Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/10011—Circoviridae
  • C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry

Definitions

• the present invention relates to the field of vaccines, more specifically, to vaccines for intradermal delivery that simultaneously protect against various microorganisms.
• the invention relates to intradermal combination vaccines comprising
• Mycoplasma antigens and Porcine circovirus antigens and their use for livestock immunization.
• Mycoplasma hyopneumoniae and Porcine Circovirus type 2 are two of the most important pathogens affecting pigs causing significant economic losses in the swine industry.
• Mycoplasma is a genus of bacteria that lack a cell wall around their cell membrane.
• Several species are pathogenic in swine, including Mycoplasma hyopneumoniae ( Mhyo ), which is known to cause porcine enzootic pneumonia, a highly contagious and chronic disease affecting pigs.
• Mhyopneumoniae is small in size (400- 1200 nm), has a small genome (893-920 kilo-base pairs) and lacks a cell wall.
• Porcine enzootic pneumonia is an important cause of economic loss in the swine industry due to reduced weight gain and poor feed efficiency.
• this disease causes a chronic cough, dull hair coat, retarded growth and unthrifty appearance lasting several weeks. Characteristic lesions of purple to gray areas of consolidation, particularly in ventral apical and cardiac lobes, are observed in infected animals. Although the disease causes little mortality, affected swine are often prone to secondary infections by opportunistic pathogens, resulting in death or stress.
• Mhyo infection Treatment of Mhyo infection is limited to antibiotics, which are currently ineffective as they do not completely remove the infection. Vaccines have been found to reduce the severity of the disease but do not prevent the disease from occurring in infected pigs.
• Porcine circoviruses belong to the virus family Circovihdae and are small non-enveloped DNA viruses containing a unique single-stranded circular genome.
• the viral capsid is icosahedral and approximately 17 nm in diameter.
• PCV type 1 PCV1
• PCV type 2 PCV2
• PCV2 shares approximately 80% sequence identity with porcine circovirus type 1 (PCV1 ).
• PCVAD Porcine Circovirus-Associated Diseases
• PMWS Postweaning Multisystemic Wasting Syndrome
• PMWS is generally regarded to be the major clinical manifestation of PCVAD.
• PMWS affects pigs between 5-18 weeks of age.
• PMWS is clinically characterized by wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, and jaundice.
• a combination of all symptoms will be apparent while other affected swine will only have one or two of these symptoms.
• necropsy microscopic and macroscopic lesions also appear on multiple tissues and organs, with lymphoid organs being the most common site for lesions.
• Mortality rates for swine infected with PCV2 can approach 80%.
• Intradermal vaccines deliver antigens directly into the dermis, an anatomical space that contains large numbers of antigen-presenting cells and thus, has the potential for greater immunogenicity than subcutaneous or intramuscular injection of a given amount of antigen.
• ID vaccines there are currently on the market vaccines for swine immunization against either Mhyo (Porcilis® M Hyo ID Once) or porcine circovirus (Porcilis® PCV ID).
• Mhyo Panes® M Hyo ID Once
• porcine circovirus porcine circovirus
• Vaccines in solution may undergo processes over time which decrease the
• immunogenicity of its antigen components for instance the degradation of the antigen or the desorption of the antigens from the adjuvant to which they had been adsorbed.
• Another problem associated with combination vaccines is that they add an extra layer of complexity when a change in the route of administration or formulation, or a dose sparing strategy wants to be implemented, since the safety and activity of every antigen has to be maintained in the novel conditions.
• a combination vaccine is provided in W02014009586, wherein it is disclosed the use of an intramuscular recombinant combination vaccine to protect animals against Mycoplasma and porcine circovirus infections.
• the formulation of the vaccine disclosed in said patent application is not suitable for intradermal injection due to its high volume and the reactogenic nature of its adjuvants.
• changing the formulation and/or route of administration is not straightforward, requiring extensive testing to determine the safety and efficacy.
• the international application WO2017162741 disclosed an intradermal combination vaccine against Mycoplasma and porcine circovirus.
• the authors show that only some specific combinations of adjuvant emulsions provide efficient and safe vaccines.
• the experimental data presented in said document reveals that high concentrations of PCV2 antigens are needed in order to achieve immunization.
• the present inventors have discovered that a combination vaccine that combines
• Mycoplasma hyopneumoniae antigens and porcine circovirus antigens is capable of efficiently mounting an immune response that protects animals from the infection of both microorganisms, even when administered intradermically.
• the administration through the intradermal route is commonly carried out through needle-less injectors, which greatly facilitates vaccination procedure while reducing health risks to both animals and workers.
• the vaccine of the invention presents the advantage that it contains antigens from both pathogens in a single working composition, which allows reducing the rounds of administration and the risk associated to them.
• the present invention provides a combination vaccine comprising one or more antigens of Mycoplasma hyopneumoniae, one or more antigens of porcine circovirus, and pharmaceutically acceptable excipients and/or carriers, for use in the prevention and/or treatment of porcine enzootic pneumonia and/or Porcine Circovirus- Associated Diseases (PCVAD) by administration of the vaccine into the dermis of livestock, wherein the one or more antigens of Porcine circovirus comprises the PCV2 ORF2 protein in an amount from 0.1 pg/dose to 10 pg/dose.
• PCVAD Porcine Circovirus- Associated Diseases
• This aspect can also be formulated as the use of a combination vaccine comprising one or more antigens of Mycoplasma hyopneumoniae and one or more antigens of Porcine circovirus, wherein the one or more antigens of porcine circovirus comprises the PCV2 ORF2 protein in an amount from 0.1 pg/dose to 10 pg/dose, for the manufacture of a medicament for the prevention and/or treatment of porcine enzootic pneumonia and/or Porcine Circovirus-Associated Diseases (PCVAD) by administration of the medicament into the dermis of livestock.
