multivalent immunogenic and vaccine compositions and their use for immunization against mycoplasma h

专利摘要:
PCV/MYCOPLASMA HYOPNEUMONIAE COMBINATION VACCINE. This invention provides a multivalent immunogenic composition, including a soluble portion of a total cell preparation of Mycoplasma hyopneumoniae (M.hyo); and a porcine circovirus type 2 (PCV2) antigen, wherein the soluble portion of the M.hyo preparation is substantially free of (i) IgG and (ii) immunocomplexes comprised of immunoglobulin-bound antigen.
公开号:BR112014024785B1
申请号:R112014024785-4
申请日:2013-04-03
公开日:2021-06-08
发明作者:Jeffrey E. Galvin；John Keith Garrett；James R. Kulawik Ii；Tracy L. Ricker；Megan Marie Smutzer；Gregory P. Nitzel
申请人:Zoetis Services Llc；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention relates to the porcine circovirus and Mycoplasma hyopneumoniae (M. hyopneumoniae or M.hyo). More particularly, the invention relates to a multivalent immunogenic composition including a soluble portion of a M.hyo whole cell preparation and a PCV2 antigen and its use in a vaccine to protect pigs against enzootic pneumonia and Multisystemic Wasting Piglet Syndrome Weaned (PMWS). FUNDAMENTALS OF THE INVENTION
[002] Enzootic pneumonia in pigs, also called mycoplasma pneumonia, is caused by M.hyo. The disease is a chronic, non-fatal disease that affects pigs of all ages. Infected pigs show only mild symptoms of coughing and fever, but the disease has a significant economic impact, due to the effectiveness of reduced nutrition and reduced weight gain. Enzootic pneumonia is transmitted from one pig to another through the nasal passages by airborne organisms expelled from the lungs of infected pigs. Primary M.hyo infection may be followed by secondary infection with another mycoplasma species (Mycoplasma hyorhinis and Mycoplasma flocculare) as well as other bacterial pathogens.
[003] M.hyo is a small, prokaryotic microbe capable of a free living existence, although it is often found in association with eukaryotic cells, as it has absolute requirements for sterols and exogenous fatty acids. These requirements generally require growth in serum containing medium. M.hyo is bound by a cell membrane, but not by a cell wall.
[004] The physical association of mycoplasmas with the host cell surface is the basis for the development and persistence of enzootic pneumonia. M.hyo infects the respiratory tract of pigs, colonizing the trachea, bronchi and bronchioles. Mycoplasma produces a ciliostatic factor that causes the cilia to cover the respiratory passages until they stop beating. Eventually, the cilia degenerate, leaving the pig prone to infection by secondary pathogens. Characteristic lesions of purple to gray patches of consolidation are seen in infected animals. Analyzes of slaughtered animals revealed lesions in 30 to 80% of the pigs. Results from 37 herds in 13 states indicated that 99% of the herds had pigs with pneumonia lesions typical of enzootic pneumonia. Therefore, the need for effective preventive and treatment means is great.
[005] Antibiotics such as tiamulin, trimethoprim, tetracyclines and lincomycin have some benefit but are expensive and require long-term use. Additionally, antibiotics have been shown to effectively eliminate M.hyo diffusion or reinfection. Prevention by keeping herds free of pathogen is sometimes possible, but reintroduction of M.hyo often occurs. Due to the serious economic consequences of swine pneumonia, vaccines against M.hyo were sought. Vaccines containing preparations of mycoplasma organisms grown in serum-containing medium have been commercialized but raise concerns regarding adverse reactions induced by serum components (such as immune complexes or specific non-immunogenic proteins) present in the immunizing material. Other attempts to provide vaccines against M. hyo have been successful, but the disease remains widespread.
[006]M.hyo and porcine circovirus type 2 (PCV2) are the two most prevalent pathogens that are found in the swine industry. Pigs infected with PCV2 exhibit a syndrome commonly referred to as Multisystemic Weaning Piglet Wasting Syndrome (PMWS). PMWS is clinically characterized by wasting, pale skin, respiratory distress, diarrhea and jaundice. In addition to PMWS, PCV2 has been associated with several other infections, including pseudorabies, porcine reproductive and respiratory syndrome (PRRS), Glasser's disease, streptococcal meningitis, salmonellosis, post-weaning colibacillosis, dietary hepatosis, and suppurative bronchopneumonia. M.hyo is associated with enzootic pneumonia and has also been implicated as a major cofactor in the development of Porcine Circovirus Associated Disease (PCVAD).
[007] Porcine reproductive and respiratory syndrome (PRRS) is caused by an arterivirus, which has a particular affinity for macrophages, particularly those found in the lung (alveolar macrophages). These macrophages ingest and remove invading bacteria and viruses, but not in the case of PRRS virus (PRRSV). In the case of PRRS virus, it multiplies inside macrophages producing more virus and killing macrophages. Once PRRSV enters a herd, it tends to remain present and indefinitely active. Up to 40% of macrophages are destroyed, which allows bacteria and other viruses to proliferate and do damage. A common example of this is the remarkable increase in the severity of enzootic pneumonia in grower/finisher units when they become infected with PRRS virus. More than half of PRRS-negative pigs of weaning age become infected before going to market.
What is needed is a PCV2/M.hyo combination vaccine against PCV2 and mycoplasma infection in pigs. Preferably, this multivalent vaccine will be compatible with other swine antigens, such as the PRRS virus antigen. It would be highly desirable to provide a ready-to-use PCV2/M.hyo combination vaccine in a single-dose vial. SUMMARY OF THE INVENTION
[009] The present invention provides a multivalent immunogenic composition, including a soluble portion of a total cell preparation of Mycoplasma hyopneumoniae (M.hyo); and a porcine circovirus type 2 (PCV2) antigen, wherein the soluble portion of the M.hyo preparation is substantially free of (i) IgG and (ii) immunocomplexes comprised of immunoglobulin-bound antigen. In one aspect, the soluble portion of the M.hyo total cell preparation was treated with protein A or protein G before being added to the immunogenic composition. In another aspect, the composition is in the form of a ready-to-use liquid composition.
In one embodiment, the soluble portion of the M.hyo preparation includes at least one M.hyo protein antigen. In another embodiment, the soluble portion of the M.hyo preparation includes two or more M.hyo protein antigens.
[0011] In some embodiments, the composition of the present invention induces a protective immune response against M.hyo and PCV2. In one embodiment, the PCV2 antigen is in the form of a chimeric type-1-type 2 circovirus, the chimeric virus, including an inactivated recombinant type 1 porcine circovirus that expresses the ORF2 protein of the porcine circovirus type 2. In another embodiment, the PCV2 antigen is in the form of a recombinant ORF2 protein. In yet another embodiment, the recombinant ORF2 protein is expressed from a baculovirus vector.
[0012] In some embodiments, the PCV2/M.hyo composition of the present invention further includes at least one additional antigen. In one embodiment, at least one additional antigen is protective against a microorganism that can cause disease in pigs.
[0013] In one embodiment, the microorganism includes bacteria, viruses or protozoa. In another embodiment, the microorganism is selected from, but not limited to, the following: porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), Haemophilus parasuis, Pasteurella multocida, Streptococcum suis, Staphylococcus hyicus, Actinobacilllus pleuropneumoniae, Bordetella bronchiseptica, Salmonella choleraesuis, Salmonella enteritidis, Erysipelothrix rhusiopathiae, Mycoplama hyorhinis, Mycoplasma hyosynoviae, leptospira bacteria, Lawsonia intracellularis, Influenza swine influenzae virus, Erythromycine, Erysphine, Erysphine, Erysphine, swine influenzae, E. coli, E. coli. Swine (PED), Rotavirus, Torque Teno Virus (TTV), Porcine Cytomegalovirus, Porcine Enterovirus, Encephalomyocarditis Virus, a pathogen causing Aujesky's Disease, Classical Swine Fever (CSF) and a pathogen causing Transmissible Gastroenteritis to Swine, or combinations thereof.
[0014] In some embodiments, the composition of the present invention further includes an adjuvant. In one embodiment, the adjuvant is selected from, but not limited to, the following: an oil-in-water adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an aluminum hydroxide adjuvant, an vitamin E adjuvant and combinations thereof. In another embodiment, the composition of the present invention further includes a pharmaceutically acceptable carrier.
In certain embodiments, the composition of the present invention induces a protective immune response against M.hyo and PCV2 when administered as a single dose administration. In other embodiments, the composition induces a protective immune response against M.hyo, PCV2, and at least one additional microorganism that can cause disease in pigs when administered as a single dose administration. In yet other embodiments, a composition of the present invention induces a protective response against M.hyo and PCV2 when administered as a two-dose administration.
The present invention also provides a method for immunizing a pig against M.hyo and PCV2. This method includes administering to the pig an immunogenic composition, including a soluble portion of a whole cell preparation of Mycoplasma hyopneumoniae (M.hyo); and a porcine circovirus type 2 (PCV2) antigen, wherein the soluble portion of the M.hyo preparation is substantially free of (i) IgG and (ii) immunocomplexes comprised of immunoglobulin-bound antigen. In one embodiment, the soluble portion of the M.hyo preparation of the administered composition includes at least one M.hyo protein antigen.
[0017] In one embodiment of the method of the present invention, the composition is administered intramuscularly, intradermally, transdermally or subcutaneously. In another embodiment of the method of this invention, the composition is administered in a single dose. In yet another embodiment of the method of this invention, the composition is administered as two doses.
[0018] In a further embodiment of the method of the present invention, the PCV2/M.hyo composition is administered in combination with at least one additional antigen that is protective against a microorganism that can cause disease in pigs, such as one or more of the microorganisms described above. Such other antigens can be supplied concurrently with the PCV2/M.hyo composition (ie, as separate single vaccines) or combined into a ready-to-use vaccine.
[0019] In a further modality, the composition is administered to pigs that have maternally derived antibodies against at least one between M.hyo and PCV2. In yet a further modality, the composition is administered to pigs that have maternally derived antibodies against M.hyo and PCV2.
In one embodiment, the composition is administered to pigs 3 weeks of age or older.
[0021] The present invention further provides a kit. This kit contains a vial including an immunogenic composition. This immunogenic composition includes a PCV2 antigen and the soluble portion of a total cell preparation of Mycoplasma hyopneumoniae (M.hyo), wherein the soluble portion of the M.hyo preparation is substantially free of (i) IgG and (ii) immune complexes of antigen/immunoglobulin. In one embodiment, this kit further includes an instruction manual that contains information for administering the immunogenic composition. In another embodiment, the immunogenic composition in the vial is provided as a ready-to-use liquid composition.
[0022] Additionally, the present invention provides a method for preparing an immunogenic composition. This method includes: i) cultivating M.hyo in a suitable medium for periods ranging from 18 to 144 hours; ii) subsequently inactivating the M. hyo culture; iii) collecting the inactivated culture fluid, wherein the inactivated culture fluid comprises a whole cell preparation of M.hyo comprising a soluble liquid fraction and insoluble cell material; iv) separating the soluble liquid fraction from the insoluble cellular material; v) substantially removing IgG and antigen/immunoglobulin immunocomplexes from the separated soluble liquid fraction to form a soluble portion of the total M.hyo cell preparation; and vi) subsequently combining the soluble portion of the total M.hyo cell preparation with a PCV2 antigen. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 is a graph showing the efficacy of M.hyo monovalent vaccines prepared with M.hyo antigens from different treatments (T02 to T10 described in Example 3) vs. a placebo (T01). Results are presented as % Least Squares Mean Lung Injury values.
[0024] Figure 2 is a graph showing the results of PCV2 antigen potency (PCV2 ELISA antigen) of vaccines in combination of M.hyo with killed PCV Type1-Type2 chimeric virus. The chimeric virus was included in the compositions at an initial level of about 1.6<RP. The state of each sample is expressed as relative potency (RP).
Figure 3 is a graph showing PCV2 viremia (PCV2 Quantitative PCR) results observed with PCV/M.hyo vaccine formulations using different adjuvant platforms.
Figure 4 is a graph showing the PCV2 antibody (S/P) ELISA serological results observed with PCV/M.hyo vaccine formulations using different adjuvant platforms on days 1, 20 and 42 of challenge.
[0027] Figure 5 is a graph showing the fecal excretion of PCV2 obtained with treatments T02 to T04 described in Example 7 vs. a placebo (T01). Results are expressed as PCV2/ml DNA copies.
[0028] Figure 6 is a graph showing nasal excretion of PCV2 obtained with treatments T02 to T04 described in Example 7 vs. the placebo (T01). Results are expressed as PCV2/ml DNA copies.
[0029] Figure 7 (A and B) are graphs showing the results of an interferon-gamma (IFN-Y) assay that measures PCV2-specific cellular mediated immune responses (CMI). The post-vaccination/pre-challenge results are shown in Figure 7A, and the post-vaccination/post-challenge results are shown in Figure 7B. The 5 x 106 cell stimulus was considered significant.
Figure 8 represents the M.hyo efficacy of PCV2/M.hyo experimental vaccine formulations in SP-oil. Lung scores for formulations using treatments from M.hyo T02 to T08 vs. a placebo (T01) are graphically represented in Figure 8A. The table in Figure 8B represents the contrast of treatments T02 to T08 with placebo.
[0031] Figure 9 is a flowchart showing one embodiment of a manufacturing process used to prepare M.hyo antigen treated with PCV2-compatible Protein A.
[0032] Figure 10 is a table showing the assessment of adjuvant for virucidal activity against PRRS virus. BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 is an embodiment of a nucleotide sequence encoding p46 from M.hyo strain P-5722;
SEQ ID NO: 2 is one embodiment of an amino acid sequence corresponding to p46 from M.hyo strain P-5722;
SEQ ID NO:3 is an embodiment of a nucleotide sequence encoding p97 from M.hyo strain P-5722;
SEQ ID NO: 4 is an embodiment of an amino acid sequence corresponding to p97 from M.hyo strain P-5722;
SEQ ID NO: 5 is an embodiment of a genomic sequence encoding a chimeric PCV1-2 virus;
SEQ ID NO: 6 is an embodiment of a nucleotide sequence corresponding to ORF2 of a porcine circovirus;
SEQ ID NO: 7 is an embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 8 is an embodiment of a genomic sequence encoding a chimeric PCV1-2 virus;
SEQ ID NO: 9 is an embodiment of a nucleotide sequence corresponding to ORF2 of a porcine circovirus;
SEQ ID NO: 10 is an embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 11 is an embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 12 is an embodiment of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11;
SEQ ID NO: 13 is an embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 14 is an embodiment of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13;
SEQ ID NO: 15 is an embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 16 is an embodiment of a genomic sequence of a non-virulent form of the North American PRRS virus isolate designated P129; and
SEQ ID NO: 17 is an embodiment of a nucleotide sequence corresponding to ORF2 to ORF5 of the PRRS virus isolate designated ISU-55.
SEQ ID NO: 18 is an embodiment of a nucleotide sequence corresponding to ORF6 and ORF7 of the PRRS virus isolate designated ISU-55. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a multivalent immunogenic composition, including a soluble portion of a total cell preparation of Mycoplasma hyopneumoniae (M. hyo); and a porcine circovirus type 2 (PCV2) antigen, wherein the soluble portion of the M. hyo preparation is substantially free of (i) IgG and (ii) immunocomplexes comprised of immunoglobulin-bound antigen. In one embodiment, the composition induces a protective immune response in a pig against PCV2 and M.hyo.
Applicants surprisingly found that the insoluble fraction of the total cell preparation of M.hyo is non-immunogenic. In contrast, the IgG-free soluble M.hyo preparation is immunogenic and can be effectively combined with antigens from other pathogens, such as PCV2, without analytical or immunological interference between the antigens. This makes the soluble M.hyo preparation an effective platform for the multivalent vaccines of this invention, including one-vial and ready-to-use formulations. Applicants have also surprisingly found that the removal of immunoglobulin and insoluble cellular debris from the M.hyo preparation enhances the safety of the immunogenic composition.
[0053] As used in the specification and claims, the singular form “a”, “an” and “the”, “a” include plural references, unless the context clearly indicates otherwise. For example, the term "a protein antigen" includes a plurality of protein antigens, including mixtures thereof.
[0054] As used in this report, the term “comprising” is intended to mean that compositions and methods include the elements cited but do not exclude other elements.
[0055] As defined in this report, a soluble portion of a total M.hyo cell preparation refers to a liquid soluble fraction of a total M.hyo cell preparation after separation of insoluble material and substantial removal of IgG and bound immune complexes to the antigen. The soluble portion of M.hyo may alternatively be referred to in this report as the supernatant fraction, culture supernatant and the like. It includes soluble M.hyo-expressed proteins (M.hyo protein antigens) that have been separated or isolated from insoluble proteins, whole bacteria, and other insoluble M.hyo cellular material by conventional means such as centrifugation, filtration, or precipitation. In addition to including soluble M.hyo specific proteins, the soluble portion of the total M.hyo cell preparation also includes heterologous proteins, such as those contained in the culture medium used for M.hyo fermentation.
The term "antigen" refers to an immunogenic compound, composition or substance that can stimulate the production of antibodies or a T cell response, or both, in an animal, including compositions that are injected or absorbed into an animal. The immune response can be generated to the whole molecule or to a portion of the molecule (eg, an epitope or hapten).
[0057] As defined in this report, an "immunogenic or immunological composition" refers to a composition of matter that comprises at least one antigen that induces an immune response in the host to a cellular and/or antibody-mediated immune response to the composition or vaccine of interest.
[0058]The term “immune response”, as used in this report, refers to a response induced in an animal. An immune response can refer to cellular immunity (CMI); humoral immunity or may involve both. The present invention also contemplates a limited response to a part of the immune system. Usually, an "immune response" includes, but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells and/or cytotoxic T cells and/or T cells yd, specifically directed to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will exhibit a therapeutic or protective immune response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by a reduction or lack of symptoms normally exhibited by an infected host, a faster recovery time and/or a decreased viral titer in the infected host.
[0059] As used in this report, the term “immunogenicity” means capable of producing an immune response in a host animal against an antigen or antigens. This immune response forms the basis of the protective immunity induced by a vaccine against a specific infectious organism.
[0060] An "adjuvant", as used in this report, means a composition comprised of one or more substances that enhance the immune response to an antigen(s). The mechanism of how an adjuvant operates is not fully understood. Some adjuvants are believed to enhance the immune response through slow release of the antigen, while other adjuvants are strongly immunogenic and believed to work synergistically.
[0061] As used in this report, the term “multivalent” means a vaccine containing more than one antigen from the same species (ie, different Mycoplasma hyopneumoniae isolates), from a different species (ie, isolates a from Pasteurella hemolytica and Pasteurella multocida), or a vaccine containing a combination of antigens from different genera (eg a vaccine comprising antigens from Pasteurella multocida, Salmonella, Escherichia coli, Haemophilus somnus and Clostridium).
[0062] The term “pig” or “piglet”, as used in this report, means an animal of swine origin, while “sow” refers to a female of age and reproductive capacity. A "pig" is a female pig that has never been pregnant.
[0063] As used in this report, the term “virulent” means an isolate that retains its ability to be infectious in a host animal.
[0064]“Inactivated vaccine” means a vaccine composition containing an infectious organism or pathogen that is no longer capable of replication or growth. The pathogen can be bacterial, viral, protozoal or fungal in origin. Inactivation can be accomplished by a variety of methods, including freeze-thaw, chemical treatment (eg, treatment with thimerosal or formalin), sonication, radiation, heat or any other means of convention sufficient to prevent the organism from replicating or growing, maintaining its immunogenicity.
[0065] The term "variant", as used in this report, refers to a polypeptide or a nucleic acid sequence that encodes a polypeptide, which has one or more conservative amino acid variations or other minor modifications such that the corresponding polypeptide has substantially equivalent function when compared to wild-type polypeptide.
[0066] "Conservative variation" denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically residue residue similar. Examples of conservative variations include replacing a hydrophobic residue such as isoleucine, valine, leucine or methionine with another hydrophobic residue, or replacing a polar residue such as replacing arginine with lysine, glutamic acid with aspartic acid or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted precursor amino acid, provided that antibodies recruited to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
[0067] As used in this report, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable carrier" are interchangeable and refer to a fluid vehicle to contain vaccine antigens that can be injected into a host without adverse effects. Suitable pharmaceutically acceptable carriers known in the art include, but are not limited to sterile water, saline, glucose, dextrose or buffered solutions. Carriers can include auxiliary agents, including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colorants, and the like.
[0068] As used in this report, the term "vaccine composition" includes at least one antigen or immunogen in a pharmaceutically acceptable carrier useful for inducing an immune response in a host. Vaccine compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary field, taking into account such factors as the age, sex, weight, species and condition of the recipient animal, and the route of administration. The route of administration can be percutaneous, mucosal route of administration (for example, oral, nasal, anal, vaginal) or via a parenteral route (intradermal, transdermal, intramuscular, subcutaneous, intravenous or intraperitoneal). Vaccine compositions can be administered alone or can be co-administered or sequentially administered with other treatments or therapies. Forms of administration may include suspensions, syrups or elixirs and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (for example, injectable administration) such as sterile suspensions or emulsions. Vaccine compositions can be administered as a spray or mixed with food and/or water or released in admixture with a suitable carrier, diluent or excipient such as sterile water, physiological saline, glucose or the like. The compositions may contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colorants, and the like, depending on the route of administration and the preparation desired. Standard pharmaceutical texts, such as “Remington's Pharmaceutical Sciences,” 1990 can be consulted to manufacture adequate preparations without undue experimentation.
[0069] "North American PRRS virus" means any PRRS virus that exhibits genetic characteristics associated with a North American PRRS virus isolate, such as, but not limited to, the PRRS virus that was first isolated in the United States at the beginning the 1990s (see, for example, Collins, JE, et al., 1992, J. Vet. Diagn. Invest. 4:117 - 126); North American PRRS virus isolate MN-1b (Kwang, J. et al., 1994, J. Vet. Diagn. Invest. 6:293 - 296); the Quebec LAF-exp91 strain of PRRS virus (Mardassi, H. et al., 1995, Arch. Virol. 140:1405 - 1418); and North American PRRS virus isolate VR 2385 (Meng, X.-J et al., 1994, J. Gen. Virol. 75:1795 - 1801). Additional examples of North American PRRS virus strains are described in this report. Genetic traits refer to the genomic nucleotide sequence similarity and the amino acid sequence similarity shared by the strains of the North American PRRS virus. Chinese PRRS virus strains generally show about 80 to 93% nucleotide sequence similarity to North American strains.
[0070]“European PRRS virus” refers to any strain of PRRS virus that exhibits the genetic characteristics associated with the PRRS virus that was first isolated in Europe around 1991 (see, for example, Wensvoort, G., et al., 1991, Vet. Q. 13:121 - 130). The "European PRRS virus" is also sometimes referred to in the art as "Lelystad virus". Other examples of European PRRS virus strains are described in this report.
[0071] A genetically modified virus is "attenuated" if it is less virulent than its unmodified parental strain. A strain is “less virulent” if it shows a statistically significant decrease in one or more parameters determining disease severity. Such parameters may include level of viremia, fever, severity of respiratory distress, severity of reproductive symptoms or number or severity of lung lesions, etc.
[0072] An "infectious clone" is an isolated or cloned genome of the disease agent (eg, virus) that can be specifically and determinedly modified in the laboratory and then used to recreate the genetically modified living organism. A genetically modified live virus produced from the infectious clone can be used in a live viral vaccine. Alternatively, inactivated virus vaccines can be prepared by treating live virus derived from the infectious clone with inactivating agents such as formalin or hydrophobic solvents, acids, etc., by irradiation with ultraviolet light or X-rays, by heating, etc.
All currently available M.hyo and M.hyo combination vaccines are prepared from killed total cell mycoplasma (bacterins) preparations. Rather, the present invention envisages a soluble portion of a total cell preparation of Mycoplasma hyopneumoniae (M.hyo) for combination with the PCV2 antigen, wherein the soluble portion of the M.hyo preparation is substantially free of (i) IgG and (ii) immunocomplexes comprised of immunoglobulin-bound antigen.
[0074]M.hyo has absolute requirements for sterols and exogenous fatty acids. These requirements generally call for growth of M.hyo in serum containing medium such as porcine serum. Separating the insoluble material from the soluble portion of the M.hyo total cell preparation (eg, by centrifugation, filtration or precipitation) does not remove IgG or porcine immune complexes. In one embodiment of the present invention, the soluble portion of M.hyo is treated with protein A or protein G so as to substantially remove IgG and immune complexes contained in the culture supernatant. In this embodiment, it is understood that treatment with protein A occurs after fermentation of M.hyo. This is alternatively referred to in this report as downstream protein A treatment. In another embodiment, treatment with protein A upstream of the growth medium (ie, prior to M.hyo fermentation) can be used. Protein A binds to the Fc portion of IgG. Protein G preferentially binds to the Fc portion of IgG, but can also bind to the Fab region. Methods to purify/remove total IgG from crude protein mixtures such as tissue culture supernatant, serum and ascitic fluid, are known in the art.
In some embodiments, the soluble portion of the M.hyo preparation includes at least one M.hyo protein antigen. In other embodiments, the soluble portion of the M.hyo preparation includes two or more M.hyo protein antigens.
In one embodiment, the M.hyo supernatant fraction includes one or more of the following M.hyo-specific protein antigens: M.hyo proteins of molecular weights of approximately 46 kD (p46), 64 kD ( p64) and 97 kD (p97). In another embodiment, the supernatant fraction at least includes the M.hyo protein antigens p46, p64 and p97. The approximately 64 kD M.hyo protein (p64) may alternatively be referred to in this report as the p65 surface antigen from M.hyo described by Kim et al. [Infected Immun. 58(8):2637 - 2643 (1990)], as well as in U.S. Patent No. 5,788,962.
[0077] Futo et al. described the cloning and characterization of a 46 kD surface protein from M.hyo, which can be used in the compositions of this invention [J. Bact 177: 1915 - 1917 (1995)]. In one embodiment, the M.hyo culture supernatant includes p46, whose corresponding nucleotide and amino acid sequences from strain P-5722 are shown in SEQ ID NOs: 1 and 2, respectively. It is further contemplated that variants of such p46 sequences can be used in the compositions of the present invention, as described below.
[0078]Zhang et al. described and characterized an M.hyo p97 adhesin protein [Infect. Immun. 63: 1013 - 1019, 1995]. Additionally, King et al. described a 124 kD protein called Mhp1 from the M.hyo strain P-5722 and presented data suggesting that Mhp1 and p97 are the same protein [Vaccine 15:25 - 35 (1997)]. Such p97 proteins can be used in the compositions of this invention. In one embodiment, the M.hyo culture supernatant includes p97, whose corresponding nucleotide and amino acid sequences from strain P-5722 are shown in SEQ ID NOs: 3 and 4, respectively. It is further contemplated that variants of such p97 sequences can be used in the compositions of the present invention, as described below.
The M.hyo culture supernatant may include other M.hyo-specific protein antigens, such as, but not limited to, the approximately 41 kD (p41), 42 kD (p42), 89 kD (p89) proteins and 65 kD (p65). See, Okada et al., 2000, J. Vet. Med. B 47:527 - 533 and Kim et al., 1990, Infect. Immun. 58(8):2637 - 2643. In addition, the M.hyo culture supernatant may include M.hyo-specific protein antigens of approximately 102 kD (p102) and 216 kD (p216). See, U.S. Patent Nos. 6,162,435 and 7,419,806 to Minnion et al.
[0080] Any strain of M.hyo can be used as a starting material to produce the soluble portion of the M.hyo preparation of the compositions of the present invention. Suitable strains of M.hyo can be obtained from commercial or academic sources, including depositories such as the American Type Culture Collection (ATCC) (Manassas, Va.) and the NRRL Culture Collection (Agricultural Research Service, US Department of Agriculture , Peoria, Ill.). ATCC alone lists the following six strains of M.hyo for sale: M.hyo ATCC 25095, M.hyo ATCC 25617, M.hyo ATCC 25934, M.hyo ATCC 27714, M.hyo ATCC 27715 and M.hyo ATCC 25934D . A preferred strain of M.hyo for use in the embodiments of this invention is identified as strain P-5722-3, ATCC #55052, filed May 30, 1990, pursuant to accessibility rules required by the US Patent and Trademark Office. In view of the widespread spread of the disease, strains can also be obtained through M.hyo recovery from lung or tissue secretions from pigs infected with strains known to cause mycoplasma pneumonia in pigs.
[0081] It is understood by those of skill in the art that variants of the M.hyo sequences can be used in the compositions of the present invention. Such variants can vary by 10 to 20% in sequence identity and still retain the antigenic characteristics that make them useful in immunogenic compositions. Preferably the M.hyo variants have at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% sequence identity with the full length genomic sequence of the strain. of wild type M.hyo. The antigenic characteristics of an immunological composition can be, for example, estimated by the challenge experiment as provided in the Examples. Furthermore, the antigenic characteristic of a modified M.hyo antigen is still retained when the modified antigen confers at least 70%, preferably 80%, more preferably 90% of protective immunity compared to the M.hyo-like protein wild.
[0082] In one embodiment, the soluble M.hyo p46 antigen is included in the compositions of the invention at a final concentration of about 1.5 µg/ml to about 10 µg/ml, preferably about 2 µg/ml at about 6 µg/ml. It is noted that p46 is the protein used for the M.hyo potency test (see example section below). In another embodiment, the M.hyo antigen may be included in the compositions in a final amount of from about 5.5% to about 35% of the total M.hyo protein A-treated supernatant.
[0083] The soluble M.hyo preparation of the present invention is safe and effective against M.hyo and is suitable for single dose administration. Furthermore, Applicants surprisingly found that the soluble M.hyo preparation can be effectively combined with antigens from other pathogens, including PCV2, without immunological interference between the antigens. This makes the soluble M.hyo preparation an effective platform for multivalent vaccines, including the PCV2/M.hyo combination vaccine of this invention. The PCV2 antigen can be supplied concurrently with the M.hyo composition (ie, as separate single vaccines), but is preferably combined in a ready-to-use single vial vaccine.
