Inactivated APXIA, APXIIA, and APXIIIA toxins

Abstract

Disclosed are inactivated ApxIA, ApxIIA and ApxIIIA toxins, vaccines comprising said inactivated toxins, and their use for immunizing and protecting mammals.

Description

Technical Field

[0001] The present invention relates to inactivated ApxIA, ApxIIA and ApxIIIA toxins, vaccines containing the inactivated toxins, and their use for the immunization and protection of mammals.

Background Art

[0002] Actinobacillus pleuropneumoniae (APP) is a Gram-negative bacterium and a member of the family Pasteurellaceae. APP is the causative agent of porcine pleuropneumonia, a severe lung disease in pigs that causes significant economic losses in pig production worldwide. This disease is characterized by hemorrhagic, fibrinous and necrotic lung lesions. Pigs that survive this disease often become asymptomatic carriers of APP and are a major cause of spreading the bacteria.

[0003] So far, 18 serotypes have been identified based on the antigenic properties of capsular polysaccharides (CPS) and the results of genotypic analysis. The main virulence factors of APP are exotoxins, CPS, lipopolysaccharides (LPS), and membrane proteins. The most important virulence factor is the Apx exotoxin belonging to the membrane pore-forming RTX (repeats in toxin) family. This toxin is known to be highly immunogenic and is very important for obtaining protective immunity against APP-related pleuropneumonia. At least four different Apx toxins, designated ApxI, ApxII, ApxIII, and ApxIV, are produced by APP. ApxI shows strong hemolytic activity, and ApxII shows low hemolytic activity. Both are cytotoxic and active against a wide range of cells from various host species. ApxIII does not show hemolytic activity but has strong cytotoxicity against porcine alveolar macrophages and neutrophils as the main targets. ApxIV does not show cytotoxicity but shows only weak hemolytic activity. Among the serotypes of APP, there is no one that produces all four Apx toxins. Most serotypes produce three Apx toxins. The pattern of Apx toxin production is related to virulence, and serotypes 1, 5, 9, and 11 that produce ApxI and ApxII are the most virulent.

[0004] The production and secretion of active Apx toxins involve at least four genes. Gene A encodes the structural part of the toxin, and gene C encodes an acyltransferase essential for the post-translational activation of the toxin. Genes B and D encode two membrane proteins required for the secretion of the mature toxin. The Apx genes are organized as an operon. The operons of ApxI and ApxIII toxins consist of the CABD genes, while the ApxII operon contains only the CA genes. Therefore, the secretion of ApxIIA depends on the activities of genes B and D of the ApxI operon.

[0005] Currently, the treatment of pleuropneumonia caused by APP infection in pigs is usually carried out with antibiotics. However, APP has often been found to show antibiotic resistance to at least one of the antibiotics commonly used for the treatment of APP infection.

[0006] Vaccination against APP is a promising preventive strategy for preventing pleuropneumonia. Several vaccines have been commercially available. Commercially available vaccines are either chemically inactivated whole cell vaccines or subunit vaccines, or a combination of both. The immunological responses of animals vaccinated with whole cell vaccines are mainly directed against surface structures such as CPS and LPS. Since there are no secreted proteins such as Apx toxins, which are highly immunogenic and known to be essential for defense, their defensive ability is only observed limitedly in bacteria. Furthermore, the APP whole cell vaccine confers only homologous defensive ability to the serotype used in the preparation of the vaccine.

[0007] Commercially available subunit vaccines (European Patent No. EP0453024 (B1)) contain chemically inactivated ApxA toxin and outer membrane proteins. Inactivation of ApxA toxin with denaturing substances such as formaldehyde can lead to a decrease in the immunogenicity of the toxoid. Disadvantages of such vaccines include inadequate defense by the denatured toxin and accompanying severe side effects, possibly due to residual toxicity, resulting from incomplete inactivation of the ApxA toxin. Moreover, the decrease in immunogenicity after inactivation necessitates an increase in the dosage of the toxin, resulting in an increase in the amount of LPS contaminating the vaccine. A high LPS content can cause side effects as seen in commercially available APP subunit vaccines.

[0008] In conclusion, it can be said that currently commercially available vaccines do not have a satisfactory safety and / or efficacy profile against APP infections.

[0009] Furthermore, there are several other experimental approaches regarding APP vaccines.

