# Patent

## Title
PCV/Mycoplasma hyopneumoniae combination vaccine

## Abstract

This invention provides a multivalent immunogenic composition including a soluble portion of a Mycoplasma hyopneumoniae (M. hyo) whole cell preparation; and a porcine circovirus type 2 (PCV2) antigen, wherein the soluble portion of the M. hyo preparation is substantially free of both (i) IgG and (ii) immunocomplexes comprised of antigen bound to immunoglobulin.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 13/850,329, filed Mar. 26, 2013, which claims the benefit of U.S. Provisional Application No. 61/620,175, filed Apr. 4, 2012, the contents each of which are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates to 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 an M. hyo whole cell preparation and a PCV2 antigen and its use in a vaccine for protecting pigs against enzootic pneumonia and Post-weaning Multisystemic Wasting Syndrome (PMWS).
BACKGROUND OF THE INVENTION
Enzootic pneumonia in swine, also called mycoplasmal pneumonia, is caused by M. hyo. The disease is a chronic, non-fatal disease affecting pigs of all ages. Infected pigs show only mild symptoms of coughs and fever, but the disease has significant economic impact due to reduced feed efficiency and reduced weight gain. Enzootic pneumonia is transmitted from pig to pig through the nasal passages by airborne organisms expelled from the lungs of infected pigs. The primary infection by M. hyo may be followed by secondary infection by other mycoplasma species (Mycoplasma hyorhinis and Mycoplasma flocculare) as well as other bacterial pathogens.
M. hyo is a small, prokaryotic microbe capable of a free living existence, although it is often found in association with eukaryotic cells because it has absolute requirements for exogenous sterols and fatty acids. These requirements generally necessitate growth in serum-containing media. M. hyo is bounded by a cell membrane, but not a cell wall.
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 swine, colonizing the trachea, bronchi, and bronchioles. The mycoplasma produces a ciliostatic factor which causes the cilia lining the respiratory passages to stop beating. Eventually, the cilia degenerate, leaving the pig prone to infection by secondary pathogens. Characteristic lesions of purple to gray areas of consolidation are observed in infected animals. Surveys of slaughtered animals revealed lesions in 30 to 80% of swine. Results from 37 herds in 13 states indicated that 99% of the herds had hogs with pneumonia lesions typical of enzootic pneumonia. Therefore, the need for effective preventative and treatment measures are great.
Antibiotics such as tiamulin, trimethoprim, tetracyclines and lincomycin have some benefit, but are expensive and require prolonged use. Additionally, antibiotics have not been shown to effectively eliminate spread or reinfection of M. hyo. Prevention by maintaining pathogen-free herds is sometimes possible but reintroduction of M. hyo often occurs. Due to the serious economic consequences of swine pneumonia, vaccines against M. hyo have been sought. Vaccines containing preparations of mycoplasmal organisms grown in serum-containing medium have been marketed, but raise concerns regarding adverse reactions induced by serum components (such as immunocomplexes or non-immunogenic specific proteins) present in the immunizing material. Other attempts to provide M. hyo vaccines have been successful, but the disease remains widespread.
M. hyo and porcine circovirus type 2 (PCV2) are the two most prevalent pathogens that are encountered in the pig industry. Swine infected with PCV2 exhibit a syndrome commonly referred to as Post-weaning Multisystemic Wasting Syndrome (PMWS). PMWS is clinically characterized by wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, 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, postweaning colibacillosis, dietetic hepatosis, and suppurative bronchopneumonia. M. hyo is associated with enzootic pneumonia and has also been implicated as one of the major co-factors in the development of Porcine Circovirus Associated Disease (PCVAD).
Porcine reproductive and respiratory syndrome (PRRS) is caused by an arterivirus, which has a particular affinity for the macrophages particularly those found in the lung (alveolar macrophages). These macrophages ingest and remove invading bacteria and viruses, but not in the case of the PRRS virus (PRRSV). In the case of the PRRS virus, it multiplies inside the macrophages producing more virus and kills the macrophages. Once PRRSV has entered a herd, it tends to remain present and active indefinitely. Up to 40% of the macrophages are destroyed, which allows bacteria and other viruses to proliferate and do damage. A common example of this is the noticeable increase in severity of enzootic pneumonia in grower/finisher units when they become infected with PRRS virus. More than half of weaning-age PRRS virus-negative pigs become infected before going to market.
What is needed is a PCV2/M. hyo combination vaccine against both PCV2 and mycoplasma infection in swine. Preferably, this multivalent vaccine will be compatible with other porcine antigens, such as PRRS virus antigen. It would be highly desirable to provide a ready-to-use in one bottle, single-dose PCV2/M. hyo combination vaccine.
SUMMARY OF THE INVENTION
The present invention provides a multivalent immunogenic composition including a soluble portion of a Mycoplasma hyopneumoniae (M. hyo) whole cell preparation; and a porcine circovirus type 2 (PCV2) antigen, wherein the soluble portion of the M. hyo preparation is substantially free of both (i) IgG and (ii) immunocomplexes comprised of antigen bound to immunoglobulin. In one aspect, the soluble portion of the M. hyo whole cell preparation has been treated with protein-A or protein-G prior to being added to the immunogenic composition. In a further 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.
In some embodiments, the composition of the present invention elicits 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 porcine circovirus type 1 expressing the porcine circovirus type 2 ORF2 protein. In another embodiment, the PCV2 antigen is in the form of a recombinant ORF2 protein. In still another embodiment, the recombinant ORF2 protein is expressed from a baculovirus vector.
In some embodiments, the PCV2/M. hyo composition of the present invention further includes at least one additional antigen. In one embodiment, the at least one additional antigen is protective against a microorganism that can cause disease in pigs.
In one embodiment, the microorganism includes bacteria, viruses, or protozoans. In another embodiment, the microorganism is selected from, but is 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, swine influenza virus (SIV), Escherichia coli antigen, Brachyspira hyodysenteriae, porcine respiratory coronavirus, Porcine Epidemic Diarrhea (PED) virus, rotavirus, Torque teno virus (TTV), Porcine Cytomegalovirus, Porcine enteroviruses, Encephalomyocarditis virus, a pathogen causative of Aujesky's Disease, Classical Swine fever (CSF) and a pathogen causative of Swine Transmissable Gastroenteritis, or combinations thereof.
In some embodiments, the composition of the present invention further includes an adjuvant. In one embodiment, the adjuvant is selected from, but is not limited to, the following: an oil-in-water adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an aluminum hydroxide adjuvant, a 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 elicits a protective immune response against both M. hyo and PCV2 when administered as a single dose administration. In further embodiments, the composition elicits 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 still further embodiments, a composition of the present invention elicits a protective response against both M. hyo and PCV2 when administered as a two dose administration.
The present invention also provides a method of immunizing a pig against M. hyo and PCV2. This method includes administering to the pig an immunogenic composition including a soluble portion of a Mycoplasma hyopneumoniae (M. hyo) whole cell preparation; and a porcine circovirus type 2 (PCV2) antigen, wherein the soluble portion of the M. hyo preparation is substantially free of both (i) IgG and (ii) immunocomplexes comprised of antigen bound to immunoglobulin. In one embodiment, the soluble portion of the M. hyo preparation of the administered composition includes at least one M. hyo protein antigen.
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.
In a further embodiment of the method of the present invention, the PCV2/M. hyo composition is administered in conjunction 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 given concurrently with the PCV2/M. hyo composition (i.e., as separate single vaccines) or combined in a ready-to-use vaccine.
In a further embodiment, the composition is administered to pigs having maternally derived antibodies against at least one of M. hyo and PCV2. In a still further embodiment, the composition is administered to pigs having maternally derived antibodies against both M. hyo and PCV2.
In one embodiment, the composition is administered to pigs at 3 weeks of age or older.
The present invention further provides a kit. This kit contains a bottle including an immunogenic composition. This immunogenic composition includes both a PCV2 antigen and the soluble portion of a Mycoplasma hyopneumoniae (M. hyo) whole cell preparation, wherein the soluble portion of the M. hyo preparation is substantially free of both (i) IgG and (ii) antigen/immunoglobulin immunocomplexes. In one embodiment, this kit further includes an instruction manual which contains the information to administer the immunogenic composition. In another embodiment, the immunogenic composition in the bottle is provided as a ready-to-use liquid composition.
Additionally, the present invention provides a method for preparing an immunogenic composition. This method includes: i) culturing M. hyo in a suitable media over periods ranging from 18-144 hours; ii) subsequently inactivating the M. hyo culture; iii) harvesting the inactivated culture fluid, wherein the inactivated culture fluid comprises an M. hyo whole cell preparation comprising both a soluble liquid fraction and insoluble cellular material; iv) separating the soluble liquid fraction from the insoluble cellular material; v) substantially removing both IgG and antigen/immunoglobulin immunocomplexes from the separated soluble liquid fraction to form a soluble portion of the M. hyo whole cell preparation; and vi) subsequently combining the soluble portion of the M. hyo whole cell preparation with a PCV2 antigen.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the efficacy of M. hyo monovalent vaccines prepared with M. hyo antigens from different treatments (T02-T10 described in Example 3) vs. a placebo (T01). The results are presented as % Lung Lesion Least Square Mean values.
FIG. 2 is a graph showing the PCV2 antigen potency results (PCV2 antigen ELISA) of M. hyo vaccines in combination 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 status of each sample is expressed as relative potency (RP).
FIG. 3 is a graph showing the PCV2 viremia results (PCV2 Quantitative PCR) observed with PCV/M. hyo vaccine formulations employing different adjuvant platforms.
FIG. 4 is a graph showing the PCV2 antibody ELISA (S/P) serological results observed with PCV/M. hyo vaccine formulations employing different adjuvant platforms on days 1, 20, and 42 of challenge.
FIG. 5 is a graph showing the PCV2 fecal shed obtained with the T02-T04 treatments described in Example 7 vs. a placebo (T01). The results are expressed as PCV2 DNA copies/ml.
FIG. 6 is a graph showing the PCV2 nasal shed obtained with the T02-T04 treatments described in Example 7 vs. the placebo (T01). The results are expressed as PCV2 DNA copies/ml.
FIGS. 7 (A & B) are graphs showing the results of an interferon-gamma (IFN-γ) test that measures PCV2-specific cellular mediated immune (CMI) responses. The results of pos-vaccination/pre-challenge are presented in FIG. 7A, and the results of post-vaccination/post-challenge are presented in FIG. 7B. Stimulation of 5×106 cells was considered significant.
FIG. 8 depicts the M. hyo efficacy of the PCV2/M. hyo experimental vaccine formulations in SP-oil. The lung scores for formulations employing M. hyo treatments T02-T08 vs. a placebo (T01) are depicted graphically in FIG. 8A. The table in FIG. 8B depicts the contrast of treatments T02-T08 with the placebo.
FIG. 9 is a flowchart which shows one embodiment of a manufacturing process used to prepare PCV2-compatible Protein-A treated M. hyo antigen.
FIG. 10 is a table showing the adjuvant evaluation for virucidal activity against PRRS virus.