• a combination vaccine comprising one or more antigens of Mycoplasma hyopneumoniae and one or more antigens of Porcine circovirus, wherein the one or more antigens of porcine circovirus comprises the PCV2 ORF2 protein in an amount from 0.1 pg/dose to 10 pg/dose, for the manufacture of a medicament for the prevention and/or treatment of porcine e
• This embodiment can also be formulated as a method of intradermically immunizing livestock in need thereof with a combination vaccine comprising one or more antigens of Mycoplasma hyopneumoniae and one or more antigens of Porcine circovirus, wherein the one or more antigens of porcine circovirus comprises the PCV2 ORF2 protein in an amount from 0.1 pg/dose to 10 pg/dose, in particular for preventing and/or treating porcine enzootic pneumonia and/or Porcine
• PCVAD Circovirus-Associated Diseases
• Example 1 Surprisingly, and as can be observed in Example 1 , the inventors found out that the combination vaccine of the invention was efficient and safe even when the PCV2 antigen amount was greatly reduced (even below 1 pg per dose). Moreover, the combination vaccine was effective in a single dose. This was completely unexpected since
• combination vaccines commonly require higher antigen amounts than monovalent vaccines to counteract antigen interference—i.e. the reduction of antigen immunogenicity when combined with other antigens.
• a second aspect of the invention provides an intradermal combination vaccine comprising one or more antigens of Mycoplasma hyopneumoniae and one or more antigens of porcine circovirus, wherein the one or more antigens of porcine circovirus comprises the porcine circovirus PCV2 ORF2 protein in an amount from 0.1 pg/dose to 10 pg/dose.
• the present invention provides a vaccination kit of parts comprising (a) an intradermal combination vaccine as defined in the second aspect of the invention, (b) optionally a delivery device suitable for intradermal delivery of vaccines; and (c) optionally, instructions for its use.
• the invention refers to an intradermal combination vaccine comprising one or more antigens of Mycoplasma hyopneumoniae, one or more antigens of Porcine circovirus, an adjuvant emulsion comprising mineral oil and an additive selected from a hydrophobic additive, an amphiphilic additive and combinations thereof, and pharmaceutically acceptable excipients and/or carriers.
• the invention provides the intradermal combination vaccine of the fourth aspect for use as a medicament in livestock.
• the invention provides a vaccination kit of parts comprising (a) an intradermal combination vaccine as defined in the fourth aspect; (b) optionally a delivery device suitable for intradermal delivery of vaccines; and (c) optionally, instructions for its use.
• Fig. 1 shows the body temperature of vaccinated piglets at different times after vaccination with the vaccine of the invention.
• DO (+3h) corresponds to three hours after vaccination;
• DO (+6h) corresponds to six hours after vaccination;
• D1 one day after vaccination; and D2, two days after vaccination.
• the groups of vaccinated piglets A to G are described in Example 1.
• Fig. 2 shows the local reactions score of piglets at various times after vaccination with the vaccine of the invention.
• DO (+3h) corresponds to three hours after vaccination;
• DO (+6h) corresponds to six hours after vaccination;
• D1 , D2, D7, D14 and D21 correspond to one, two, seven, fourteen and twenty one days after vaccination, respectively.
• the groups of vaccinated piglets A to G are described in Example 1.
• Fig. 3, related to Example 1 shows the pulmonary lesion score of vaccinated pigs at day 27 after the challenge infection with Mycoplasma hyopneumoniae.
• the groups of vaccinated piglets A to G are described in Example 1.
• Fig. 4 related to Example 2, shows the local reactions score of piglets at various times after vaccination with the vaccine of the invention.
• DO (+3h) corresponds to three hours after vaccination
• DO (+6h) corresponds to six hours after vaccination
• D1 , D2, D7, D14 and D21 correspond to one, two, seven, fourteen and twenty one days after vaccination, respectively.
• the groups of vaccinated piglets A to E are described in Example 2.
• Fig. 5, related to Example 2 shows the percentage of median lung surface affected area of vaccinated pigs at day 28 after the challenge infection with Mycoplasma
• Fig. 6 related to Example 2, shows the seroconversion to PCV2 ORF2 (S/P ratio) of vaccinated pigs at days 7 (D7), 14 (D14), 21 (D21 ), and 28 (D28) after the PCV2 challenge.
• the groups of vaccinated piglets A to E are as described in Example 2.
• antigen refers to a molecule against which a subject can initiate an immune response, e.g. a humoral and/or cellular immune response. Depending on the intended function of the composition, one or more antigens may be included.
• medicament is synonymous of a pharmaceutical or veterinary drug (also referred to as medicine, medication, or simply drug) used to cure, treat or prevent disease in animals, including humans, as widely accepted.
• Drugs are classified in various ways. One key distinction is between traditional small-molecule drugs, usually derived from chemical synthesis, and biopharmaceuticals, which include recombinant proteins, vaccines, blood products used therapeutically (such as IVIG), gene therapy, monoclonal antibodies and cell therapy (for instance, stem-cell therapies).
• medicament preferably is a veterinary medicament, and even more preferably is a vaccine for veterinary use.
• vacuna means an immunogenic composition accompanied by adequate excipients and/or carriers, that when administered to an animal, elicits, or is able to elicit, directly or indirectly, an immune response in the animal.
• the vaccines of the present invention elicit an immunological response in the host of a cellular or antibody-mediated type upon administration to the subject that it is protective.
• intradermal vaccine refers to vaccines administered into the dermis.
• “combination vaccine” means that the vaccine contains various antigens in a single preparation, protecting against two or more diseases or against one disease caused by two or more microorganisms.
• the vaccine includes as“active principle” an “immunogenic composition”, which as used herein, refers to a material that elicits an immunological response in the host of a cellular or antibody-mediated immune response type to the composition upon administration to a vertebrate, including humans.
• the immunogenic composition comprises molecules with antigenic properties, such as killed or attenuated bacteria or virus, among others, and also immunogenic polypeptides.
• An immunogenic polypeptide is generally referred to as antigenic.
• a molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
• An antigenic polypeptide contains an epitope of at least about five, and particularly at least about 10, at least 15, at least 20 or at least 50 amino acids.
• An antigenic portion of a polypeptide, also referred to as an epitope can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier polypeptide for
• the immunogenic composition relates according to this description, to the active molecule, composition comprising said molecule, or composition comprising more than one antigenic molecule to which a particular immune reaction is desired.
• immunologically effective amount means the administration of that amount or dose of antigen, either in a single dose or as part of a series, that elicits, or is able to elicit, an immune response that reduces the incidence of or lessens the severity of infection or incident of disease in an animal for either the treatment or prevention of disease.
• the immunologically effective amount or effective dose is also able for inducing the production of antibody for either the treatment or prevention of disease. This amount will vary depending upon a variety of factors, including the physical condition of the subject, and can be readily determined by someone of skill in the art.
