Combination vaccine for intradermal administration

Abstract

The present invention relates to the field of veterinary vaccinology, specifically combination vaccines for swine. In particular, the present invention relates to a combination vaccine for protection against infection with the pathogens porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhyo), comprising a non-replicating immunogen for PCV2 and a non-replicating immunogen for Mhyo. The vaccine is characterized in that it is an oil-in-water emulsion comprising squalane, vitamin E acetate, and silica. In another embodiment, the present invention relates to a combination vaccine for protection against infection with the pathogens PCV2 and Mhyo by intradermal administration.

Description

Technical Field

[0001] The present invention relates to the field of veterinary vaccinology, specifically combination vaccines for swine. In particular, the present invention relates to a combination vaccine for protection against infection with the pathogens porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhyo), comprising a non-replicating immunogen for PCV2 and a non-replicating immunogen for Mhyo. The vaccine is characterized in that it is an oil-in-water emulsion comprising squalane, vitamin E acetate, and silica. In another embodiment, the present invention relates to a combination vaccine for protection against infection with the pathogens PCV2 and Mhyo by intradermal administration. Background Art

[0002] Today, intensive pig farming relies heavily on veterinary medical products to maintain animal health and economical operation. Besides optimizing feed and farm management systems, various treatments are often used: medications (such as hormones or antibiotics) and vaccinations against bacterial or viral pathogens. Some of the most prominent diseases affecting pigs from an early age are caused by bacteria (such as Mycoplasma hyopneumoniae) and viruses (such as porcine circovirus type 2).

[0003] Mycoplasma hyopneumoniae (Mhyo) is the leading pathogen causing enzootic pneumonia (a chronic respiratory disease of pigs) worldwide. Young piglets are particularly susceptible to this highly contagious disease. This bacterium is relatively small, lacks a cell wall, and belongs to the genus Mollicutes. It maintains a parasitic lifestyle on or within host cells. The lung disease caused by Mhyo is primarily an immune-mediated pathology, leading to consolidative pneumonia. The bacterium colonizes and damages the ciliated lung epithelium, resulting in loss of ciliary activity. The most problematic consequence of this disease is its susceptibility to various secondary infections of the pig's respiratory system caused by other bacterial and viral pathogens, depending on housing conditions and environmental stressors. This results in the so-called porcine respiratory disease complex (PRDC), characterized by severe lung lesions. In addition to the discomfort experienced by the animals, enzootic pneumonia and PRDC cause significant economic losses to the pig industry due to reduced growth rate and feed conversion, as well as the costs of veterinary care and antibiotic use.

[0004] Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS) observed in piglets. Clinical symptoms and pathology were described in 1996 and include progressive emaciation, dyspnea, tachypnea, and occasional jaundice and icterus. The new agent, termed PCV2, is distinct from known PCVs, which are natural contaminants of PK-15 cells. PCV2 is a very small, non-enveloped virus in the genus Circovirus that contains a circular, single-stranded DNA genome with two major genes. The ORF2 gene encodes a viral capsid protein of approximately 233 amino acids. Recombinantly expressed PCV2 ORF2 protein forms virus-like particles that are highly effective as a subunit vaccine.

[0005] There are several commercial vaccines against Mhyo that are routinely used in most commercial pig farming operations. Typically, these vaccines contain non-replicating immunogens such as subunit proteins and / or bacterins (i.e., whole or non-whole killed bacteria) that are usually administered by parenteral injection. Some examples are: (Zoetis), M.hyo (Boehringer Ingelheim) and (Merck Animal Health).

[0006] Conventional vaccines for the prophylactic treatment of animals (particularly pigs) against PCV2 infection are based on whole, inactivated PCV2 viruses as (non-replicating) immunogens. Furthermore, it has been found in the art that the capsid protein encoded by ORF2 (e.g., when recombinantly expressed) is suitable as a subunit immunogen for PCV2 in appropriate vaccines. This is understandable, as this subunit behaves in vivo in the same manner as the virus itself (forming virus-like particles), differing only in that DNA and nonstructural proteins are not present within the capsid. Several vaccines against PCV2 are commercially available in the art. PCV (available from MSD Animal Health, Boxmeer, The Netherlands) is a vaccine used to protect pigs against porcine circovirus type 2 and is indicated for use in pigs three weeks of age and older. When administered as two injections (two doses), the duration of immunity (DOI) is 22 weeks, which almost completely covers the fattening period of pigs. Vaccine (available from Boehringer Ingelheim, Ingelheim) is a vaccine for the protection of pigs against porcine circovirus type 2. It is used in pigs two weeks of age and older and is registered as a single-shot (one-dose) vaccine. (available from Merial, Lyon, France) is a product for the protection of pigs against porcine circovirus type 2 and is used in pigs three weeks of age and older. PCV (available from Zoetis, Capelle a / d IJssel, The Netherlands) is a vaccine for the protection of pigs against porcine circovirus type 2 and is used in pigs three weeks of age and older. Other PCV2 vaccines are described in, for example, WO 2007 / 028823, WO 2007 / 094893 and WO 2008 / 076915.

[0007] To limit stress on the animals and cost and labor for the caregiver, some pig vaccines have been prepared as combination vaccines. Examples are: Ingelvac CircoFLEX and Ingelvac MycoFLEX (Boehringer) which can be mixed shortly before administration, PCV MH (Zoetis) and PCV MHyo (MSD Animal Health), which combines antigens from PCV2 and Mhyo.

[0008] An important component of vaccines containing non-replicating immunogens is the adjuvant. This provides an immune stimulus to the non-replicating immunogen, which would otherwise not be immunogenic. This triggers different pathways of the immune system, the underlying mechanisms of which are still unclear. In veterinary vaccines, a variety of compounds can be used as adjuvants, for example: mineral oil, e.g. or or paraffin oil; non-mineral oils such as squalene, squalane, or vegetable oils such as ethyl oleate; aluminum salts such as aluminum hydroxide or aluminum phosphate; polypeptides such as dimethylglycine or tuftsin; bacterial cell wall components such as lipid A and muramyl dipeptide; (synthetic) polymers such as pluronics, dextran, carbomer, pyran or saponin; cytokines; and stimulators of toll-like receptors such as immunostimulatory oligodeoxynucleotides containing unmethylated cpg groups, etc.

[0009] A major challenge to overcome in developing adjuvanted combination vaccines is preventing interactions between the various vaccine components that could negatively impact the immune response or vaccine safety or stability. Such interactions can occur, for example, between the immunogens themselves, as some are relatively crude products, such as the Mhyo bacterin. However, for PCV2 and Mhyo, the art has shown that various types of non-replicating immunogens from these pathogens can be combined into effective vaccines. However, adjuvants may interfere with or even impair the vaccine immunogens. This is also recognized by the regulatory bodies providing marketing authorizations, for example, USDA implementing regulations 9 CFR 113.35 for testing virucidal activity in inactivated vaccines containing live viruses. Secondly, the specific route of administration can have a significant impact on the safety of the adjuvanted composition: an adjuvant may be safe when administered intramuscularly, but may lead to unacceptable safety issues when administered subcutaneously.

[0010] These potential problems in the development of combination vaccines are generally recognized; see, for example, the publication from the EMEA: "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL); and the U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research: "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies," filed No. 97N-0029, April 1997. These publications all warn of the effects of interference on the efficacy and safety of vaccines when immunogens and adjuvants are combined.

[0011] It is therefore difficult to develop a combination vaccine that induces an effective immune response against a complex combination of immunogens associated with multiple pathogen species. Furthermore, the combination vaccine should be safe when used in animals, i.e., not produce significant side effects such as fever, local swelling, loss of appetite, etc. Furthermore, more practical properties are relevant: the combination vaccine should ideally be economical to produce, sufficiently stable during formulation and storage, and allow for efficacy testing methods for each immunogen in the presence of the other immunogen.