In one embodiment, the immunogenic PCV2/M.hyo compositions of the present invention include at least one additional antigen. In one embodiment, at least one additional antigen is protective against a microorganism that can cause disease in pigs.
[0085] In some embodiments, at least one additional antigen component is protective against bacteria, viruses or protozoa that infect pigs. Examples of such microorganisms include, but are not limited to the following: porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), Haemophilus parasuis, Pasteurella multocida, Streptococcum suis, Staphylococcus hyicus, Actinobacilllus pleuropneumoniae, Bordetella choleraesuis, Salmonella enteritidis, Erysipelothrix rhusiopathiae, Mycoplama hyorhinis, Mycoplasma hyosynoviae, leptospira bacteria, Lawsonia intracellularis, swine influenza virus (SIV), Escherichia coli antigen, Brachyspira hysenteriae, swine rotademic virus, Diarin respiratory virus Torque Teno virus (TTV), Porcine Cytomegalovirus, porcine enteroviruses, Encephalomyocarditis virus, an Aujesky Disease-causing pathogen, Classical Swine Fever (CSF) and a pathogen causing Transmissible Swine Gastroenteritis, or combinations thereof.
[0086] In one embodiment, a PCV2/M.hyo combination vaccine, in accordance with the present invention, is provided as a single-dose vaccine, ready for use in a single vial. Such a ready-to-use combination vaccine does not require mixing of separate vaccines, thus there is no risk of contamination or additional labor associated with mixing and no requirement to use the mixture within a few hours. In addition, a single-vial PCV2/M.hyo combination vaccine cuts waste and refrigerator space for storage in half. Furthermore, single dose administration eliminates the labor associated with administering a second dose to the animal. It is noted that although PCV2/M.hyo combination vaccines currently exist, they are supplied as a ready-to-use two-dose vaccine (Circumvent®PCVM) or as a single-dose, 2-vial vaccine that requires simultaneous administration of separate vaccines (eg Ingelvac CircoFLEX® and Ingelvac MycoFLEX®). Preferably, the PCV2/M.hyo combination according to the present invention would be compatible with other antigens, such as the PRRS virus antigen, such that all antigens can be administered in a single dose.
In some embodiments, the PCV2 antigen component of a PCV2/M.hyo combination vaccine is in the form of a chimeric type-1-type-2 circovirus. The chimeric virus includes an inactivated recombinant porcine circovirus type 1 that expresses the porcine circovirus type 2 ORF2 protein. Chimeric porcine circoviruses and methods for their preparation are described in WO 03/049703 A2, and also in US Patent Nos. 7,279,166 and 7,575 .752, which are fully incorporated herein by reference.
In one embodiment, the full-length DNA sequence of the chimeric PCV1-2 virus genome corresponds to SEQ ID NO: 5. or variants thereof, as described below. In another embodiment, the immunogenic ORF2 capsid gene of the chimeric PCV1-2 virus corresponds to SEQ ID NO: 6. In a further embodiment, the amino acid sequence of the immunogenic ORF2 protein expressed by the chimeric PCV1-2 virus corresponds to SEQ ID NO: 7.
[0089] In yet another embodiment, the full-length DNA sequence of the chimeric PCV1-2 virus genome corresponds to SEQ ID NO: 8. In one embodiment, the chimeric PCV1-2 virus immunogenic ORF2 capsid gene corresponds to SEQ ID NO: 9. In a further embodiment, the amino acid sequence of the immunogenic ORF2 protein expressed by the chimeric PCV1-2 virus corresponds to SEQ ID NO: 10.
However, PCV2 ORF2 DNA and chimeric PCV1-2 virus protein are not limited to the sequences described above, as PCV2 ORF2 DNA and protein is a highly conserved domain in PCV2 isolates.
In some embodiments, the PCV2 antigen component of a M.hyo/PCV2 combination vaccine is in the form of a recombinant ORF2 protein. In one embodiment, the recombinant ORF2 protein is expressed from a baculovirus vector. Alternatively, other known expression vectors can be used, such as, including, but not limited to, parapox vectors.
In one embodiment, the recombinant PCV2 ORF2 protein is that of SEQ ID NO: 11, which is encoded by SEQ ID NO: 12 (GenBank Accession No AF086834). In another embodiment, the recombinant ORF2 protein is that of SEQ ID NO:13, which is encoded by SEQ ID NO:14. In yet another embodiment, the recombinant ORF2 protein corresponds to SEQ ID NO:15. In another embodiment, the recombinant PCV2 ORF2 protein corresponds to SEQ ID NO: 7. In a further embodiment, the recombinant PCV2 ORF2 protein corresponds to SEQ ID NO: 10.
[0093] However, the present invention is not limited to the particular DNA and ORF2 protein sequences described above. Since the PCV2 ORF2 DNA and protein is a highly conserved domain within PCV2 isolates, any PCV2 ORF2 is likely to be highly effective as the source of the PCV2 ORF2 DNA and/or polypeptide as used in the PCV1 virus. 2 chimeric or recombinant PCV2 protein.
[0094] An example of a suitable PCV2 isolate from which PCV2 ORF2 DNA and protein sequences can be derived is PCV2 isolate number 40895 (deposited with ATCC on December 7, 2001 and ATCC Patent Deposit Designation PTA -3914 determined). The genomic sequence (nucleotide) of PCV2 isolate number 40895 is available under GenBank accession number AF264042. Other examples of suitable PCV2 isolates from which the PCV2 ORF2 DNA and protein sequences can be derived include, but are not limited to the following: Imp.999, Imp.1010-Stoon, Imp.1011-48121 and Imp. 1011-48285. The GenBank accession numbers of the genomic sequences corresponding to such PCV2 isolates are AF055391, AF055392, AF055393 and AF055394, respectively.
[0095] In some forms, the immunogenic portions of the PCV2 ORF2 protein are used as the antigenic component in the composition. For example, truncated and/or substituted forms or fragments of the PCV2 ORF2 protein can be used in the compositions of the present invention.
[0096] It is understood by those of skill in the art that variants of the PCV2 sequences can be used in the compositions of the present invention. Such variants can vary by 10 to 20% in sequence identity and still retain the antigenic characteristics that make them useful in immunogenic compositions. Preferably the PCV2 variants have at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% sequence identity with the full length genomic sequence of the PCV2 isolate of the wild type. The antigenic characteristics of an immunological composition can be, for example, estimated by the challenge experiment provided in the Examples. Furthermore, the antigenic characteristic of a modified PCV2 antigen is still retained when the modified antigen confers at least 70%, preferably 80%, more preferably 90% protective immunity compared to wild-type PCV2 ORF2 protein.
[0097] The PCV2 antigen component is provided in the immunogenic composition at an antigen inclusion level effective to induce the desired immune response, ie, reducing the incidence or decreasing the severity of clinical signs resulting from PCV2 infection.
[0098] In one embodiment, a chimeric PCV1-2 virus is included in the compositions of the invention at a level of at least 1.0 < RP < 5.0, where RP is the unit of Relative Potency determined by ELISA antigen quantification (in vitro potency test) compared to a reference vaccine. In another embodiment, a chimeric PCV1-2 virus is included in the composition of the invention at a final concentration of from about 0.5% to about 5% of 20 times (20X) the PCV1-2 antigen in bulk concentrate.
[0099] In another embodiment, the recombinant PCV2 ORF2 protein is included in the compositions of the invention at a level of at least 0.2 µg of antigen/ml of the final immunogenic composition (µg/ml). In a further embodiment, the inclusion level of the recombinant PCV2 ORF2 protein is from about 0.2 to about 400 µg/ml. In yet another embodiment, the inclusion level of the recombinant PCV2 ORF2 protein is from about 0.3 to about 200 µg/ml. In yet a further embodiment, the inclusion level of the recombinant PCV2 ORF2 protein is from about 0.35 to about 100 µg/ml. In yet another embodiment, the inclusion level of the recombinant PCV2 ORF2 protein is from about 0.4 to about 50 µg/ml.
[00100] In one embodiment, an immunogenic composition of the present invention includes the inventive combination of at least one soluble M.hyo antigen (for example, two or more) and a porcine circovirus type 2 (PCV2) antigen, as well as a PRRS virus antigen. In another modality, the composition induces a protective immune response in a pig against M.hyo, PCV2 and PRRS virus.
[00101] In one embodiment, a PCV2/M.hyo/PRRS combination vaccine is provided as a single-dose, 2-vial vaccine. For example, in some embodiments, a PCV2/M.hyo combination is provided as a stable liquid composition in a first vial and a PRRS virus is provided in a lyophilized state in a second vial. In some embodiments, additional swine antigens can be added to the first or second vial.
[00102] In one embodiment, the PRRS virus component is provided as a genetically modified live lyophilized virus. Prior to administration, PCV2/M.hyo liquid from a first vial can be used to rehydrate the PRRS virus in a second vial so that all three antigens can be administered to the animal in a single dose. It is noted that although PCV2/M.hyo/PRRS combination vaccines currently exist, they are supplied as a single-dose, 3-vial vaccine that requires simultaneous administration of the three separate vaccines (eg, Ingelvac CircoFLEX®, Ingelvac MycoFLEX® and Ingelvac®PRRS MLV).
[00103]The etiologic agent of PRRS was first isolated in the Netherlands, and named as Lelystad virus. This virus has been described in WO 92/21375 (Stichting Centraal Diegeneeskundig Instituut). A European PRRS virus isolate has been deposited at the Institut Pasteur de Paris, number I-1102. The North American type virus was isolated almost simultaneously with the isolation of the European type virus, and is described in WO 93/03760 (Collins et al.) A North American type virus isolate has been deposited with the American Type Culture Collection (ATCC), number VR-2332.
[00104]Different strains have been isolated from European and North American virus types. WO 93/07898 (Akzo) describes a European strain, and vaccines derived therefrom, deposited with the CNCM (Institut Pasteur), number I-1140. Furthermore, WO 93/14196 (Rhone-Merieux) describes a new strain isolated in France, deposited at the CNCM (Institut Pasteur), number I-1153. Furthermore, EP0595436 B1 (Solvay) describes a new North American type strain, more virulent than that initially described, and vaccines thereof. This strain has been filed with the ATCC, but the filing number is not detailed in the patent application. Furthermore, ES2074950 BA (Cyanamid Iberica) and its counterpart GB2282811 B2 describe a so-called “Spanish strain”, which is different from other European and North American strains. This "Spanish strain" has been deposited with the European Animal Cell Culture Collection (EACCC), number V93070108.
PRRS virus antigens suitable for use in the PCV2/M.hyo/PRRS compositions of the present invention include North American PRRS virus isolates, Chinese PRRS virus strains and European PRRS virus strains as well. as genetically modified versions of such isolates/strains. In one embodiment, the PRRS virus antigen component used in the compositions in accordance with the present invention is a North American PRRS virus.
[00106] In some embodiments, the PRRS virus antigen component used in the compositions of this invention is the North American PRRS virus isolate designated P129 or a live, genetically modified version thereof. Preferably, the genetically modified PRRS virus is incapable of producing a pathogenic infection and is capable of inducing an effective immunoprotective response against wild-type PRRS virus infection.
[00107] A genetically modified PRRS virus for use in the compositions of the invention can be produced from an infectious clone. The preparation of an infectious cDNA clone from the North American PRRS virus isolate designated P129 is described in U.S. Patent No. 6,500,662 which is incorporated in its entirety by reference. The cDNA sequence of P129 is disclosed in Genbank Accession Number AF494042 and in U.S. Patent No. 6,500,662.
[00108] In one embodiment, the nucleotide sequence of a non-virulent form of P129 for use in the compositions of the present invention is represented by SEQ ID NO: 16. However, the present invention is not limited to this sequence. This sequence and the sequences of other non-virulent forms of P129 are described in International Application No. PCT/IB2011/055003, filed November 9, 2011, the contents of which (including any US National Stage deposits based on this International Application) are fully incorporated in this report for reference. Preferably, the PRRS virus is modified to prevent interferon-mediated downregulation of function.
[00109] In other embodiments, the PRRS virus antigen component used in the compositions of the invention is the PRRS virus isolate designated ISU-55. Isolated ISU-55 has been deposited with the American Type Culture Collection (ATCC) under accession number VR2430. The nucleotide sequence of the ORF2 to ORF5 genes of the ISU-55 isolate is represented by SEQ ID NO:17. The nucleotide sequence of the ORF6 and ORF7 genes of the ISU-55 isolate is represented by SEQ ID NO: 18.
[00110] Another suitable North American PRRS virus isolate that can be used in the compositions is ISU-12, which has been deposited with the ATCC under accession numbers VR2385 [3 x purified plaque] and VR2386 [unpurified plaque]. Other suitable North American PRRS virus isolates that can be used in the compositions of this invention are as follows: ISU-51, ISU-3927, ISU-1894, ISU-22 and ISU-79, which have been deposited with the ATCC under the numbers of access VR2498, VR2431, VR2475, VR2429 and VR2474, respectively. Genetically modified versions of any one of these ISU isolates can be used in the compositions of this invention. These ISU isolates and the ISU-55 isolate are described in detail in the following US Patents to Paul, et al.: US 5,695,766, 6,110,467, 6,251,397, 6,251,404, 6,380,376, 6,592,873, 6,773 .908, 6,977,078, 7,223,854, 7,264,802, 7,264,957 and 7,517,976, all of which are fully incorporated herein by reference.
[00111] Still in other modalities, the component of the PRRS virus antigen used in the compositions, according to the present invention, is the North American type deposited in the American Type Culture Collection (ATCC), number VR-2332 or a genetically modified version modified of the same. For example, the PRRS virus can be a live virus modified based on the isolate identified as ATCC VR2332, which is used in INGELVAC® PRRS ATP and INGELVAC® PRRS MLV, from Boehringer Ingelheim Vetmedica, Inc.
[00112] In still other embodiments, the PRRS virus antigen component used in the compositions of the present invention is a European PRRS virus isolate or Lelystad virus or a genetically modified version thereof. An example of a suitable PRRS virus strain is identified as deposit No. I-1102, described above. Nucleotide and amino acid sequences corresponding to deposit I-1102 are described in U.S. Patent No. 5,620,691 to Wensvoort et al., which is incorporated in this report in its entirety by reference. The preparation of an infectious clone of an isolate of European PRRS virus or Lelystad virus is described in U.S. Patent No. 6,268,199 which is incorporated in its entirety by reference herein.
[00113]Other examples of suitable PRRS virus isolates include, but are not limited to those described above. Furthermore, live and genetically modified versions of the PRRS virus isolates can be used in the compositions of the present invention. An infectious clone can be used to recreate such genetically modified living organisms.
It is understood by those of skill in the art that variants of the PRRS virus sequences can be used in the compositions of the present invention. Such variants can vary by 10 to 20% in sequence identity and still retain the antigenic characteristics that make them useful in immunogenic compositions. Preferably, the PRRS virus variants have at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% sequence identity with the full length genomic sequence of the isolate of wild-type PPRS virus. The antigenic characteristics of an immunological composition can be, for example, estimated by challenge experiments. Furthermore, the antigenic characteristic of a modified PRRS virus antigen is still retained when the modified antigen confers at least 70%, preferably 80%, more preferably 90% of protective immunity compared to PRRS virus-type antigen wild.
[00115] In one embodiment, the PRRS virus antigen component is a genetically modified live virus that is included in the compositions of the invention at a level of at least 2.1 < TCID50 < 5.2, where TCID50 is 50% of the tissue culture infectious dose determined by antigen quantification (in vitro potency test).
The PCV2 antigen component of the PCV2/M.hyo/PRRS compositions of the invention may be in the form of a chimeric type-1-type 2 circovirus, the chimeric virus including an inactivated recombinant type 1 swine circovirus that expresses the Porcine circovirus type 2 ORF2 protein. In another embodiment, the PCV2 antigen component of the PCV2/M.hyo/PRRS compositions of the invention is in the form of a recombinant ORF2 protein.
PCV2 antigens suitable for use in the PCV2/M.hyo/PRRS compositions can be derived from any of the PCV2 isolates described above, as well as other PCV2 isolates. Suitable PCV2 antigens that can be used in the compositions of the invention include, but are not limited to, the PCV2 sequences described above and variants thereof.
[00118] The vaccines of the present invention may be formulated following accepted convention to include acceptable carriers for animals, including humans (if applicable), such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and may also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Other suitable vaccine carriers and additives, including those that are particularly useful in the formulation of modified live vaccines, are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
The vaccines of the present invention may further comprise one or more additional immunomodulatory components, such as, for example, an adjuvant or cytokine, among others. Types of adjuvants suitable for use in the compositions of the present invention include the following: an oil-in-water adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an aluminum hydroxide adjuvant, a vitamin adjuvant. And and combinations thereof. Some specific examples of adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, Corynebacterium parvum, Bacillus Calmette Guerin, aluminum hydroxide gel, glycan, dextran sulfate, iron oxide, sodium alginate, Bacto -Adjuvant, certain synthetic polymers such as polyamino acids and amino acid copolymers, block copolymer (CytRx, Atlanta, Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Massa.), SAF-M (Chiron, Emeryville Calif. .), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A and Avridine lipid-amine adjuvant (N,N-dioctadecyl-N',N'--bis(2-hydroxyethyl)-propanediamine) , "REGRESSIN" (Vetrepharm, Athens, Ga.), paraffin oil, RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), muramyl dipeptide, and the like.
[00120] Non-limiting examples of oil-in-water emulsions useful in the vaccine of the invention include modified SEAM62 and SEAM 1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) of TWEEN® 80 detergent (ICI Surfactants), 2.5 % (v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol and 0.5 % (v/v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween 80 detergent , 2.5% (v/v) ethanol, 100 µg/ml Quil A and 50 µg/ml cholesterol.
[00121] Another example of an adjuvant useful in the compositions of the invention is SP-oil. As used in the specification and claims, the term "SP oil" designates an oil emulsion comprising a polyoxyethylene-polyoxypropylene block copolymer, squalane, polyoxyethylene sorbitan monooleate and a buffered saline solution. Polyoxyethylene-polyoxypropylene block copolymers are surfactants that aid in suspending solid and liquid components. These surfactants are commercially available as polymers under the trade name Pluronic®. The preferred surfactant is poloxamer 401 which is commercially available under the trade name Pluronic® L-121. In general, the SP oil emulsion is an immunostimulating adjuvant mixture which will comprise about 1 to 3% vol/vol block copolymer, about 2 to 6% vol/vol squalane, more particularly about 3 to 6% of squalane, and about 0.1 to 0.5% vol/vol of polyoxyethylene sorbitan monooleate, with the remainder being a buffered saline solution. In one embodiment, the SP-oil emulsion is present in the final composition in v/v amounts of about 1% to 25%, preferably about 2% to 15%, more preferably about 5% to 12% v/ v.
[00122] Another example of an adjuvant suitable for use in the compositions of the invention is AMPHIGEN™ adjuvant which consists of oil-free lecithin dissolved in an oil, usually light liquid paraffin.
[00123] Other examples of adjuvants useful in the compositions of the invention are the following patented adjuvants: Microsol Diluvac Forte® duel emulsion adjuvant system, Emunade adjuvant and Xsolve adjuvant. Emunade and Xsolve adjuvants are light mineral oil-in-water emulsions, but Emunade also contains alhydrogel, and d,l-α-tocopheryl acetate is part of the XSolve adjuvant. Another example of an adjuvant suitable for use in the compositions of the invention is the ImpranFLEX™ adjuvant (a water-in-oil adjuvant). Another example of a suitable adjuvant is a Carbomer based adjuvant (Carbopol®). Preferred Carbopol® adjuvants include Carbopol® 934 polymer and Carbopol® 941 polymer.
[00124] In one embodiment, the adjuvant or adjuvant mixture is added in an amount from about 100 µg to about 10 mg per dose. In another embodiment, the adjuvant/adjuvant mixture is added in an amount from about 200 µg to about 5 mg per dose. In yet another embodiment, the adjuvant/adjuvant mixture is added in an amount from about 300 µg to about 1 mg/dose.
The adjuvant or adjuvant mixture is typically present in the vaccine composition of the invention in v/v amounts of about 1% to 25%, preferably about 2% to 15%, more preferably about 5% to 12% v/v.
[00126] Other "immunomodulators" that can be included in the vaccine include, for example, one or more interleukins, interferons or other known cytokines. In one embodiment, the adjuvant can be a cyclodextrin derivative or a polyanionic polymer, such as that described in U.S. Patent Nos. 6,165,995 and 6,610,310, respectively.
[00127] Another aspect relates to a method for preparing an immunogenic composition, according to the present invention. This method comprises i) cultivating M.hyo in a suitable medium for periods ranging from 18 to 144 hours; ii) subsequently inactivating the M. hyo culture; iii) collecting the inactivated culture fluid, wherein the inactivated culture fluid comprises a whole cell preparation of M.hyo comprising a soluble liquid fraction and insoluble cell material; iv) separating the soluble liquid fraction from the insoluble cellular material; v) substantially removing IgG and antigen/immunoglobulin immunocomplexes from the separated soluble liquid fraction to form a soluble portion of the total M.hyo cell preparation; and vi) subsequently combining the soluble portion of the M.hyo total cell preparation with a PCV2 antigen.
[00128]An example of a suitable medium to cultivate M.hyo is the PPLO Broth (Mycoplasma Broth Base), which, when supplemented with nutritional enrichments, is used to isolate and cultivate Mycoplasma.
[00129] In some modalities, the M.hyo culture is grown until the last log phase of growth, after which the culture is inactivated. In other embodiments, the culture is inactivated by raising the pH (eg, to about 7.8). This occurs by exposing the production crop to an inactivating agent such as binary ethyleneimine (BEI). BEI is generated in situ during the incubation of L-bromoethylamine hydrobromide (BEA) in the production culture. Subsequently, the pH of the inactivated culture is neutralized, such as by adding an equivalent amount of an agent that neutralizes the inactivating agent in the solution. In some embodiments, the inactivating agent is BEI and the neutralizing agent is sodium thiosulfate. In one embodiment, the pH of the inactivated culture is adjusted to about 7.4 through the addition of sodium thiosulfate.
[00130] In some embodiments, the soluble liquid fraction of the total cell preparation of M.hyo is separated from the insoluble cellular material using conventional methods. In one embodiment, this separation takes place through a filtration step. In another modality, this separation takes place through a centrifugation step. In yet another modality, the separation takes place through a precipitation step.
In one embodiment, the liquid soluble fraction of an inactivated and neutralized M.hyo whole cell preparation is treated with Protein A resin to substantially remove IgG and antigen/immunoglobulin immune complexes therein. In other embodiments, the Protein G resin can be used to substantially remove IgG and antigen/immunoglobulin immune complexes contained in the soluble liquid fraction. Methods to remove IgG and antigen/immunoglobulin immunocomplexes with Protein A or Protein G resins are well known in the art.
[00132] According to another aspect, the method for preparing a multivalent immunogenic composition according to the present invention comprises preparing the soluble M.hyo antigen, as described above, and mixing this with a PCV2 antigen, a suitable adjuvant and one or more pharmaceutically acceptable carriers. This method optionally includes adding at least one additional swine antigen, such as, but not limited to, the PRRS virus antigen, as described above.
[00133] Another aspect of the present invention relates to a kit. A “kit” refers to a plurality of components that are grouped together. In one embodiment, a kit according to the present invention includes a vial (or other suitable container) comprising an immunogenic composition. This immunogenic composition includes a PCV2 antigen and the soluble portion of a total cell preparation of Mycoplasma hyopneumoniae (M.hyo), wherein the soluble portion of the M.hyo preparation is substantially free of (i) IgG and (ii) immune complexes of antigen/immunoglobulin. Optionally, the kit can also include an instruction manual. The instruction manual includes information for administering the immunogenic composition.
[00134] In some embodiments, the PCV2/M.hyo combination in the kit vial is supplied as a ready-to-use liquid composition. In other embodiments, the kit includes a second vial comprising PRRS virus antigen. In some embodiments, the PRRS virus antigen is in the form of a genetically modified live virus that is supplied in a lyophilized state. In such examples, the instruction manual will include directions to rehydrate the PRRS virus component with the liquid contents from the vial containing the PCV2/M.hyo combination. The instruction manual will also include information for administering the resulting trivalent PCV2/M.hyo/PRRS formulations.
[00135] In some embodiments, an immunogenic composition, in accordance with this invention, is administered to pigs that have maternally derived antibodies against M.hyo. In other embodiments, an immunogenic composition of the present invention is administered to pigs that have maternally derived antibodies against M.hyo and PCV2.
[00136] In some embodiments, a multivalent immunogenic composition, in accordance with the present invention, is administered to a piglet 3 weeks or older. However, it is considered that a multivalent vaccine composition according to the invention can also be used to re-vaccinate pre-generated gilts. As is known in the art, a sow is a female pig that has never been pregnant. Vaccinated gilts will pass maternally derived antibodies during breastfeeding to the newborn via colostrum.
[00137] It is still considered that a multivalent vaccine, according to the invention, can be used to annually revaccinate production herds. Preferably, a multivalent vaccine according to the present invention is administered to pigs (eg piglets or gilts) in one dose. In one embodiment, a multivalent vaccine according to the present invention does not require mixing of separate monovalent vaccines prior to administration, i.e. it is supplied as a ready-to-use PCV2/M.hyo formulation contained in a vial. In another embodiment, a multivalent formulation requires mixing a divalent vaccine, according to the present invention, contained in a first vial with a monovalent vaccine contained in a second vial. In one embodiment, the monovalent vaccine contained in the second vial includes PRRS virus antigen. Optionally, additional antigens can be added to any of these vials.
[00138] In some embodiments, the onset of immunity is 2 to 3 weeks post-vaccination with a multivalent vaccine composition in accordance with the present invention. In other embodiments, the duration of immunity is about 17 to 23 weeks post-vaccination with a multivalent vaccine composition in accordance with the present invention.
[00139] The following examples present preferred materials and procedures according to the present invention. However, it should be understood that these examples are provided by way of illustration only, and should not be a limitation on the overall scope of the invention.EXAMPLESEXAMPLE 1: METHODS OF PRODUCING MYCOPLASMA HYOPNEUMONIAE FOR M.HYO ANTIGEN COMBINABLE WITH PCV2FERMENTATION AND M INACTIVATION .HYO
[00140]The medium for seed scaling and antigen production was prepared as follows. Organism Broth (PPLO) of the Pleuropenumonia type derived from porcine heart (BD Biosciences catalog No. 21498) was prepared per manufacturer's directions (ie 21 g/L) and yeast extract solution was prepared at 21 g/L in USP . The yeast extract solution was then added to the PPLO at 6.25% and the mixture was sterilized by heating at 121°C for >30 minutes. Cysteine hydrochloride was prepared at 90 g/L and filter sterilized. Dextrose solution was prepared by adding 450 g of dextrose per liter of USP water, followed by heat sterilization. To prepare the final medium, swine serum was added to the base medium at 10%, followed by cysteine at 0.01% and dextrose at 1.0%. The medium was inoculated with 10% v:v of a log phase culture of M. hyopeumoniae (strain P-5722-3). The culture was maintained at 37 °C and pH and dO were maintained at 7.0 and 25%, respectively. In the last log phase growth, the culture was inactivated by binary ethylenimine (BEI), an aziridine compound produced from 2-bromoethylamine hydrobromide. Specifically, inactivation occurred by raising the pH to 7.8 by adding 2-bromoethylamine hydrobromide (BEA) to a final concentration of 4 mM and incubating for 24 hours. BEI was neutralized by addition of sodium thiosulfate in a 1:1 molar ratio, followed by an additional 24 hour incubation. The inactivated culture fluid was held at 2 to 8°C until further processing. EXAMPLE 2: METHODS OF PRODUCTION OF SWINE CIRCOVIRUS (CPCV)1-2 CHIMERIC
[00141] The cPCV1-2 was constructed by cloning the immunogenic capsid gene of the pathogenic porcine circovirus type 2 (PCV2) into the genomic structure of the non-pathogenic porcine circovirus type 1 (PCV1). The procedure for constructing the chimeric DNA clone is described, for example, in U.S. Patent No. 7,279,166, which is incorporated in this report in its entirety by reference. An infectious stock of chimeric virus was purchased from Dr. XJ Meng, Virginia Polytechnic Institute and State University, Blacksburg, VA, and was used to infect porcine kidney (PK)-15 cells grown in supplemented Minimum Essential Medium (MEM) with 0.05% lactalbumin hydrolyzate (LAH), 30 μg/mL gentamicin sulfate and 5% fetal bovine serum. The resulting cPCV1-2 infected PK-15 cells were further expanded by serial passage four more times using the same growth medium, except with 2-3% fetal bovine serum. The fifth pass was frozen, thawed and filtered, and the resulting lysates were used to prepare a pre-inoculum and subsequent inoculum.
[00142] The medium that was used to produce virus seeds was the same used in the production of virus stock. For growth medium, MEM, OptiMEM or equivalent is the basal medium that can be used to plant the PK-15 cell line for growth. The growth medium can be supplemented with up to 10% bovine serum, up to 0.5% lactalbumin hydrolyzate, up to 0.5% bovine serum albumin and up to 30 μg/ml gentamicin. For virus propagation medium, MEM, OptiMEM, or equivalent is used. Virus propagation medium can be supplemented with up to 0.5% lactalbumin hydrolyzate, up to 2% bovine serum, up to 0.5% bovine serum albumin and up to 30 μg/mL gentamicin. Up to 5 g/L glucose and up to 5 mmols/L L-glutamine can be added to the growth medium and/or virus propagation medium as required to sustain the cells.
The cPCV1-2 inoculum virus is added to a cell suspension of PK-15 cells and adsorbed within 3 hours. Seed virus is diluted in basal growth medium to provide a multiplicity of infection (MOI) of 0.1 to 0.0001.
[00144]PK-15 cell cultures are initially inoculated with working seed virus in the cell planting period, or when cells reach approximately 20% to 50% confluence. This initial passage can be referred to as the “One Step Infection Method” for the production of antigen stock, or it can be further used for serial passages. For serial passages, cPCV1-2 infected PK-15 cells are further expanded to passage 7 by serial separations in the ratio of 1:5 to 20 for virus propagation. Culture medium containing a cell suspension infected from the previous passage serves as seed material for the next passage. Cells infected with cPCV1-2 are incubated for three (3) to 14 days for each passage at 36 ± 2°C when cells reach >90% confluency. The cPCV1-2 virus causes observable cytopathic changes during viral replication. On collection, cell rounding and considerable floating debris are observed. Cultures are also observed for visual evidence of bacterial or fungal contamination. The incubation time between collections for cPCV antigen is given in Table 1 below: Table 1: Minimum and Maximum Times for cPCV Antigen Collection