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 is one embodiment of a nucleotide sequence encoding p46 from the P-5722 strain of M. hyo;
SEQ ID NO: 2 is one embodiment of an amino acid sequence corresponding to p46 from the P-5722 strain of M. hyo;
SEQ ID NO: 3 is one embodiment of a nucleotide sequence encoding p97 from the P-5722 strain of M. hyo;
SEQ ID NO: 4 is one embodiment of an amino acid sequence corresponding to p97 from the P-5722 strain of M. hyo;
SEQ ID NO: 5 is one embodiment of a genomic sequence encoding a chimeric PCV1-2 virus;
SEQ ID NO: 6 is one embodiment of a nucleotide sequence corresponding to ORF2 of a porcine circovirus;
SEQ ID NO: 7 is one embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 8 is one embodiment of a genomic sequence encoding a chimeric PCV1-2 virus;
SEQ ID NO: 9 is one embodiment of a nucleotide sequence corresponding to ORF2 of a porcine circovirus;
SEQ ID NO: 10 is one embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 11 is one embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 12 is one embodiment of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11;
SEQ ID NO: 13 is one embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 14 is one embodiment of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13;
SEQ ID NO: 15 is one embodiment of an amino acid sequence corresponding to the ORF2 polypeptide of a porcine circovirus;
SEQ ID NO: 16 is one 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 one embodiment of a nucleotide sequence corresponding to ORF2 to ORF5 of the PRRS virus isolate designated ISU-55.
SEQ ID NO: 18 is one 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 Mycoplasma hyopneumoniae (M hyo) whole cell preparation; and a porcine circovirus type 2 (PCV2) antigen, wherein the soluble portion of the M. hyo preparation is substantially free of both (i) IgG and (ii) immunocomplexes comprised of antigen bound to immunoglobulin. In one embodiment, the composition elicits a protective immune response in a pig against both PCV2 and M. hyo.
Applicants have surprisingly discovered that the insoluble fraction of the M. hyo whole cell preparation is non-immunogenic. In contrast, the IgG-free M. hyo soluble 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 M. hyo soluble preparation an effective platform for the multivalent vaccines of this invention, including one-bottle, ready-to-use formulations. Applicants have also surprisingly discovered that removing the immunoglobulin and the insoluble cell debris from the M. hyo preparation enhances the safety of the immunogenic composition.
As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein antigen” includes a plurality of protein antigens, including mixtures thereof.
As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements.
As defined herein, a soluble portion of an M. hyo whole cell preparation refers to a soluble liquid fraction of an M. hyo whole cell preparation after separation of the insoluble material and substantial removal of IgG and antigen-bound immunocomplexes. The M. hyo soluble portion may alternatively be referred to herein as the supernatant fraction, culture supernatant and the like. It includes M. hyo-expressed soluble 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 M. hyo-specific soluble proteins, the soluble portion of the M. hyo whole cell preparation also includes heterologous proteins, such as those contained in the culture medium used for M. hyo fermentation.
The term “antigen” refers to a compound, composition, or immunogenic 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 may be generated to the whole molecule, or to a portion of the molecule (e.g., an epitope or hapten).
As defined herein, an “immunogenic or immunological composition”, refers to a composition of matter that comprises at least one antigen which elicits an immunological response in the host of a cellular and or antibody-mediated immune response to the composition or vaccine of interest.
The term “immune response” as used herein refers to a response elicited in an animal. An immune response may refer to cellular immunity (CMI); humoral immunity or may involve both. The present invention also contemplates a response limited to a part of the immune system. Usually, an “immunological 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 yd T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological 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 either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.
As used herein, 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 elicited by a vaccine against a specific infectious organism.
An “adjuvant” as used herein means a composition comprised of one or more substances that enhances the immune response to an antigen(s). The mechanism of how an adjuvant operates is not entirely known. Some adjuvants are believed to enhance the immune response by slowly releasing the antigen, while other adjuvants are strongly immunogenic in their own right and are believed to function synergistically.
As used herein, the term “multivalent” means a vaccine containing more than one antigen whether from the same species (i.e., different isolates of Mycoplasma hyopneumoniae), from a different species (i.e., isolates from both Pasteurella hemolytica and Pasteurella multocida), or a vaccine containing a combination of antigens from different genera (for example, a vaccine comprising antigens from Pasteurella multocida, Salmonella, Escherichia coli, Haemophilus somnus and Clostridium).
The term “pig” or “piglet” as used herein means an animal of porcine origin, while “sow” refers to a female of reproductive age and capability. A “gilt” is a female pig who has never been pregnant.
As used herein, the term “virulent” means an isolate that retains its ability to be infectious in an animal host.
“Inactivated vaccine” means a vaccine composition containing an infectious organism or pathogen that is no longer capable of replication or growth. The pathogen may be bacterial, viral, protozoal or fungal in origin. Inactivation may be accomplished by a variety of methods including freeze-thawing, chemical treatment (for example, treatment with thimerosal or formalin), sonication, radiation, heat or any other convention means sufficient to prevent replication or growth of the organism while maintaining its immunogenicity.
The term “variant” as used herein refers to a polypeptide or a nucleic acid sequence encoding a polypeptide, that has one or more conservative amino acid variations or other minor modifications such that the corresponding polypeptide has substantially equivalent function when compared to the wild-type polypeptide.
“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 similar residue. Examples of conservative variations include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue, such as the substitution of arginine for lysine, glutamic acid for 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 parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
As used herein, the terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable vehicle” are interchangeable and refer to a fluid vehicle for containing 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 may 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, colors and the like.
As used herein, the term “vaccine composition” includes at least one antigen or immunogen in a pharmaceutically acceptable vehicle 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 arts, taking into consideration 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, via mucosal administration (e.g., 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 (e.g., injectable administration) such as sterile suspensions or emulsions. Vaccine compositions may be administered as a spray or mixed in food and/or water or delivered in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard pharmaceutical texts, such as “Remington's Pharmaceutical Sciences,” 1990 may be consulted to prepare suitable preparations, without undue experimentation.
“North American PRRS virus” means any PRRS virus having 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 around the early 1990's (see, e.g., Collins, J. E., 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 herein. Genetic characteristics refer to genomic nucleotide sequence similarity and amino acid sequence similarity shared by North American PRRS virus strains. Chinese PRRS virus strains generally evidence about 80-93% nucleotide sequence similarity with North American strains.
“European PRRS virus” refers to any strain of PRRS virus having the genetic characteristics associated with the PRRS virus that was first isolated in Europe around 1991 (see, e.g., Wensvoort, G., et al., 1991, Vet. Q. 13:121-130). “European PRRS virus” is also sometimes referred to in the art as “Lelystad virus”. Further examples of European PRRS virus strains are described herein.
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.
An “Infectious clone” is an isolated or cloned genome of the disease agent (e.g. viruses) that can be specifically and purposefully modified in the laboratory and then used to re-create the live genetically modified organism. A live genetically modified virus produced from the infectious clone can be employed in a live viral vaccine. Alternatively, inactivated virus vaccines can be prepared by treating the 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 made from killed whole cell mycoplasma preparations (bacterins). In contrast, the present invention employs a soluble portion of a Mycoplasma hyopneumoniae (M. hyo) whole cell preparation for combination with the PCV2 antigen, wherein the soluble portion of the M. hyo preparation is substantially free of both (i) IgG and (ii) immunocomplexes comprised of antigen bound to immunoglobulin.
M. hyo has absolute requirements for exogenous sterols and fatty acids. These requirements generally necessitate growth of M. hyo in serum-containing media, such as porcine serum. Separation of the insoluble material from the soluble portion of the M. hyo whole cell preparation (e.g., by centrifugation, filtration, or precipitation) does not remove the porcine IgG or immune complexes. In one embodiment of the present invention, the M. hyo soluble portion is treated with protein-A or protein-G in order to substantially remove the IgG and immune complexes contained in the culture supernatant. In this embodiment, it is understood that protein A treatment occurs post-M. hyo fermentation. This is alternatively referred to herein as downstream protein A treatment. In another embodiment, upstream protein A treatment of the growth media (i.e., before M. hyo fermentation) can be employed. Protein A binds to the Fc portion of IgG. Protein G binds preferentially to the Fc portion of IgG, but can also bind to the Fab region. Methods for purifying/removing total IgG from crude protein mixtures, such as tissue culture supernatant, serum and ascites 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 approximately 46 kD (p46), 64 kD (p64) and 97 kD (p97) molecular weights. In another embodiment, the supernatant fraction at least includes the p46, p64 and p97 M. hyo protein antigens. The M. hyo protein of approximately 64 kD (p64) may be alternatively referred to herein as the p65 surface antigen from M. hyo described by Kim et al. [Infect. Immun. 58(8):2637-2643 (1990)], as well as in U.S. Pat. No. 5,788,962.
Futo et al. described the cloning and characterization of a 46 kD surface protein from M. hyo, which can be employed in the compositions of this invention [J. Bact 177: 1915-1917 (1995)]. In one embodiment, the M. hyo culture supernatant includes the p46 whose corresponding nucleotide and amino acid sequences from the P-5722 strain are set forth in SEQ ID NOs: 1 and 2, respectively. It is further contemplated that variants of such p46 sequences can be employed in the compositions of the present invention, as described below.
Zhang et al. described and characterized a p97 adhesin protein of M. hyo [Infect. Immun. 63: 1013-1019, 1995]. Additionally, King et al. described a 124 kD protein termed Mhp1 from the P-5722 strain of M. hyo and presented data suggesting that Mhp1 and p97 are the same protein [Vaccine 15:25-35 (1997)]. Such p97 proteins can be employed in the compositions of this invention. In one embodiment, the M. hyo culture supernatant includes the p97 whose corresponding nucleotide and amino acid sequences from the P-5722 strain are set forth in SEQ ID NOs: 3 and 4, respectively. It is further contemplated that variants of such p97 sequences can be employed in the compositions of the present invention, as described below.
The M. hyo culture supernatant may include further M. hyo specific protein antigens such as, but not limited to, proteins of approximately 41 kD (p41), 42 kD (p42), 89 kD (p89), 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 can include M. hyo specific protein antigens of approximately 102 kD (p102) and 216 kD (p216). See, U.S. Pat. Nos. 6,162,435 and 7,419,806 to Minnion et al.
Any M. hyo strain may 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 may 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, U.S. Department of Agriculture, Peoria, Ill.). The 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, deposited on May 30, 1990 pursuant to the accessibility rules required by the U.S. Patent and Trademark Office. In view of the widespread dissemination of the disease, strains may also be obtained by recovering M. hyo from lung secretions or tissue from swine infected with known strains causing mycoplasmal pneumonia in swine.
It is understood by those of skill in the art that variants of the M. hyo sequences can be employed in the compositions of the present invention. Such variants could vary by as much as 10-20% in sequence identity and still retain the antigenic characteristics that render it 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 identify with the full-length genomic sequence of the wild-type M. hyo strain. The antigenic characteristics of an immunological composition can be, for example, estimated by the challenge experiment as provided in the Examples. Moreover, 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 the protective immunity as compared to the wild-type M. hyo protein.
In one embodiment, M. hyo soluble 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 at about 2 μg/ml to 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 can be included in the compositions at a final amount of about 5.5% to about 35% of the M. hyo whole culture protein A-treated supernatant.
The M. hyo soluble preparation of the present invention is both safe and efficacious against M. hyo and is suitable for single dose administration. In addition, Applicants have surprisingly discovered that the M. hyo soluble preparation can be effectively combined with antigens from other pathogens, including PCV2, without immunological interference between the antigens. This makes the M. hyo soluble preparation an effective platform for multivalent vaccines, including the PCV2/M. hyo combination vaccine of this invention. The PCV2 antigen may be given concurrently with the M. hyo composition (i.e., as separate single vaccines), but is preferably combined in a ready-to-use, one-bottle vaccine.
In one embodiment, the immunogenic PCV2/M. hyo compositions of the present invention include at least one additional antigen. In one embodiment, the at least one additional antigen is protective against a microorganism that can cause disease in pigs.
In some embodiments, the at least one additional antigen component is protective against bacteria, viruses, or protozoans that are known to 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 bronchiseptica, Salmonella choleraesuis, Salmonella enteritidis, Erysipelothrix rhusiopathiae, Mycoplama hyorhinis, Mycoplasma hyosynoviae, leptospira bacteria, Lawsonia intracellularis, swine influenza virus (SIV), Escherichia coli antigen, Brachyspira hyodysenteriae, porcine respiratory coronavirus, Porcine Epidemic Diarrhea (PED) virus, rotavirus, Torque teno virus (TTV), Porcine Cytomegalovirus, Porcine enteroviruses, Encephalomyocarditis virus, a pathogen causative of Aujesky's Disease, Classical Swine fever (CSF) and a pathogen causative of Swine Transmissable Gastroenteritis, or combinations thereof.
In one embodiment, a PCV2/M. hyo combination vaccine according to the present invention is provided as a single-dose, ready-to-use in one bottle vaccine. Such a ready-to-use combination vaccine requires no mixing of separate vaccines, so there is no risk of contamination or additional labor associated with mixing and no requirement to use the mixture within a few hours. Also, a one-bottle PCV2/M. hyo combination vaccine cuts waste and refrigerator storage space in half. Furthermore, one-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 provided as either a two-dose, ready-to-use vaccine (Circumvent®PCVM) or as a single-dose, 2-bottle vaccine which requires the simultaneous administration of separate vaccines (e.g., Ingelvac CircoFLEX® and Ingelvac MycoFLEX®). Preferably, the PCV2/M. hyo combination according to the present invention would be compatible with other antigens, such as PRRS virus antigen, such that all antigens can be administered in a single-dose.
In some embodiments, the PCV2 antigen component of an 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 expressing 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 U.S. Pat. Nos. 7,279,166 and 7,575,752, which are incorporated herein by reference in their entirety.
In one embodiment, the full-length DNA sequence of the genome of the chimeric PCV1-2 virus 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.
In yet another embodiment, the full-length DNA sequence of the genome of the chimeric PCV1-2 virus corresponds to SEQ ID NO: 8. In one embodiment, the immunogenic ORF2 capsid gene of the chimeric PCV1-2 virus 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, the PCV2 ORF2 DNA and protein of the chimeric PCV1-2 virus are not limited to the sequences described above since PCV2 ORF2 DNA and protein is a highly conserved domain within PCV2 isolates.
In some embodiments, the PCV2 antigen component of an 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 still another embodiment, the recombinant PCV2 ORF2 protein corresponds to SEQ ID NO: 7. In a still further embodiment, the recombinant PCV2 ORF2 protein corresponds to SEQ ID NO: 10.
However, the present invention is not limited to the particular ORF2 DNA and protein sequences described above. Since PCV2 ORF2 DNA and protein is a highly conserved domain within PCV2 isolates, any PCV2 ORF2 is highly likely to be effective as the source of the PCV2 ORF2 DNA and/or polypeptide as used in the chimeric PCV1-2 virus or in the recombinant PCV2 protein.
An example of a suitable PCV2 isolate from which the PCV2 ORF2 DNA and protein sequences can be derived is PCV2 isolate number 40895 (deposited in the ATCC on Dec. 7, 2001 and assigned ATCC Patent Deposit Designation PTA-3914). The genomic (nucleotide) sequence of the 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 these PCV2 isolates are AF055391, AF055392, AF055393 and AF055394, respectively.
In some forms, immunogenic portions of PCV2 ORF2 protein are used as the antigenic component in the composition. For example, truncated and/or substituted forms or fragments of PCV2 ORF2 protein may be employed in the compositions of the present invention.
It is understood by those of skill in the art that variants of the PCV2 sequences can be employed in the compositions of the present invention. Such variants could vary by as much as 10-20% in sequence identity and still retain the antigenic characteristics that render it 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 identify with the full-length genomic sequence of the wild-type PCV2 isolate. The antigenic characteristics of an immunological composition can be, for example, estimated by the challenge experiment as provided in the Examples. Moreover, the antigenic characteristic of a modified PCV2 antigen is still retained when the modified antigen confers at least 70%, preferably 80%, more preferably 90% of the protective immunity as compared to the wild-type PCV2 ORF2 protein.
The PCV2 antigen component is provided in the immunogenic composition at an antigen inclusion level effective for inducing the desired immune response, namely reducing the incidence of or lessening the severity of clinical signs resulting from PCV2 infection.
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, wherein RP is the Relative Potency unit 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 about 0.5% to about 5% of 20-times (20×) concentrated bulk PCV1-2 antigen.
In another embodiment, the PCV2 ORF2 recombinant protein is included in the compositions of the invention at a level of at least 0.2 μg antigen/ml of the final immunogenic composition (μg/ml). In a further embodiment, the PCV2 ORF2 recombinant protein inclusion level is from about 0.2 to about 400 μg/ml. In yet another embodiment, the PCV2 ORF2 recombinant protein inclusion level is from about 0.3 to about 200 μg/ml. In a still further embodiment, the PCV2 ORF2 recombinant protein inclusion level is from about 0.35 to about 100 μg/ml. In still another embodiment, the PCV2 ORF2 recombinant protein inclusion level is from about 0.4 to about 50 μg/ml.