[0012] In summary, it is well known that combination vaccination against multiple pathogens is not simple and requires experiments to determine safety and efficacy, especially when the combined vaccination is an adjuvanted combination vaccine.

[0013] Therefore, there is a need to overcome one or more disadvantages of the prior art and to provide effective and safe combination vaccines against diseases associated with PCV2 and Mhyo infection.

[0014] In the vaccination of large numbers of animals, it is important to limit stress on the animals and humans being vaccinated. In addition, vaccination via the conventional intramuscular route is often associated with pain and stress for the animals, and is associated with an increased risk of side effects and infections. One possibility to overcome the problems associated with intramuscular administration is to vaccinate by administering the vaccine intradermally, also known as intradermal administration. Intradermal vaccination allows for a wider range of administration sites in the animal, providing increased flexibility for the user. This is particularly useful when vaccinating large herds of pigs, as it allows for a quick and non-invasive application, causing less stress for the pigs and the administrator.

[0015] P. Martelli et al., Vet Microbiol. 2014; 168(2-4): 357-64 describe the intradermal administration of a commercial inactivated Mycoplasma hyopneumoniae whole-cell vaccine ( Comparison of systemic and respiratory local immune responses induced by MHYO ID ONCE - MSD Animal Health) with two commercial vaccines administered by the intramuscular route and a negative control.

[0016] In particular, intradermal administration has the advantage that it can be performed by needle-free vaccination devices, e.g. Vaccinator (available from MSD Animal Health, Boxmeer, The Netherlands). "Intradermal" administration itself should not be equated with "needle-free" administration. Only when the needle-free device is "configured for intradermal vaccination" is the vaccine actually delivered (at least partially) into the skin. Needle-free intradermal administration is less invasive than needle injection and produces fewer adverse systemic effects in the animal and induces a good immune response. It also reduces the risk of needle-transmitted disease between pigs during vaccination.

[0017] However, providing safe and effective vaccines suitable for intradermal administration is difficult because the vaccine volume needs to be very small, typically in the range of about 0.1 to 0.5 ml. Consequently, the immunogen and other vaccine components (such as possible adjuvants) need to be very concentrated, which increases the risk of interactions between the various vaccine components. As mentioned above, vaccines combining immunogens from PCV2 and Mhyo are commercially available. However, these combination vaccines are intended for intramuscular administration and are not suitable for intradermal administration.

[0018] Purpose of the Invention

[0019] The object of the present invention is to provide a combination vaccine suitable for intradermal administration against diseases associated with PCV2 and Mhyo infection. In particular, the object of the present invention is to provide a safe and effective vaccine that can be used for the prophylactic treatment of animals against infection with PCV2 and Mhyo. Summary of the Invention

[0020] Surprisingly, it was found that these objectives could be met and one or more disadvantages of the prior art could be overcome by designing a specific adjuvant that enables a combination vaccine comprising a non-replicating immunogen of porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma hyopneumoniae (Mhyo), wherein the combination vaccine is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica.

[0021] In particular, it was surprisingly discovered that a combination vaccine comprising a non-replicating immunogen of PCV2 and a non-replicating immunogen of Mhyo, wherein the combination vaccine is an oil-in-water emulsion (i.e., wherein the continuous phase is an aqueous phase having a hydrophobic liquid dispersed therein, a discontinuous phase, wherein the latter phase itself may have a second or additional phase dispersed therein) comprising squalane, vitamin E acetate and silica, can be used for the safe prophylactic treatment of animals against PCV2 infection and Myho infection by intradermal administration.

[0022] The present invention also embodies the adjuvant composition itself, particularly an adjuvant composition for formulating a non-live vaccine, wherein the composition is an oil-in-water emulsion comprising squalane, vitamin E acetate and silica.

[0023] definition

[0024] A "combination vaccine" is a vaccine that contains immunogens from more than one microbial species. The combination vaccine according to the present invention contains at least immunogens from porcine circovirus type 2 and Mycoplasma hyopneumoniae. Therefore, the combination vaccine according to the present invention can be colloquially referred to as a vaccine "against" PCV2 and Mycoplasma hyopneumoniae.

[0025] A "vaccine" is generally referred to as a pharmaceutical composition that can be safely administered to a subject animal, such as a pig, and that is capable of inducing protective immunity against a pathogenic microorganism in that animal. Vaccines typically comprise an immunologically active component and a pharmaceutically acceptable carrier. The "immunologically active component" is one or more immunogenic molecules, such as non-replicating immunogens from PCV2 and Mhyo. These are recognized by the target animal's immune system and induce a protective immune response.

[0026] Vaccines are generally effective in reducing the severity of infection, for example by reducing the number of pathogens, or shortening the duration of replication of pathogens in the host animal. In addition, or possibly as a result thereof, vaccines are generally effective in alleviating or ameliorating the (clinical) symptoms of disease that may be caused by such infection or replication, or by the animal's response to such infection or replication.

[0027] A "non-replicating immunogen" of a pathogen is any substance or compound corresponding to the pathogen, other than the live replicating pathogen as a whole (wild-type or attenuated form), against which an immune response is elicited, such that the corresponding virulent pathogen or one or more virulence factors thereof are recognized by the host's immune system as a result of the immune response and ultimately at least partially neutralized. Typical examples of non-replicating immunogens are inactivated whole pathogens (this term includes lysed forms of these pathogens) and subunits of these pathogens, such as capsid proteins, surface-expressed molecules (e.g., recombinantly expressed proteins or lipopolysaccharides), and secreted molecules, such as toxins.

[0028] "Prophylactic treatment" against pathogen infection generally helps to prevent, ameliorate or cure the pathogen infection or the condition caused by the infection caused by the pathogenic pathogen after treatment, particularly to reduce its load in the host after such a challenge, and optionally helps to prevent or ameliorate one or more clinical manifestations caused by the post-treatment infection with the pathogen. DETAILED DESCRIPTION

[0029] In a first embodiment, the present invention relates to a combination vaccine comprising a non-replicating immunogen of PCV2 and a non-replicating immunogen of Mhyo, characterized in that the combination vaccine is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silicon dioxide.

[0030] Each "non-replicating immunogen" in the combination vaccine according to the present invention may be of a single type, or may be of multiple types, such as one or more strains from the corresponding pathogen. For the present invention, the non-replicating immunogen of PCV2 is preferably an inactivated whole PCV2 virus. Even more preferably, the ORF2 protein is used as a subunit, which is usually obtained from a recombinant expression system, or delivered and expressed by replicon particles. PCV2 ORF2 can be expressed and harvested in insect cell culture by recombinant baculovirus. Replicon particles are defective virus particles, such as the alpha virus particles developed by AlphaVax. The parent PCV2 of the expressed ORF2 sequence can be any PCV2 serotype a, b, c or d, or can be a chimera from one or more of these serotypes.

[0031] The non-replicating immunogen of Mycoplasma hyopneumoniae typically comprises inactivated whole Mycoplasma hyopneumoniae, i.e., inactivated Mhyo bacterin. The Mhyo bacterin is preferably from strain 11 or strain J.

[0032] An "oil-in-water emulsion" is an emulsion in which the continuous phase is an aqueous phase having a hydrophobic liquid dispersed therein, and a discontinuous phase, the latter of which itself may have a second phase or another phase dispersed therein. Such an emulsion can be formed by selecting an emulsifier of the appropriate type and concentration. The procedures and equipment for preparing oil-in-water emulsions for use as vaccines are well known in the art and are described, for example, in textbooks such as: "Remington: the science and practice of pharmacy" (2000, Lippincot, USA, ISBN: 683306472), and: "Veterinary vaccinology" (P. Pastoret et al., ed., 1997, Elsevier, Amsterdam, ISBN 0444819681).

[0033] In the present invention, the external aqueous phase may comprise non-replicating immunogens from PCV2 and Myho and silica; and the oil phase may comprise squalane and vitamin E-acetate.