• inactivated cells refers to cells that have been grown in culture and then killed by physical means (such as heat) or chemical means (such as contact with formaldehyde, BEI, or any other well-known inactivation agent) as the main ways of inactivation methods which the skilled man perfectly knows among others.
• The“inactivated cells” may be whole cells or partial cells, including cell lysates and supernatant recovered cells for example.
• the term“attenuated cells” refers to viable and replication competent cells the virulence of which has been reduced.
• livestock relates to domesticated or farm animals raised to produce
• commodities such us food.
• it relates to food-producing animals such as cattle, sheep, goats, swine, poultry (including egg-producing poultry), and equine animal. More in particular, it relates to food-producing animals such as cattle, sheep, goats, swine, poultry and equine animals. Even more in particular, in the present invention relates to swine species.
• carrier is to be understood as a pharmaceutically acceptable component other than the immunogenic component.
• the carrier can be organic, inorganic, or both.
• Suitable carriers well known to those of skill in the art and include, without limitation, large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles.
• compositions or vehicles pharmaceutically acceptable materials, compositions or vehicles.
• Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio. In particular, it must be suitable for use in the intradermal route.
• the term“host” or“subject” is intended for the target individuals in need thereof to whom the immunogenic composition or vaccine of the invention are administered.
• compositions of the invention administered, among others humans, mammals, livestock, or any other animal species susceptible to be vaccinated with the compositions of the invention.
• the mammal is from porcine species, more particularly is swine.
• pig or“swine” is intended for porcine species including, among others, pigs, boars, sows, gilts and piglets of any age or in any phase of their production cycle.
• preventing include without limitation decreasing, reducing or ameliorating the risk of a symptom, clinical sings, disorder, condition, or disease, and protecting an animal from a symptom, clinical signs, disorder, condition, or disease.
• a prevention may be applied or administered prophylactically.
• treating include without limitation restraining, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, clinical sign, disorder, condition, or disease.
• a treatment may be applied or administered therapeutically.
• %w/w or “percentage weight per weight” of a component refers to the amount of the single component relative to the total weight of the composition or, if specifically mentioned, of other component.
• mineral oil refers to a variety of complex mixtures of hydrocarbons derived from inorganic matter, such as petroleum and/or liquid paraffin of different pharmaceutical grades. These mixtures are marketed under commercial names such as Marcol 52, Drakeol 5, Eolane 130, for example.
• amphiphilic compound or additive is to be understood a compound possessing both hydrophilic (water-affinity, polar) and lipophilic (fat-affinity) properties. Such a compound is called amphiphilic or amphipathic. Common amphiphilic substances are soaps, detergents and lipoproteins. Other examples of amphiphilic compounds are: Saponins, phospholipids, glycolipids, polysorbates. Amphiphilic compounds have lipophilic (typically hydrocarbon) structures and hydrophilic polar functional groups (either ionic or uncharged). In the particular case of block copolymers, the monomer units of one of the blocks have lipophilic structures meanwhile the monomer units of other blocks have polar functional groups.
• amphiphilic block copolymer is poloxamer (block copolymer comprising units of polyethylene and polyoxypropylene).
• poloxamer block copolymer comprising units of polyethylene and polyoxypropylene.
• some amphiphilic compounds may dissolve in water and to some extent in non-polar organic solvents.
• the amphiphilic compound When placed in an immiscible biphasic system consisting of aqueous and organic solvents, the amphiphilic compound will partition the two phases.
• the extent of the hydrophobic and hydrophilic portions determines the extent of partitioning.
• For“hydrophobic additive or compound” is to be understood as a compound that deters water. In chemistry, it refers to the property of a substance to repel water.
• hydrophobic substance exhibits hydrophobicity and may be termed a hydrophobe.
• Hydrophobic molecules tend to be nonpolar molecules that group together to form micelles rather than be exposed to water.
• Common hydrophobic substances are oils, cholesterol, lecithin, or polymer of isoprene units.
• An example of a hydrophobic polymer of isoprene units is squalene.
• hydroophobicity can be tested, among others, using the Hildebrand solubility parameter d. The term
• “Hildebrand solubility parameter” refers to a parameter indicating the cohesive energy density of a substance. The d parameter is determined as follows:
• d is the solubility parameter, (cal/cm 3 ) 1/2 ;
• hydrophilic additives are those with a logP ⁇ 0.
• a partition- coefficient (P) or distribution-coefficient (D) is the ratio of concentrations of a compound in a mixture of two immiscible phases at equilibrium. This ratio is therefore a measure of the difference in solubility of the compound in these two phases.
• the partition-coefficient generally refers to the concentration ratio of un-ionized species of compound whereas the distribution-coefficient refers to the concentration ratio of all species of the compound (ionized plus un-ionized). The skilled man in the art will know how to calculate this paremeter.
• Block copolymers are polymers that comprise two or more homopolymer subunits linked by covalent bonds. The union of the homopolymer subunits may require an intermediate non-repeating subunit, known as a junction block. Block copolymers with two or three distinct blocks are called diblock copolymers and triblock copolymers, respectively. These di- and triblock-copolymers, can be sequential di- and triblock-copolymers or grafted di- and triblock-copolymers.
• a sequential diblock copolymer has the following structure -(A)n-(B)m-; a sequential triblock copolymer corresponds to the structure -(A)n- (B)m-(A)p-; a grafted copolymer is a segmented copolymer with a linear backbone of one composite and randomly distributed branches of another composite.
• the present invention discloses an intradermal combination vaccine comprising one or more antigens of Mycoplasma hyopneumoniae and one or more antigens of PCV2 for use in the prevention and/or treatment of porcine enzootic pneumonia and/or Porcine Circovirus-Associated Diseases (PCVAD).
• PCVAD Porcine Circovirus-Associated Diseases
• PCV2a Different genotypes of PCV2 are encompassed in particular embodiments of the first aspect, such as PCV2a, PCV2b, PCV2c and PCV2d.
• PCV2a there are currently five different PCV2 genotypes, designated as a-e.
• Genotypes of PCV2 are determined by the number of nucleotide substitutions in ORF 2 (capsid) divided by the total nucleotides in ORF2 (702 nt); this fraction is called p-distance. If the p-distance is greater than 0.035, the viruses are given a new genotype (Segales J. et al.,“PCV-2 genotype definition and nomenclature”, Vet Rec., 2008; vol. 162(26), pp. 867-8).