In one embodiment, an immunogenic composition of the present invention includes the inventive combination of at least one M. hyo soluble antigen (e.g., two or more) and a porcine circovirus type 2 (PCV2) antigen, as well as a PRRS virus antigen. In another embodiment, the composition elicits a protective immune response in a pig against M. hyo, PCV2 and PRRS virus.
In one embodiment, a PCV2/M. hyo/PRRS combination vaccine is provided as a single-dose, 2-bottle vaccine. For example, in some embodiments, a PCV2/M. hyo combination is provided as a stable liquid composition in a first bottle and a PRRS virus is provided in a lyophilized state in a second bottle. In some embodiments, additional porcine antigens can be added to either the first or the second bottle.
In one embodiment, the PRRS virus component is provided as a lyophilized, genetically modified live virus. Prior to administration, the PCV2/M. hyo liquid from a first bottle can be used to re-hydrate the PRRS virus in a second bottle 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 provided as a single-dose, 3-bottle vaccine which requires the simultaneous administration of three separate vaccines (e.g., Ingelvac CircoFLEX®, Ingelvac MycoFLEX® and Ingelvac®PRRS MLV).
The PRRS etiological agent was isolated for the first time in The Netherlands, and named as Lelystad virus. This virus was described in WO 92/21375 (Stichting Centraal Diegeneeskundig Instituut). An isolate of the European PRRS virus was deposited in the Institut Pasteur of Paris, number 1-1102. The North American type was isolated almost simultaneously with the isolation of the European type virus, and is described in WO-93/03760 (Collins et al.) An isolate of the North American type virus was deposited in the American Type Culture Collection (ATCC), number VR-2332.
Different strains have been isolated from both the European and North American virus types. WO 93/07898 (Akzo) describes a European strain, and vaccines derived from it, deposited in CNCM (Institut Pasteur), number 1-1140. Also, WO 93/14196 (Rhone-Merieux) describes a new strain isolated in France, deposited in CNCM (Institut Pasteur), number 1-1153. Furthermore, EP0595436 B1 (Solvay) describes a new North American type strain, more virulent than the one initially described, and vaccines thereof. This strain has been deposited in ATCC, but the deposit number is not detailed in the patent application. In addition, ES2074950 BA (Cyanamid Iberica) and its counterpart GB2282811 B2 describe a so-called “Spanish strain”, that is different from other European and North American strains. This “Spanish strain” has been deposited in European Animal Cell Culture Collection (EACCC), number V93070108.
Suitable PRRS virus antigens 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 employed in the compositions according to the present invention is a North American PRRS virus.
In some embodiments, the PRRS virus antigen component employed 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 unable to produce a pathogenic infection yet is able to elicit an effective immunoprotective response against infection by the wild-type PRRS virus.
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 of the North American PRRS virus isolate designated P129 is described in U.S. Pat. No. 6,500,662 which is hereby incorporated fully by reference. The sequence of P129 cDNA is disclosed in Genbank Accession Number AF494042 and in U.S. Pat. No. 6,500,662.
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 Nov. 9, 2011, the contents of which (including any US National Stage filings based on this International Application) are incorporated herein by reference in their entirety. Preferably, the PRRS virus is modified to prevent downregulation of interferon-mediated function.
In other embodiments, the PRRS virus antigen component employed in the compositions of the invention is the PRRS virus isolate designated ISU-55. The ISU-55 isolate was deposited in the American Type Culture Collection (ATCC), under the 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.
Another suitable North American PRRS virus isolate which can be used in the compositions is ISU-12, which was deposited in the ATCC under the accession numbers VR2385 [3×plaque purified] and VR2386 [non-plaque purified]. Still other suitable North American PRRS virus isolates which can be employed in the compositions of this invention are the following: ISU-51, ISU-3927, ISU-1894, ISU-22 and ISU-79, which were deposited in the ATCC under the accession numbers VR2498, VR2431, VR2475, VR2429 and VR2474, respectively. Genetically modified versions of any of these ISU isolates can be employed in the compositions of this invention. These ISU isolates and the ISU-55 isolate are described in detail in the following U.S. patents to Paul, et al: U.S. Pat. Nos. 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 incorporated herein by reference in their entirety.
In still other embodiments, the PRRS virus antigen component employed 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 thereof. For example, the PRRS virus can be a modified live virus based on the isolate identified as ATCC VR2332, which is employed in INGELVAC® PRRS ATP and INGELVAC® PRRS MLV, from Boehringer Ingelheim Vetmedica, Inc.
In still other embodiments, the PRRS virus antigen component employed 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. 1-1102, described above. Nucleotide and amino acid sequences corresponding to the 1-1102 deposit are described in U.S. Pat. No. 5,620,691 to Wensvoort et al, which is hereby fully incorporated herein by reference. The preparation of an infectious clone of a European PRRS virus isolate or Lelystad virus is described in U.S. Pat. No. 6,268,199 which is hereby fully incorporated herein by reference.
Other examples of suitable PRRS virus isolates include, but are not limited to, those described above. Also, live, genetically modified versions of the PRRS virus isolates can be employed in the compositions of the present invention. An infectious clone can be used to re-create such live genetically modified organisms.
It is understood by those of skill in the art that variants of the PRRS virus sequences can be employed in the compositions of the present invention. Such variants could vary by as much as 10-20% in sequence identity and still retain the antigenic characteristics that render it 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 identify with the full-length genomic sequence of the wild-type PPRS virus isolate. The antigenic characteristics of an immunological composition can be, for example, estimated by challenge experiments. Moreover, 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 the protective immunity as compared to the wild-type PRRS virus antigen.
In one embodiment, the PRRS virus antigen component is a genetically modified, live virus which is included in the compositions of the invention at a level of at least 2.1≦TCID50≦5.2, wherein TCID50 is the tissue culture infectious dose 50% determined by antigen quantification (in vitro potency test)
The PCV2 antigen component of the PCV2/M. hyo/PRRS compositions of the invention can be in the form of a chimeric type-1-type 2 circovirus, the chimeric virus including an inactivated recombinant porcine circovirus type 1 expressing 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.
Suitable PCV2 antigens 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 to be employed in the compositions of the invention include, but are not limited to, the PCV2 sequences described above and variants thereof.
Vaccines of the present invention can 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 can 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 vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
Vaccines of the present invention can further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others. Types of suitable adjuvants 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 E adjuvant and combinations thereof. Some specific examples of adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, Corynebacterium parvum, Bacillus Calmette Guerin, aluminum hydroxide gel, glucan, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, certain synthetic polymers such as poly amino acids and co-polymers of amino acids, Block copolymer (CytRx, Atlanta, Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), 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.
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) 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.
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 salt 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 of block copolymer, about 2 to 6% vol/vol of 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 salt 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.
Yet another example of a suitable adjuvant for use in the compositions of the invention is AMPHIGENTM adjuvant which consists of de-oiled lecithin dissolved in an oil, usually light liquid paraffin.
Other examples of adjuvants useful in the compositions of the invention are the following proprietary adjuvants: Microsol Diluvac Forte® duel emulsion adjuvant system, Emunade adjuvant, and Xsolve adjuvant. Both the Emunade and Xsolve adjuvants are emulsions of light mineral oil in water, but Emunade also contains alhydrogel, and d,l-α-tocopheryl acetate is part of the XSolve adjuvant. A still further example of a suitable adjuvant for use in the compositions of the invention is ImpranFLEXTM adjuvant (a water-in-oil adjuvant). A still further example of a suitable adjuvant is a Carbomer (Carbopol®) based adjuvant. Preferred Carbopol® adjuvants include Carbopol® 934 polymer and Carbopol® 941 polymer.
In one embodiment, the adjuvant or adjuvant mixture is added in an amount of about 100 μg to about 10 mg per dose. In another embodiment, the adjuvant/adjuvant mixture is added in an amount of about 200 μg to about 5 mg per dose. In yet another embodiment, the adjuvant/adjuvant mixture is added in an amount of 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.
Other “immunomodulators” that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines. In one embodiment, the adjuvant may be a cyclodextrin derivative or a polyanionic polymer, such as those described in U.S. Pat. Nos. 6,165,995 and 6,610,310, respectively.
A further aspect relates to a method for preparing an immunogenic composition according to the present invention. This method comprises i) culturing M. hyo in a suitable media over periods ranging from 18-144 hours; ii) subsequently inactivating the M. hyo culture; iii) harvesting the inactivated culture fluid, wherein the inactivated culture fluid comprises an M. hyo whole cell preparation comprising both a soluble liquid fraction and insoluble cellular material; iv) separating the soluble liquid fraction from the insoluble cellular material; v) substantially removing both IgG and antigen/immunoglobulin immunocomplexes from the separated soluble liquid fraction to form a soluble portion of the M. hyo whole cell preparation; and vi) subsequently combining the soluble portion of the M. hyo whole cell preparation with a PCV2 antigen.
An example of a suitable media for culturing M. hyo is PPLO Broth (Mycoplasma Broth Base), which when supplemented with nutritive enrichments, is used for isolating and cultivating Mycoplasma. 
In some embodiments, the culture of M. hyo is grown until late log phase growth, after which the culture is inactivated. In some other embodiments, the culture is inactivated by raising the pH (e.g., to about 7.8). This occurs by exposing the production culture to an inactivation agent, such as binary ethylenimine (BEI). The BEI is generated in situ during 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 inactivation agent within the solution. In some embodiments, the inactivation agent is BEI and the neutralization agent is sodium thiosulfate. In one embodiment, the pH of the inactivated culture is adjusted to about 7.4 by adding sodium thiosulfate.
In some embodiments, the soluble liquid fraction of the M. hyo whole cell preparation is separated from the insoluble cellular material using conventional methods. In one embodiment, this separation is by a filtration step. In another embodiment, this separation is by a centrifugation step. In yet another embodiment, the separation is by a precipitation step.
In one embodiment, the soluble liquid fraction of an inactivated, neutralized M. hyo whole cell preparation is treated with Protein A resin to substantially remove both the IgG and antigen/immunoglobulin immunocomplexes therein. In other embodiments, Protein G resin can be used to substantially remove both the IgG and antigen/immunoglobulin immunocomplexes contained in the soluble liquid fraction. Methods for removing both IgG and antigen/immunoglobulin immunocomplexes with either Protein A or Protein G resins are well known in the art.
According to a further 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 porcine antigen, such as, but not limited to, PRRS virus antigen as described above.
A further aspect of the present invention relates to a kit. A “kit” refers to a plurality of components which are grouped together. In one embodiment, a kit according to the present invention includes a bottle (or other suitable receptable) comprising an immunogenic composition. This immunogenic composition includes both a PCV2 antigen and the soluble portion of a Mycoplasma hyopneumoniae (M. hyo) whole cell preparation, wherein the soluble portion of the M. hyo preparation is substantially free of both (i) IgG and (ii) antigen/immunoglobulin immunocomplexes. Optionally, the kit can further include an instruction manual. The instruction manual includes the information to administer the immunogenic composition.
In some embodiments, the PCV2/M. hyo combination in the bottle of the kit is provided as a ready-to-use liquid composition. In other embodiments, the kit includes a second bottle comprising PRRS virus antigen. In some embodiments, the PRRS virus antigen is in the form of a genetically modified, live virus which is provided in a lyophilized state. In such instances, the instruction manual will include the directions for re-hydrating the PRRS virus component with the liquid contents from bottle containing the PCV2/M. hyo combination. The instruction manual will also include the information to administer the resultant PCV2/M. hyo/PRRS trivalent formulation(s).
In some embodiments, an immunogenic composition according to this invention is administered to pigs having maternally derived antibodies against M. hyo. In other embodiments, an immunogenic composition of the present invention is administered to pigs having maternally derived antibodies against both M. hyo and PCV2.
In some embodiments, a multivalent immunogenic composition according to the present invention is administered to a piglet aged 3 weeks or older. However, it is contemplated that a multivalent vaccine composition according to the invention may also be used to re-vaccinate gilts pre-breeding. As is known in the art, a gilt is a female pig that has never been pregnant. Vaccinated gilts will pass maternally derived antibodies onto their suckling newborns via colostrum.
It is further contemplated that a multivalent vaccine according to the invention can be used to annually re-vaccinate breeding herds. Preferably, a multivalent vaccine according to the present invention is administered to pigs (e.g., 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 provided as a ready-to-use PCV2/M. hyo formulation contained in one bottle. In another embodiment, a multivalent formulation requires mixing of a divalent vaccine according to the present invention contained in a first bottle with a monovalent vaccine contained in a second bottle. In one embodiment, the monovalent vaccine contained in the second bottle includes PRRS virus antigen. Optionally, additional antigens can be added to either of these bottles.
In some embodiments, the onset of immunity is from 2-3 weeks post-vaccination with a multivalent vaccine composition according to the present invention. In other embodiments, the duration of immunity is about 17-23 weeks post-vaccination with a multivalent vaccine composition according to the present invention.
The following examples set forth preferred materials and procedures in accordance with the present invention. However, it is to be understood that these examples are provided by way of illustration only, and nothing therein should be deemed a limitation upon the overall scope of the invention.
EXAMPLES
Example 1
Mycoplasma hyopneumoniae Production Methods for PCV2 Combinable M. Hyo Antigen
M. Hyo Fermentation and Inactivation
Media for seed scale and antigen production was prepared as follows. Porcine heart derived Pleuropenumonia-like Organism (PPLO) Broth (BD Biosciences catalog No. 21498) was made per manufacturer's directions (i.e., 21 g/L) and yeast extract solution was made at 21 g/L in USP. Yeast extract solution was then added to the PPLO at 6.25% and the mixture was sterilized by heating to 121° C. for ≧30 minutes. Cysteine hydrochloride was prepared at 90 g/L and filter sterilized. Dextrose solution was made by adding 450 g of dextrose per liter of USP water followed by heat sterilization. To prepare the final medium, porcine 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 a 10% v:v of a log phase culture of M. hyopeumoniae (strain P-5722-3). The culture was held at 37° C. and pH and dO were maintained at 7.0 and 25%, respectively. At late log phase growth, the culture was inactivated was inactivated by binary ethylenimine (BEI), an aziridine compound, produced from 2-bromoethylamine hydrobromide. Specifically, the inactivation occurred by raising the pH to 7.8 by adding 2-bromoethylaminehydrobromide (BEA) to a final concentration of 4 mM and incubating for 24 hours. The BEI was neutralized by addition of sodium thiosulfate at a 1:1 molar ratio followed by additional 24 hour incubation. The inactivated culture fluid was held at 2-8° C. until further processing.
Example 2
Chimeric Porcine Circovirus (cPCV) 1-2 Production Methods
The cPCV1-2 was constructed by cloning the immunogenic capsid gene of the pathogenic porcine circovirus type 2 (PCV2) into the genomic backbone of the nonpathogenic porcine circovirus type 1 (PCV1). The procedure for construction of the chimeric DNA clone is described, for example, in U.S. Pat. No. 7,279,166, which is incorporated herein by reference in its entirety. An infectious stock of the chimeric virus was acquired from Dr. X. J. Meng, Virginia Polytechnic Institute and State University, Blacksburg, Va., and was used to infect Porcine Kidney (PK)-15 cells grown in Minimum Essential Medium (MEM) supplemented with 0.05% lactalbumin hydrolysate (LAH), 30 μg/mL gentamicin sulfate, and 5% fetal bovine serum. The resulting cPCV1-2 infected PK-15 cells were further expanded by serial passing four more times using the same growth medium except with 2-3% fetal bovine serum. The fifth passage was frozen, thawed and filtered, and the resulting lysates were used to prepare a pre-master seed and subsequent master seed.
The medium which was used for producing virus seeds was the same as that used in producing virus stock. For the growth medium, MEM, OptiMEM, or equivalent is the basal medium which can be used for planting the PK-15 cell line for outgrowth. The growth medium can be supplemented with up to 10% bovine serum, up to 0.5% lactalbumin hydrolysate, up to 0.5% bovine serum albumin, and up to 30 μg/mL gentamicin. For the virus propagation medium, MEM, OptiMEM, or equivalent is used. The virus propagation medium can be supplemented with up to 0.5% lactalbumin hydrolysate, 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 mmol/L L-glutamine can be added to the growth medium and/or the virus propagation medium as required to sustain the cells.
The cPCV1-2 master seed virus are added to a cell suspension of PK-15 cells and adsorbed for up to 3 hours. Seed virus is diluted in growth basal medium to provide a multiplicity of infection (MOI) of 0.1-0.0001.
Cultures of PK-15 cells are initially inoculated with working seed virus at the time of cell planting, or when cells reach approximately 20% to 50% confluency. This initial passage may be referred as “One-Step Infection Method” for the production of antigen stock, or may be further used for serial passages. For serial passages, the cPCV1-2 infected PK-15 cells are further expanded up to passage 7 by serial splits at the ratio of 1:5-20 for virus propagation. Culture medium containing an infected cell suspension from the previous passage serves as seed material for the next passage. The cPCV1-2 infected cells 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. At harvest, rounding of cells and considerable floating debris is observed. Cultures are also observed for visual evidence of bacterial or fungal contamination. The incubation time between harvests for the cPCV antigen is provided in Table 1 below:







TABLE 1







Minimum and Maximum Times for Harvesting cPCV Antigen










Minimum/
Temperature


Method
Maximum Time
Range





One-Step Infection
5 to 16 days
36 ± 2° C.


Serial Passage (MSV + 3 to MSV + 7)
16 to 36 Days
36 ± 2° C.









The cPCV1-2 culture fluids are harvested into sterile vessels and are sampled for mycoplasma testing using known methods. Multiple harvests may be conducted from roller bottles, bioreactors and perfusion vessels.
Prior to inactivation of the harvested cPCV1-2 virus, one or more antigen lots may be concentrated (e.g., up to 60×) by ultrafiltration. The concentrates may be washed with balanced salt solution to reduce serum proteins.
The method of inactivation, attenuation, or detoxification of the cPCV1-2 virus will now be described. After cPCV antigen concentration, Beta-propiolactone (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 inactivating bulk antigen fluids are transferred to a second sterile vessel. The transferred antigen fluids are maintained at 2-7° C., with constant agitation, for a minimum of 24 hours. After a minimum of 24 hours, a second addition of 0.2% v/v of BPL is added to the pooled suspension. The contents are subsequently agitated, transferred to a third vessel, and maintained at 2-7° C., with constant agitation, for an additional time of not less than 84 hours. In general, the total inactivation time is not less than 108 hours and not more than 120 hours. The inactivation method is summarized in Table 2 below.







TABLE 2







Inactivation Method











Final
Temp.
Time-Hours


Inactivant
Concentration
Range
(Min/Max)





Beta-propiolactone
0.4% v/v (2 ×
2-7° C.
108-120


(BPL)
0.2% v/v additions)
(w/




Agitation)









The inactivation is terminated by the addition of a final concentration of not more than 0.1 M solution of sodium thiosulfate. The pH of the inactivated antigen stock is adjusted to about 6.8 using NaOH or HCl. Following inactivation, a representative sample is taken from the pool and tested for completion of inactivation. The inactivated cPCV1-2 antigen product is standardized to a meet a target of greater than 1.0 RP as measured via potency ELISA.
Example 3
Down Stream Processing of M. Hyo Antigens and Analytical Testing of these Processed Antigens
Down Stream Processing of M. Hyo Antigens:
Inactivated fermentation fluid (prepared as described above in Example 1) was treated for each indicated group as follows. These processed M. hyo antigens were employed in Example 4 below.
T02: (Whole Bulk) not processed.
T03: (10×UF concentrated) Concentrated via tangential flow filtration via a 100 KDa molecular weight cutoff membrane (hollow fiber). Final volume reduction was equal to 10×.
T04 & T05: (10×UF concentrated & centrifuged) Concentrated mycoplasma cells (from T03) were collected and washed one time with PBS via centrifugation at  ̃20,000×g (Sorvall model RC5B).
T06 & T07: (10× centrifuged) Inactivated fermentation fluid was centrifuged at  ̃20,000×g (Sorvall RC5B) and washed one time by resuspending the cells in PBS followed by an additional centrifugation. Final volume reduction was equal to 10×.
T08: (10× centrifuged & Heated) Mycoplasma cells were concentrated and washed per T06 and heated to 65° C. for 10 minutes.
T09: (Cell-free supernatant) Supernatant collected from the first centrifugation as described for T06 was filter sterilized through a 0.2 micron filter (Nalgene).
T10: (Cell-free supernatant-Protein-A treated) Sterile supernatant (prepared per T09) was mixed with Protein A resin (Protein A Sepharose, Pharmacia Inc) at a 10:1 volume ratio for 4 hours. Resin was removed sterile filtration and filtered fluid was stored at 2-8° C. This process uses post-fermentation “downstream” protein A treatment to remove antibodies and immunocomplexes. Although the present invention does not preclude upstream protein A treatment, the present inventors have found that in the case of M. hyo, upstream protein A treatment of the growth media led to p46 results which were lower and inconsistent as compared to untreated media (data not shown).

Analytical Testing of M. Hyo Downstream Processed Antigens

The downstream processed M. hyo antigens preparations (prepared as described above) were tested for the recovery of M. hyo specific p46 antigen, 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 presented below in Table 3.







TABLE 3







Characterization of M.hyo Downstream Processed Antigens











Bulk M.Hyo
PCV2 ab
qPCR DNA











Treatment
p46 RU/mL
S/P ratio
g1TTV
g2TTV














Whole bulk
809
0.248
1.00E+03
1.78E+03


10 × UF
6666
0.819
1.00E+03
9.94E+03


concentrated






10 × UF conc. +
614
0.019
0
0


Centrifuge






10 × 
763
−0.015
1.90E+02
1.91E+02


Centrifuged






10 × 
690
−0.012
0
2.07E+02


Centrifuged +






Heated






Cell-free supe 
719
0.242
4.20E+02
3.23E+03


Cell-free supe 
826
−0.014
0
2.06E+03


(Prot A)









With reference to Table 3 above, recovery of the M. hyo-specific p46 antigen was demonstrated for each of the M. hyo downstream processed antigen preparations. In addition, the following treatments successfully removed PCV2 antibody: 10×UF concentrated & centrifuged, 10× centrifuged, 10× centrifuged & heated and Cell-free supernatant (Protein-A treated). With respect to TTV, the following treatments successfully removed g1TTV: 10×UF concentrated & centrifuged, 10× centrifuged & heated, and Cell-free supernatant (Protein-A treated). Only the treatment designated 10×UF concentrated & centrifuged removed g2TTV. Torque teno virus isolates, including genotypes 1 and 2 are described in US20110150913, which is incorporated herein by reference in its entirety.
Since it is known in the art that Protein A binds IgG, it is understood by those of ordinary skill in the art that not only PCV2 antibody, but other swine antibodies, including PRRS antibody, HPS antibody, and SIV antibody will be effectively removed by the Protein-A treatment. This makes the Cell-free Protein-A treated M. hyo supernatant of this invention compatible not only with PCV2 antigen, but also with other porcine antigens due to the lack of immunological interference between the antigens. Additionally, the removal of the non-protective cell debris and removal of the immunoglobulin and antigen/immunoglobulin complexes is reasonably expected to make a safer vaccine.
Example 4
Preparation of M. Hyo Experimental Vaccine Formulations
All experimental M. hyo vaccines were formulated with a final concentration of 5% Amphigen adjuvant. In addition, all vaccines were standardized with a p46 ELISA and preserved with thimerosol. The experimental vaccine formulations were prepared with M. hyo antigens processed according to treatments T02-T10 above. In addition, Treatment T01 corresponded to a placebo (no M. hyo antigen, only 5% Amphigen adjuvant) whereas Treatment T11 is a positive control corresponding to an expired bacterin-based M. hyo vaccine (RespiSure-ONE®, Pfizer Animal Health). These formulations are described in Table 4 below.