[0034] The combination vaccine according to the present invention was found to be highly effective, safe and stable when prepared as an oil-in-water emulsion. Embodiments and preferred embodiments for preparing an oil-in-water emulsion for the combination vaccine according to the present invention will be described below.

[0035] "Squalane" is a non-mineral oil also known as hydrogenated shark liver oil, hexamethyleicosane, or perhydrosqualene. This should not be confused with squalene (CAS nr. 111-02-4), which is a polyunsaturated C30 oil and metabolizable as a compound of the cholesterol pathway. However, squalane is the fully hydrogenated form of squalene and is therefore not susceptible to oxidation. Therefore, although squalane can be transported from the injection site (so it "disappears" from the injection site) and is therefore sometimes referred to as "metabolizable," it is actually an inert, non-metabolizable oil (it is only physically transported away from the injection site without being metabolized).

[0036] The precursor of squalane was originally obtained from shark liver, but due to environmental concerns, it has been transferred to other natural sources (such as olive oil) or chemical synthesis. Therefore, the definition of squalane includes natural, synthetic or semi-synthetic forms or mixtures thereof. Squalane is commercially available in various purities, for example: vegetable sources, such as Worlee (Squalane, Vegetable), or Croda (Pripure Squalane); or synthetic, such as Kuraray (Squalane-PE). For the present invention, high-purity squalane is preferred: preferably greater than 75% purity, more preferably greater than 80, 90 or even greater than 95% purity, in order of preference.

[0037] Squalane is typically present in the combination vaccine according to the present invention in an amount of 1-15% w / v of the vaccine. More preferably, squalane is present in an amount of 3-12% w / v, or even 5-9% w / v of the vaccine, in order of preference, such as 5%, 6%, 7%, 8% or 9% w / v. Most preferably, squalane is present in an amount of about 6.8% w / v of the vaccine.

[0038] Therefore, in one embodiment of the combination vaccine according to the invention, the vaccine comprises squalane in an amount of 1-15% w / v.

[0039] "Vitamin E acetate" is the acetate ester of vitamin E (tocopherol), which may be derived from plant materials such as seeds, nuts, fruits or leaves, or from fatty meats, but may also be produced synthetically. Some alternative names are: tocopheryl acetate, or alpha-tocopheryl acetate. The definition of vitamin E acetate includes natural, synthetic or semi-synthetic forms or mixtures thereof. Vitamin E acetate is commercially available in varying degrees of purity. The vitamin E acetate used in the combination vaccine according to the present invention may be DL-alpha-tocopherol-acetate, which is the racemate of the chemical with CAS number 7695-91-2.

[0040] Vitamin E acetate is typically present in the combination vaccine according to the present invention in an amount of 2-20% w / v of the vaccine. More preferably, vitamin E acetate is present in an amount of 4-16% w / v, or even 6-10% w / v of the vaccine, in order of preference, such as 6%, 7%, 8%, 9% or 10% w / v. Most preferably, vitamin E acetate is present in an amount of about 8% w / v of the vaccine.

[0041] Therefore, in one embodiment of the combination vaccine according to the invention, the vaccine comprises Vitamin E-acetate in an amount of 2-20% w / v.

[0042] "Silica" is silicon dioxide. Silica is widely described as being used in adjuvant compositions and is often referred to as pharmaceutical grade silica. What all pharmaceutical grade silicas have in common is that they are colloidal silica and have been used in the pharmaceutical industry for nearly 50 years. Within this type of silica, different surface areas, hydrophilicity and hydrophobicity (e.g., methylated), crystalline or amorphous (e.g., fumed silica), and different granulation ratios are available, all of which are commonly used in adjuvant compositions. An example of a preferred type of silica for use in adjuvant compositions is amorphous silica (which, when used in the present invention, can be hydrophilic or hydrophobic, but is preferably hydrophilic). A common type of amorphous silica is fumed silica, which is also called pyrogenic silica because it is produced in a flame and consists of microscopic droplets of amorphous silica that fuse into branched, chain-like, three-dimensional secondary particles, which then aggregate into tertiary particles.

[0043] The silica used in the combination vaccine of the present invention may have a particle size of 100 to 700 m 2 / gram, more preferably 300 to 500 m 2 / g, further more preferably 350 to 410 m 2 / gram, most preferably about 395 ± 25m 2 The surface area can be determined by methods known in the art, for example, by calculation using Brunauer's nitrogen adsorption method (Brunauer, S. et al., J. Am. Chem. Soc., 60, 309 (1938)).

[0044] Such products are commercially available, for example under the trade name or (Many variants are available under this trade name, e.g. with different surface areas, hydrophobic and hydrophilic, crystalline or amorphous.) An example is the HYDROLYZER® from Evonik Resource Efficiency GmbH, Germany. 380, or S5130 from Sigma-Aldrich, which has a particle size of about 0.007 μm, a pH (4% dispersion) of 3.7 to 4.5, and a tap density of about 50 g / l.

[0045] Silica is typically present in the combination vaccine according to the present invention in an amount of 0.02-2% w / v of the vaccine. More preferably, silica is present in an amount of 0.05-1.0% w / v, or even 0.1-0.4% w / v of the vaccine, in order of preference, such as 0.1%, 0.2%, 0.3% or 0.4% w / v. Most preferably, silica is present in an amount of about 0.2% w / v of the vaccine.

[0046] Therefore, in one embodiment of the combination vaccine according to the invention, the vaccine comprises silicon dioxide in an amount of 0.02-2% w / v.

[0047] The combination vaccine of the present invention typically contains a pharmaceutically acceptable carrier, preferably water. Preferably, the water is of high purity, such as double-distilled water, microfiltered water, or reverse osmosis water. More preferably, the water is water for injection, is sterile, and is substantially free of pyrogens.

[0048] A convenient feature of vaccines based on oil-in-water emulsions is that the immunogen is typically in the aqueous phase. This means that the oil phase can be prepared and emulsified solely in water, using methods and techniques that are incompatible with maintaining the quality or viability of the vaccine immunogen; for example, using high-energy emulsification at high temperatures. This results in the oily emulsion used in the present invention, which is an oil-in-water emulsion of squalane, vitamin E acetate, and silica in water. To prepare the combination vaccine according to the present invention, the aqueous phase containing the immunogen and silica is combined with the oily emulsion containing the other adjuvants by gentle mixing at room temperature.

[0049] Combining the two compositions results in dilution of each. Therefore, each composition needs to be prepared as an intermediate composition in which the concentrations of the various components are higher than those in the final vaccine by a factor equal to the applied dilution. Typically, the aqueous phase and the oily emulsion can be mixed in a volume ratio of either 10:90 or 90:10.

[0050] The combination vaccine according to the present invention preferably comprises both an aqueous phase and an oily emulsion, as described, in a volume ratio between 20:80 and 80:20. Thus, in one embodiment, the combination vaccine according to the present invention is prepared from a mixture of an aqueous phase and an oily emulsion in a volume ratio between 20:80 and 80:20. Preferably, the volume ratio is between 30:70 and 70:30; between 40:60 and 60:40; or even a volume ratio of about 50:50, in that order of preference.

[0051] Obviously, when the combined ratio of the aqueous phase and the oily emulsion is about 50:50, each of the two compositions should contain the various components in amounts or concentrations that are twice as high as those required in the final vaccine formulation prepared by the combination of the two intermediate compositions.

[0052] In a preferred embodiment, the oily emulsion of the present invention is prepared using an emulsifier having an HLB value (hydrophile-lipophile balance) of 8 to 20; a preferred emulsifier is polysorbate 80.

[0053] Polysorbate 80 refers to the chemical with CAS number 9005-65-6, also known as polyoxyethylene sorbitan monooleate. It has an HLB value of about 15 and is widely commercially available, for example as Tween 80.

[0054] Preferably, polysorbate 80 is present in the combination vaccine according to the invention in an amount of 0.5-10% w / v of the vaccine. More preferably, polysorbate 80 is present in an amount of 0.7-7% w / v, 1.0-5%, or even 2-4% w / v of the vaccine, in that order of preference.