• PCV2 encodes a protein of about 233 amino acids. Any PCV2 ORF2 is suitable to be used in the invetion.
• PCV2 ORF2 sequences of different genotypes are disclosed in Segales et al., 2008 or Xiao et al., “Global molecular genetic analysis of porcine circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d”, J Gen Virol, 2015; vol. 96(Pt 7), pp.1830-41.
• PCV3 European porcine circovirus
• the invention is meant to encompas all PCV2 ORF2 protein variants, for example the ones with the GenBank accession numbers AAC35310 (version AAC35310.1 ),
• AAC35331 version AAC35331.1
• ABX71779 version ABX71779.1
• the one or more antigens of Mycoplasma hyopneumoniae and the one or more antigens of porcine circovirus in the intradermal combination vaccine are in an immunologically effective amount.
• the amount of the PCV2 ORF2 protein in the intradermal combination vaccine is from 0.1 pg/dose to 10 pg/dose. In a more particular embodiment, the amount of the PCV2 ORF2 protein is from 0.1 pg/dose to 5 pg/dose, and more preferably the amount of the PCV2 ORF2 proteins is from 0.5 pg/dose to 4 pg/dose.
• the one or more antigens of Mycoplasma hyopneumoniae are in a form selected from the group consisting of isolated proteins of Mycoplasma hyopneumoniae, attenuated Mycoplasma hyopneumoniae cells, inactivated Mycoplasma hyopneumoniae cells, and combinations thereof. More particularly, the one or more antigens of Mycoplasma hyopneumoniae are in the form of inactivated Mycoplasma hyopneumoniae cells. Even more particularly, the one or more antigens of Mycoplasma hyopneumoniae are in the form of inactivated Mycoplasma hyopneumoniae cell lysates.
• the antigens in the intradermal combination vaccine of the first aspect are provided as inactivated Mycoplasma hyopneumoniae mutant strain cells, said mutant cells expressing one or more proteins of porcine circovirus.
• hyopneumoniae mutant strain cells comprise, in particular, one or more exogenous DNA sequence incorporated in the genome or in the cytosol thereof, said exogenous DNA sequence coding for at least one protein of porcine circovirus.
• the inactivated Mycoplasma hyopneumoniae cell of the combination vaccine of the invention is from a mutant strain of Mhyo, deposited by Laboratories HIPRA, S.A.
• the one or more Mycoplasma hyopneumoniae antigens are in the form of attenuated and/or inactivated Mycoplasma hyopneumoniae cells, in an amount from 10 3 cells/dose to 10 11 cells/dose. More particularly, from 10 5 cells/dose to 10 11 cells/dose, and more particularly from 10 9 cells/dose to 10 11 cells/dose.
• the intradermal combination vaccine for use according to the first aspect is for use against another disease or pathological conditions affecting swine, the vaccine comprising one or more additional antigens selected from a microorganism which is selected from the group formed by Actinobacillus sp., Brachyspira sp.,
• Pasteurella multocida Salmonella sp., Streptococcus sp., Isospora sp., Erysipelothrix rhusiopathiae, Leptospira sp., Staphylococcus sp., Haemophilus parasuis, Bordetella bronchiseptica, Clostridium sp., Mycoplasma sp., Lawsonia intracellularis, Escherichia coli, porcine reproductive and respiratory syndrome virus (PRRS), swine influenza virus, contagious gastroenteritis virus, porcine parvovirus, encephalomyocarditis virus, coronavirus, rotavirus, porcine periweaning failure to thrive syndrome agent, classical swine fever virus, African swine fever virus, calicivirus, transmissible gastroenteritis coronavirus (TGEV), porcine epidemic diarrhea virus (PED) and torque teno virus (TTV).
• the inventors also discovered that using a specific selection of adjuvants in the formulation of the intradermal combination vaccine for the use according to the first aspect, the efficacy was maintained while the safety was improved.
• the specified adjuvants diminished the rise in body temperature post-vaccination and reduced the severity of local reactions— as shown below in Example 1.
• the intradermal combination vaccine further comprises an adjuvant.
• the adjuvant is an adjuvant emulsion comprising mineral oil and an additive, said additive selected from a hydrophobic additive, an amphiphilic additive and combinations thereof.
• the additive is selected from the group consisting of a polymer of isoprene units, a block copolymer, and combinations thereof. More particularly, the polymer of isoprene units is squalene and the block copolymer is a non-ionic block copolymer. More particularly, the non-ionic block copolymer is a poloxamer. Even more particularly, the poloxamer is of formula (I):
• a is an integer from 2 to130 and b is an integer from 15-67 . Even more particularly, a is 101 and b is 56.
• non-ionic block copolymer is Poloxamer 407 (Sigma), Pluronic F-127
• non-ionic block copolymer is Poloxamer 407 (Sigma).
• useful poloxamers are those with an hydrophilic-lipophilic balance (HLB) from 10 to 30, and more particularly from 18 to 24. HLB is a measure of the degree to which a compound is hydrophilic or lipophilic, determined standard procedures known by the skilled man.
• the intradermal combination vaccine further comprises an adjuvant emulsion comprising mineral oil and an additive selected from a hydrophobic additive, an amphiphilic additive and combinations thereof, wherein the concentration of mineral oil is from 5%w/w to 40%w/w and the concentration of the additive is from
• the concentration of mineral oil is from 10%w/w to 25%w/w and the concentration of the additive is from 0.5%w/w to 2%w/w. More particularly, the concentration of mineral oil is 21 2%w/w and the concentration of the additive is 0.5%w/w. All percentages are relative to the total weight of the composition.
• the intradermal combination vaccine comprises mineral oil in a concentration from 5%w/w to 40%w/w and a non-ionic block copolymer in a concentration from 0.5%w/w to 5%w/w as additive in the adjuvant.
• the non-ionic block copolymer is the poloxamer of formula (I). More particularly, the poloxamer of formula (I) wherein a is 101 and b is 56.
• the intradermal combination vaccine for use according to the first aspect further comprises a surfactant selected from the group consisting of alkylphenol ethoxylates, ethoxylated sorbitan esters, ethoxylated fatty alcohols, ethoxylated fatty acids, fatty acid alkanolamides, ethoxylated fatty acid alkanolamides, ethoxylated fatty amines, fatty amine oxides, fatty amidoamine oxides, fatty acid glycerides, sucrose esters, alkyl
• a surfactant selected from the group consisting of alkylphenol ethoxylates, ethoxylated sorbitan esters, ethoxylated fatty alcohols, ethoxylated fatty acids, fatty acid alkanolamides, ethoxylated fatty acid alkanolamides, ethoxylated fatty amines, fatty amine oxides, fatty amidoamine oxides,
• the surfactant is sorbitan monooleate.