TABLE 4







M.hyo Experimental Vaccine Formulations















M Hyo





IVP 
Target p46
antigen 
Adjuvant
Formulation


Treatment
Serial*
units/ds
(mL)
(mL)
Vol. (mL)












T01
123639 
5% Amphigen only, No Antigen



(Placebo)













T02
L100211A
452
279.36
250
1000


T03
L100211B
452
6.78
50
200


T04
L100211C
452
73.62
50
200


T05
L100211D
816
132.90
50
200


T06
L100211E
452
59.24
50
200


T07
L100211F
816
106.95
50
200


T08
L100211G
452
65.51
50
200


T09
L100211H
452
62.87
50
200


T10
L100211J
452
54.72
50
200









T11
A827870
Expired “RespiSure” vaccine





*Investigational Veterinary Product (IVP) Serial






Example 5
Evaluation of the In Vivo Efficacy 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). Pigs at 3 weeks of age 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. Animals were necropsied 28 days after challenge and the lungs were removed and scored for consolidation consistent with M. hyo infection. The primary criterion for protection against M. hyo challenge was lung consolidation scores. It is generally accepted that there is a relationship between the size of the lung lesions caused by enzootic pneumonia and an adverse effect on growth rate. Table 5 below contains the lung lesion scores for the respective treatment groups. Statistical significance was determined by a Mixed Model Analysis of lung scores for each group.







TABLE 5







Lung Lesion Results

















% Lung








p46 RP
Lesions Back
Range %







Target/
Transformed
Lung with





Treatment
Description
Observed
LS Means
Lesions
Contrast
p-value
Significant

















T01
Placebo (5%
N/A
11.7
1.2-44.3
N/A
N/A
N/A



Amphigen)








T02
Whole bulk
13/15.6
1.2
0.1-18.5
T01 vs 02 
0
Yes


T03
Whole bulk
13/11.9
0.3
0.0-2.8 
T01 vs 03 
0
Yes



UF 10x








T04
UF 10x +
13/28.1
5.9
0.0-40.5
T01 vs 04 
0.1589
No



Centrifuged








T05
UF 10x +
24/48.2
3.7
0.0-42.3
T01 vs T05
0.0309
Yes



Centrifuged








T06
10x Centrifuged
13/30.4
4.7
0.0-23.6
T01 vs 06 
0.0388
Yes


T07
10x Centrifuged
24/57.4
4.6
0.3-37.3
T01 vs T07
0.0323
Yes


T08
10x Centrifuged +
13/17.7
4.5
0.3-21.7
T01 vs T08
0.0137
Yes



Heat








T09
Supernatant
13/14.1
1.4
0.0-33.0
T01 vs T09
0.0004
Yes



(no cells)








T10
Supernatant +
13/12.1
3.1
0.0-25.8
T01 vs T10
0.0094
Yes



Prot A








T11
Expired RSO
13/12.5
2.2
0.1-32.1
T01 vs T11
0.0009
Yes









With reference to Table 5 above, the results with M. hyo antigens from different downstream processes indicated that all experimental vaccines except T04 significantly differed from the placebo. These M. hyo lesion results are depicted graphically in FIG. 1. As shown in FIG. 1, T04 gave unacceptable results. All other treatments differed significantly from the placebo (T01). The lung consolidation scores indicated that T02, T03 and T09-T11 gave the most efficacious protection against M. hyo challenge.
The p46 relative potency of the experimental vaccines was assessed by using a double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA). The p46 DAS ELISA results presented in Table 5 above indicate that all the experimental vaccines exceeded the target potency. In addition, the p46 relative potency was either maintained or increased during storage of the vaccines over a one-month period (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. While not wishing to be bound by any one theory, it is likely that cell “carcasses” are breaking up over time and released more of the membrane bound p46 antigen in the case of the centrifuged antigens.
Example 6
Evaluation of the Compatibility of the Experimental M. Hyo Vaccines with PCV2 Antigen
This study was conducted to evaluate the compatibility of the M. hyo experimental vaccines with M hyo antigens from different downstream processes with PCV2 antigen. The M. hyo experimental vaccine formulations are described in Tables 4 and 5 above. The observed p46 relative potencies for these vaccines are described in Table 5 above. These M. hyo experimental vaccines were each combined with 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, wherein the RP is the Relative Potency unit determined by PCV2 ELISA antigen quantification (in vitro potency test) compared to an efficacious reference vaccine.
The experimental M. hyo/PCV2 combination formulations were evaluated by PCV2 ELISA. The results are presented in FIG. 2. As shown in FIG. 2, only the M. hyo antigen preparations from the following downstream processes were compatible with the PCV2 antigen: Ultrafiltration & Centrifugation (T04 & T05), Centrifugation (T06 & T07), Centrifugation plus heat (T08) and Protein A-treated Supernatant (T10). Of these, the M. hyo Protein A-treated supernatant was the most compatible with PCV2 antigen when compared to the placebo control which included the chimeric virus and Amphigen adjuvant, but no M. hyo antigen. The level of chimeric PCV virus in the Protein-A treated supernatant was 1.5 RP as compared to 1.69 RP for the placebo. It was therefore concluded that there is no or minimal immunological interference between the Protein-A treated M. hyo soluble antigen preparation and PCV2 antigen of the chimeric virus.
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 M. hyo-PCV2 combinations.
Example 7
Evaluation of PCV2 Efficacy of a 1-Bottle PCV2/M. Hyo Combination Vaccine in Different Adjuvant Formulations
This study was designed to evaluate the PCV2 efficacy in a 1-bottle 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 an M. hyo soluble antigen preparation that was substantially free of IgG (i.e., Protein A-treated supernatant).
Processing of Fluids:
Inactivated M. hyo fermentation fluid (described above in Example 1) was treated for each indicated group as follows.
T02-T04: Whole fermentation fluid containing live M. hyopneumoniae cells (described above) was centrifuged at  ̃20,000×g (Sorvall RCSB) 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 the storage buffer and treatment with 2 column volumes of 1M 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 clarified/filtered M. hyopneumoniae antigen containing fluids were passed through the Protein A resin at a flow rate of 100 cm/hr. The flow through was collected and sterilized via 0.2 μM filter.

T05: This is a positive control corresponding to a Fostera PCV-like formulation (no M. hyo antigen). The level of the chimeric virus in this Fostera PCV-like formulation was approximately at Minimum Immunizing 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. The experimental vaccine formulations were prepared with M. hyo antigens processed according to treatments T02-T04 above. In addition, Treatment T01 corresponded to a placebo (sterile saline).
All vaccines were standardized with a p46 ELISA and preserved with thimerosol.
These experimental formulations are described in Table 6 below, wherein the symbol * indicates the M hyo antigen from global M hyo seed, Protein A treated supernatant and the symbol ** indicates Investigational Veterinary Product (IVP) serial.







TABLE 6







PCV2/M.hyo Experimental Vaccine Formulations Used 


for PCV2 Efficacy Study














PCV1-2
M Hyo*




Treatment
IVP Serial**
Ag
Ag
Adjuvant
Other













T01
87-244-DK
NA
Sterile













(Placebo)



Saline


T02
L0411RK08
1.6 RP
7.5 RP
10% SP Oil
NA


T03
L0411RK09


5% Amphigen



T04
L0611RK03


5% Amphigen +







5% SLCD



T05
L0611RK04

NA
20% SLCD









Pigs at 3 weeks of age 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.
FIG. 3 is a graph showing the PCV2 viremia results (PCV2 Quantitative PCR) observed with the different adjuvant platforms. It is noted that PCV2 viremia was used as the primary efficacy variable. The PCV2 viremia results are presented as DNA copies/ml. As shown in FIG. 3, all treatments had significantly less viremia compared to the placebo on days 28, 35 and 42 (challenge was day 21). The 10% SP-oil adjuvant had significantly less viremia compared to 5% Amphigen at Days 28 and 35. The 5% Amphigen plus 5% SLCD adjuvant had significantly less viremia compared to 5% Amphigen at Days 28 and 35. The 20% SLCD adjuvant platform had significantly less viremia compared to 5% Amphigen at Days 28, 35 and 42.
PCV2 Serology, PCV2 fecal shed, PCV2 nasal shed, Cell Mediated Immune (CMI) responses, lymphoid depletion, and Immunohistochemistry (IHC) were also monitored as secondary efficacy variables. These results will now be described below.
FIG. 4 is a graph showing the PCV2 ELISA results on days 1, 20 and 42 of the study (challenge was day 21). The status of each sample was expressed as a sample to positive ratio (S/P). As shown in FIG. 4, 20% SLCD was the only treatment which was significantly different from the placebo (T01) at both day 20 and day 42. Also, 5% Amphigen was the only treatment not significantly different from the placebo at day 20.
FIG. 5 is a graph showing the PCV2 fecal shed obtained with the T02-T04 treatments vs. the placebo (T01). These results are expressed as PCV2 DNA copies/ml. The results in FIG. 5 indicate that all treatments had significantly less fecal shed when compared to the placebo at day 42. In addition, 5% Amphigen & 5% SLCD (T04) had significantly less fecal shed as compared to 5% Amphigen (T03) at day 42. No other treatment differences were noted.
FIG. 6 is a graph showing the PCV2 nasal shed obtained with the T02-T04 treatments vs. the placebo (T01). These results are expressed as PCV2 DNA copies/ml. The results in FIG. 6 indicate that all treatments had significantly less nasal shed when compared to the placebo at day 42. In addition, 20% SLCD (T05) had significantly less nasal shed compared to 5% Amphigen (T03) at day 42. No other treatment differences were noted.
FIGS. 7 (A & B) are of two graphs showing the results of an interferon-gamma (IFN-γ) test that measures PCV2-specific cellular mediated immune (CMI) responses. The CMI results are shown post-vaccination/pre-challenge (FIG. 7A), and post-vaccination/post-challenge (FIG. 7B). In these graphs, stimulation of 5×106 cells was considered significant ( . . . ). All PCV2/M. hyo experiment vaccines gave a detectable IFN-γ response post-vaccination. The 10% SP-oil (T02) drove the strongest IFN-γ response post-vaccination. The 20% SLCD (T05) induced an earlier response, but the lowest response at 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 as compared to the placebo group.
Table 7 below shows the lymphoid depletion obtained with the experimental treatments contrasted to the placebo.







TABLE 7







PCV2 Histopathology (Lymphoid Depletion)










Lymphoid Depletion
Contrasted to Placebo












Treatment 
Positive
Negative
% Ever Pos.
P-value
Significant















Placebo
9
7
56%
NA
NA


10% SP-oil
1
15
6%
0.0059
Yes


5%
1
15
6%
0.0059
Yes


Amphigen







5%
0
16
0%
0.0008
Yes


Amph + 







5% SLCD







20% SLCD
1
15
6%
0.0059
Yes









The results presented in Table 7 above show that all vaccines afforded strong protection against lymphoid depletion. Also, no statistically significant vaccine treatment contrasts were observed. Table 8 below shows the immunohistochemistry obtained with the experimental treatments contrasted to the placebo.







TABLE 8







PCV2 Histopathology (Immunohistochemistry)










Immunohistochemistry
Contrasted to Placebo












Treatment
Positive
Negative 
% Ever Pos.
P-value
Significant















Placebo
12
4
75%
NA
NA


10% SP-oil
0
16
0%
0.0001
Yes


5%
1
15
6%
0.0002
Yes


Amphigen







5%
0
16
0%
0.0001
Yes


Amph + 







5% SLCD







20% SLCD
0
16
6%
0.0001
Yes









The results presented in Table 8 above show that all vaccines afforded strong protection against PCV2 colonization as evidenced by immunohistochemistry. Also, no statistically significant vaccine treatment contrasts were observed.
In conclusion, the results presented in this example demonstrate that the M. hyo soluble antigen preparation does not interfere with PCV2 efficacy. The results also show that all the PCV/M. hyo experimental 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 yielding the strongest efficacy.
Example 8
Evaluation of M. Hyo Efficacy of a 1-Bottle PCV2/M. Hyo Combination Vaccine in with Different Adjuvant Formulations
This study was designed to evaluate the M. hyo efficacy of a 1-bottle PCV2/M. hyo combination vaccine with different adjuvant formulations. The M. hyo antigen was combined with Porcine Circovirus (Type 1-Type 2 Chimera, or PCV1-2, killed virus) in one bottle.
Processing of Fluids:
Inactivated M. hyo fermentation fluid (described above in Example 1) was treated for each indicated group as follows.
T02-T04: These treatments were the same as those described for treatment groups T02-T04 in Example 7 above.
T05: This was formulated with inactivated M. hyo cells (M. hyo bacterin) as described in Example 1 above under the heading “Fermentation and Inactivation”.
All experimental PCV2/M. hyo vaccines were formulated with different adjuvant formulations. The experimental vaccine formulations were prepared with M. hyo antigens processed according to treatments T02-T04. In addition, Treatment T01 corresponded to a placebo (sterile saline). Treatment T05 is a positive control corresponding to an expired RespiSure® vaccine, which is an M. hyo bacterin-based vaccine (Pfizer Animal Health).
These experimental formulations are described in Table 9 below, wherein the symbol * indicates the M hyo antigen from global M hyo seed, Protein A treated supernatant and the symbol ** indicates Investigational Veterinary Product (IVP) serial.