[00145]CPCV1-2 culture fluids are collected in sterile vessels and are experienced for mycoplasma testing using known methods. Multiple collections can be conducted from roller bottles, bioreactors and perfusion vessels.
[00146]Prior to inactivation of the collected cPCV1-2 virus, one or more lots of antigen can be concentrated (eg up to 60X) by ultrafiltration. Concentrates can be washed with balanced salt solution to reduce whey protein.
[00147] The method of inactivating, attenuating or detoxifying the cPCV1-2 virus will now be described. After cPCV antigen concentration, Betapropiolactone (BPL) is added to the pooled cPCV1-2 viral material to obtain an approximate concentration of 0.2 % v/v. The pooled viral fluids are then agitated for a minimum of 15 minutes and then the inactivated bulk antigen fluids are transferred to a second sterile vessel. The transferred antigen fluids are kept at 2 to 7°C, with constant agitation, for a minimum of 24 hours. After a minimum of 24 hours, a second addition of 0.2% v/v GLP is added to the pooled suspension. The contents are subsequently shaken, transferred to a third vessel, and held at 2 to 7°C, with constant agitation, for an additional time of not less than 84 hours. In general, the total inactivation time is no less than 108 hours and no longer than 120 hours. The inactivation method is summarized in Table 2 below.Table 2: Inactivation Method

