Images