TABLE 9







PCV2/M.hyo Experimental Vaccine Formulations Used for M.hyo 


Efficacy Study in Different Adjuvant Formulations














PCV1-2
M Hyo*




Treatment 
IVP Serial **
Ag
Ag
Adjuvant
Other













T01 
87-244-DK
NA
Sterile













(Placebo)



Saline


T02
L0411RK08
1.6 RP
7.5 RP
10% SP Oil
NA


T03
L0411RK09


5% Amphigen



T04
L0611RK03


5% Amphigen +







5% SLCD










T05
A827870
Expired “RespiSure” vaccine









Pigs at 3 weeks of age were intramuscularly inoculated with a single dose of the different vaccine formulations described in Table 9 above. Fourteen animals were included in both the placebo and 10% SP-oil groups, thirteen animals were included in the positive control group, and sixteen animals were included in both the 5% Amphigen and 5% Amphigen+5% SLCD groups.
Animals were challenged 21 days after vaccination with a virulent M. hyo field isolate. Animals were necropsied 28 days after challenge and the lungs were removed and scored for consolidation consistent with M. hyo infection. Table 10 below contains the lung lesion scores for the respective treatment groups. Statistical significance was determined by a Mixed Model Analysis of lung scores for each group.







TABLE 10







M.hyo Lung Lesions













#
LS Mean 
Range % 



Treatment
Animal
Lung Lesion
Lung Lesion







Placebo (T01)
14
13.1%
0.1-50.5



10% SP-oil (T02) 
14
 4.3%
0.0-50.8



5% Amphigen (T03)
16
 4.7%
0.0-38.5



5% Amph + 5%
16
12.0%
0.1-55.8



SLCD (T04)






Expired RSO (T05)
13
2.28%
0.0-34.5










As indicated in Table 10 above, the placebo group had a mean lung lesion score of 13.1%, as compared to the 10% SP-oil and 5% Amphigen treatment groups which had mean lung scores of 4.3% and 4.7%, respectively. Both the 10% SP-oil and 5% Amphigen formulations reduced and/or prevented lung lesions. Thus, the 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.
In contrast, the 5% Amphigen+5% SLCD group had a mean lung lesion score of 12.0%. which was an unacceptable result in that it was not different as compared to the placebo. Consequently, the experiment PCV/M. hyo vaccine formulated with 5% Amphigen+5% SLCD was not considered as efficacious.
It is noted that due to the reduced animal number and high variability in lung lesion scoring, no statistical treatment effect could 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 the PCV/M. hyo experimental formulations in 10% SP-oil. This repeat study is presented in Example 9 below.
Example 9
Evaluation of M. Hyo Efficacy of a 1-Bottle PCV2/M. Hyo Combination Vaccine in 10% SP-Oil
This study is a proof of concept designed to evaluate the M. hyo fraction efficacy of four experimental PCV2/M. hyo vaccines (Serials L0711RK11, L0711RK12, L0711RK13 and L0711RK14 in Table 11 below) prepared by different M. hyo manufacturing processes which utilize 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.
Processing of Fluids:
T02: Inactivated M. hyopneumoniae antigen as described under “Fermentation and Inactivation” in Example 1 above.
T03 and T04: Formulated with inactivated M. hyopneumoniae cells as described under “Fermentation and Inactivation” in Example 1 above.
T05: Protein A treatment of medium used to grow M. hyopneumoniae. PPLO (porcine heart derived) was made per manufacturer's directions (i.e., 21 g/L) and yeast extract solution was made at 21 g/L in USP. Yeast extract solution was added to the PPLO at 6.25% and the mixture was sterilized by heating to 121° C. for ≧30 minutes. Cysteine hydrochloride was prepared at 90 g/L and filter sterilized. Dextrose solution was made by adding 450 g of dextrose per liter of USP water followed by heat sterilization. To prepare the final medium, porcine serum was added to the base medium at 10% followed by cysteine at 0.01% and dextrose at 1.0%. Antibodies in the complete PPLO media 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 (10×11.5 cm). After removal of storage buffer, the column was treated with 2 column volumes of 1M acetic acid. The resin was equilibrated with 5 column volumes of 50 mM NaPO4, 1M 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 column flow through was collected and filter sterilized through a 0.2 micron filter (Sartorius). The treated medium was used propagate M. hyopneumoniae cells as described under “Fermentation and Inactivation” above. Whole inactivated culture (including cells) was formulated into the final vaccine.

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×g (Sorvall RCSB) for 30 min. and the supernatant was sterilized via 0.2 uM filtration. One hundred fifteen mls of rProtein A resin (part number 12-1279-04, MAbSelect, GE Healthcare) was packed into a chromatography column (5×6 cm). After removal of the storage buffer and treatment with 2 column volumes of 1M acetic acid, the resin was equilibrated with 5 column volumes of 50 mM NaPO4/1M NaCl buffer, pH 7.01. Approximately 1.2 liters of the clarified/filtered M. hyopneumoniae antigen containing fluids were passed through the resin at a flow rate of 120 cm/hr. The flow through was collected and sterilized via 0.2 μM filter.

T07: Inactivated M. hyopneumoniae cells were prepared as described under “Fermentation and Inactivation” in Example 1 above. The inactivated fermentation fluid was clarified by via tangential flow filtration. Briefly, a polyether sulfone filter (GE HealthCare, part number 56-4102-71) with nominal pore size of 0.2 μM was sanitized with 0.5N sodium hydroxide solution followed by extensive rinsing with sterile USP water. Inactivated mycoplasma culture fluid was introduced to the apparatus at a recirculation rate targeted to 14.6 L/minute and a transmembrane pressure of 2-3.4 PSI. Clarification was performed at room temperature. Filter permeate was collected and stored at 2-8 C until further processing. One hundred fifteen mls of rProtein A resin (part number 12-1279-04, MAbSelect, GE Healthcare) was packed into a chromatography column (5×6 cm). After removal of the storage buffer and treatment with 2 column volumes of 1M 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 clarified/filtered M. hyopneumoniae antigen containing fluids were passed through the resin at a flow rate of 120 cm/hr. The flow through was collected and sterilized via 0.2 μM filter.

T08: Inactivated M. hyopneumoniae cells were prepared as described under “Fermentation and Inactivation” above. The inactivated fermentation fluid was centrifuged at  ̃20,000×g (Sorvall RCSB) for 30 min. and the supernatant was sterilized via 0.2 uM filtration. One hundred fifteen mls of rProtein A Sepharose (part number 17-5199-03 GE Healthcare) was packed into a chromatography column (5×6 cm). After removal of the storage buffer and treatment with 2 column volumes of 1M acetic acid, the resin was equilibrated with 5 column volumes of 50 mM NaPO4/1M NaCl buffer, pH 7.01. Approximately 1.2 liters of the clarified/filtered M. hyopneumoniae antigen containing fluids were passed through the resin at a flow rate of 120 cm/hr. The flow through was collected and sterilized via 0.2 uM filter.

The experimental vaccine formulations were prepared with M. hyo antigens processed according to treatments T02-T08 above. T02, T03 and T04 corresponded to positive controls. In addition, Treatment T01 corresponded to a placebo (sterile saline).
These experimental formulations are described in Table 11 below. The M. hyo antigen corresponds to the M. hyo antigen from global M. hyo seed, Protein A treated supernatant. The information in the “Protein A Treatment” column indicates whether the M. hyo supernatant was treated with Protein A either before or after fermentation.







TABLE 11







PCV2/M. hyo Experimental Vaccine Formulations Used for M. hyo Efficacy Study in SP-Oil Adjuvant




















Supernatant







PCV1-2
M. hyo
Protein A
Clarification
Protein A




Treatment
Serial No.
Ag
Ag
Treatment
Method
Brand
Adjuvant
Other













T01
L0311AS11
NA
Sterile Saline













T02
A828718
NA
13    
Expired RespiSure One
Amphigen
NA


T03
L0711RK09
1.5 RP
7.5 RP
M. hyo without Protein A treatment and with PCV-2
10% SP Oil



T04
L0711RK10
NA

M. hyo without Protein A treatment and without PCV-2

















T05
L0711RK11
1.5 RP 

Before
NA
Sepharose




T06
L0711RK12


After
Centrifuge
MAbSelect




T07
L0711RK13


After
Filter
MAbSelect




T08
L0711RK14


After
Centrifuge
Sepharose









Pigs at 3 weeks of age were intramuscularly inoculated with a single dose of the different vaccine formulations described in Table 11 above. There were 18 pigs included in each treatment group. Animals were challenged 21 days after vaccination with a virulent M. hyo field isolate. Animals were necropsied 28 days after challenge and the lungs were removed and scored for consolidation consistent with M. hyo infection. FIGS. 8 (A & B) show the lung lesion scores for the respective treatment groups. Statistical significance was determined by a Mixed Model Analysis of lung scores for each group.
The lung lesion results depicted in FIGS. 8A and 8B indicate that of all the treatments, only two (T07 and T08) had 100% of pigs in the <5% lung lesion category. It is noted that strong statistical difference were observed in this study.
The results in the present example demonstrate significant M. hyo efficacy in a 1-bottle PCV2/M. hyo experimental formulation employing the Protein A-treated M. hyo supernatant and utilizing SP-oil as the adjuvant. Additionally, Example 7 above demonstrated PCV2 efficacy in a 1-bottle PCV2/M. hyo formulation employing the Protein A-treated M. hyo supernatant and utilizing SP-oil as the adjuvant. Taken together, both M. hyo and PCV2 efficacy have been demonstrated in the 1-bottle PCV2/M. hyo combinations employing Protein A-treated M. hyo supernatant.
Example 10
In Vivo Safety of Experimental PCV2/M. Hyo Experimental Vaccines
This study was conducted to evaluate in vivo safety of experimental PCV2-M. hyo vaccines formulated at maximum antigen dose in various adjuvant formulations in the host animal when given at the youngest age (3 weeks of age). Different adjuvant platforms were evaluated in order to determine which of these platforms provided an acceptable safety profile 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 historic issues with injection site reactions observed by this investigative group and others.
Processing of Fluids:
All vaccines were prepared with inactivated M. hyopneumoniae antigen as described under “Fermentation and Inactivation” in Example 1. M. hyo whole bulk antigen was used since it was known to contain soluble and insoluble M. hyo antigens, in addition to the immunoglobulins and immunocomplexes that would be removed upon protein A treatment. It is reasonable to conclude that removal of insoluble cell debris and immunuoglobulins and immunocomplexes will only further enhance the safety of the vaccine formulations. The intention of this study was to stringently test the safety of the 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 Safety Study

















Minimum



PCV1-2
M Hyo*


Vaccine


IVP Serial
Ag
Ag
Adjuvant
Other
Vol. (mL)













87-244-DK
NA
Sterile
NA












(Placebo)



Saline



L0411RK15
7.8 RP
13 RP
10% SP Oil
NA
200


L0411RK16


5% Amphigen

200


L0611RK05 


5% Amphigen +

200





5% SLCD




L0611RK06


20% SLCD +

200





10% SP Oil





* M hyo antigen = from global M hyo seed (whole bulk antigen).






The safety parameters employed in this study were rectal temperature profile and injection site reaction. The results of this study indicated that all candidate adjuvant platforms provided an acceptable safety profile in terms of rectal temperature profile and clinical observations (results not shown). Only the 20% SLCD+10% SP-oil (i.e., positive control) was significantly different than the placebo vaccine and had a number of severe injection site reactions (results not shown).
Example 11
Preparation of Protein a Treated M. Hyo Antigen for Pivotal Studies
FIG. 9 is a flowchart which shows one embodiment of a manufacturing process used to prepare PCV2 compatible Protein-A treated M. hyo antigen. Inactivated whole cultures of M. hyo were clarified of cells via tangential flow filtration. Briefly, a polyether sulfone filter (GE Healthcare, part number 56-4102-49) with nominal pore size of 0.45 μM was sanitized with 0.5N sodium hydroxide solution followed by extensive rinsing with sterile USP water. Inactivated mycoplasma culture fluid was introduced to the apparatus at a recirculation rate targeted to 11.0 L/minute and a transmembrane pressure of  ̃5 PSI. Clarification was performed at room temperature. Filter permeate was collected and stored at 2-8° C. until further processing.
Following clarification, antigen containing fluids were treated with protein A resin to reduce antibody levels. Briefly, MAbSelect protein A resin (GE Healthcare) was packed into a glass column to 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). Antigen containing fluid, equivalent to 10 column volumes, was loaded onto the resin at a linear flow rate of 100 cm/hour. The column flow through was collected and filter sterilized through a 0.2 micron filter. Regeneration of the column was achieved by flowing 3 column volumes of 25 mM acetate solution at pH 3.7 followed by 4 column volumes of 1M acetic acid solution. Anti-PCV2 antibodies and M. hyopneumoniae antigen levels were measured in the final antigen fluid via PCV2 specific antibody ELISA and p46 antigen quantification ELISA, respectively.
Example 12
Efficacy of the PCV1-2 Chimeric Fraction Following Intramuscular Administration of a 1-Bottle PCV2/M. Hyo Combination Vaccine in 10% SP-Oil
The study presented in this example was designed to evaluate the efficacy of the PCV1-2 chimera, killed virus fraction of an experimental 1-Bottle PCV2/M. hyo combination vaccine, administered once to pigs of 21±3 days of age and challenged with a virulent PCV2 isolate at approximately 6 weeks of age.
Four experimental bivalent PCV1-2/M. hyo vaccines at different, but balanced, antigen dose levels and one experimental monovalent M. hyo (negative control) vaccine were formulated with the highest passage antigen. 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 inactivation of the chimeric virus, the PCV2 antigen lot was concentrated 20× and the concentrates were washed with a balanced salt solution. The final experimental vaccine formulations were adjuvanted using 10% SP oil. These experimental formulations are described below and in Table 13, wherein the antigen dose (% of the PCV2 and M. hyo antigen lots) is provided.
T01: Experimental preparation (L1211RK11) of M. hyo antigen (14.1%-High) without PCVType1-Type2 chimera, killed virus fraction (0%). This corresponds to a negative control (monovalent M. hyo).
T02: Experimental preparation (L1211RK09) of high passage PCVType1-Type2 chimera, killed virus (1.375%-High) and M. hyo antigen (14.1%-High).
T03: Experimental preparation (L1211RK15) of high passage PCVType1-Type2 chimera, killed virus (0.688%-Medium) and M. hyo antigen (9.4%-Medium).
T04: Experimental preparation (L0112RK03) of high passage PCVType1-Type2 chimera, killed virus (0.344%-Low) and M. hyo antigen (4.7%-Low).
T05: Experimental preparation (L1211RK17) of high passage PCVType1-Type2 chimera, killed virus (0.172%-Very Low) and M. hyo antigen (2.32%-Very Low).