[00148]Inactivation is terminated by the addition of a final concentration of not more than 0.1 M sodium thiosulfate solution. The pH of the inactivated antigen stock is adjusted to about 6.8 using NaOH or HCl. After inactivation, a representative sample is taken from the cluster and tested for inactivation completion. The inactivated cPCV1-2 antigen product is standardized to find a target greater than 1.0 RP, as measured via ELISA potency. ANTIGENS OF M. HYO:
The inactivated fermentation fluid (prepared as described above in Example 1) was treated for each group indicated as follows. These processed M.hyo antigens were used in Example 4 below.
[00150]T02: (Total Bulk) Not processed.
[00151]T03: (10X concentrated UF) Concentrated by means of tangential flow filtration via a 100 KDa molecular weight cut-off membrane (hollow fiber). The final volume reduction was equal to 10X.
[00152]T04 and T05: (10X concentrated and centrifuged UF) Concentrated mycoplasma cells (from T03) were collected and washed once with PBS by means of centrifugation at ~20,000 x g (Sorvall model RC5B).
[00153]T06 and T07: (10X centrifuged) The inactivated fermentation fluid was centrifuged at ~20,000 x g (Sorvall RC5B) and washed once by resuspension of the cells in PBS, followed by an additional centrifugation. The final volume reduction was equal to 10X.
[00154]T08: (10X centrifuged and warmed) Mycoplasma cells were concentrated and washed to T06 and heated to 65°C for 10 minutes.
[00155]T09: (Cell-free supernatant) The supernatant collected from the first centrifugation as described for T06 was filter sterilized through a 0.2 micron filter (Nalgene).
[00156]T10: (treated with cell-free supernatant-Protein-A) Sterile supernatant (prepared for T09) was mixed with Protein A resin (Protein A Sepharose, Pharmacia Inc) at a volume ratio of 10:1 during 4 hours. The resin was removed by sterile filtration and the filtered fluid was stored at 2 to 8°C. This process uses post-fermentation “downstream” protein A treatment to remove antibodies and immune complexes. Although the present invention does not eliminate treatment with upstream protein A, the present inventors found that in the case of M.hyo, treatment with protein A upstream of the growth medium led to p46 results that were smaller and inconsistent in comparison. to untreated medium (data not shown). ANALYTICAL TEST OF DOWNSTREAM M.HYO ANTIGENS
Downstream processed M.hyo antigen preparations (prepared as described above) were tested for M.hyo specific p46 antigen recovery, and the presence of PCV2 antibody. In addition, these M.hyo antigen preparations were tested for the presence of Torque Teno Virus (TTV), including genotype 1 (g1TTV) and genotype 2 (g2TTV). The results are shown below in Table 3. Table 3: Characterization of . Downstream Processed M.hyo Antigens


[00158]Referring to Table 3 above, M.hyo specific p46 antigen recovery was demonstrated for each of the downstream processed M.hyo antigen preparations. In addition, the following treatments successfully removed PCV2 antibody: 10X UF concentrated and centrifuged, 10X centrifuged, 10X centrifuged and heated, and cell-free supernatant (treated with Protein A). With respect to TTV, the following treatments successfully removed g1TTV: 10X UF concentrated and centrifuged, 10x centrifuged and heated, and cell-free supernatant (treated with Protein A). Only the treatment designated 10X concentrated and centrifuged UF removed g2TTV. Torque teno virus isolates, including genotypes 1 and 2, are described in US 20110150913, which is incorporated in this report in its entirety by reference.
[00159] Since it is known in the art that Protein A binds to IgG, it is understood by those of ordinary skill in the art that not only the PCV2 antibody, but other porcine antibodies, including PRRS antibody, HPS antibody and SIV antibody, will be effectively removed by Protein A treatment. This makes the cell-free Protein A-treated M.hyo supernatant of this invention compatible not only with the PCV2 antigen, but also with other swine antigens, due to the lack of immunological interference between the antigens . Additionally, removal of non-protective cellular debris and removal of immunoglobulin and antigen/immunoglobulin complexes is reasonably expected to prepare a safer vaccine.EXAMPLE 4: PREPARATION OF M.HYO EXPERIMENTAL VACCINE FORMULATIONS
[00160]All experimental M.hyo vaccines were formulated with a final concentration of 5% Amphigen adjuvant. Furthermore, all vaccines were standardized with a p46 ELISA and preserved with thimerosol. Experimental vaccine formulations were prepared with processed M.hyo antigens according to treatments T02 to T10 above. Furthermore, Treatment T01 was matched to a placebo (no M.hyo antigen, only 5% Amphigen adjuvant), whereas Treatment T11 was a positive control corresponding to an expired bacterin-based M.hyo vaccine ( RespiSure-ONE®, Pfizer Animal Health). These formulations are represented in Table 4 below. Table 4: Experimental M.hyo Vaccine Formulations

*Research Veterinary Product (IVP) Series EXAMPLE 5: IN VIVO EFFECTIVENESS ASSESSMENT OF M.HYO VACCINES WITH M.HYO ANTIGENS FROM DIFFERENT DOWNSTREAM PROCESSES
This study was conducted to evaluate the in vivo efficacy of Mycoplasma hyopneumoniae (M hyo) vaccines with M hyo antigens from different downstream processes (DSP). 3 week old pigs were intramuscularly inoculated with a single dose of the different vaccine formulations described in Table 4 above. Sixteen animals were included in each of the treatment groups. Animals were challenged 21 days after vaccination with a virulent M.hyo field isolate. The animals were submitted to necropsy 28 days after the challenge and the lungs were removed and recorded for consolidation compatible with M.hyo infection. The primary criterion for protection against M.hyo challenge was lung consolidation records. It is generally accepted that there is a relationship between the size of lung lesions caused by enzootic pneumonia and an adverse effect on growth rate. Table 5 below contains the lung injury records for the respective treatment groups. Statistical significance was determined by a Mixed Model Analysis of lung records for each group.Table 5: Lung Injury Results