• the intradermal combination vaccine further comprises an emulsifier selected from the group consisting of sorbitan Polysorbate 80, Montanox 80, Tween 80, Simulsol, Macrogol, and mixtures thereof. More particularly, the emulsifier is Polysorbate 80.
• the vaccine dose of an intradermal combination vaccine like the one of the invention is comprised from 0.05 to 0.5 ml, particularly from 0.1 to 0.2 ml.
• the vaccine can be administered in one or more dose.
• a multiple-dose vaccination as is well known in the art, consists of administering a first immunizing dose, followed by one or more additional doses which act as booster doses.
• the vaccine preferably is of single-dose administration.
• the livestock is swine.
• the present invention also provides an intradermal combination vaccine comprising one or more antigens of Mycoplasma hyopneumoniae and one or more antigens of porcine circovirus.
• the one or more Mycoplasma hyopneumoniae antigens are in a form of attenuated and/or inactivated Mycoplasma hyopneumoniae cells, in an amount from 10 3 cells/dose to 10 11 cells/dose. More particularly, from 10 5 cells/dose to 10 11 cells/dose, and more particularly from 10 9 cells/dose to 10 11 cells/dose.
• the one or more antigens of Mycoplasma hyopneumoniae and the one or more antigens of porcine circovirus in the combination vaccine of this second aspects are in
• the intradermal combination vaccine further comprises one or more additional antigens, wherein the additional antigen is selected from a group of microorganisms consisting of Actinobacillus, Bordetella, Borrelia, Brachyspira, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium,
• Neisseria Pasteurella, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema Vibrio and Yersinia genus, and porcine reproductive and respiratory syndrome virus (PRRS), swine influenza virus, contagious gastroenteritis virus, porcine parvovirus, encephalomyocarditis virus, coronavirus, rotavirus, porcine
• PRRS porcine reproductive and respiratory syndrome virus
• the vaccine further comprises an adjuvant.
• the adjuvant is an adjuvant emulsion comprising mineral oil and an additive selected from a hydrophobic additive, an amphiphilic additive and combinations thereof.
• the additive is selected from the group consisting of a polymer of isoprene units, block copolymers, and combinations thereof. More particularly, the polymer of isoprene units is squalene and the block copolymer is a non-ionic block copolymer. More particularly, the non-ionic block copolymer is a poloxamer. Even more particularly, the poloxamer is of formula (I):
• the non-ionic block copolymer is Poloxamer 407 (Sigma), Pluronic F-127 (Acofarma), Kolliphor P407 (BASF), Poloxamer 188 such as Koliphor P188 (BASF), Poloxamer L121 such as Pluronic L121 (BASF). More particularly, is Poloxamer 407 (Sigma).
• the intradermal combination vaccine comprises mineral oil in a concentration from 5%w/w to 40%w/w and a non-ionic block copolymer in a concentration from 0.5%w/w to 5%w/w as additive in the adjuvant.
• the non-ionic block copolymer is the poloxamer of formula (I). More particularly, the poloxamer of formula (I) wherein a is 101 and b is 56.
• the intradermal combination vaccine of the second aspect is for use as a medicament in livestock.
• the intradermal combination vaccine of the second aspect is for use in the prevention and/or treatment of porcine enzootic pneumonia and/or Porcine Circovirus- Associated Diseases (PCVAD) by administration of the vaccine into the dermis of livestock.
• PCVAD Porcine Circovirus- Associated Diseases
• the invention also provides a vaccination kit comprising the vaccine of the second aspect of the invention. More particularly, the vaccination kit is for use in the prevention and/or treatment of a disease caused by Mycoplasma sp. and/or porcine circovirus. More particularly, for use in the prevention and/or treatment of porcine enzootic pneumonia caused by Mycoplasma and/or Porcine Circovirus-Associated Diseases (PCVAD).
• PCVAD Porcine Circovirus-Associated Diseases
• the invention also provides an intradermal vaccine comprising an adjuvant emulsion comprising mineral oil and an additive selected from a hydrophobic additive, an amphiphilic additive and combinations thereof.
• the additive of the intradermal combination vaccine is selected from the group consisting of a polymer of isoprene units, a block copolymer, and combinations thereof. More particularly, the polymer of isoprene units is squalene and the block copolymer is a non-ionic block copolymer. In a particular embodiment, the additive is a block copolymer . More particularly, the block copolymer is a non-ionic block copolymer . More particularly, the non-ionic block copolymer is a poloxamer . Even more particularly, the non-ionic block copolymer is a poloxamer of formula (I):
• the non-ionic block copolymer is Poloxamer 407 (Sigma), Pluronic F-127 (Acofarma), Kolliphor P407 (BASF), Poloxamer 188 such as Koliphor P188 (BASF), Poloxamer L121 such as Pluronic L121 (BASF). More particularly, the non-ionic block copolymer is Poloxamer 407 (Sigma).
• the intradermal combination vaccine further comprises adjuvant emulsion, wherein the concentration of mineral oil is from 5%w/w to 40%w/w and the concentration of the additive is from 0.5%w/w to 5%w/w. More particularly, the concentration of mineral oil is from 10%w/w to 25%w/w and the concentration of the additive is from 0.5%w/w to 2%w/w. More particularly, the concentration of mineral oil is 21 2%w/w and the concentration of the additive is 0.5%w/w. All percentages relative to the total weight of the vaccine composition.
• the intradermal combination vaccine comprises mineral oil in a concentration from 5%w/w to 40%w/w and a non-ionic block copolymer in a concentration from 0.5%w/w to 5%w/w as additive in the adjuvant.
• the non-ionic block copolymer is the poloxamer of formula (I). More particularly, the poloxamer of formula (I) wherein a is 101 and b is 56.
• the one or more antigens of Mycoplasma hyopneumoniae in the intradermal combination vaccine are in a form selected from the group consisting of isolated proteins of Mycoplasma hyopneumoniae, for example and without limitation the P46, P65, P97, and P102 surface proteins, described in Minion et al.,“The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis”, J. Bacteriol., 2004, vol. 186(21 ), pp.