TABLE 13







Experimental Design













Vaccination

















Serial/
Antigen Dose





Group
N
CP or IVP1
Lot No.
(PCV2/M hyo)
Adjuvant
Dose
Route

















T01
24
Placebo
L1211RK11
None/High
10% SP
2 mL
IM,


T02
24
PCV2-M hyo
L1211RK09
High/High
Oil

Right


T03
24
PCV2-M hyo
L1211RK15 
Medium/Medium


neck


T04
24
PCV2-M hyo
L0112RK03
Low/Low





T05
24
PCV2-M hyo
L1211RK17
Very low/Very low





1IVP = Porcine Circovirus Type 1-Type 2 Chimera (PCV2), Killed Virus vaccine- Mycoplasma Hyopneumoniae (M hyo) Bacterial Extract


CP = Mycoplasma Hyopneumoniae bacterial extract adjuvanted with SP-oil 10% (without Porcine Circovirus Type 1-Type 2 Chimera fraction)


IM = Intramuscularly






On Day 0 (3 weeks of age) a single 2 mL dose of the assigned vaccine was administered by IM injection in the right neck of each pig enrolled in the study. No adverse events were observed post vaccination. A serum sample was collected from all pigs weekly prior to challenge. Any pig detected with PCV2 viremia prior to challenge was removed from the study. On the day prior to challenge fecal swabs and serum samples were collected. The pigs were subsequently challenged with a PCV2a challenge virus. Challenge was conducted around 3 weeks after the vaccination (Day 21). Each pig was inoculated with a total 3 mL of PCV2a challenge virus (Isolate #40895, pre-diluted to 5.10 log10 FAID50/mL) with 2 mL intranasally and 1 mL intramuscularly in the left neck. A reserved aliquot of the challenge virus was titrated following the challenge to confirm the actual challenge dose. The undiluted bulk was prediluted 2-fold and the back-titer results achieved a 5.10 log10 FAID50/mL challenge level. Prior to necropsy, serum and fecal swabs were collected weekly during the three-week challenge phase. At three weeks post challenge all pigs were euthanized and necropsied. Serum samples and fecal swabs were collected, along with 4 different lymphoid tissues. During necropsy, sections of three lymph nodes (tracheobronchial, mesenteric, inguinal), and tonsil were collected for each pig and individually identified and fixed in 10% buffered formalin solution. The results of testing are provided below.
Vaccine Potency Testing
The L1211RK15 PCV/M. hyo vaccine serial described above was considered to be the reference candidate. Consequently, relative potency for both the M. hyo fraction and the PCV2 fraction were determined versus this candidate reference. These results are presented in Table 14 below. Serial L1211RK11 corresponds to the placebo (no PCV2 fraction).







TABLE 14







Potency Results









Reference L1211RK15










M. hyo
PCV


Serial
Potency
Potency












L1211RK11
1.57
0.00


L1211RK09

2.08


L1211RK15

1.05


L0112RK03

0.56


L1211RK17

0.27





Results shown for each serial are averages of all replicates tested.






PCV2 Viremia

Following challenge, when compared to the placebo group, all vaccinated groups had a significant reduction in the percent of viremic pigs [P≦0.05], and throughout the study at least 47% of pigs in the treated groups (T02-T05) stayed negative for PCV2 viremia (Table 15 below). Similarly, all vaccine groups had significantly lower (P=0.0001) PCV2 DNA copy numbers than the placebo group post challenge (data not shown).







TABLE 15







qPCR Qualitative Serum Viremia—Percent Ever Positive 


and Estimate of Prevented Fraction













Ever Positive?
Total















Pos
Neg
Observations















Trt
Vaccine
#
%
#
%
Number
P-Value

















T01
L1211RK11
23
95.8
1
4.2
24



T02
L1211RK09
2
8.3
22
91.7
24
<0.0001


T03
L1211RK15
7
31.8
15
68.2
22
0.0006


T04
L0112RK03
12
52.2
11
47.8
23
0.0063


T05
L1211RK17
7
29.2
17
70.8
24
0.0004









PCV2 Fecal Shedding

Post-challenge fecal swabs revealed 83.3% of the placebo group pigs (T01) were positive for PCV2 fecal shedding. In contrast, all vaccine groups (T02-T05) had a significant reduction in the percent of pigs shedding PCR detectable PCV2 DNA (P≦0.0061). These results are presented in Table 16 below. Similarly, all vaccine groups had significantly lower PCV2 DNA copy numbers than the placebo group post challenge (data not shown).







TABLE 16







Fecal Shedding Ever Present after Challenge (Day >21)













Fecal Ever Present?
Total















Pos
Neg
Observations















Trt
Vaccines
#
%
#
%
Number
P-Value

















T01
L1211RK11
20
83.3
4
16.7
24



T02
L1211RK09
6
25.0
18
75.0
24
0.0002


T03
L1211RK15
9
40.9
13
59.1
22
0.0049


T04
L0112RK03
10
43.5
13
56.5
23
0.0061


T05
L1211RK17
6
25.0
18
75.0
24
0.0002









Serum Antibody Response

All pigs were PCV2 seronegative 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) of PCV2 antibody titer on Day 20 post vaccination when compared to placebo, indicating the active immune response to PCV2 following vaccination. PCV2 ELISA antibody titers are summarized in Table 17 below. Pre-challenge titers indicated a significant difference (P≦0.0393) in the T02 and T03 groups from the T01 group on Days 7-20 and between the T01 and T04 groups on Day 20 (P≦0.0287). On Days 28 through 42, all vaccine groups had significantly higher titers than T01 (P≦0.0129; Table 17 below).







TABLE 17





PCV2 ELISA S/P Titers Treatment Comparison by Study Day




















Contrast
Day −1
Day 07
Day 14
Day 20
Day 28





T01 vs T02
ns
0.0229
0.0005
0.0001
<0.0001


T01 vs T03
ns
0.0302
0.0393
0.0060
<0.0001


T01 vs T04
ns

ns
0.0287
<0.0001


T01 vs T05
ns

ns
ns
<0.0001












Contrast 
Day 35
Day 42





T01 vs T02
<0.0001
0.0056


T01 vs T03
<0.0001
0.0024


T01 vs T04
<0.0001
0.0114


T01 vs T05
<0.0001
0.0129





*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

At the time of necropsy, when compared to the placebo group, all vaccinated groups had a significant reduction in total amount of PCV2 antigen detected in tissues. The data concerning PCV2 infection in Lymphoid Tissues (IHC scores) are summarized in Table 18 below. As shown in Table 18, all vaccine groups had significantly lower IHC scores than the T01 placebo group.







TABLE 18







PCV2 IHC Scores: If lymphoid or tonsil tissues ever abnormal













Compared to L12111RK11



Treatment
LSM
P-value















L1211RK11
0.75




L1211RK09
0.15
0.0003



L1211RK15
0.30
0.0055



L0112RK03
0.34
0.0106



L1211RK17
0.41
0.0273







* Animal was considered abnormal if score was >0 in any lymphoid tissue or tonsil sample.






All vaccinated groups also saw a significant reduction in PCV2 Lymphoid Depletion as shown in Table 19 below.







TABLE 19







PCV2 Lymphoid Depletion: If lymphoid or tonsil tissues ever abnormal











Compared to L12111RK11


Treatment
LSM
P-value












L1211RK11
0.48



L1211RK09
0.04
0.0053


L1211RK15
0.13
0.0181


L0112RK03
0.08
0.0069


L1211RK17
0.11
0.0116





* Animal was considered abnorma if score was >0 in any lymphoid tissue or tonsil sample.






Additionally, all vaccinated groups saw a significant reduction in Histiocytic Replacement as shown in Table 20 below.







TABLE 20







PCV2 Histiocytic Replacement: if lymphoid or tonsil tissues ever abnormal











Compared to L12111RK11


Treatment
LSM**
P-value












L1211RK11
ND



L1211RK09
ND
0.0006


L1211RK15
ND
0.0098


L0112RK03
ND
0.0098


L1211RK17
ND
0.0173





*Animal was considered abnormal if score was >0 in any lymphoid tissue or tonsil sample.


**Fisher exact test was used due to non-convergence, therefore least square means were not determined (ND).






The data presented in this Example indicates that the vaccine groups:

    
    
        Significantly protected and aided in the prevention of PCV2 viremia post-challenge;
        Significantly aided in the prevention of fecal shedding of PCV2 post-challenge in all vaccinated animals;
        Elicited a statistically significant serological response 28 days post vaccination in groups T02-T05. In addition, T02 and T03 demonstrated a statistically significant response as early as 7 days post-vaccination when compared to T01;
        Significantly reduced microscopic lesions (Lymphoid Depletion and Histiocytic replacement) in all vaccinated animals; and
        All vaccines proved to be efficacious and vaccine serial L1211RK15 was selected as a reference candidate.
    
    


Example 13
Efficacy of the M. Hyo Fraction Following Intramuscular Administration of a 1-Bottle PCV2/M. Hyo Combination Vaccine in 10% SP-Oil
The objective of the study presented in this example was to evaluate efficacy of the Mycoplasma hyopneumoniae (M. hyopneumoniae) fraction of an experimental Porcine Circovirus (PCV) Type 1-Type 2 Chimera, Killed Virus-Mycoplasma Hyopneumoniae (M hyo) Bacterial Extract, administered intramuscularly once to pigs of 21±3 days of age and challenged with a virulent lung homogenate of M. hyopneumoniae 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 (negative control) vaccine 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 inactivation of the chimeric virus, the PCV2 antigen lot was concentrated 20× and the concentrates were washed with a balanced salt solution. The final experimental vaccine formulations were adjuvanted using 10% SP oil. These experimental formulations are described below and in Table 21 below, wherein the antigen dose (% of the PCV2 and M. hyo antigen lots) is provided.
T01: Experimental preparation (L1211RK10) of high passage PCVType1-Type2 chimera, killed virus (1.375%-High) without M. hyo fraction (0%). This corresponds to a negative control (monovalent PCV2).
T02: Experimental preparation (L1211RK09) of high passage PCVType1-Type2 chimera, killed virus (1.375%-High) and M. hyo antigen (14.1%-High).
T03: Experimental preparation (L1211RK15) of high passage PCVType1-Type2 chimera, killed virus (0.688%-Medium) and M. hyo antigen (9.4%-Medium).







TABLE 21







Experimental Design













Vaccination

















Serial/Lot
Antigen dose RP





Group
N
CP or IVP1
No.
(PCV2/M hyo)
Adjuvant
Dose
Route














NTX
9
Sentinel
NA
No Vaccination














T01
38
Placebo—PCV2
L1211RK10
High/None
10% SP
2 mL
IM, 


T02
38
PCV2-M hyo
L1211RK09
High/High
Oil

Left


T03
38
PCV2-M hyo
L1211RK15
Medium/Medium


neck





1IVP = Investigational Veterinary Product = Porcine Circovirus Type 1-Type 2 Chimera (PCV2), Killed Virus vaccine- Mycoplasma Hyopneumoniae (M hyo) Bacterial Extract


CP = Control Product = Killed Chimeric PCV1-2 fraction adjuvanted with SP-oil 10% adjuvanted (without M. hyopneumoniae fraction)


IM = Intramuscularly






On Day 0, 123 clinically healthy, susceptible pigs were enrolled in this study at three weeks of age. Pigs were blocked by litter and randomly assigned to a sentinel (NTX) group or one of the three treatment groups (T01-T03); administered intramuscularly 2 mL of either an experimental PCV1-2/M. hyopneumoniae vaccine at the minimum immunizing dose (MID), an experimental PCV1-2/M. hyopneumoniae vaccine at a dose slightly higher than MID or a placebo containing PCV1-2 only at MID. Seven weeks after vaccination the sentinel pigs were euthanized and necropsied to confirm 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 lungs were scored for lesions typical of M. hyopneumoniae. Post-challenge lung lesions are the primary outcome variable. Vaccination is considered effective if the lower 95% confidence interval of the mitigated fraction is >0.
Vaccine Potency Testing
The L1211RK15 PCV/M. hyo vaccine serial described above was considered to be the reference candidate. Consequently, relative potency for both the M. hyo fraction and the PCV2 fraction used in this M. hyo efficacy study were determined versus this candidate reference. These results are presented in Table 22 below. Serial L1211RK10 corresponds to the placebo (no M. hyo fraction).