[00162] Referring to Table 5 above, results with M.hyo antigens from different downstream processes indicated that all experimental vaccines except T04 were significantly different from placebo. These M.hyo injury results are graphically represented in Figure 1. As shown in Figure 1, T04 provided unacceptable results. All other treatments were significantly different from placebo (T01). Lung consolidation records indicated that T02, T03 and T09 to T11 provided the most effective protection against M.hyo challenge.
The relative potency of p46 of the experimental vaccines was assessed through the use of an antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA). The p46 DAS ELISA results shown in Table 5 above indicate that all experimental vaccines exceeded target potency. Furthermore, the relative potency of p46 was maintained or increased during storage of the vaccines for a period of one month (data not shown). A perceived increase in potency over time was observed in centrifuged antigens with the exception of those antigens that were subjected to heat. Without limiting the invention to any theory, it is likely that cellular “carcasses” will break down over time and release more of the membrane-bound p46 antigen in the case of centrifuged antigens.EXAMPLE 6: EVALUATION OF COMPATIBILITY OF EXPERIMENTAL M.HYO VACCINES WITH PCV2 ANTIGEN
[00164]This study was conducted to evaluate the compatibility of experimental M.hyo vaccines with M hyo antigens from different downstream processes with the PCV2 antigen. Experimental M.hyo vaccine formulations are described in Tables 4 and 5 above. The relative potencies of p46 observed for these vaccines are described in Table 5 above. These experimental M.hyo vaccines were combined with the PCV2 antigen. In this example, the PCV2 antigen was a killed PCV Type 1-Type 2 chimeric virus (Fostera PCV) prepared as described above in Example 2. The chimeric virus was included in the compositions at an initial level of about 1.6 < RP, where RP is the unit of relative potency determined by quantification of PCV2 antigen ELISA (in vitro potency test) compared to an effective reference vaccine.
Experimental M.hyo/PCV2 combination formulations were evaluated by PCV2 ELISA. The results are shown in Figure 2. As shown in Figure 2, only M.hyo antigen preparations from the following downstream processes were compatible with the PCV2 antigen: Ultrafiltration and Centrifugation (T04 and T05), Centrifugation (T06 and T07), Centrifugation plus heat (T08) and Protein A treated supernatant (T10). Among these, the M.hyo supernatant treated with Protein A was the most compatible with PCV2 antigen when compared to the placebo control that included the chimeric virus and Amphigen adjuvant, but no M.hyo antigen. The level of PCV chimeric virus in the Protein A-treated supernatant was 1.5 PR compared to 1.69 PR for placebo. Therefore, it was concluded that there is minimal or no immunological interference between the soluble M.hyo antigen preparation treated with Protein A and the chimeric virus PCV2 antigen.
The in vivo efficacy of the Protein A treated M.hyo supernatant demonstrated in Example 5 above together with the results described in the present example indicated that the Protein A treated supernatant was a potentially effective platform for the M.hyo combinations -PCV2.EXAMPLE 7: EVALUATION OF THE EFFECTIVENESS OF PCV2 OF A 1-BOTTLE PCV2/M.HYO COMBINATION VACCINE IN DIFFERENT ADJUVANT FORMULATIONS
This study was designed to evaluate the efficacy of PCV2 in a 1-vial PCV2/M.hyo combination vaccine in different adjuvant formulations. In this example, the PCV2 antigen was a killed PCV Type 1-Type 2 chimeric virus (Fostera PCV). The chimeric virus was combined with a soluble M.hyo antigen preparation that was substantially free of IgG (ie, Protein A-treated supernatant).FLUID PROCESSING:
Inactivated M.hyo fermentation fluid (described above in Example 1) was treated for each indicated group as follows.
[00169]T02-T04: Total fermentation fluid containing live M. hyopneumoniae cells (described above) was centrifuged at ~20,000 x g (Sorvall RC5B) and the supernatant collected and sterilized through a 0.2 μM filter. rProtein A Sepharose (part number 17-5199-03, GE Healthcare) was packed into a 1 L chromatography column. After removal of storage buffer and treatment with 2 column volumes of 1 M acetic acid, the resin was equilibrated with 5 column volumes of 50 mM NaPO4/1M NaCl buffer, pH 7.04. Approximately 2 liters of the fluids containing clean/filtered M. hyopneumoniae antigen were passed through the Protein A resin at a flow rate of 100 cm/h. The through flow was collected and sterilized through a 0.2 μM filter.
[00170]T05: This is a positive control corresponding to a Fostera PCV type formulation (no M.hyo antigen). The level of chimeric virus in this Fostera PCV-like formulation occurred approximately at Minimum Immunization Dose (MID) formulation levels. The chimeric virus was included in the PCV2/M.hyo experimental vaccines at similar formulation levels.
All experimental PCV2/M.hyo vaccines were formulated with different adjuvant formulations. Experimental vaccine formulations were prepared with processed M.hyo antigens according to treatments T02 to T04 above. In addition, Treatment T01 corresponded to a placebo (sterile saline solution).
All vaccines were standardized with a p46 ELISA and preserved with thimerosal.
[00173] These experimental formulations are described in Table 6 below, where the symbol * indicates the M hyo antigen from the global M hyo seed, supernatant treated with Protein A and the symbol ** indicates the Research Veterinary Product series (IVP).Table 6: PCV2/M.hyo Experimental Vaccine Formulations Used for PCV2 Efficacy Study


3 week old pigs were intramuscularly inoculated with a single dose of the different vaccine formulations described in Table 6 above. Sixteen animals were included in each of the treatment groups. Animals were challenged 21 days after vaccination with a virulent PCV2 field isolate.
[00175] Figure 3 is a graph showing the results of PCV2 viremia (PCV2 Quantitative PCR) observed with the different adjuvant platforms. It is noted that PCV2 viremia was used as the primary efficacy variable. PCV2 viremia results are presented as DNA copies/ml. As shown in Figure 3, all treatments had significantly less viremia compared to placebo on days 28, 35, and 42 (the challenge was on day 21). Adjuvant SP-Oil 10% had significantly less viremia compared to Amphigen 5% on Days 28 and 35. Adjuvant Amphigen 5% plus SLCD 5% had significantly less viremia compared to Amphigen 5% on Days 28 and 35. The 20% SLCD adjuvant platform had significantly less viremia compared to the 5% Amphigen on Days 28, 35 and 42.
PCV2 serology, PCV2 fecal excretion, PCV2 nasal excretion, Cell-Mediated Immune Responses (CMI), lymphoid depletion and Immunohistochemistry (IHC) were also monitored as secondary efficacy variables. These results will now be described below.
Figure 4 is a graph showing PCV2 ELISA results on days 1, 20 and 42 of the study (challenge was on day 21). The status of each sample was expressed as a sample to positive ratio (S/P). As shown in Figure 4, SLCD 20% was the only treatment that was significantly different from placebo (T01) on days 20 and 42. In addition, Amphigen 5% was the only treatment not significantly different from placebo on day 20.
[00178] Figure 5 is a graph showing the fecal excretion of PCV2 obtained with treatments T02 to T04 vs. the placebo (T01). These results are expressed as PCV2/ml DNA copies. The results in Figure 5 indicate that all treatments had significantly less fecal shedding compared to placebo on day 42. In addition, Amphigen 5% and SLCD 5% (T04) had significantly less fecal shedding compared to Amphigen 5% (T03) on day 42. No other treatment differences were observed.
[00179] Figure 6 is a graph showing the nasal excretion of PCV2 obtained with treatments T02 to T04 vs. the placebo (T01). These results are expressed as PCV2/ml DNA copies. The results in Figure 6 indicate that all treatments had significantly less nasal excretion when compared to placebo on day 42. In addition, SLCD 20% (T05) had significantly less nasal excretion compared to Amphigen 5% (T03) on day 42. No other differences in treatment were observed.
[00180] Figure 7 (A and B) are two graphs showing the results of an interferon-gamma (IFN-Y) test that measures PCV2-specific cell-mediated (CMI) immune responses. MIC results are shown post-vaccination/pre-challenge (Figure 7A) and post-vaccination/post-challenge (Figure 7B). In these graphs, the stimulus of 5 x 106 cells was considered significant (...). All experimental PCV2/M.hyo vaccines provided a detectable post-vaccination IFN-Y response. 10% SP-oil (T02) directed the strongest post-vaccination IFN-Y response. 20% SLCD (T05) induced an early response, but the lowest response on day 20. There was a large post-challenge response, especially seen in the placebo group. Additionally, the post-challenge response was lower in the vaccinated pig treatment groups compared to the placebo group.
[00181]Table 7 below shows the lymphoid depletion obtained with experimental treatments contrasted with placebo.Table 7: Histopathology of PCV2 (Lymphoid Depletion)

[00182] The results presented in Table 7 above show that all vaccines provided strong protection against lymphoid depletion. Furthermore, no statistically significant contrast from vaccine treatment was observed.
[00183] Table 8 below shows the immunohistochemistry obtained with experimental treatments contrasted with placebo.Table 8: Histopathology of PCV2 (Immunohistochemistry)

[00184] The results presented in Table 8 above show that all vaccines provided strong protection against PCV2 colonization, as evidenced by immunohistochemistry. Furthermore, no statistically significant contrast from vaccine treatment was observed.
In conclusion, the results presented in this example demonstrate that the soluble M.hyo antigen preparation does not interfere with the efficacy of PCV2. The results also show that all experimental PCV/M.hyo vaccine formulations provide efficacy against PCV2 challenge. Additionally, the results indicate that there are some statistical and numerical differences obtained with the different adjuvant formulations, with 10% SP-oil producing the strongest efficacy.EXAMPLE 8: EVALUATION OF THE EFFECTIVENESS OF M.HYO OF A PCV2 COMBINATION VACCINE /M.HYO 1 BOTTLE WITH DIFFERENT ADJUVANT FORMULATIONS
[00186]This study was designed to evaluate the M.hyo efficacy of a 1-vial PCV2/M.hyo combination vaccine with different adjuvant formulations. The M.hyo antigen was combined with porcine circovirus (Chimera Type 1-Type 2, or PCV1-2, killed virus) in a vial. FLUID PROCESSING:
Inactivated M.hyo fermentation fluid (described above in Example 1) was treated for each indicated group as follows.
[00188]T02 to T04: These treatments were the same as those described for treatment groups T02 to T04 in Example 7 above.
[00189]T05: This was formulated with inactivated M.hyo cells (M.hyo bacterin) as described in Example 1 above under the heading "Fermentation and Inactivation".
[00190]All experimental PCV2/M.hyo vaccines were formulated with different adjuvant formulations. Experimental vaccine formulations were prepared with processed M.hyo antigens according to treatments T02 to T04. In addition, Treatment T01 corresponded to a placebo (sterile saline solution). Treatment T05 is a positive control corresponding to an expired RespiSure® vaccine, which is a bacterin-based M.hyo vaccine (Pfizer Animal Health).
[00191] These experimental formulations are described in Table 9 below, where the symbol * indicates the M hyo antigen from the global M hyo seed, supernatant treated with Protein A and the symbol ** indicates the Research Veterinary Product series (IVP).Table 9: PCV2/M.hyo Experimental Vaccine Formulations Used for the Study of M.hyo Efficacy in Different Adjuvant Formulations


The 3 week old Pigs were intramuscularly inoculated with a single dose of the different vaccine formulations described in Table 9 above. Fourteen animals were included in the placebo and 10% SP-oil groups, thirteen animals were included in the positive control group, and sixteen animals were included in the 5% Amphigen and 5% Amphigen + 5% SLCD groups.
[00193] Animals were challenged 21 days after vaccination with a virulent M.hyo field isolate. The animals were submitted to necropsy 28 days after the challenge and the lungs were removed and recorded for consolidation compatible with M.hyo infection. Table 10 below contains the lung injury records for the respective treatment groups. Statistical significance was determined by a Mixed Model Analysis of lung records for each group.Table 10: Lung Injuries by M.hyo

[00194] As indicated in Table 10 above, the placebo group had a mean lung injury record of 13.1%, compared to the 10% SP-oil and 5% Amphigen treatment groups who had mean lung injury records of 4.3 % and 4.7 %, respectively. The 10% SP-oil and 5% Amphigen formulations reduced and/or prevented lung damage. Thus, experimental PCV/M.hyo vaccines formulated with 10% SP-oil or 5% Amphigen were considered efficacious. The PCV2 antigen did not appear to interfere with the M.hyo efficacy of these formulations.
[00195] In contrast, the 5% Amphigen + 5% SLCD group had a mean lung injury record of 12.0% which was an unacceptable result in that it was no different compared to placebo. Consequently, the experimental PCV/M.hyo vaccine formulated with 5% Amphigen + 5% SLCD was not considered effective.
[00196] It is observed that, due to the reduced animal number and high variability in the recording of lung injury, no statistical treatment effect can be conclusively demonstrated in this study. For this reason, it was decided that another study would be designed to test the M.hyo efficacy of experimental PCV/M.hyo formulations in 10% SP-oil. This repeat study is presented in Example 9 below.EXAMPLE 9: EVALUATION OF THE EFFECTIVENESS OF M.HYO OF A 1 BOTTLE PCV2/M.HYO COMBINATION VACCINE IN 10% SP-OIL
[00197] This study is a proof of concept designed to evaluate the efficacy of the M.hyo fraction of four experimental PCV2/M.hyo vaccines (Series L0711RK11, L0711RK12, L0711RK13 and L0711RK14 in Table 11 below) prepared by manufacturing processes of different M.hyo vaccines using Protein A for IgG removal compared to control vaccines prepared with the standard M.hyo manufacturing process. Each of these four experimental PCV2/M.hyo vaccines included 10% SP-oil as the adjuvant.FLUID PROCESSING:
[00198]T02: M. hyopneumoniae antigen inactivated as described under "Fermentation and Inactivation" in Example 1 above.
[00199]T03 and T04: Formulated with inactivated M. hyopneumoniae cells as described under "Fermentation and Inactivation" in Example 1 above.
[00200]T05: Treatment with Protein A of medium used to develop M. hyopneumoniae. PPLO (porcine heart derivative) was prepared per manufacturer's directions (ie, 21 g/L) and yeast extract solution was prepared at 21 g/L in USP. The yeast extract solution was added to the PPLO at 6.25% and the mixture was sterilized by heating at 121°C for >30 minutes. Cysteine hydrochloride was prepared at 90 g/L and filter sterilized. Dextrose solution was prepared by adding 450 g of dextrose per liter of USP water, followed by heat sterilization. To prepare the final medium, swine serum was added to the base medium at 10%, followed by cysteine at 0.01% and dextrose at 1.0%. Antibodies in complete PPLO medium were removed by treatment with protein A. Briefly, one liter of rProtein A Sepharose (part number 17-5199-03 GE Healthcare) was packed into a glass column (10 X 11.5 cm). After removal of storage buffer, the column was treated with 2 column volumes of 1 M acetic acid. The resin was equilibrated with 5 column volumes of 50 mM NaPO4, 1 M NaCl buffer (pH 7.0). Fifteen liters of complete PPLO medium was loaded onto the resin at a linear flow rate of 140 cm/hour. The flow through column was collected and filter sterilized through a 0.2 micron filter (Sartorius). The treated medium was used to propagate M. hyopneumoniae cells as described under "Fermentation and Inactivation" above. The whole inactivated culture (including the cells) was formulated into the final vaccine.
[00201]T06: Inactivated M. hyopneumoniae cells were prepared as described under "Fermentation and Inactivation" in Example 1 above. The inactivated fermentation fluid was centrifuged at ~20,000 x g (Sorvall RC5B) for 30 min and the supernatant was sterilized via 0.2 µM filtration. One hundred fifteen ml of rProtein A resin (part number 12-1279-04, MAbSelect, GE Healthcare) was packed into a chromatography column (5 x 6 cm). After removal of storage buffer and treatment with 2 column volumes of 1 M acetic acid, the resin was equilibrated with 5 column volumes of 50 mM NaPO4/1M NaCl buffer, pH 7.01. Approximately 1.2 liter of the cleaned/filtered M. hyopneumoniae antigen-containing fluids were passed through the resin at a flow rate of 120 cm/h. The through flow was collected and sterilized through a 0.2 μM filter.
[00202]T07: Inactivated M. hyopneumoniae cells were prepared as described under "Fermentation and Inactivation" in Example 1 above. The inactivated fermentation fluid was cleaned via tangential flow filtration. Briefly, a polyether sulfone filter (GE HealthCare, part number 56-4102-71) with a nominal pore size of 0.2 µM was sterilized with 0.5 N sodium hydroxide solution followed by extensive rinsing with sterile USP water. Inactivated mycoplasma culture fluid was introduced into the instrument at a targeted recirculation rate of 14.6L/minute and a transmembrane pressure of 2 to 3.4 PSI. Cleaning was carried out at room temperature. Filter permeate was collected and stored at 2 to 8 °C until further processing. One hundred and fifteen ml of rProtein A resin (part number 12-1279-04, MAbSelect, GE Healthcare) was packed into a chromatography column (5 x 6 cm). After removal of storage buffer and treatment with 2 column volumes of 1 M acetic acid, the resin was equilibrated with 5 column volumes of 50 mM NaPO4/1M NaCl buffer, pH 7.01. Approximately 2.3 liters of the cleaned/filtered M. hyopneumoniae antigen-containing fluids were passed through the resin at a flow rate of 120 cm/h. The through flow was collected and sterilized through a 0.2 μM filter.
[00203]T08: Inactivated M. hyopneumoniae cells were prepared as described under “Fermentation and Inactivation” above. The inactivated fermentation fluid was centrifuged at ~20,000 x g (Sorvall RC5B) for 30 min. and the supernatant was sterilized via 0.2 µM filtration. One hundred and fifteen ml of rProtein A Sepharose (part number 17-5199-03 GE Healthcare) was packed into a chromatography column (5 x 6 cm). After removal of storage buffer and treatment with 2 column volumes of 1 M acetic acid, the resin was equilibrated with 5 column volumes of 50 mM NaPO4/1M NaCl buffer, pH 7.01. Approximately 1.2 liter of the fluids containing clean/filtered M. hyopneumoniae antigen were passed through the resin at a flow rate of 120 cm/h. The through flow was collected and sterilized via a 0.2 µM filter.
[00204] Experimental vaccine formulations were prepared with M.hyo antigens processed according to treatments T02 to T08 above. T02, T03 and T04 corresponded to the positive controls. In addition, Treatment T01 corresponded to a placebo (sterile saline solution).
[00205] These experimental formulations are described in Table 11 below. The M.hyo antigen corresponds to the M.hyo antigen from the global M.hyo seed, supernatant treated with Protein A. The information in the column “Treatment with Protein A” indicates whether the M.hyo supernatant was treated with Protein A before or after fermentation.Table 11: PCV2/M.hyo Experimental Vaccine Formulations Used for Studying the Efficacy of M.hyo in SP-Oil Adjuvant