• the one or more antigens of Mycoplasma hyopneumoniae are in the form of inactivated Mycoplasma hyopneumoniae cells. Even more particularly, the one or more antigens of Mycoplasma hyopneumoniae are in the form of inactivated Mycoplasma hyopneumoniae cell lysates.
• the one or more antigens of porcine circovirus are in a form selected from the group consisting of inactivated porcine circovirus particles, attenuated porcine circovirus particles, isolated proteins of porcine circovirus, and combinations thereof. More particularly, the one or more antigens of porcine circovirus are isolated proteins of porcine circovirus . Even more particularly, the one or more antigens are selected from the group comprising ORF1 , ORF2, and ORF3 proteins. More particularly, the antigen is ORF2 protein.
• the porcine circovirus is a porcine circovirus type 2 (PCV2).
• PCV2a, PCV2b, PCV2c and PCV2d Different genotypes of PCV2 are encompassed in particular embodiments of the first aspect, such as PCV2a, PCV2b, PCV2c and PCV2d.
• PCV2a, PCV2b, PCV2c and PCV2d There are currently five different PCV2 genotypes, designated as a-e.
• Genotypes of PCV2 are determined by the number of nucleotide substitutions in ORF 2 (capsid) divided by the total nucleotides in ORF2 (702 nt); this fraction is called p-distance. If the p-distance is greater than 0.035, the viruses are given a new genotype (Segales et al., 2008). While this can be somewhat confusing, at least the nomenclature is standardized.
• PCV2 encodes a protein of about 233 amino acids. Any PCV2 ORF2 is suitable to be used in the invetion. PCV2 ORF2 sequences of different genotypes are disclosed in Segales et al., 2008 or Xiao et al.,
• the invention is meant to encompas all PCV2 ORF2 protein variants, for example the ones with the GenBank accession numbers AAC35310 (version AAC35310.1 ),
• AAC35331 version AAC35331.1
• ABX71779 version ABX71779.1
• the antigens of the intradermal combination vaccine are inactivated Mycoplasma hyopneumoniae mutant strain cells, said cells expressing one or more protein of porcine circovirus.
• the inactivated Mycoplasma hyopneumoniae (Mhyo) cell is from a mutant strain of Mhyo deposited in Leibniz-lnstitut DSMZ and selected from a mutant strain of Mhyo with the accession number DSM 26020; a mutant strain of Mhyo with the accession number DSM 26034; a mutant strain of Mhyo with the accession number DSM 26027; and a mutant strain of Mhyo with the accession number DSM 26033.
• the one or more antigens of Mycoplasma hyopneumoniae and the one or more antigens of porcine circovirus in the intradermal combination vaccine of this fourth aspect are in immunologically effective amounts.
• the amount of porcine circovirus antigens is from 0.1 pg/dose to 10 pg/dose, more particularly from 0.1 pg/dose to 5 pg/dose, even more particularly from 0.5 pg/dose to 4 pg/dose.
• the amount of antigens of Mycoplasma hyopneumoniae in the combination vaccine is from 10 3 cells/dose to 10 11 cells/dose. More particularly, from 10 5 cells/dose to 10 11 cells/dose, and more particularly from 10 9 cells/dose to 10 11 cells/dose.
• the vaccine dose of an intradermal combination vaccine like the one of the invention is comprised from 0.05 to 0.5 ml, particularly from 0.1 to 0.2 ml.
• the intradermal combination vaccine further comprises one or more additional antigens wherein the additional antigen is selected from a group of microorganisms consisting of Actinobacillus, Bordetella, Borrelia, Brachyspira, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium,
• Enterococcus Erysipelothrix, Escherichia, Francisella, Haemophilus, Helicobacter, Isospora, Lawsonia, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema Vibrio and Yersinia genus, and porcine reproductive and respiratory syndrome virus (PRRS), swine influenza virus, contagious gastroenteritis virus, porcine parvovirus, encephalomyocarditis virus, coronavirus, rotavirus, porcine
• PRRS porcine reproductive and respiratory syndrome virus
• the invention also provides an intradermal combination vaccine for use as a medicament in livestock.
• the vaccine is for use in the prevention and/or treatment of porcine enzootic pneumonia caused by Mycoplasma hyopmeumoniae and/or Porcine Circovirus-Associated Diseases (PCVAD) in livestock.
• This embodiment can also be formulated as the use of the intradermal combination vaccine of the fifth aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of porcine enzootic pneumonia and/or Porcine Circovirus-Associated Diseases (PCVAD) by administration of the medicament into the dermis of livestock.
• This embodiment can also be formulated as a method for intradermically immunizing livestock in need thereof with the combination vaccine of the fifth aspect, in particular for the prevention and/or treatment of porcine enzootic pneumonia caused by Mycoplasma and/or Porcine Circovirus-Associated Diseases (PCVAD).
• PCVAD Porcine Circovirus-Associated Diseases
• the livestock is swine.
• the vaccine is for use in the prevention and/or treatment of an additional disease, wherein the vaccine comprises one or more additional antigens selected from a microorganism selected from the group formed by Actinobacillus sp., Brachyspira sp., Pasteurella multocida, Salmonella sp., Streptococcus sp., Isospora sp., Erysipelothrix rhusiopathiae, Leptospira sp., Staphylococcus sp., Haemophilus parasuis, Bordetella bronchiseptica, Clostridium sp., Mycoplasma sp., Lawsonia intracellularis, Escherichia coli, porcine reproductive and respiratory syndrome virus (PRRS), swine influenza virus, contagious gastroenteritis virus, porcine parvovirus, encephalomyocarditis virus, coronavirus,
• a microorganism selected from
• the invention also refers to a vaccination kit of parts comprising (a) an intradermal combination vaccine of the fourth aspect; (b) optionally a delivery device suitable for intradermal delivery of vaccines; and (c) optionally, instructions for its use.
• the vaccination kit is for use in the prevention and/or treatment of a disease caused by Mycoplasma sp. and/or porcine circovirus. More particularly, for use in the prevention and/or treatment of porcine enzootic pneumonia caused by Mycoplasma and/or Porcine Circovirus-Associated Diseases (PCVAD).