[0055] Most preferably: Polysorbate 80 is present in an amount of about 3.2% w / v of the vaccine.

[0056] Thus, in one embodiment, the combination vaccine according to the invention comprises polysorbate 80 in an amount of 0.5-10% w / v.

[0057] The oily emulsions of the present invention can be prepared on any scale and using any suitable homogenizing equipment, for example by: Microfluidizer TM , Silverson TM 、Ultra Turrax TM , or Dispax reactor (IKA). Those skilled in the art can perform and optimize such an emulsification process to control the size of the dispersed phase (here: oil adjuvant) particles. Together with the type and concentration of the emulsifier, this controls the pharmaceutical properties of the emulsion and its stability. The main parameters of the emulsification process itself are: energy input (power and rpm), temperature, duration, and number of repetitions. Details of an embodiment of the emulsification process are as follows.

[0058] The size of the dispersed phase particles is preferably very small. When the diameter of the dispersed phase particles is less than about 1 micron, such emulsions are generally referred to as "submicron emulsions." In one embodiment of the oil-in-water emulsion of the combination vaccine according to the present invention, the emulsion is a submicron emulsion.

[0059] Equipment is generally available to measure particle sizes of 1 micron or less, for example by laser diffraction measurement. Typically, particle sizes are expressed in nanometers (nm) and as the average particle size, also called the median diameter, expressed as the D50 of the cumulative particle size distribution.

[0060] For the present invention, the particle size is expressed in nm of D50, such as using (Malvern Instruments). Particle size measurements can be performed in (concentrated) oily emulsions or combination vaccines; the particle refractive index of the oil phase of the present invention is 1.48. The Malvern Mastersizer size analysis report indicates D50 as D(0.50). Therefore, in embodiments of the submicron oil-in-water emulsion of the combination vaccine according to the present invention, the oil droplets have a D50 of 500 nm or less; preferably, a D50 of 250 nm or less. More preferably, a D50 of 150 nm or less.

[0061] There are many methods for producing such submicron emulsions, typically by using high energy emulsification methods, such as using: high pressure homogenizers, rotor-stator devices, stirrers, ultrasound, microporous membranes or microchannel devices.

[0062] The preferred method for high energy emulsification used in the present invention is to use a high pressure homogenizer, preferably a Microfluidizer TM (Microfluidics). Typically, three channels at a pressure of 500 to 1500 bar (i.e., 7000 to 22000 psi) will be sufficient. Emulsions prepared in this manner typically have dispersed phase particles with a D50 of 500 nm or less and a narrow particle size distribution; for the present invention, the dispersed phase is droplets of oily adjuvant.

[0063] Typically, emulsions with such very finely sized dispersed phase particles are prepared in multiple steps. In this manner, an initial relatively coarse oil emulsion is prepared by low-energy mixing, followed by one or more subsequent high-energy treatments to achieve further reduction in particle size. Next, a "microfluidized" oil emulsion containing an adjuvant and, optionally, an emulsifier in water is combined with an aqueous phase containing the immunogen to prepare a combination vaccine according to the invention.

[0064] For reasons of product consistency and quality, it is advantageous not only to monitor and control the median particle diameter, but also to monitor and control the distribution (also referred to as size distribution) in the particle diameter. The size distribution of the oil droplets in the submicron oil-in-water emulsion of the combined vaccine according to the present invention is preferably relatively narrow. One index of particle size distribution is the D90 of the cumulative particle size distribution.

[0065] Thus, in embodiments of the submicron oil-in-water emulsion of the combination vaccine according to the invention, the oil droplets have a D90 below 900 nm, more preferably a D90 below 500 nm, 400 nm or even below 300 nm, in that order of preference. Most preferably: a D90 of about 150 to 250 nm.

[0066] One of the advantages of an emulsion with such a small particle size and distribution is that it can be sterilized by filtration without significant loss of material, since typical sterilizing filters have a pore size of about 0.2 microns. This filter sterilization overcomes the need for other sterilization methods that may compromise the quality of the components of the oil emulsion, such as by heat, chemicals, or irradiation.

[0067] Thus, a combination vaccine according to the present invention typically comprises non-replicating immunogens from PCV2 and Mhyo in amounts capable of inducing a protective immune response in an animal target against their associated diseases, as described above.

[0068] Those skilled in the art of the present invention will be better able to determine the effectiveness of the combination vaccine according to the present invention, for example by monitoring the immune response after vaccination or after challenge infection, for example by monitoring the target's disease symptoms, clinical scores or by re-isolation of the pathogen, and comparing these results with the vaccination-challenge response observed in mock-vaccinated animals.

[0069] As an indicator, the amounts of immunogens used in combination vaccines according to the present invention can be based on those used in the respective monovalent or combination vaccines containing these immunogens. For example, a combination vaccine according to the present invention may contain per milliliter: 1 to 150 μg of PCV2 ORF2; and Mhyo: 2-50% w / v inactivated concentrated Mhyo culture. Methods for quantifying these immunogens are well known in the art and may also rely on ELISA-based quantification against specific standards.

[0070] The combination vaccine according to the invention may advantageously be combined with one or more replicating or non-replicating, intact or destroyed other antigens or immunogens.Thus, in one embodiment, the combination vaccine according to the invention may comprise at least one other antigen or immunogen.

[0071] Other antigens or immunogens are either attenuated forms of microorganisms that are pathogenic to pigs or non-replicating antigens or immunogens derived from microorganisms that are pathogenic to pigs. The microorganisms can be any virus, bacterium, parasite, fungus, rickettsia, protozoa and / or parasite that is pathogenic to pigs. Examples of such microorganisms that are pathogenic to pigs are: pseudorabies virus, porcine parvovirus, classical swine fever virus, swine influenza virus, foot-and-mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, vesicular stomatitis virus, Lawsonia intracellularis, Actinobacillus pleuropneumoniae, Brachyspira, Escherichia coli, Haemophilus, Streptococcus, Salmonella, Clostridium, Pasteurella, Erysipelothrix, Leptospira, Bordetella, Toxoplasma, Isospora and Trichinella spiralis. Preferred additional antigens or immunogens are from one or more of the following: Lawsonia intracellularis, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Brachyspira hyodysenteriae, and swine influenza virus.

[0072] The observed effects of the combined vaccine according to the invention are:

[0073] For Mhyo: Prevent or reduce lung damage caused by Mhyo, such as consolidation pneumonia and chronic respiratory disease. For Mhyo, the most reliable measure of vaccine efficacy is the reduction of lung lesion scores after Mhyo challenge infection. This lesion is usually scored during autopsy based on the Goodwin scale (Goodwin et al., 1969, J. Hyg. Camb., vol. 67, p. 465-476) by macroscopic assessment of lung consolidation; for all affected lungs, the scale ranges from zero to a maximum of 55 points per animal.

[0074] For PCV2: prevention or reduction of clinical signs of wasting or ill health, the presence of gross and microscopic lesions characteristic of the disease, and the presence of viral antigen or DNA in microscopic lymphoid lesions. For PCV2, the most reliable measure of vaccine efficacy is the presence of viral nucleic acid in serum, stool swab material, nasal swab material, inguinal lymph nodes, mesenteric lymph nodes, tonsils, and lungs by qPCR. The induction of antibodies against PCV2 after vaccination correlates with protection.

[0075] In a preferred embodiment, the combination vaccine is for use in pigs. The term "pig" refers to animals of the family Sus domestica, preferably animals of the genus Sus, which are also known as porcines. Examples are: wild or domesticated pigs, hogs, wild boars, babirusa or warthogs. This also includes pigs referred to by any name, for example referring to their sex or age, such as: sow, queen, boar, barrow, pig, gilt, weaner or piglet. Furthermore, the term pig refers to any type of porcine animal, such as a breeding type or a fattening type, as well as any parental line of these types.