The 3 week old pigs were intramuscularly inoculated with a single dose of the different vaccine formulations described in Table 11 above. 18 pigs were included in each treatment group.
Animals were challenged 21 days after vaccination with a virulent M.hyo field isolate. The animals were submitted to necropsy 28 days after the challenge and the lungs were removed and recorded for consolidation compatible with M.hyo infection. Figure 8 (A and B) shows the lung injury records for the respective treatment groups. Statistical significance was determined by a Mixed Model Analysis of lung records for each group.
The results of lung injury represented in Figures 8A and 8B indicate that of all treatments, only two (T07 and T08) had 100% of pigs in the lung injury category <5%. A strong statistical difference was observed in this study.
The results in the present example demonstrate significant M.hyo efficacy in an experimental 1-vial PCV2/M.hyo formulation using the M.hyo supernatant treated with Protein A and using SP-oil as the adjuvant. Additionally, Example 7 above demonstrated the efficacy of PCV2 in a 1 vial PCV2/M.hyo formulation using the M.hyo supernatant treated with Protein A and using SP-oil as the adjuvant. Together, the efficacy of M.hyo and PCV2 was demonstrated in the 1-vial PCV2/M.hyo combinations using the Protein A treated M.hyo supernatant.
[00210]EXAMPLE 10: IN VIVO SAFETY OF PCV2/M.HYO EXPERIMENTAL VACCINES
This study was conducted to evaluate the in vivo safety of experimental PCV2-M.hyo vaccines formulated at maximum antigen dose in various host animal adjuvant formulations when given to younger animals (3 weeks of age). Different adjuvant platforms were evaluated in order to determine that between these platforms an acceptable safety profile was provided based on temperature, injection site reactions and clinical observations. A 20% SLCD/10% SP-oil formulation was used as a positive (“unsafe”) control, due to historical problems with injection site reactions observed by this investigative group and others.FLUID PROCESSING:
[00212] All vaccines were prepared with inactivated M. hyopneumoniae antigen as described under “Fermentation and Inactivation” in Example 1. Bulk total M.hyo antigen was used as it was known to contain M.hyo antigens soluble and insoluble cells, in addition to immunoglobulins and immune complexes that would be removed after treatment with protein A. It is reasonable to conclude that removal of insoluble cell debris and immunoglobulins and immune complexes will only enhance the safety of vaccine formulations. The intent of this study was to rigorously test the safety of various adjuvant formulations containing PCV2 antigen and M.hyo antigen. The PCV2 and M.hyo antigens were formulated at maximum release levels to further assess safety. These experimental formulations are described in Table 12 below. IVP indicates Investigational Veterinary Product (IVP).Table 12: PCV2/M.hyo Experimental Vaccine Formulations Used for Study of . Safety

M hyo antigen = from global M hyo seed (total mass antigen).
[00213] The safety parameters used in this study were rectal temperature profile and injection site reaction. The results of this study indicated that all candidate adjuvant platforms provide an acceptable safety profile in terms of rectal temperature profile and clinical observations (results not shown). Only 20% SLCD + 10% SP-oil (ie, positive control) were significantly different than the placebo vaccine and had a number of severe injection site reactions (results not shown).EXAMPLE 11: ANTIGEN PREPARATION OF M.HYO TREATED WITH PROTEIN A FOR ESSENTIAL STUDIES
[00214] Figure 9 is a flowchart showing one embodiment of a manufacturing process used to prepare PCV2-compatible Protein A-treated M.hyo antigen. Total inactivated cultures of M.hyo were cleaned from cells via tangential flow filtration. Briefly, a polyether sulfone filter (GE Healthcare, part number 56-4102-49) with a nominal pore size of 0.45 µM was sterilized with 0.5 N sodium hydroxide solution, followed by extensive rinsing with sterile USP water . Inactivated mycoplasma culture fluid was introduced into the instrument at a targeted recirculation rate of 11.0 L/minute and a transmembrane pressure of ~5 PSI. Cleaning was carried out at room temperature. Filter permeate was collected and stored at 2 to 8 °C until further processing.
[00215]After cleaning, the antigen-containing fluids were treated with protein A resin to reduce antibody levels. Briefly, MAbSelect protein resin A (GE Healthcare) was packed into a glass column at a height of 12 cm. The resin was equilibrated with 5 column volumes of 50 mM sodium phosphate, 250 mM NaCl buffer (pH 7.0). The antigen-containing fluid, equivalent to 10 column volumes, was loaded onto the resin at a linear flow rate of 100 cm/hour. The flow through the column was collected and filter sterilized through a 0.2 micron filter. Column regeneration was achieved by flowing 3 column volumes of 25 mM acetate solution at pH 3.7, followed by 4 column volumes of 1 M acetic acid solution. Anti-PCV2 antibody and M. hyopneumoniae were measured in the final antigen fluid via PCV2-specific antibody ELISA and p46 antigen quantitation ELISA, respectively.EXAMPLE 12: EFFECTIVENESS OF PCV1-2 CHIMERIC FRACTION AFTER INTRAMUSCULAR ADMINISTRATION OF A PCV2/M.HYO COMBINATION VACCINE 1 BOTTLE IN 10% SP-OIL
The study presented in this example was designed to evaluate the efficacy of the killed PCV1-2 chimera virus fraction of an experimental 1 Bottle PCV2/M.hyo combination vaccine administered once to 21±3 day old pigs. aged and challenged with a virulent PCV2 isolate at approximately 6 weeks of age.
[00217] Four experimental bivalent PCV1-2/M.hyo vaccines at different but balanced antigen dose levels and one experimental monovalent M.hyo vaccine (negative control) were formulated with the highest passage antigen. The control M.hyo antigen lot was prepared as described in Example 11 above. The PCV2 antigen was a killed cPCV1-2 antigen prepared as described in Example 2 above. Prior to chimeric virus inactivation, the PCV2 antigen pool was concentrated 20X and the concentrates were washed with a balanced salt solution. Final experimental vaccine formulations were adjuvanted using 10% SP-Oil. These experimental formulations are described below and in Table 13, where the antigen dose (% of PCV2 and M.hyo antigen lots) is given.
[00218]T01: Experimental preparation (L1211RK11) of the M.hyo antigen (14.1 %-High) without killed PCVTipo1-Type2 chimera virus fraction (0%). This corresponds to a negative control (monovalent M.hyo).
[00219]T02: Experimental preparation (L1211RK09) of dead virus PCVType1-Type2 chimera, high passage (1.375 %-High) and M.hyo antigen (14.1 %-Alta).
[00220]T03: Experimental preparation (L1211RK15) of PCVTipo1-Type2 chimera high passage killed virus (0.688%-Medium) and M.hyo antigen (9.4%-Medium).
[00221]T04: Experimental preparation (L0112RK03) of killed virus of high passage PCVType1-Type2 chimera (0.344%-Low) and M.hyo antigen (4.7%-Low).
[00222]T05: Experimental preparation (L1211RK17) of dead virus PCVType1-Type2 chimera (0.172 %-Very Low) and M.hyo antigen (2.32 %-Very Low).Table 13: Experimental design

1 IVP = Porcine Circovirus Virus Type 1-Type 2 Chimera (PCV2), killed - Bacterial Extract of Mycoplasma Hyopneumoniae (M hyo)CP = Bacterial Extract of Mycoplasma Hyopneumoniae adjuvanted with 10% SP-Oil (without fraction of Porcine Circovirus Type 1-Type 2 Chimera) IM = Intramuscularly
[00223] On Day 0 (3 weeks of age), a single 2 mL dose of the designated vaccine was administered by IM injection into the right neck of each pig involved in the study. No adverse events were observed after vaccination. A serum sample was collected from all pigs weekly before challenge. Any pigs detected with PCV2 viremia prior to challenge were removed from the study. On the day before challenge, faecal cotton swabs and serum samples were collected. Pigs were subsequently challenged with a PCV2a challenge virus. The challenge was conducted around 3 weeks after vaccination (Day 21). Each pig was inoculated with a total of 3 ml PCV2a challenge virus (Isolate #40895, prediluted to 5.10 log10 FAID50/ml) with 2 ml intranasally and 1 ml intramuscularly in the right neck. A reserved aliquot of challenge virus was titrated after challenge to confirm the actual challenge dose. The undiluted mass was prediluted 2-fold and re-titer results obtained a challenge level of 5.10 log10 FAID50/ml. Before necropsy, serum and faecal cotton swabs were collected weekly during the three-week challenge phase. Three weeks post-challenge, all pigs were euthanized and necropsied. Serum samples and faecal cotton swabs were collected, along with 4 different lymphoid tissues. During necropsy, sections of three lymph nodes (tracheobronchial, mesenteric, inguinal), and tonsils were collected from each pig and individually identified and fixed in a 10% buffered formalin solution. Test results are provided below. VACCINE POWER TEST
[00224] The PCV/M.hyo L1211RK15 series vaccine described above was considered a reference candidate. Consequently, the relative potency for the M.hyo fraction and PCV2 fraction was determined versus this reference candidate. These results are shown in Table 14 below. The L1211RK11 series corresponds to placebo (no PCV2 fraction).Table 14: Potency Results
Results shown for each series are averages of all replicates tested. PCV2 VIREMIA
[00225] After the challenge, when compared to the placebo group, all vaccinated groups showed a significant reduction in the percentage of viraemic pigs [ P<0.05] and throughout the study at least 47% of the pigs in the treated groups (T02- T05) remained negative for PCV2 viremia (Table 15 below). Similarly, all vaccine groups had significantly lower PCV2 DNA copy numbers (P=0.0001) compared to post-challenge placebo group (data not shown). Table 15: qPCR Qualitative Serum Viremia - Percent Always Positive , and Predicted Fraction Estimate
PCV2 FECAL EXCRETION
Post-challenge faecal cotton swabs revealed that 83.3% of the placebo group pigs (T01) were positive for PCV2 fecal excretion. In contrast, all vaccine groups (T02 to T05) showed a significant reduction in the percentage of PCV2 DNA detectable by pig excretion PCR (P<0.0061). These results are shown in table 16 below. Similarly, all vaccine groups had significantly lower PCV2 DNA copy numbers compared to the post-challenge placebo group (data not shown). Table 16: Fecal Excretion Always Present After Challenge (Day>21)

Serum Antibody Response
[00227]All pigs were seronegative for PCV2 prior to vaccination. Pigs in the placebo group remained seronegative prior to challenge. In contrast, pigs in all vaccine groups except for the T05 group showed significant increases (P<0.0287) in PCV2 antibody titer on day 20 post-vaccination when compared to placebo, indicating an active immune response to PCV2 after vaccination. PCV2 ELISA antibody titers are summarized in table 17 below. Pre-challenge titers indicated a significant difference (P < .0393) in groups T02 and T03 from group T01 on days 7 to 20 and between groups T01 and T04 on day 20 (P < .0287). On days 28 to 42, all vaccine groups had significantly higher titers relative to T01 (P <.0129; Table 17 below).

*When contrast is significant (<0.05) a P value is reported, P values > 0.05 are designated as ns (not significant) LYMPHOID LESIONS AND COLONIZATION
[00228] During necropsy, when compared to the placebo group, all vaccinated groups showed a significant reduction in the total amount of PCV2 antigen detected in the tissues. Data with respect to PCV2 infection in lymphoid tissues (IHC records) are summarized in table 18 below. As shown in table 18, all vaccine groups had significantly lower IHC records compared to the T01 placebo group.
[00229]Table 18: PCV2 IHC records: If lymphoid or tonsil tissues always abnormal
* The animal was considered abnormal if the record was > 0 in any lymphoid tissue or tonsil sample.
[00230]All vaccinated groups also showed a significant reduction in PCV2 lymphoid depletion, as shown in table 19 below.
[00231]Table 19: PCV2 Lymphoid Depletion: If lymphoid tissues or tonsils always abnormal
1 The animal was considered abnormal if the recording was > 0 in any lymphoid tissue or tonsil sample.
[00232]In addition, all vaccinated groups showed a significant reduction in histiocytic substitution, as shown in table 20 below.Table 20: Histiocytic substitution of PCV2: If lymphoid or tonsil tissues always abnormal

The animal was considered abnormal if the recording was > 0 in any lymphoid tissue or tonsil sample.2 * Fisher's exact test was used due to non-convergence, therefore, least squares means were not determined (ND).
[00233] The data presented in this example indicated that the vaccine groups:
[00234]Protected and significantly aided in the prevention of post-challenge PCV2 viremia;
[00235] Significantly helped prevent post-challenge PCV2 fecal excretion in all vaccinated animals;
[00236] induced a statistically significant serological response 28 days after vaccination in groups T02 to T05. Furthermore, T02 and T03 demonstrated a statistically significant response 7 days post-vaccination when compared to T01;
[00237] Significantly reduced microscopic lesions (lymphoid depletion and histiocytic replacement) in all vaccinated animals; and
[00238] All vaccines have been proven to be effective and the L1211RK15 series vaccine was selected as a reference candidate.EXAMPLE 13: EFFECTIVENESS OF THE M.HYO FRACTION AFTER INTRAMUSCULAR ADMINISTRATION OF A PCV2/M.HYO DE COMBINATION VACCINE 1 BOTTLE IN 10% SP-OIL
[00239] The aim of the study presented in this example was to evaluate the efficacy of the Mycoplasma hyopneumoniae (M. hyopneumoniae) fraction of a Porcine Circovirus (PCV) Type 1-Type 2 Chimera, killed - Bacterial Extract of Mycoplasma Hyopneumoniae (M hyo) experimental, administered intramuscularly once to 21 ± 3 day old pigs and challenged with a virulent M. hyopneumoniae lung homogenate at 7 weeks after vaccination.
Four experimental bivalent PCV1-2/M.hyo vaccines at different but balanced antigen dose levels and one experimental monovalent PCV2 vaccine (negative control) were formulated. The M.hyo antigen control lot was prepared as described in Example 11 above. The PCV2 antigen was a killed cPCV1-2 antigen prepared as described in example 2 above. Prior to chimeric virus inactivation, the PCV2 antigen pool was concentrated 20X and the concentrates were washed with a balanced salt solution. Final experimental vaccine formulations were adjuvanted using 10% SP oil. These experimental formulations are described below and in table 21 below, where the antigen dose (% PCV2 and M.hyo antigen lots) is given.
[00241]T01: Experimental preparation (L1211RK10) of killed virus PCVTipo1-Type2 high passage chimera (1.375 %-High) without M.hyo fraction (0%). This corresponds to a negative control (monovalent PCV2).
[00242]T02: Experimental preparation (L1211RK09) of PCVType1-Type2 high passage chimera killed virus (1.375 %-High) and M.hyo antigen (14.1 %-High).
[00243]T03: Experimental preparation (L1211RK15) of dead virus PCVTType1-Type2 chimera, high passage (0.688%-Medium) and M.hyo antigen (9.4%-Medium). Tabe a 21: Experimental Design