TABLE 22







Potency Results











Reference L1211RK15











Serial
M. hyo1 Potency
PCV Potency2







L1211RK10
0.00
2.33



L1211RK09
1.48
2.20



L1211RK15
1.00
1.07







Results shown for each serial are averages of all replicates tested.



1M. hyopneumoniae potency tested in five replicates.



2PCV potency tested in one replicate (L1211RK10), or five (L1211RK09, L1211RK15) replicates.






Serology

M. hyo antibody titers indicated that all pigs were serologically M. hyopneumoniae negative on Day 0 and remained negative prior to challenge. At all time points post vaccination (Days 21, 47 and 75) T02 and T03 had significantly higher (P≦0.0004) geometric least squares mean M. hyopneumoniae antibody titers compared to T01 (serology data not shown).
Percentage of Total Lung with Lesions
Frequency distributions of lung lesion scores for each lung lobe were calculated by treatment. Percentage of total lung with lesions was calculated using the following formula: Percentage of total lung with lesions={(0.10×left cranial)+(0.10×left middle)+(0.25×left caudal)+(0.10×right cranial)+(0.10×right middle)+(0.25×right caudal)+(0.10×accessory)}. The arcsine square root transformation was applied to the percentage of total lung with lesions prior to analysis. Percentage of Total Lung with Lesions was analyzed using a mixed linear model. Pair-wise comparisons were made between treatment groups if the treatment effect was significant. Back transformed least squares means of percentage of total lung with lesions, and their 95% confidence intervals were calculated as well as the minimums and maximums. Lung lesions were summarized with the stratified mitigated fraction and 95% confidence limits. The lung lesion results are presented in Table 23 below.







TABLE 23





Analysis of Percent of Total Lung with Lesion


Summary of Least Squares Means
























% Lung








with





Group
Serial
N
Lesion1
Range





NTX

9
0.1
0.0 to 0.8


















% Lung

Mitigated Fraction
















with

Contrast



Group
Serial
N
Lesion1
Range
vs T01
95% Cl4





T01
L1211RK10
36
6.1b
0.0 to 32.3




T02
L1211RK09
36
2.7a
0.0 to 23.5
40.9
13.3 to 61.8


T03
L1211RK15
38
2.6a
0.0 to 49.0
46.9
18.2 to 68.3





1Back Transformed Least Squares Mean


Confidence Interval


Treatment groups with the same letter are not significantly different at P-value 0.05.






A few low percentage lung lesions were observed in the NTX pig lungs which were attributed to a known incidence of Bordetella in this herd. Bacterial culture on lung tissue swabs confirmed several pigs were B. bronchiseptica culture positive and M. hyopneumoniae culture confirmed all NTX pigs were M. hyopneumoniae culture negative prior to challenge administration.
The protocol met the validity criteria in that the LS mean lung lesions for T01 were >4%. The LS Mean lung lesions for T02 and T03 were significantly (P≦0.05) lower than T01 and both met the mitigated fraction criteria that the lower 95% confidence interval 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 back-transformed mean lung lesion scores in placebo pigs (T01) was >4%.
Compared to the negative control group (T01), treatment groups T02 and T03 demonstrated a significant reduction (P≦0.05) in percent lung with lesion compared to T01. The mitigated fractions for T02 and T03 compared to T01 met the protocol criteria for efficacy.
Under the conditions of this study, both vaccines (T02 & T03) helped to mitigate lung lesions, the primary variable for efficacy. The results in the present example demonstrate significant M. hyo efficacy in a 1-bottle PCV2/M. hyo experimental formulation.
Example 14
Evaluation of Virucidal Activity Against PRRS Virus
The studies presented in this example were designed to evaluate the various adjuvant platforms for virucidal activity against PRRS virus. Initial experiments focused on adjuvant alone (i.e., the formulations did not contain PCV or M. hyo antigens). The adjuvant evaluation for PRRS virucidal activity is presented in FIG. 10. Preliminary virucidal assessment indicated that 10% SP-oil, 0.2% Carbopol and 2.5% Amphigen are non-virucidal to PRRS virus. In contrast, the 20% SLCD adjuvant appeared to be virucidal to PRRS virus.
Further studies were performed to evaluate whether the PCV/M. hyo formulations adjuvanted with the different adjuvant platforms were non-virucidal to PRRS virus. These results are presented in Table 24 below, wherein the symbol * indicates those vaccine serials which were virucidal to PRRS virus.







TABLE 24







Results of PRRS Virucidal Assay with Different Formulations














Potency












Vaccine Serial Used in Studies of


PCV2
PRRS


Examples 7, 8, 10

p46 RP
NVSL
Virucidal













Study
Description
Serial #
(ru/ds)
RP
A
B
















Examples
Sterile Saline (0.9% Sodium
87-244-DK






7, 8, 10
chloride)
(Placebo)






Examples
cPCV (RP 1.6) + M Hyo Prot
L0411RK08
7.1
1.29
−0.10
−0.13


7, 8
A treated (RP 7.5) in 10% SP








Oil







Examples
cPCV (RP 1.6) + M Hyo Prot
L0411RK09
7.3
1.33
−0.10
+0.14


7, 8
A treated (RP 7.5) in 5%








Amphigen







Examples
cPCV (RP 1.6) + M Hyo Prot
L0611RK03
6.9
1.15
−0.36
−0.33


7, 8
A treated (RP 7.5) in 5%








Amph + 5% SLCD







Example 7
cPCV (RP 1.6) monovalent
L0611RK04

1.50
−1.86*
−0.50



in 20% SLCD







Example 8
Expired RespiSure One serial
A827870
12.6





Example
cPCV (RP 7.8) + M Hyo
L0411RK15
14
1.03
−0.32
−0.03


10
Whole Bulk (RP 13.3) in








10% SP Oil







Example
cPCV (RP 7.8) + M Hyo
L0411RK16
15.5
1.12
−0.36
−0.53


10
Whole Bulk (RP 13.3) in 5%








Amphigen







Example
cPCV (RP 7.8) + M Hyo
L0611RK05
17.5
1.50
−0.54
−0.33


10
Whole Bulk (RP 13.3) in 5%








Amph + 5% SLCD







Example
cPCV (RP 7.8) + M Hyo
L0611RK06
15.9
1.13
−1.93*
−0.99*


10
Whole Bulk (RP 13.3) in








20% SLCD + 10% SP Oil





*Indicates Virucidal (>0.7 log loss)


A—Virucidal assay control GMT ~5.53 log/mL


B—Virucidal assay control GMT ~6.42 log/mL






The results presented in Table 24 above indicate that 10% SP-oil is non-virucidal to PRRS virus. Further PCV/M. hyo vaccine serials were prepared using 10% SP-oil as the adjuvant (Table 25). The antigenic potency of these vaccine serials was compared to a Reference PCV/M. hyo vaccine serial (L1211RK15) described above. The results shown in Table 25 below further indicate that 10% SP-oil is non-virucidal to PRRS virus. The test sample values in Table 25 were each higher (+ sign) than the virucidal assay control, which had a geometric mean titer (GMT) of about 5.9±0.5 log/ml.







TABLE 25







Results of Virucidal Assay with Different PCV/M.hyo Formulations 


Adjuvanted with 10% SP-oil












Potency












Vaccine 

p46 RP
PCV2
PRRS


Serial 

(ru/ds)
NVSL
Virucidal


Used

Reference
Reference
log10


Description
Serial #
L1211RK15
L1211RK15
TCID50/mL














Sterile Diluent 
1949122
na
na



(sterile water)






cPCV + M Hyo 
L0912RK12
1.62
2.60
+0.58


Prot A treated 






in 10% SP Oil






cPCV + M Hyo 
L0912RK10
0.88
1.23
+0.58


Prot A treated 






in 10% SP Oil






cPCV + M Hyo 
L0912RK11
1.24
2.62
+0.58


Prot A treated 






in 10% SP Oil






cPCV + M Hyo 
L0912RK08
1.08
1.03
+0.91


Prot A treated 






in 10% SP Oil






cPCV + M Hyo 
L0912RK09
1.65
2.06
+0.50


Prot A treated 






in 10% SP Oil





Virucidal Assay control GMT~5.9 ± 0.5 log/ml






The results presented in this example demonstrate that 10% SP-oil is non-virucidal to PRRS virus. The results presented in this example further demonstrate that the PCV/M. hyo formulation adjuvanted with 10% SP-oil was among those vaccine serials which were considered non-virucidal to PRRS virus (Table 24 and Table 25). In conclusion, the PCV/M. hyo formulation adjuvanted with 10% SP-oil was considered an effective platform on which to base a trivalent combination including PCV, M. hyo, and PRRS virus.
Example 15
Preparation of a PCV/M. Hyo/PRRS Combination Vaccine
A PCV/M. hyo formulation adjuvanted with an adjuvant platform which is non-virucidal to PRRS virus (see Tables 24 and 25 above), is provided as a ready-to-use in one-bottle liquid composition. This 1-bottle PCV/M. hyo formulation employs Protein A-treated M. hyo supernatant. Both M. hyo and PCV2 efficacy have been demonstrated in such PCV2/M. hyo formulations employing M. hyo Protein A-treated supernatant (see Examples 7-9). In the present example, this divalent PCV2/M. hyo formulation is combined with a monovalent PRRS virus antigen.
In one embodiment, a PCV/M. hyo combination in 10% SP-oil and corresponding to one of the vaccine serials L0711RK11, L0711RK12, L0711RK13 and L0711RK14 in Table 11 above or L1211RK09, L1211RK15, L0112RK03 and L1211RK17 in Table 13 above is provided as a ready-to-use in one bottle liquid composition. The results presented in Example 14 above demonstrated that 10% SP-oil is non-virucidal to PRRS virus. Example 14 also demonstrated that PCV2/M. hyo formulations adjuvanted with 10% SP-oil were among those vaccine serials which were considered non-virucidal to PRRS virus. In the present example, such a 1-bottle PCV2/M. hyo liquid composition is used to re-hydrate a lyophilized genetically modified live PRRS virus composition contained in a second bottle, such that all antigens are contained in a single bottle prior to being administered to a pig of a suitable age (e.g., at 3 weeks of age or older).
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 was deposited in the ATCC under the accession number VR 2430. Suitable amounts of the respective antigens are described herein. Desirably, all antigens are administered in a single dose to the pig.

## Claims

1. A method for preparing a multivalent immunogenic composition including a soluble portion of a Mycoplasma hyopneumoniae (M. hyo) whole cell preparation, the method comprising:
i) culturing M. hyo in a suitable media over periods ranging from 18-144 hours;
ii) subsequently inactivating the M. hyo culture;
iii) harvesting the inactivated culture fluid, wherein the inactivated culture fluid comprises an M. hyo whole cell preparation comprising both a soluble liquid fraction and insoluble cellular material;
iv) separating the soluble liquid fraction from the insoluble cellular material;
v) substantially removing both IgG and antigen/immunoglobulin immunocomplexes from the separated soluble liquid fraction to form a soluble portion of the M. hyo whole cell preparation; and
vi) subsequently combining the soluble portion of the M. hyo whole cell preparation with a PCV2 antigen.

2. The method of claim 1, wherein the M. hyo culture is inactivated by exposing the culture to an inactivation agent.

3. The method of claim 2, wherein the inactivation agent is binary ethylenimine (BEI).

4. The method of claim 2, further comprising neutralizing the inactivation agent.

5. The method of claim 1, wherein the soluble liquid fraction is separated from the insoluble cellular material by a centrifugation step.

6. The method of claim 1, wherein the soluble liquid fraction is separated from the insoluble cellular material by a filtration step.

7. The method of claim 1, wherein the soluble liquid fraction is separated from the insoluble cellular material by a precipitation step.

8. The method of claim 1, wherein the separated soluble liquid fraction is treated with a Protein A or Protein G resin to substantially remove both the IgG and antigen/immunoglobulin immunocomplexes contained in the separated soluble liquid fraction.

9. The method of claim 8, wherein the separated soluble liquid fraction is treated with a Protein A resin to substantially remove both the IgG and antigen/immunoglobulin immunocomplexes contained in the separated soluble liquid fraction.

10. The method of claim 1, further comprising admixing the immunogenic composition with an adjuvant.

11. The method of claim 10, wherein the adjuvant is an oil emulsion comprising a polyoxyethylene-polyoxypropylene block copolymer, squalane, polyoxyethylene sorbitan monooleate and a buffered salt solution (SP-oil).

12. The method of claim 1, further comprising admixing the immunogenic composition with a pharmaceutically-acceptable carrier.

13. The method of claim 1, further comprising combining the immunogenic composition with at least one additional porcine antigen.