[0076] As described herein, the combination vaccines according to the present invention can be constituted in different ways.

[0077] In one embodiment, the combination vaccine according to the invention is provided as a ready-to-use formulation, ie as a formulation in which all components of the vaccine are readily mixed, such that the combination vaccine can be used directly for vaccination without any further mixing or reconstitution steps.

[0078] In an alternative embodiment, the combination vaccine according to the present invention can be produced from a kit comprising at least two containers: a container comprising all the components of the combination vaccine according to the present invention except the Myho immunogen; and a container comprising the Myho immunogen. The PCV or Myho immunogen can be provided, for example, in lyophilized form, or as a sterile suspension (e.g., an aqueous suspension). The lyophilized form can be a lyophilized cake in a container (e.g., a bottle), but can also be in a Sphereon TMLyospheres used in the technology.

[0079] The components of the partial kit together embody the combination vaccine according to the present invention. The contents of at least two containers can be reconstituted in situ directly before use (i.e., before vaccination). After reconstitution of the non-replicating immunogen from Mhyo, the complete combination vaccine according to the present invention is formed. This is also called "on-site" mixed vaccine, or "field-side" mixing.

[0080] Thus, in another aspect, the present invention relates to a kit of parts comprising at least two containers: a container comprising a non-replicating immunogen from PCV2 in an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica; and a container comprising a non-replicating immunogen from Mhyo.

[0081] In an alternative embodiment, both PCV2 and Mhyo immunogens may be provided in one container, for example as an aqueous solution or dispersion or in lyophilized form, optionally also containing silicon dioxide, and reconstituted before use with the components of a second container containing (other) adjuvants in the form of an o / w emulsion.

[0082] The kit-of-parts according to the invention and elements thereof may comprise any embodiment (preferred or not preferred) as described herein for a combination vaccine according to the invention, or any combination of two or more of those embodiments of a combination vaccine according to the invention.

[0083] Therefore, in another aspect, the present invention relates to a method for preparing a combination vaccine according to the present invention, comprising the following steps:

[0084] - preparing an aqueous phase comprising non-replicating immunogens from PCV2 and Mhyo, and

[0085] - The aqueous phase is mixed with an oily emulsion comprising squalane, vitamin E-acetate and silica to form an oil-in-water emulsion.

[0086] In an alternative aspect, the present invention relates to a method for preparing a combination vaccine according to the present invention, comprising the following steps:

[0087] - preparing an aqueous phase comprising a non-replicating immunogen from PCV2,

[0088] - mixing the aqueous phase with an oily emulsion comprising squalane, vitamin E-acetate and silica to form an oil-in-water emulsion, and

[0089] - The oil-in-water emulsion is mixed with a non-replicating immunogen from Mhyo.

[0090] In another aspect, the present invention relates to a method for preparing a combination vaccine according to the present invention, comprising the following steps:

[0091] - preparation of a non-replicating immunogen from Mhyo in lyophilized form,

[0092] - preparing an aqueous phase comprising a non-replicating immunogen from PCV2,

[0093] - mixing the aqueous phase with an oily emulsion comprising squalene, vitamin E-acetate and silicon dioxide, and

[0094] - Reconstitution of the freeze-dried non-replicating immunogen from Mhyo with a mixture of the aqueous phase and the oily emulsion.

[0095] In another aspect, the present invention relates to a method for preparing a combination vaccine according to the present invention, comprising the following steps:

[0096] - preparing a non-replicating immunogen from PCV in lyophilized form,

[0097] - preparing an aqueous phase comprising a non-replicating immunogen from Mhyo and silica,

[0098] - mixing the aqueous phase with an oily emulsion comprising squalane and vitamin E-acetate, and

[0099] - reconstitution of said freeze-dried non-replicating immunogen from PCV with a mixture of said aqueous phase and oily emulsion.

[0100] At different points in these methods, additional steps may be added, for example for additional processing, such as for purification or storage. The method for preparation may also include mixing with additional antigens or immunogens, or pharmaceutically acceptable excipients (such as stabilizers or preservatives).

[0101] As described above, the combination vaccine of the present invention, which can be prepared by the method according to the present invention, can be advantageously used for intradermal administration to pigs to combat PCV2 and Mhyo infection and / or diseases associated with PCV2 and Mhyo infection.

[0102] Thus, in another aspect, the present invention relates to an oil-in-water emulsion comprising squalane, vitamin E-acetate, silica, non-replicating immunogens from PCV2 and Mhyo for use in vaccination in the skin of animals (e.g., pigs) against PCV2 and Mhyo.

[0103] In another aspect, the present invention relates to the use of non-replicating immunogens from PCV2 and Mhyo in the preparation of a combined vaccine for animals (e.g., pigs), characterized in that the vaccine is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica.

[0104] The combination vaccine according to the invention can be used for vaccination of pigs against PCV2 and Mhyo.

[0105] Thus, in another aspect, the present invention relates to a method for vaccinating animals (e.g., pigs) against PCV2 and Mhyo by intradermally administering to said animals an oil-in-water emulsion comprising squalane, vitamin E-acetate, silica, and a non-replicating immunogen from PCV2 and Mhyo.

[0106] Alternatively, in a similar embodiment: the present invention relates to a method for vaccinating an animal (eg, a pig) against PCV2 and Mhyo by intradermally administering to said animal a combination vaccine according to the present invention.

[0107] Therefore, the combination vaccine according to the present invention is usually administered to the skin of the animal, i.e. applied by intradermal administration. This can be achieved in different ways, for example using a typical syringe and hypodermic needle. Alternatively, parenteral administration can be performed by some needle-free injection methods, by means of an intradermal applicator (e.g. from MSD Animal Health). Applicator) to deliver the vaccine.

[0108] For intradermal administration, the volume of the animal dose of the combination vaccine according to the present invention is typically 0.05 to 1.0 ml per animal; preferably 0.1 to 0.5 ml, more preferably about 0.2, 0.3 or 0.4 ml, most preferably a dose of about 0.2 ml per animal, in that order of preference.

[0109] The administration regimen for the vaccination method according to the present invention to the target pigs may be a single dose or multiple doses, or in a manner compatible with aspects of pig husbandry practice.

[0110] When necessary, later in life, the animal target can be administered a second or further administration of the combination vaccine according to the invention, a so-called booster vaccination. However, the combination vaccine according to the invention is optimized in such a way that a single vaccination dose will generally be sufficient to provide immune protection over the relevant life cycle of the animal (e.g. the fattening period of pigs up to 6 months of age).

[0111] Therefore, in a preferred embodiment, the combination vaccine according to the invention is administered only once to each animal target, ie it is a single-dose vaccine.

[0112] Preferably, to further reduce stress on the animals and reduce labor costs, the protocol for the vaccination method is integrated into an existing vaccination schedule for other vaccines that the target pigs may need. These other vaccines can be administered simultaneously, in parallel, or sequentially in a manner compatible with their registered uses. Thus, in one embodiment of the method for vaccination of pigs according to the present invention, the combination vaccine according to the present invention is administered in combination with another pig vaccine.

[0113] In the case where the target animal is a pig, the target pigs for vaccination according to the present invention can be any age at which they are susceptible to vaccination and / or susceptible to the disease or infection protected by the vaccine. Thus, in one embodiment of the porcine vaccination method according to the present invention, the combination vaccine according to the present invention is administered to young pigs, i.e., pigs of about 2 months of age. Alternatively, the combination vaccine according to the present invention is administered to adult pigs, i.e., pigs of about 6 months of age.

[0114] Due to the high prevalence of Mhyo and PCV2 and the widespread use of vaccines against one or more of these pathogens, many sows will be seropositive for antibodies against one or more of Mhyo and PCV2. Therefore, piglets that consume colostrum from such sows may be MDA+ (maternally derived antibody positive). This does not hinder the effectiveness of the combination vaccine according to the present invention, as it is also effective in MDA+ pigs. Therefore, in one embodiment of the vaccination method according to the present invention, the combination vaccine according to the present invention is administered to MDA+ pigs.