1 IVP = Research Veterinary Product = Porcine Circovirus Virus Vaccine Type 1- Type 2 Chimera (PCV2), killed- Bacterial Extract of Mycoplasma Hyopneumoniae (M hyo)CP = Control Product = Fraction of killed chimeric PCV1-2 with SP-adjuvant 10% Oil (without M. hyopneumoniae fraction) IM = Intramuscularly
[00244]On day 0, 123 clinically healthy and susceptible pigs were enrolled in this study at three weeks of age. Pigs were blocked per litter and randomly assigned to either a sentinel group (NTX) or one of three treatment groups (T01-T03); intramuscularly administered 2 ml of an experimental PCV1-2/M vaccine. hyopneumoniae at the minimum immunization dose (MID), an experimental PCV1-2/M vaccine. hyopneumoniae at a dose slightly higher than MID or a placebo containing PCV1-2 only in MID. Seven weeks after vaccination, sentinel pigs were euthanized and necropsied to confirm the absence of M. hyopneumoniae and all treated pigs were challenged twice (on two successive days) with a live virulent M. hyopneumoniae lung homogenate. All remaining pigs were euthanized and necropsied four weeks after challenge. At necropsy, the lungs were recorded for typical M. hyopneumoniae lesions. Post-challenge lung lesions are the primary outcome variable. Vaccination is considered effective if the lowest 95% confidence interval of the mitigated fraction is >0. VACCINE POWER TEST
The L1211RK15 series of the PCV/M.hyo vaccine described above was considered the reference candidate. Consequently, the relative potency for the M.hyo fraction and PCV2 fraction used in this M.hyo efficacy study was determined versus this reference candidate. These results are shown in table 22 below. The L1211RK10 series corresponds to placebo (no fraction of M.hyo).Table 22: Potency Results
Results shown for each series are means of all replicates tested.1 M. hyopneumoniae potency tested in five replicates.2 PCV potency tested in one replicate (L1211RK10) or five replicates (L1211RK09, L1211RK15). SEROLOGY
[00246] M.hyo antibody titers indicated that all pigs were M. hyopneumoniae serologically negative on day 0 and remained negative prior to challenge. At all post-vaccination time points (days 21, 47 and 75), T02 and T03 had significantly higher M. hyopneumoniae antibody titers with geometric least squares mean (P<0.0004) compared to T01 (data of serology not shown). PERCENTAGE OF TOTAL LUNGS WITH INJURIES
[00247] Lung injury frequency distributions recorded for each lung lobe were calculated across treatment. The percentage of total lung with injuries was calculated using the following formula: Percent of total lung with injuries = {(0.10 x left cranial) + (0.10 x left middle) + (0.25 x left caudal) + ( 0.10 x right cranial) + (0.10 x middle right) + (0.25 x right caudal) + (0.10 x accessory)}. The square root transformation of the arc function was applied to the percentage of the total lung with lesions before analysis. The percentage of the total lung with lesions was analyzed using a mixed linear model. Pairwise comparisons were made between treatment groups if the treatment effect was significant. The retransformed least squares means of the percentage of the total lung with lesions and their 95% confidence intervals were calculated as well as the minimum and maximum. Lung lesions were summarized with stratified and mitigated fraction and 95% confidence limits. The results of lung injury are shown in Table 23 below.Table 23: Analysis of Percentage of Total Lung with Injury

Retransformed least squares mean Treatment groups with the same letter are not significantly different at the P 0.05 value.
[00248] A low percentage of lung lesions was observed in the lungs of the NTX pig which has been attributed to a known incidence of Bordetella in this herd. Bacterial culture on cotton pads with lung tissue confirmed that several pigs had positive culture for B. bronchiseptica and culture for M. hyopneumoniae confirmed that all NTX pigs had negative culture for M. hyopneumoniae prior to challenge administration.
[00249] The protocol met the validity criteria in which the mean LS lung lesions for T01 were >4%. The mean LS lung lesions for T02 and T03 were significantly lower (P<0.05) than T01 and met the fraction mitigated criteria relative to the 95% confidence interval that was >0. The validity requirements of this study were met in that there was no evidence of pre-challenge exposure to M. hyopneumoniae. The challenge was valid in that the mean lung injury records retransformed in placebo pigs (T01) was >4%.
[00250] Compared with the negative control group (T01), the treatment groups T02 and T03 demonstrated a significant reduction (P < 0.05) in the percentage of injured lung compared to T01. The fractions mitigated for T02 and T03 in relation to T01 met the protocol criteria for efficacy.
[00251] Under the conditions of this study, vaccines (T02 and T03) helped to attenuate lung lesions, the primary efficacy variable. The results of the present example demonstrate the significant efficacy of M.hyo in a 1-vial PCV2/M.hyo experimental formulation.EXAMPLE 14: EVALUATION OF VIRUCID ACTIVITY AGAINST PRRS VIRUS
[00252] The studies presented in this example were designed to evaluate the various adjuvant platforms for virucidal activity against PRRS virus. Initial experiments focused only on the adjuvant (ie, the formulations did not contain PCV or M.hyo antigens). Adjuvant evaluation for PRRS virucidal activity is shown in Figure 10. Preliminary virucidal evaluation indicated that 10% SP-Oil, 0.2% Carbopol, and 2.5% Amphigen are not virucidal for PRRS virus. In contrast, the 20% SLCD adjuvant appeared to be virucidal for PRRS virus.
[00253] Other studies were performed to assess whether PCV / M.hyo formulations adjuvanted with the different adjuvant platforms were non-virucidal to PRRS virus. These results are shown in Table 24 below, where the symbol * indicates the serial numbers of vaccines that were virucidated for PRRS virus.Table 24: Results of PRRS Virucidal Assay with Different Formulations

*Indicates Virucidal (>0.7 log loss) A - Virucidal control assay GMT ~5.53 log/mlB - Virucidal control assay GMT ~6.42 log/mL
[00254] The results presented in Table 24 above indicate that 10% SP-Oil is non-virucidal to PRRS virus.
[00255] Other PCV/M.hyo vaccine series were prepared using 10% SP-Oil as the adjuvant (Table 25). The antigenic potency of these vaccine series was compared to a PCV/M.hyo Reference vaccine series (L1211RK15) described above. The results shown in Table 25 further indicate that 10% SP-Oil is not virucidal for PRRS virus. The test sample values in Table 25 were higher (+ sign) compared to the control of the virucidal assay, which had a geometric mean titer (GMT) of about 5.9 ± 0.5 log/ml. Virucidal Assay with Different PCV/M.hyo Formulations Adjuvated with 10% SP-Oil
Virucida control assay GMT ~ 5.9 + 0.5 log/ml
[00256]The results presented in this example demonstrate that 10% SP-oil is not virucidal for PRRS virus. The results presented in this example further demonstrate that the PCV / M.hyo formulation adjuvanted with 10% SPoil occurred among the series of vaccines that were considered non-virucidal for the PRRS virus (Table 24 and Table 25). In conclusion, the PCV/M.hyo formulation augmented with 10% SP-oil was found to be an effective platform over one to base a trivalent combination including PCV, M.hyo and PRRS virus. EXAMPLE 15: PREPARATION OF A PCV/M.HYO/PRRS COMBINATION VACCINE
[00257] An adjuvanted PCV / M.hyo formulation with an adjuvant platform, which is non-virucidal for PRRS virus (see Tables 24 and 25 above), is provided as a ready-to-use solution in the liquid composition of a bottle. This 1 vial PCV-1/M.hyo formulation employs M.hyo supernatant treated with Protein A. The efficacy of M.hyo and PCV2 has been demonstrated in such PCV2/M.hyo formulations employing the M.hyo supernatant treated with Protein A (see Examples 7 to 9). In the present example, this divalent PCV2/M.hyo formulation is combined with a monovalent PRRS virus antigen.
[00258] In one embodiment, a combination of PCV / M.hyo in 10% SP-oil and which corresponds to one of the L0711RK11, L0711RK12, L0711RK13 L0711RK14 in Table 11 above or L1211RK09, L1211RK15, L0112RK03 L1211RK17 in the Table vaccine series 13 above is provided as a ready-to-use solution in the 1-vial liquid composition. The results presented in Example 14 above demonstrate that 10% SP-oil is non-virucidal for PRRS virus. Example 14 also demonstrated that 10% SP-oil-adjuvanted PCV2/M.hyo formulations were among those vaccine series that were considered non-virucidal to PRRS virus. In the present example, a 1 vial liquid PCV2/M.hyo composition is used to rehydrate a lyophilized composition of genetically modified live PRRS virus contained in a second vial such that all antigens are contained in a single vial prior to administration to pigs of the appropriate age (eg 3 weeks of age or older).
[00259] In one embodiment, the PRRS virus has the genomic sequence corresponding to SEQ ID NO: 16 or a variant thereof. In another embodiment, the PRRS virus employed in the trivalent composition is the PRRS virus isolate designated ISU-55, which has been deposited with the ATCC under accession number VR 2430. Appropriate amounts of the respective antigens are described in this report. Desirably, all antigens are administered in a single dose to the pig.

权利要求:
Claims (17)
[0001]
1. Multivalent immunogenic composition CHARACTERIZED by the fact that it comprises a combination of a soluble portion of a total cell preparation of Mycoplasma hyopneumoniae (M.hyo); and a porcine circovirus type 2 (PCV2) antigen, alone or in combination with at least one additional antigen, wherein the soluble portion of the Mycoplasma hyopneumoniae (M.hyo) preparation comprises specific M.hyo soluble protein antigens and is separated from insoluble cellular material and substantially free of both IgG and antigen/immunoglobulin immune complexes, and wherein the at least one additional antigen is protective against a microorganism that can cause disease in pigs, wherein the microorganism is selected from the group that consists of swine parvovirus (PPV), Haemophilus parasuis, Pasteurella multocida, Streptococcum suis, Staphylococcus hyicus, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Salmonella choleraesuis, Salmonella enteritidis, Erysipelapathia suis, Erysipelathrix, Erysipelathrix, Erysipelathrix bacteria, intracellular hyusspirals (SIV), Escherichia coli antigen, Brachyspira hyodysenteriae, corona swine respiratory virus, Swine Epidemic Diarrhea virus (PED), rotavirus, torque tenus virus (TTV), swine cytomegalovirus, swine enterovirus, encephalomyocarditis virus, a pathogen causing Aujesky Disease, Classical Swine Fever (CSF) and a causative pathogen of Transmissible Gastroenteritis to Swine, or combinations thereof.
[0002]
2. Composition according to claim 1, CHARACTERIZED by the fact that the soluble portion of the M.hyo preparation was treated with protein A or protein G before being added to the immunogenic composition.
[0003]
3. Composition according to claim 2, CHARACTERIZED by the fact that the soluble portion of the M.hyo preparation was treated with protein A before being added to the immunogenic composition.
[0004]
4. Composition, according to any one of claims 1 to 3, CHARACTERIZED by the fact that the PCV2 antigen is in the form of a type1-type2 chimeric circovirus, said chimeric virus comprising an inactivated recombinant type 1 swine circovirus expressing the ORF2 protein of porcine circovirus type 2.
[0005]
5. Composition according to any one of claims 1 to 4, CHARACTERIZED by the fact that the PCV2 antigen is in the form of a recombinant ORF2 protein.
[0006]
6. Composition according to claim 5, CHARACTERIZED by the fact that the recombinant protein ORF2 is expressed from a baculovirus vector.
[0007]
7. Composition according to any one of claims 1 to 6, CHARACTERIZED by the fact that the composition further comprises an adjuvant.
[0008]
8. Composition according to claim 7, CHARACTERIZED by the fact that the adjuvant is selected from the group consisting of an oil-in-water adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an adjuvant of aluminum hydroxide, a vitamin E adjuvant and combinations thereof.
[0009]
9. Use of a composition as defined in any one of claims 1 to 8, CHARACTERIZED by the fact that it is for the manufacture of a drug to elicit a protective immune response against both M.hyo and PCV2, in which the drug is formulated to be administered as a single dose and, if present, the at least one additional microorganism that is known to infect pigs, when formulated to be administered as a single dose.
[0010]
10. Use of a composition as defined in any one of claims 1 to 8, CHARACTERIZED by the fact that it is for the manufacture of a drug to immunize a pig against Mycoplasma hyopneumoniae (M.hyo) and PCV2.
[0011]
11. Use, according to claim 10, CHARACTERIZED by the fact that the drug is formulated to be administered in a single dose.
[0012]
12. Use, according to claim 10 or 11, CHARACTERIZED by the fact that the drug is formulated to be administered to pigs having antibodies of maternal origin against at least one of M.hyo and PCV2, or both M.hyo and PCV2 .
[0013]
13. Use, according to any one of claims 10 to 12, CHARACTERIZED by the fact that the drug is formulated to be administered to pigs 3 weeks of age or older.
[0014]
14. Vaccine composition CHARACTERIZED by the fact that it comprises an immunogenic composition as defined in any one of claims 1 to 8, wherein the composition further comprises a pharmaceutically acceptable carrier.
[0015]
15. Vaccine composition according to claim 14, CHARACTERIZED by the fact that it is for use in the protection of pigs against enzootic pneumonia, post-weaning multisystemic wasting syndrome (PMWS) and porcine circovirus-associated disease (PCVAD).
[0016]
16. Kit CHARACTERIZED in that it comprises: a vial comprising an immunogenic or vaccine composition including both a PCV2 antigen and the soluble portion of a total cell preparation of Mycoplasma hyopneumoniae (M.hyo), wherein the soluble portion of the preparation of M.hyo comprises specific M.hyo soluble protein antigens and is separated from insoluble cellular material and substantially free of both IgG and antigen/immunoglobulin immunocomplexes as defined in any one of claims 1 to 15, and optionally further includes a manual instructions that contain the information for administering the immunogenic composition.
[0017]
17. Method for preparing an immunogenic or vaccine composition, as defined in any one of claims 1 to 15, CHARACTERIZED by the fact that the method comprises: i) cultivating M.hyo in a suitable medium for periods ranging from 18 to 144 hours ;ii) subsequently inactivating the M.hyo culture;iii) collecting the inactivated culture fluid, wherein the inactivated culture fluid comprises a total cell preparation of M.hyo comprising a soluble liquid fraction and insoluble cellular material, wherein the soluble liquid fraction comprises specific M.hyo soluble protein antigens; iv) separating the soluble liquid fraction from the insoluble cellular material; v) substantially removing IgG and antigen/immunoglobulin immunocomplexes from the separated soluble liquid fraction to form a soluble portion the total cell preparation of M.hyo; evi) subsequently combining the soluble portion of the total M.hyo cell preparation with a PCV2 antigen.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US11154609B2|2021-10-26|PCV/mycoplasma hyopneumoniae vaccine

---

US11141472B2|2021-10-12|Mycoplasma hyopneumoniae vaccine

---

US9650601B2|2017-05-16|PCV/mycoplasma hyopneumoniae/PRRS combination vaccine

同族专利:

---

公开号 | 公开日

---

US9649369B2|2017-05-16|

---

HK1206609A1|2016-01-15|

---

CN104334186B|2018-11-13|

---

CL2014002675A1|2014-12-12|

---

US20190209673A1|2019-07-11|

---

CO7160025A2|2015-01-15|

---

ZA201406974B|2016-07-27|

---

WO2013152083A2|2013-10-10|

---

NI201400119A|2015-03-05|

---

TWI602574B|2017-10-21|

---

HRP20140954A2|2015-01-30|

---

CA2869594C|2018-06-19|

---

BR112014024785A2|2017-07-11|

---

PL2833910T3|2019-10-31|

---

UA114503C2|2017-06-26|

---

AU2013243537B2|2017-10-12|

---

US20150283222A1|2015-10-08|

---

MY174622A|2020-05-02|

---

US20170216423A1|2017-08-03|

---

US20200338187A1|2020-10-29|

---

KR20150003259A|2015-01-08|

---

EP2833910B1|2019-02-13|

---

DK2833910T3|2019-04-15|

---

AU2013243537A1|2014-10-09|

---

JP2015512449A|2015-04-27|

---

KR102003620B1|2019-07-24|

---

RU2615443C2|2017-04-04|

---

LT2833910T|2019-04-25|

---

EP2833910A2|2015-02-11|

---

TW201345549A|2013-11-16|

---

RS58481B1|2019-04-30|

---

KR20180049221A|2018-05-10|

---

US20130266602A1|2013-10-10|

---

AR090615A1|2014-11-26|

---

EP3508218A1|2019-07-10|

---

PT2833910T|2019-05-23|

---

CN104334186A|2015-02-04|

---

TR201904603T4|2019-04-22|

---

US10206993B2|2019-02-19|

---

CA2869594A1|2013-10-10|

---

HRP20190563T1|2019-05-17|

---

US11154609B2|2021-10-26|

---

MX336503B|2016-01-21|

---

MX2014012015A|2014-11-10|

---

MX354759B|2018-03-20|

---

HUE043322T2|2019-08-28|

---

US10668145B2|2020-06-02|

---

ES2721925T3|2019-08-06|

---

RU2014140106A|2016-05-27|

---

CR20140437A|2014-11-10|

---

PH12014502251B1|2014-12-15|

---

SI2833910T1|2019-05-31|

---

US9125885B2|2015-09-08|

---

PH12014502251A1|2014-12-15|

---

WO2013152083A3|2013-12-19|

---

KR20170051528A|2017-05-11|

---

JP6271503B2|2018-01-31|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

GB228811A|1924-11-04|1925-02-12|Congoleum Company Inc|Improvements in stands or racks for displaying rolled merchandise|