• PCVAD Porcine Circovirus-Associated Diseases
• An intradermal combination vaccine comprising:
• an adjuvant emulsion comprising mineral oil and an additive selected from a hydrophobic additive, an amphiphilic additive and combinations thereof;
• Clause 2 The intradermal combination vaccine according to clause 1 , wherein the additive is selected from the group consisting of a polymer of isoprene units, a block copolymer, and combinations thereof.
• a is an integer from 2 to130 and b is an integer from 15-67;
• Clause 7. The intradermal combination vaccine according to clause 6, wherein in the poloxamer a is 101 and b is 56.
• Clause 8. The intradermal combination vaccine according to any of clauses 1-7, wherein the one or more antigens of Mycoplasma hyopneumoniae are in a form selected from the group consisting of isolated proteins of Mycoplasma hyopneumoniae, attenuated
• Mycoplasma hyopneumoniae cells inactivated Mycoplasma hyopneumoniae cells, and combinations thereof.
• Clause 9 The intradermal combination vaccine according to clause 8, wherein the one or more antigens of Mycoplasma hyopneumoniae are in the form of inactivated Mycoplasma hyopneumoniae cells.
• Clause 10 The intradermal combination vaccine according to any of clauses 1-9, wherein the one or more antigens of porcine circovirus are in a form selected from the group consisting of inactivated porcine circovirus particles, attenuated porcine circovirus particles, isolated proteins of porcine circovirus, and combinations thereof.
• Clause 11 The intradermal combination vaccine according to any of clauses 1-10, wherein the one or more antigens of porcine circovirus are in the form of isolated proteins of porcine circovirus.
• Clause 12 The intradermal combination vaccine according to any of clauses 1-11 , wherein the antigens are inactivated Mycoplasma hyopneumoniae mutant strain cells, said cells expressing one or more proteins of porcine circovirus.
• the inactivated Mycoplasma hyopneumoniae cell is from a mutant strain of Mhyo deposited in Leibniz-lnstitut DSMZ and selected from a mutant strain of Mhyo with the accession number DSM 26020; a mutant strain of Mhyo with the accession number DSM 26034; a mutant strain of Mhyo with the accession number DSM 26027; and a mutant strain of Mhyo with the accession number DSM 26033.
• the intradermal combination vaccine according to any of the previous clauses, further comprising one or more additional antigens wherein the additional antigen is selected from a group of microorganisms consisting of Actinobacillus, Bordetella, Borrelia, Brachyspira, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Erysipelothrix, Escherichia, Francisella, Haemophilus, Helicobacter, Isospora, Lawsonia, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema Vibrio and Yersinia genus, and porcine reproductive and respiratory syndrome virus (PRRS), swine
• Clause 15 An intradermal combination vaccine as defined in any of clauses 1-14, for use as a medicament in livestock.
• Clause 16 The intradermal combination vaccine for use according to clause 15, which is for use in the prevention and/or treatment of porcine enzootic pneumonia caused by Mycoplasma hyopneumoniae and/or Porcine Circovirus-Associated Diseases (PCVAD), and optionally against another disease or pathological conditions affecting swine.
• PCVAD Porcine Circovirus-Associated Diseases
• the vaccine comprising one or more additional antigens selected from a microorganism selected from the group formed by Actinobacillus sp., Brachyspira sp., Pasteurella multocida, Salmonella sp., Streptococcus sp., Isospora sp., Erysipelothrix rhusiopathiae, Leptospira sp., Staphylococcus sp., Haemophilus parasuis, Bordetella bronchiseptica, Clostridium sp., Mycoplasma sp., Lawsonia intracellularis, Escherichia coli, porcine reproductive and respiratory syndrome virus (PRRS), swine influenza virus, contagious gastroenteritis virus, porcine parvovirus, encephalomyocarditis virus, coronavirus, rotavirus, porcine periweaning
• a microorganism selected from the group formed by Actin
• a vaccination kit of parts comprising:
• Example 1 Safety and efficacy of different intradermal combined vaccines against porcine circovirus associated disease (PCVAD) and enzootic pneumonia: at fixed PCV2 ORF2 concentration per dose.
• PCVAD porcine circovirus associated disease
• enzootic pneumonia at fixed PCV2 ORF2 concentration per dose.
• the objective of this study was to assess the safety and efficacy of different combined M. hyopneumoniae and PCV2 intradermal vaccine formulations against enzootic pneumonia (EP) and porcine circovirus associated disease (PCVAD) in piglets.
• EP enzootic pneumonia
• PCVAD porcine circovirus associated disease
• All piglets were vaccinated at day 0 of study by intradermal route with a single dose of 0.2 ml of the corresponding formulae applied with a needle-free device (such as the one disclosed in W02016102619, HIPRA SCIENTIFIC, S.L.U., Spain).
• the combined vaccines of the group A to E contained the same antigenic material, 0.6 pg/dose of PCV2 ORF2 protein and 1.57x10 10 M. hyopneumoniae inactivated cells/dose.
• Group F received a single dose of 0.2 ml of the commercial available vaccine PORCIUS® MHYO ID ONCE (monovalent vaccine containing M.hyo antigen only) applied with the IDAL vaccinator (MSD Animal Health) and a single dose of 2 ml of the commercial available vaccine PORCIUS® PCV (monovalent vaccine containing PCV2 ORF2 antigen only) given simultaneously by intramuscular route with a needle-syringe system. Finally, Group G received a 0.2 ml of placebo (PBS) by intradermal route in the same way as Groups A to E.
• PBS placebo
• Group A M.hyo/PCV2 combined vaccine containing 21.2 % of mineral oil.
• Group B M.hyo/PCV2 combined vaccine containing 21.2 % mineral oil and 2% of block copolymer (Poloxamer 407, BASF).
• Group C M.hyo/PCV2 combined vaccine containing 30 % mineral oil and 5% of squalene.
• Group D M.hyo/PCV2 combined vaccine containing 21.2 % mineral oil and 5% of squalene.
• Group E M.hyo/PCV2 combined vaccine containing Montanide IMS 251 (SEPPIC).
• Group F PORCIUS® MHYO ID ONCE and PORCIUS® PCV (M.hyo monovalent ID vaccine and PCV2 ORF2 monovalent IM vaccine, respectively; MSD Animal Health), both containing mineral oil and dl-a-tocopheryl acetate as adjuvants according to their respective EU Marketing Authorisation, Summary of Product Characteristics.
• Group G placebo, injected with PBS.