[0115] Administration of the combination vaccine according to the present invention can be applied as a preventive treatment or a therapeutic treatment, or both, because it interferes with the establishment and progression of Mhyo and PCV2 infections. Use of the combination vaccine according to the present invention will help reduce infection with one or both of Mhyo and PCV2 in pigs in a herd, farm, or geographic area. Thus, in another aspect, the present invention relates to a method for reducing Mhyo and PCV2 infection or disease-related symptoms in pigs, characterized in that the method comprises administering a combination vaccine according to the present invention intradermally to the pigs.

[0116] The invention will now be further described by way of the following non-limiting examples.

[0117] Example

[0118] Example 1: Preparation of combined vaccine

[0119] The combined vaccine according to the present invention is prepared according to the following steps:

[0120] The oil emulsion was prepared at 2x concentration according to the following subsequent process steps:

[0121] -Weigh the required amounts of Vitamin E-acetate and Squalane and mix in a beaker.

[0122] - homogenize the vitamin E acetate / squalane mixture by low energy mixing (magnetic stirrer) at room temperature,

[0123] - Weigh the required amount of polysorbate 80 and add it to the homogenized vitamin E acetate / squalane mixture,

[0124] - homogenizing the mixed mixture again by low-energy mixing at room temperature,

[0125] - Passed through a 0.2 micron filter (Pall, Ultipor TM N66) Filter sterilize the homogenized mixture,

[0126] - weighing the required amount of (heat sterilized) silicon dioxide and adding it to the homogenized mixture, after which the mixed mixture is homogenized again by low energy mixing at room temperature,

[0127] - Heat the mixture to 65-75°C,

[0128] - Heat the water for injection (sterile) to 65-75°C,

[0129] - Premix the heated oil phase with water using high energy mixing via Ultra Turrax equipped with N18 rod for 5-15 minutes; reduce the temperature from 65°C to 55°C,

[0130] - Pass the premix through a Microfluidizer at 800 bar TM Three times; the temperature was kept below 50°C with a cooling screw.

[0131] In the final oil emulsion, the integrity and level of homogenization were checked by optical microscopy. Further pH (7.34) and osmotic pressure (221 mOsm / kg) were also checked. Particle size measurements showed: D100 = 300 nm; D99 = 250 nm; D90 = 200 nm and D50 = 130 nm.

[0132] The aqueous phase (2x concentration) was prepared by taking the required amount of each non-replicating immunogen: Mhyo: 10x concentrated killed culture at 6% v / v and PCV: 50 μg ORF2.

[0133] Next, the two concentrate compositions (oil emulsion with adjuvant and aqueous phase with immunogen) were mixed in an approximately 50:50 volume ratio by low energy mixing at room temperature.

[0134] The following vaccine adjuvant compositions were prepared as oil-in-water (o / w) emulsions using the above procedure (all percentages are % w / v). In preparing the oil emulsions, specific amounts of aluminum hydroxide (as a double concentrate) as well as polysorbate 80 and squalane were added to some compositions:

[0135] Table 1

[0136]

[0137] *Phosphate buffered saline

[0138] Example 2: Efficacy of PCV2 / Mhyo ID formulations against Mhyo challenge infection in pigs

[0139] The intradermal (ID) formulation of Mhyo (0.2 ml, with The efficacy of vaccination (administered on the right side of the neck) against Mhyo challenge was investigated. Formulations were prepared using various treatments of the Mhyo immunogen and formulated at 5 PCVU / ml (approximately 25% w / v inactivated Mhyo culture). Animals were vaccinated at 3 weeks of age according to the following schedule (Table 2). Four weeks after vaccination, all animals were infected with Mhyo. At 7 weeks of age (i.e., 4 weeks after vaccination), 10 ml of Mhyo strain 98 was administered intratracheally at 10 [units unclear] for each of the two doses. 9 and 10 9 All animals were challenged with CCU / ml. Three weeks after the challenge, the animals were sacrificed and the extent of Mhyo-induced consolidative pneumonia was scored according to Goodwin (maximum score: 55).

[0140] Table 2

[0141]

[0142] * Proprietary adjuvant for PCV ID (MSD Animal Health)

[0143] result:

[0144] The efficacy of vaccination was determined by lung lesion score (LLS, mean), which was recorded for each pig and compared with that of the unvaccinated control group.

[0145] Table 3

[0146] Group Lung Lesion Score (LLS) 1 13.2 2 11.5 3 15.4

[0147] Compared with the control group, lung lesion scores were not significantly reduced for any of the vaccines tested in Groups 1 and 2. Instead, lung lesion scores were similar to those in the unvaccinated control group.

[0148] Therefore, the combination of adjuvants heterogeneous hydrogel, squalane, vitamin E-acetate and silicon dioxide was combined with the immunogens of PCV2 and Mhyo and the commercially available PCV vaccine as adjuvant. Neither the combination of PCV ID nor silicon dioxide is suitable for safe and effective intradermal administration.

[0149] Example 3: Efficacy of PRRS vaccine reconstituted in PCV2 / Mhyo ID formulation in pigs

[0150] The aim of this study was to compare the efficacy of 5-week-old piglets administered intradermally (ID) in the neck of Safety and serological efficacy of different PCV2 and / or Mhyo vaccines reconstituted with PRRS containing Aerosil 200 or Aerosil 380. Piglets were assigned to the treatment groups as shown below (Table 4). Piglets were vaccinated intradermally at approximately 5 weeks of age. 10 [mu]ltiple doses of ... 4.5 TCID 50 Piglets from groups 4 and 5 were inoculated with the virus.

[0151] Table 4

[0152]

[0153] - Group 4 piglets were intradermally vaccinated with a single dose (0.2 ml) of a vaccine formulated with PCV2 (10000 AU / ml; approximately 80 μg / ml) + M.Hyo (10 PCVU / ml) and adjuvant Aerosil 200.

[0154] - Piglets of group 5 were intradermally vaccinated with a single dose (0.2 ml) of the vaccine formulated with PCV2 (10000 AU / ml) + M.Hyo (10 PCVU / ml) and adjuvant Aerosil 380.

[0155] - Group 6 piglets were intradermally inoculated with a single dose (0.2 ml each) of unmixed PCV PRRS vaccine.

[0156] - Piglets from Group 7 were not vaccinated (negative control group). All animals were examined for injection site reactions.

[0157] All piglets were observed daily for clinical signs after vaccination. Temperatures were taken and serum samples were collected from all animals. The samples were tested for antibodies against PCV2, Mycoplasma hyopneumoniae, and PRRSV.

[0158] result:

[0159] After vaccination, body temperatures were comparable in all groups. On the day of vaccination, all animals had comparable PCV2 antibody titers. PCV2 antibody titers in all groups remained at a constant level until the end of the study. Antibody titers in control group 7 decreased over time. On the day of vaccination, all animals were negative for PRRS antibodies. All following groups showed 0-20% PRRS responders. Control group 7 remained negative. At the beginning of the study, all animals were seronegative for Mycoplasma hyopneumoniae. All groups showed 0-20% responders on SD21. On SD28, most groups showed positive animals. Control group 7 showed no positive reactions for Mycoplasma hyopneumoniae. The results of IgM antibody responses to PCV2 and Mhyo (predicted by vaccination) are shown in Tables 5 and 6.

[0160] Table 5: Percentage of animals positive for PCV2-specific IgM after vaccination

[0161] Group SD0 SD14 SD21 4 0 80 40 5 20 50 80 6 0 100 100 7 20 20 20

[0162] Table 6: Percentage of animals with positive Mhyo serum antibody response after vaccination

[0163] Group SD0 SD14 SD21 SD28 4 0 0 20 20 5 0 0 0 10 6 0 0 0 10 7 0 0 0 0

[0164] From this study it can be concluded that the vaccinated group did not show an acceptable antibody response against PCV2 or Mhyo. When reconstituted with PRRS, PCV2, and Mhyo immunogens, neither the adjuvants Aerosil A380 nor Aerosil A200 were suitable for safe and effective intradermal administration in pigs.