---

US4606918A|1983-08-22|1986-08-19|Syntex Inc.|Polyoxypropylene-polyoxyethylene block polymer based adjuvants|

---

US4681870A|1985-01-11|1987-07-21|Imre Corporation|Protein A-silica immunoadsorbent and process for its production|

---

JPH0832637B2|1985-02-14|1996-03-29|アクゾ・エヌ・ヴエー|Synthetic immunogen|

---

US5084269A|1986-11-06|1992-01-28|Kullenberg Fred W|Adjuvant for dose treatment with antigens|

---

ZA881694B|1987-03-17|1988-09-06|Akzo N.V.|Adjuvant mixture|

---

US5252328A|1987-03-26|1993-10-12|Martin Marietta Energy Systems, Inc.|Mycoplasma hyopneumoniae antigen and uses therefor|

---

EP0283840A3|1987-03-26|1989-08-09|Ml Technology Ventures, L.P.|Mycoplasma hyopneumoniae antigen and uses therefor|

---

JP2746272B2|1987-11-03|1998-05-06|シンテツクス（ユーエスエイ）インコーポレーテツド|Vaccine adjuvant|

---

US4985243A|1988-01-20|1991-01-15|Ml Technology Ventures, L.P.|Composition and method for protecting against diseases caused by microorganisms|

---

US5240706A|1989-04-07|1993-08-31|Ml Technology Ventures, L.P.|Intranasal administration of Mycoplasma hyopneumoniae antigen|

---

ZA906806B|1989-08-31|1991-06-26|Ici Australia Operations|Plants|

---

US6113916A|1990-05-29|2000-09-05|American Cyanamid Company|Method of enhancing cell mediated immune responses|

---

AT160699T|1990-05-29|1997-12-15|American Cyanamid Co|VACCINE AGAINST PNEUMONIA IN PIGS AND METHOD FOR THE PRODUCTION THEREOF|

---

US5695769A|1990-06-13|1997-12-09|Pfizer Inc.|Pasteurella multocida toxoid vaccines|

---

US5565205A|1990-08-16|1996-10-15|Solvay Animal Health, Inc.|Inactivated Mycoplasma hypopneumoniae bacterin and method of use thereof|

---

AU2196592A|1991-06-06|1993-01-08|Stichting Centraal Diergeneeskundig Instituut|Causative agent of the mystery swine disease, vaccine compositions and diagnostic kits|

---

DE601062T1|1991-08-26|1995-05-18|James Edward Collins|VACCINE AGAINST AND DIAGNOSTIC METHOD FOR THE PIG INFERTILITY AND BREATHING SYNDROME .|

---

DK0610250T4|1991-10-14|2009-03-02|Intervet Int Bv|Vaccine with Porcine Reproductive Respiratory Syndrome and diagnosis thereof|

---

AU667858B2|1991-11-15|1996-04-18|Smithkline Beecham Corporation|Gram-negative bacterial vaccines|

---

FR2686097B1|1992-01-14|1994-12-30|Rhone Merieux|PREPARATION OF ANTIGENS AND MYSTERY DISEASE VIRUS VACCINES, ANTIGENS AND VACCINES OBTAINED FOR THE PREVENTION OF THIS DISEASE.|

---

US5338543A|1992-02-27|1994-08-16|Ambico, Inc.|Thimerosal inactivated mycoplasma hyopneumoniae vaccine|

---

US5534256A|1992-07-02|1996-07-09|University Of Saskatchewan|Haemophilus somnus outer membrane protein extract enriched with iron-regulated proteins|

---

US6592873B1|1992-10-30|2003-07-15|Iowa State University Research Foundation, Inc.|Polynucleic acids isolated from a porcine reproductive and respiratory syndrome virus and proteins encoded by the polynucleic acids|

---

US6380376B1|1992-10-30|2002-04-30|Iowa State University Research Foundation|Proteins encoded by polynucleic acids of porcine reproductive and respiratory syndrome virus |

---

US6251397B1|1992-10-30|2001-06-26|Iowa State University Research Foundation, Inc.|Proteins encoded by polynucleic acids isolated from a porcine reproductive and respiratory syndrome virus and immunogenic compositions containing the same|

---

US5695766A|1992-10-30|1997-12-09|Iowa State University Research Foundation|Highly virulent porcine reproductive and respiratory syndrome viruses which produce lesions in pigs and vaccines that protect pigs against said syndrome|

---

US6773908B1|1992-10-30|2004-08-10|Iowa State University Research Foundation, Inc.|Proteins encoded by polynucleic acids of porcine reproductive and respiratory syndrome virus |

---

ES2074950B1|1993-09-17|1996-03-16|Iberica Cyanamid|VACCINE FOR THE PREVENTION OF REPRODUCTIVE AND RESPIRATORY DISEASE OF THE SOW.|

---

ES2429401T3|1994-05-10|2013-11-14|Boehringer Ingelheim Vetmedica, Inc.|Modified live vaccine against BRSV, improved|

---

BE1008978A5|1994-12-27|1996-10-01|Solvay|Adjuvants for vaccines.|

---

US5788962A|1995-01-17|1998-08-04|The Curators Of The University Of Missouri|DNA sequences coding for mycoplasma hyopneumoniae surface antigens, corresponding proteins and use in vaccines and diagnostic procedures|

---

AUPN178995A0|1995-03-16|1995-04-13|University Of Melbourne, The|Antigen composition|

---

US5820869A|1995-06-07|1998-10-13|American Home Products Corporation|Recombinant raccoon pox viruses and their use as an effective vaccine against feline immunodeficiency virus infection|

---

DE19601754A1|1996-01-19|1997-07-24|Hoechst Ag|Dictyocaulus viviparus antigen for the diagnosis of lung worm infestation and for vaccination|

---

US5846735A|1996-04-18|1998-12-08|University Of Iowa Research Foundation|Hepatitis C virus Fc-binding function|

---

GB9622159D0|1996-10-24|1996-12-18|Solvay Sociutu Anonyme|Polyanionic polymers as adjuvants for mucosal immunization|

---

EP0839912A1|1996-10-30|1998-05-06|Instituut Voor Dierhouderij En Diergezondheid |Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof|

---

EP0948625B1|1996-12-20|2011-01-26|The Board Of Regents, The University Of Texas System|Uspa1 and uspa2 antigens of moraxella catarrhalis|

---

PL341797A1|1997-11-26|2001-05-07|Univ Iowa State Res Found Inc|Recombined vaccine against mycoplasma hyopneumoniae|

---

FR2772047B1|1997-12-05|2004-04-09|Ct Nat D Etudes Veterinaires E|GENOMIC SEQUENCE AND POLYPEPTIDES OF CIRCOVIRUS ASSOCIATED WITH PIGLET LOSS DISEASE , APPLICATIONS TO DIAGNOSIS AND TO PREVENTION AND / OR TREATMENT OF INFECTION|

---

US6342231B1|1998-07-01|2002-01-29|Akzo Nobel N.V.|Haemophilus parasuis vaccine and diagnostic|

---

NZ513289A|1998-12-22|2003-04-29|Pfizer Prod Inc|Infectious cDNA clone of north american procine reproductive and respiratory syndrome virus and uses thereof|

---

US6632439B2|1999-09-29|2003-10-14|Novartis Animal Health, Inc.|Fusobacterium necrophorum vaccine and method for making such vaccine|

---

US6585981B1|2000-07-27|2003-07-01|Regents Of The University Of Minnesota|Temperature-sensitive live vaccine for Mycoplasma hyopneumoniae|

---

US7018638B2|2000-12-19|2006-03-28|Wyeth|Mycoplasma hyopneumoniae bacterin vaccine|

---

MY129765A|2000-12-19|2007-04-30|Wyeth Corp|Improved mycoplasma hyopneumoniae bacterin vaccine|

---

EP1474067B2|2001-07-02|2017-08-16|Zoetis Services LLC|One dose vaccination withi mycoplasma hyopneumoniae /i|

---

BR0211049A|2001-07-02|2004-07-20|Pfizer Prod Inc|Method of treatment or prevention of disease or dysfunction and use of mycoplasma hyopneumoniae|

---

US7279166B2|2001-12-12|2007-10-09|Virginia Tech Intellectual Properties, Inc.|Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof|

---

US7276353B2|2001-12-12|2007-10-02|Virginia Tech Intellectual Properties, Inc.|Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof|

---

WO2004003161A2|2002-06-28|2004-01-08|Iowa State University Research Foundation, Inc.|Immunogenic mycoplasma hyopneumoniae polypeptides|

---

AT392905T|2003-07-24|2008-05-15|Merial Ltd|VACCINE FORMULATIONS WITH OIL-IN-WATER EMULSION|

---

AT462445T|2005-01-13|2010-04-15|Boehringer Ingelheim Vetmed|PRRS VACCINES|

---

WO2007028823A1|2005-09-09|2007-03-15|Intervet International B.V.|Pcv-2 vaccine|

---

EP1792996A1|2005-12-01|2007-06-06|Consejo Superior de Investigaciones Cientificas|Nucleic acid sequences encoding vaccines against Porcine reproductive and respiratory syndrome virus |

---

EP2371385B1|2005-12-29|2015-08-19|Boehringer Ingelheim Vetmedica, Inc.|Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs|

---

DK1968630T5|2005-12-29|2018-08-13|Boehringer Ingelheim Vetmedica Inc|MULTIVALENT PCV2 IMMUNOGENIC COMPOSITIONS.|

---

CN101420975B|2006-04-10|2012-06-27|英特威国际有限公司|Vaccine against mycoplasma PRRSV|

---

WO2007116032A1|2006-04-10|2007-10-18|Intervet International B.V.|Vaccine against mycoplasma prrsv|

---

WO2008076915A2|2006-12-15|2008-06-26|Boehringer Ingelheim Vetmedica, Inc.|Treatment of pigs with pcv2 antigen|

---

MX2010005014A|2007-11-06|2010-06-30|Wyeth Llc|Mycoplasma hyopneumoniae avirulent -adjuvanted live vaccine.|

---

US20090317423A1|2008-01-23|2009-12-24|Boehringer Ingelheim Vetmedica, Inc.|Pcv2 mycoplasma hyopneumoniae immunogenic compositions and methods of producing such compositions|

---

US8444989B1|2008-04-18|2013-05-21|Boehringer Ingelheim Vetmedica Gmbh|One dose vaccination against mycoplasma infections of pigs|

---

TWI449533B|2008-04-18|2014-08-21|Intervet Int Bv|Vaccine for protection against lawsonia intracellularis, mycoplasma hyopneumoniae and porcine circo virus|

---

KR20130054466A|2008-10-16|2013-05-24|화이자 인코포레이티드|Torque teno virus isolates and compositions|

---

FI20096134A0|2009-11-03|2009-11-03|Pulse Finland Oy|Adjustable antenna|

---

WO2011141443A1|2010-05-11|2011-11-17|Intervet International B.V.|Vaccine against mycoplasma hyopneumoniae, suitable for administration in the presence of maternally derived antibodies|

---

US8546149B2|2010-08-27|2013-10-01|Intervet Inc.|Potency test for vaccine formulations|

---

CA2808285C|2010-08-27|2016-09-06|Intervet International B.V.|Potency test for vaccine formulations|

---

EP2637688B1|2010-11-10|2017-01-11|Zoetis Services LLC|North american porcine reproductive and respiratory syndrome virus and uses thereof|

---

WO2013131565A1|2012-03-07|2013-09-12|Ceva Sante Animale|Novel veterinary vaccine|

---

US9120859B2|2012-04-04|2015-09-01|Zoetis Services Llc|Mycoplasma hyopneumoniae vaccine|

---

US9125886B2|2012-04-04|2015-09-08|Zoetis Services Llc|PCV/mycoplasma hyopneumoniae/PRRS combination vaccine|

---

US9125885B2|2012-04-04|2015-09-08|Zoetis Services Llc|PCV/Mycoplasma hyopneumoniae combination vaccine|CN1298694C|2005-03-02|2007-02-07|张本田|Process for producing trihydroxy methyl propane triacrylate|

---

US9125886B2|2012-04-04|2015-09-08|Zoetis Services Llc|PCV/mycoplasma hyopneumoniae/PRRS combination vaccine|

---

US9125885B2|2012-04-04|2015-09-08|Zoetis Services Llc|PCV/Mycoplasma hyopneumoniae combination vaccine|

---

US9120859B2|2012-04-04|2015-09-01|Zoetis Services Llc|Mycoplasma hyopneumoniae vaccine|

---

KR102355614B1|2012-12-28|2022-01-27|베링거잉겔하임베트메디카게엠베하|Immunogenic composition comprising mycoplasma antigens|

---

HUE045015T2|2012-12-28|2019-11-28|Boehringer Ingelheim Vetmedica Gmbh|Method of making a mycoplasma vaccine|

---

PL2994162T3|2013-05-08|2021-10-18|Pharmgate Biologics Inc.|Vaccine for pcv2 and mycoplasma|

---

CN104248759B|2013-11-19|2017-05-10|普莱柯生物工程股份有限公司|Vaccine composition, preparation method and application thereof|

---

EP3076996B1|2013-12-03|2020-01-15|Intervet International B.V.|Vaccine against porcine circo virus type 2|

---

RU2672251C1|2013-12-03|2018-11-13|Интервет Интернэшнл Б.В.|Vaccine against lawsonia intracellularis and porcine circovirus 2|

---

US9713639B2|2014-05-19|2017-07-25|Merial, Inc.|Recombinant spike protein subunit based vaccine for porcine epidemic diarrhea virus |

---

US10568954B2|2014-12-11|2020-02-25|Intervet Inc.|Process for ready-to-use PCV/M.Hyo combination vaccine|

---

EP3432920A1|2016-03-23|2019-01-30|Intervet International B.V.|A vaccine for intradermal application against pcv2 and prrs virus infection|

---

BR112018069100A2|2016-03-23|2019-01-29|Intervet Int Bv|a combined vaccine against pcv2 virus infection and mycoplasma hyopneumoniae|

---

BR112019012504A2|2016-12-23|2019-11-19|Intervet Int Bv|pig combination vaccine|

---

CN106668855B|2017-02-03|2020-11-24|四川省华派生物制药有限公司|Mycoplasma hyopneumoniae inactivated vaccine and classical swine fever live vaccine combined vaccine and preparation method thereof|

---

WO2018189290A1|2017-04-13|2018-10-18|Intervet International B.V.|Vaccines containing swine pathogens for associated non-mixed use|

---

CN109010818B|2017-06-09|2021-11-09|普莱柯生物工程股份有限公司|Bivalent vaccine composition for preventing and/or treating porcine circovirus infection and preparation method and application thereof|

---

CN109125719A|2017-06-19|2019-01-04|普莱柯生物工程股份有限公司|It is a kind of containing 3 type of pig circular ring virus, the immunogenic composition of porcine circovirus 2 type antigen and its application|

---

CN109207502B|2018-10-10|2021-08-10|浙江洪晟生物科技股份有限公司|Porcine mycoplasma pneumonia and porcine circovirus type 2 recombinant protein and preparation of bivalent vaccine|

---

CN109234418B|2018-11-20|2021-08-13|湖南中净生物科技有限公司|Primer, kit and method for identifying mycoplasma hyopneumoniae wild strain and vaccine strain|

---

CN112294953A|2020-12-31|2021-02-02|北京科牧丰生物制药有限公司|PCV2 type baculovirus vector, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine and preparation method thereof|

法律状态:
2017-08-29| B25A| Requested transfer of rights approved|Owner name: ZOETIS SERVICES LLC (US) |

---

2017-09-12| B25G| Requested change of headquarter approved|Owner name: ZOETIS SERVICES LLC (US) |

---

2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

---

2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2019-04-24| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|Free format text: NOTIFICACAO DE DEVOLUCAO DO PEDIDO POR NAO SE ENQUADRAR NO ART. 229-C DA LPI. |

---

2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2021-01-12| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

---

2021-05-11| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-06-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 03/04/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

US201261620175P| true| 2012-04-04|2012-04-04|

---

US61/620,175|2012-04-04|

---

PCT/US2013/035088|WO2013152083A2|2012-04-04|2013-04-03|Pcv/mycoplasma hyopneumoniae combination vaccine|

---