• Body temperatures were observed and recorded for each piglet at the day of vaccination (before vaccination, and 3 and 6 hours post-vaccination) and until day 2 post-vaccination.
• Local reactions of the vaccines were observed at day 0 (at 3 and 6 hours after vaccination) and at days 1 , 2, 7, 14 and 21 days post-vaccination. These reactions were assessed by the average of local reactions score. The score was calculated based on the size, colour and appearance of lesion (if any) at the point of vaccination.
• Group B (21.2 % mineral oil + Poloxamer 407) recorded a temperature increase of only 0.5 °C very similar with the values obtained with control group G and group F (vaccinated with the monovalent Porcilis® MHYO ID ONCE and Porcilis® PCV, simultaneously). It was demonstrated that the group that received the vaccine adjuvanted with 2 % of Poloxamer 407 plus mineral oil reduced the body temperature of an equivalent combination protocol of the vaccines without this component.
• the main variable for challenge efficacy evaluation was the pulmonary lesion scoring.
• Each lung lobe was scored between 0 and 5 according to the proportion of affected tissue with M. hyopneumoniae-Wke lesions (/ ' .e. lung consolidation):
• Example 2 Safety and efficacy of intradermal one-shot combined vaccines against porcine circovirus associated disease (PCV AD) and enzootic pneumonia: at different PCV2 ORF2 concentration/dose.
• PCV AD porcine circovirus associated disease
• enzootic pneumonia at different PCV2 ORF2 concentration/dose.
• the objective of this study was to assess the safety and efficacy of different combined M. hyopneumoniae and PCV2 intradermal vaccines when formulated with different concentrations of PCV2 ORF2 protein per dose and also with different concentrations of mineral oil against enzootic pneumonia (EP) and porcine circovirus associated disease (PCVAD) in piglets.
• EP enzootic pneumonia
• PCVAD porcine circovirus associated disease
• a total of 50 piglets, either male or female, of 4 weeks of age free of antibodies against M.hyopneumoniae and with low levels of maternal derived antibodies against PCV2 were used in this study.
• the piglets were allotted to 4 treatment groups of 10 animals each (A to D), Group E acted as a sentinel group. Each group received a different vaccination treatment at day 0 of the study.
• Group A vaccinated intradermally with a single dose of 0.2 ml of a combined vaccine comprising 3x10 10 inactivated M. hyopneumoniae cells and 1.25 pg of PCV2 ORF2 protein per dose, adjuvanted with 21.2 % mineral oil and 0.5 % of a block copolymer.
• Group B vaccinated intradermally with a single dose of with 0.2 ml of a vaccine comprising 2.2x10 10 inactivated M. hyopneumoniae cells and 1.49 pg of PCV2 ORF2 protein per dose, containing as adjuvant 15 % of mineral oil and 0.5 % of block copolymer.
• Group C the piglets were co-administered of two commercially available vaccines.
• the piglets received 0.2 ml of the vaccine PORCIUS® MHYO ID ONCE (MSD Animal Health) administered intradermally, and 2 ml of PORCIUS® PCV2 (MSD Animal Health) given intramuscularly in the opposite side of the neck.
• PORCIUS® MHYO ID ONCE MSD Animal Health
• PORCIUS® PCV2 MSD Animal Health
• Group D this group received 2 ml of PBS intramuscularly, as non-vaccinated challenged group.
• Group E this group received 0.2 ml of PBS intradermally, as non-vaccinated non- challenged group.
• a co-infection model of PCV2 and M. hyopneumoniae was used to evaluate the vaccines efficacy.
• the experimental challenge was carried out by intranasal infection four weeks after vaccination for groups A, B, C and D.
• Group E was not infected and served as non- vaccinated non-challenged control.
• the piglets received an inoculum of (i) 3 ml of virulent PCV2b strain inoculated intranasally by cannula to each animal (1.5 ml to each nostril), and (ii) 5 ml/piglet of virulent M.hyopneumoniae strain inoculated by cannula (2.5 ml to each nostril).
• the M.hyopneumoniae challenge was repeated for 3 consecutive days. At day 28 after challenge (study day 56) pigs were humanely euthanized. In order to assess the efficacy of the combined vaccines. M. hyopneumoniae lung lesions were scored. Tonsil, inguinal, mesenteric and mediastinal lymph nodes were collected for PCV2 quantification by Q-PCR. Furthermore, local and systemic reactions were evaluated during the study.
• piglets were observed for general health. Body temperatures were recorded for all piglets on day 0 before vaccination, at 4 and 6 hours after vaccination, and on days 1 , 2 and 3 post-vaccination. Local reactions were also recorded at day 0 (at 4 and 6 hours after vaccination), and on days 1 , 2, 3, 7 and 14 post-vaccination. The mean temperature increase was calculated for each group. All vaccinated groups had a slight increase in the average body temperature at 6 hours post vaccination. The maximum temperature increase was 1.03 °C in Group A, which is acceptable for swine vaccines.
• the combined Mhyo and PCV2 intradermal vaccines A and B resulted in good safety profile.
• the combined Mhyo and PCV2 intradermal A and B vaccines are even safer than when vaccinating with commercially available monovalent vaccines, as the monovalent Porcilis® MHYO ID ONCE vaccine led to important local reactions until day 14 in comparison to the bivalent intradermal A and B vaccines, in which local lesions were seen to be reduced at day 2 post vaccination.
• the main variable for challenge efficacy evaluation was the pulmonary lesion scoring.
• the score was calculated as described in Example 1.
• the main variable for efficacy evaluation was the reduction of PCV2 viremia determined by Q-PCR. Seroconversion to PCV2, proportion of PCR positive animals, and clinical signs were also evaluated. PCV2 IgG antibodies in sera were analysed at days 7, 14, 21 and 28 post-infection using Porcine Circovirus type 2 Antibody Test Kit (BioChek). S/P ratios were calculated ( Figure 6). The results demonstrate the efficacy of all vaccines, as all vaccinated groups increased significantly the seroconversion when compared to control groups.
• Q-PCR was performed on sera and nasal swabs at days 7, 14, 21 and 28 post-infection (days of study 35, 42, 49, 56). PCV2 genomic DNA in each sample was quantified by PCR. At the end of the study all animals were necropsied and PCV2 titer in different tissues such as lung, tonsil, mediastinal, mesenteric, and inguinal lymph nodes were analyzed.
• PCV2 porcine circovirus type 2

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