[0165] Example 4: Efficacy of PRRS vaccine reconstituted in MhyoID formulation against Mhyo challenge infection in pigs

[0166] Tested in SPF piglets with PRRS (A212D, 10 5.1 The efficacy of vaccination with an intradermal (ID) formulation of Mhyo mixed with TCID50 / dose (100 mg / dose) against Mhyo challenge infection was evaluated. Formulations were prepared with various adjuvants. ID vaccination (0.2 ml) was administered at 3 weeks of age on the right side of the neck according to the following protocol (Table 7). Three weeks after vaccination, all animals were infected with Mhyo.

[0167] Table 7:

[0168]

[0169] All animals were infected at 6 weeks of age with 10 ml intratracheal Mhyo strain 98 at 9 and 8 CCU, respectively, on two consecutive days.

[0170] result:

[0171] The efficacy of vaccination was determined by lung lesion scoring (LLS), which was recorded for each pig and compared with that of the unvaccinated control group.

[0172] Table 8

[0173]

[0174] As can be seen, intradermal administration of the combination of Mhyo and PRRS immunogens with aluminum hydroxide, squalane, and vitamin E-acetate as an adjuvant composition (as in control composition 3) showed very low and therefore unacceptable lung lesion scores, i.e., a 22% reduction compared to 74% in the positive control group using the commercial Mhyo vaccine. Therefore, the combination of the immunogen and the adjuvants aluminum hydroxide, squalane, and vitamin E-acetate is not suitable for safe and effective intradermal administration.

[0175] Example 5: Efficacy of PCV2-Mhyo ID formulation against Mhyo challenge infection in SPF pigs

[0176] Groups of 12 pigs were inoculated intradermally at three weeks of age (+ / - three days) according to the following protocol (Table 9). Group 17 was not inoculated and served as a control for the Mhyo challenge. Four weeks after vaccination, all animals were infected with the virulent Mhyo strain. Three weeks after challenge, all animals were necropsyed for lung lesions. Blood samples were collected before vaccination, before challenge, and at necropsy.

[0177] Table 9

[0178]

[0179] *Seppic ISA 28 with microfluidized vitamin E acetate

[0180] **Mhyo positive control group

[0181] ***Unvaccinated control group

[0182] Experimental procedures

[0183] Serology

[0184] Blood samples (jugular vein) were collected before vaccination (T = 0, 3 weeks of age), before challenge (T = 4, 7 weeks of age), and at necropsy (T = 7, 10 weeks of age). Samples were transported at ambient temperature. Serum was obtained from the blood samples in duplicate. All serum samples were tested for the presence of relevant antibodies in ELISA tests for M.hyo and PCV according to standard procedures.

[0185] Clinical observations and rectal temperature

[0186] Conduct clinical observations and measure rectal temperatures before vaccination, 4 hours after vaccination, and 1 and 2 days after vaccination. Record observations and temperatures.

[0187] autopsy

[0188] At the end of the experiment, 4 weeks after challenge, the pigs were sedated by electric stunning and subsequently sacrificed by hemorrhage. Lung lesions were scored according to Goodwin.

[0189] result:

[0190] No unacceptable injection site reactions were observed. No unacceptable temperatures were observed.

[0191] The efficacy of vaccination was determined by lung lesion scoring (LLS), which was recorded for each pig and compared to the unvaccinated control group. The results are shown in Table 10:

[0192] Table 10

[0193]

[0194] The above data of lung lesion scores show that groups 11, 12 (compositions of the present invention) and 13 (reference composition 2) gave acceptable scores because they were reduced by more than 45% (which served as the critical value for significant efficacy) compared to the unvaccinated control group (group 17). In contrast, groups 14 and 15 gave unacceptable scores because the LLS of group 15 was even higher than that of the unvaccinated control group, and the LLS of group 14 was only slightly reduced by 20% compared to the negative control group.

[0195] Therefore, it could be demonstrated that the vaccine composition of the present invention comprising the adjuvant combination of squalane, vitamin E-acetate and silica gave an acceptable lung lesion score indicating effective vaccination.

[0196] Example 6: Efficacy of PCV2-Mhyo ID formulation against PCV challenge infection in SPF pigs

[0197] The piglets were divided into 5 treatment groups of 10 piglets each and were vaccinated intradermally when they were about 3 weeks old. The piglets in groups 1 to 3 were vaccinated with the following vaccines. Group 4 served as a positive control group with PCV ID and Group 5 was not vaccinated (negative control). Three weeks after vaccination (6 weeks of age), all animals were challenged intranasally (3 ml / nostril) with 5.0 log10 TCID50 / mL of wild-type PCV2b challenge virus strain I12 / 11. The treatment schedule is shown in Table 11 below. Three weeks after challenge, all animals were necropsied and inguinal lymph nodes, mesenteric lymph nodes, tonsils, and lungs were sampled for PCV2.

[0198] All piglets were observed daily for clinical signs following vaccination. Serum samples were collected on the day of vaccination and on SD14, SD20, SD35, and SD42 (during necropsy). Samples were tested for antibodies against PCV2 viral nucleic acid by qPCR. Fecal swabs were collected from all animals on SD20, SD35, and SD41 and tested for PCV2 viral nucleic acid by qPCR.

[0199] Table 11

[0200]

[0201]

[0202] *Seppic ISA 28 with non-microfluidized mineral oil

[0203] **Mhyo positive control group

[0204] ***Unvaccinated control group

[0205] deal with

[0206] Dosage and administration

[0207] Vaccination was performed intradermally on the right side of the neck (0.2 ml). Group 21 was vaccinated twice on the right side. Challenge was performed intranasally using the MAD applicator, 6 ml, 3 ml per nostril.

[0208] Test system

[0209] Only healthy animals will be used. To exclude unhealthy animals, they will be examined prior to vaccination (general physical condition and absence of clinical abnormalities or disease). Prior to vaccination, all animals will be individually numbered with ear tags. All pigs will be observed daily for clinical signs of disease. Observations will consist of systemic effects such as loss of appetite, reluctance to move, tendency to lie down, listlessness or lethargy, tremors, irritability, edema (especially around the eyes), vomiting and diarrhea, and difficulty breathing.

[0210] Experimental procedures

[0211] Blood sampling

[0212] Blood samples were collected from all animals on the day of vaccination, one day before challenge, two weeks later, and on the day of necropsy. A minimum of 4 ml of blood was drawn from each animal, and a maximum of 8 ml was drawn. All pigs were individually administered according to standard procedures. Blood samples were collected without the addition of an anticoagulant.

[0213] stool swab

[0214] Fecal swabs were collected one day before challenge infection, 2 weeks before necropsy, and placed in culture medium containing antibiotics.

[0215] autopsy

[0216] Three weeks after challenge, the animals were transported to the necropsy facility. They were anesthetized using an electric shock device and exsanguinated according to standard procedures. The animals were dissected according to standard procedures. During necropsy, the animals were opened and the internal organs were examined in situ, with particular attention to the following organs: lungs, inguinal and mesenteric lymph nodes, tonsils, thymus, spleen, liver, and kidneys. Subsequently, samples from the tonsils, lungs, mesenteric lymph nodes, and inguinal lymph nodes were removed and divided into two parts: one for freezing and analysis by PCV2 qPCR, and one for fixation and subsequent (immuno)histochemical analysis.

[0217] Sample processing

[0218] Prepare serum from clotted blood samples and fill aliquots (e.g., 2 x 0.8 ml). Samples are not heat inactivated. Prepare stool samples from swabs and fill aliquots (e.g., 2 x 0.8 ml). Store samples at ≤ -15°C until use. The time between sampling and storage should not exceed 36 (serum) or 48 (swab) hours.

[0219] PCV2 antibody ELISA

[0220] Sera were tested for antibodies against PCV2 according to standard procedures. Briefly, serially diluted serum samples were incubated on microtiter plates coated with baculovirus-expressed PCV2 ORF2 antigen. After serum removal, all wells were incubated with a fixed amount of biotinylated PCV2-specific monoclonal antibody (MoAb). The bound MoAb was then incubated with peroxidase-conjugated streptavidin, followed by chromogenic detection. The titer was defined as the reciprocal of the interpolated serum dilution that had an extinction value equal to 50% of the maximum extinction tested. Results were expressed as log2 titers. Titers below 2.0 log2 were considered negative. For negative samples, a value of 1.0 log2 was used for calculation purposes.

[0221] Quantitative PCR

[0222] Quantitative PCR (qPCR) for PCV2 nucleic acid was performed on all serum and fecal swabs, as well as on 10% tissue homogenates of tonsils, lungs, mesenteric lymph nodes, and inguinal lymph nodes, according to standard procedures. Briefly, DNA was extracted from the samples using commercially available kits. PCV2 genomic DNA in each sample was quantified by polymerase chain reaction (PCR) using primers and probes specific for PCV2-ORF2. The cycle number at which specific fluorescence exceeded the threshold was correlated with the cycle number of a panel of samples containing known amounts of plasmid containing PCV2-ORF2. Results are expressed as log of the extracted DNA. 10 copies / μl(log 10 c / μl). Less than 1.00log 10 The value of c / μl was considered negative and treated as 0.00 log for calculation purposes. 10 c / μl.

[0223] Immunohistology (IHC)

[0224] Tonsil and lymph node samples were prepared for histological examination. Samples were fixed in 10% formalin, embedded in paraffin, and subjected to immunohistochemistry to detect PCV2 antigen on slides. Anti-PCV2 rabbit serum was used as the primary antibody, and Envision+ (DAKO, Denmark) was used as the detection system according to the manufacturer's instructions. Slides were counterstained with hematoxylin. Microscopic examination was performed. For tonsils and lymph nodes, characteristic brown staining was scored according to the following sequence:

[0225] 0 No specific positive staining cells were observed

[0226] 1. Scattered (single) positively stained cells are present in (less than) 10% of lymph nodes

[0227] 2 Positively stained (single) cells were observed in 10-50% of lymph nodes, or focal aggregates of >15 positively stained cells were observed in <10% of lymph nodes.

[0228] Specific staining in >50% of lymph nodes

[0229] The results were recorded as a total score, which was the sum of the ratings from each organization.

[0230] result:

[0231] PCV2 serology:

[0232] At the start of measurement on SD1, all groups had log2 titers between 4 and 5. The positive control (group 21) gave a log2 titer of 10 between SD34 and SD41. In the same week, groups 18, 19, and 20 had log2 titers between 8 and 10. The unvaccinated negative control group 22 resulted in a log2 titer below 4.

[0233] PCV2 viral load in serum (qPCR serum)

[0234] Between SD0 (start of measurement) and SD19, no viral load was detected in all groups (log10 c / μl=0).

[0235] The viral load in the positive control (Group 21) increased to approximately 1.0 log10 c / μl at SD34 and further increased to approximately 1.6 at SD41.

[0236] The viral load in the unvaccinated negative control (Group 22) increased to approximately 4.1 log10 c / μl on SD34 and decreased slightly to approximately 3.6 on SD41.

[0237] The viral loads in groups 18, 19 and 20 were between 1.3 and 1.8 log10 c / μl at SD34 and between 1.6 and 2.1 at SD41.

[0238] PCV2 viral load in stool swabs (qPCR stool swabs)

[0239] On SD19 (start of measurement), no viral load was detected in all groups (log10c / μl=0).

[0240] The viral load in the positive control (Group 21) increased to approximately 1.5 log10 c / μl at SD34 and further increased to approximately 2.2 at SD41.

[0241] The viral load in the unvaccinated negative control (Group 22) increased to approximately 3.6 log10 c / μl at SD34 and remained essentially constant until SD41.

[0242] Viral loads in Groups 18, 19, and 20 were within the range of the positive control group (between approximately 1.5 and 1.8 for SD34 and between approximately 1.8 and 2.2 for SD41).

[0243] Security

[0244] No unacceptable injection site reactions were observed. At T = 0 + 4 h, the maximum average temperature increase was 0.4 ° C. Therefore, it can be demonstrated that the vaccine composition of the present invention comprising an adjuvant combination of squalane, vitamin E acetate, and silica gave acceptable results in terms of vaccination efficacy without causing unacceptable site reactions.

[0245] Example 7: Application of different pharmaceutical grade silicon dioxide in the preparation of combined vaccines

[0246] In this example, various different types of silica, all pharmaceutical grade (colloidal) amorphous silica, are shown to be useful in the preparation of the combination vaccine according to the invention. The method used to prepare the vaccines is largely the same as in Example 1, although for some vaccines the silica is only used in the presence of a microfluidizer. TM This had no substantial effect on the final composition, with all silica being roughly distributed in the final composition with an average particle size of approximately 120 nm. However, when TM When silica was added later, a very small percentage of the total silica volume was present in the form of large aggregates (solid particles) greater than 10 μM (up to 200 μM). However, this had no negative impact on the performance of the vaccine. The data for the various compositions are given in Table 12.

[0247] Table 12

[0248]

[0249]

[0250] Other silicas suitable for use in the present invention are and

[0251] in conclusion

[0252] Examples 1-7 demonstrate that, of the compositions tested, only the novel adjuvant combination of squalane, vitamin E acetate, and silica gave acceptable results in terms of vaccination efficacy against PCV2 and Mhyo infection without causing unacceptable site reactions. Thus, using this adjuvant composition, it is possible to provide a safe and effective intradermal combination vaccine against PCV2 and Mhyo using non-replicating immunogens of porcine circovirus type 2 and Mycoplasma hyopneumoniae.

Claims

1. A combination vaccine comprising a non-replicating immunogen, characterized in that The combination vaccine is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silicon dioxide, and the non-replicating immunogen is a combination of a non-replicating immunogen of porcine circovirus type 2 and a non-replicating immunogen of Mycoplasma hyopneumoniae.

2. The combined vaccine according to claim 1, characterized in that It contains 1-15% w / v squalane.

3. The combined vaccine according to claim 1 or 2, characterized in that It contains 2-20% w / v vitamin E-acetate.

4. The combined vaccine according to claim 1 or 2, characterized in that It contains emulsifiers with an HLB value of 8-20.

5. The combined vaccine according to claim 4, characterized in that The emulsifier is polysorbate 80.

6. The combined vaccine according to claim 4 or 5, characterized in that The emulsifier is present in an amount of 0.5-10% w / v.

7. The combined vaccine according to claim 1, characterized in that It contains 0.02-2% w / v silicon dioxide.

8. The combined vaccine according to claim 1, characterized in that The non-replicating immunogen of porcine circovirus type 2 is a recombinantly expressed protein encoded by the ORF2 gene of porcine circovirus type 2.

9. The combined vaccine according to claim 1, characterized in that The non-replicating Mycoplasma hyopneumoniae immunogen comprises inactivated whole Mycoplasma hyopneumoniae.

10. The combined vaccine according to any one of claims 1 to 9, characterized in that The combination vaccine is administered intradermally to animals for the prophylactic treatment of animals against porcine circovirus type 2 infection and Mycoplasma hyopneumoniae infection.

11. The combination vaccine according to claim 10, wherein the volume of the combination vaccine administered intradermally to an animal is 0.1-0.5 ml.

12. Use of the combined vaccine according to any one of claims 1 to 9 in the preparation of a medicament for the prophylactic treatment of animals against porcine circovirus type 2 infection and Mycoplasma hyopneumoniae infection, characterized in that The combination vaccine is administered intradermally to the animals.

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