[0010] International Patent Application No. WO2004 / 045639 (A1) discloses a live attenuated vaccine against swine pleuropneumonia, which includes an APP strain in which the transmembrane domains of the genes encoding ApxIA and ApxIIA toxins are modified. To test the degree of attenuation, three-month-old pigs were inoculated with the modified live APP strain. Seven days after inoculation, the animals were sacrificed and macroscopic lesions in the respiratory organs were recorded. All animals showed behavioral changes and lung lesions at autopsy. The efficacy of such a live vaccine has not been tested.

[0011] European Patent Application No. EP0810283 (A2) and European Patent No. EP0861319 (B1) describe live attenuated APP strains lacking the gene ApxC, which is an activator of ApxA. The modified APP strains do not produce the activating protein ApxC in a functional form, and thus, the toxins ApxIA and ApxIIA are not activated by acylation. Mice were vaccinated with the ΔapxC strain and exposed to a virulent type of APP wild strain. The vaccinated mice were protected from homologous exposure and partially protected from heterologous exposure. One test was conducted in pigs. One out of six vaccinated pigs had lung lesions after heterologous exposure and autopsy. These live attenuated vaccines are considered effective, but there are risks in terms of safety for this vaccine. Since the Apx toxins produced by the vaccine strain lack ApxC, they are not activated by acylation. However, these toxins have the amino acid sequence of toxic ApxA in its original form. It is quite conceivable that a heterologous acyltransferase can acylate ApxA and convert it into an active toxic form. In the majority of pig breeding facilities around the world, there are asymptomatic carriers of virulent APP strains, as well as pigs infected with low-virulence APP strains. When such pigs are vaccinated with the ΔapxC vaccine strain, the inactivated ApxA toxin of the vaccine strain can be activated by the functional ApxC protein of the wild strain. Furthermore, there is also a possibility that the apxC deletion can be complemented by the incorporation of a functional apxC gene. There is also a possibility that the attenuated strain may recover its pathogenicity and cause disease in the vaccinated animals.

[0012] To date, live attenuated vaccines have not been commercially available.

[0013] Therefore, there is a need for an improved vaccine against APP that is highly safe and can induce cross-protection against all relevant serotypes in pigs and / or piglets.

[0014] Accordingly, an object of the present invention is to provide the use of inactivated ApxIA, ApxIIA and ApxIIIA toxins, as well as safe and effective subunits and live vaccines of these toxoids, against porcine pleuropneumonia.

Summary of the Invention

[0015] The present invention discloses modifying the acylation sites of ApxIA, ApxIIA and ApxIIIA toxins to produce toxoid forms of these proteins that are inactive but have complete immunogenicity. Substitution of the amino acids at both acylation sites with another amino acid prevents their acylation. Thereby, these modified Apx toxins are unable to initiate binding to the target cell membrane and have no cytotoxic or hemolytic activity. Since these toxins do not need to be chemically inactivated, a highly immunogenic protein is obtained. The amount of these proteins required to produce an effective subunit vaccine is relatively small. Accordingly, the content of contaminating LPS is also relatively small, resulting in a reduction of side effects such as vomiting and lethargy. Due to having superior immunogenicity compared to chemically inactivated Apx subunits, the subunit vaccine according to the present invention has a superior safety profile.

[0016] Furthermore, since the substitution of two amino acids is based on genetic recombination of the ApxA gene, it can also be used in live vaccines. That is, by introducing it into the APP strain, next, an inactivated Apx protein with high immunogenicity can be produced. These strains can be used as live vaccines that produce ApxA toxoid and a wide range of other APP proteins. These vaccines induce complex immune responses and cross-protection against a wide range of APP serotypes. Due to the genetic modification of the ApxA gene, the produced Apx toxoid cannot be activated by heterologous acyltransferase. When using live vaccines of APP strains, many advantages can be obtained. When using live vaccines, the influence of maternal antibodies on the vaccine is expected to be lower compared to subunit vaccines consisting of highly immunogenic antigens. Therefore, this vaccine can be used in pigs before 6 weeks of age and is highly likely to avoid existing immunological gaps in available vaccines. Furthermore, by using gene knockout, a more attenuated vaccine strain can be obtained, and gene knockout due to deletion of, for example, the apxIVA gene can be used as a marker for differentiating infected animals from vaccinated animals (DIVA). Therefore, the present invention provides a safety and efficacy profile superior to any known vaccination approach. Since APP strongly adapts to pigs, an attenuated APP vaccine strain can also be used as a vector for antigens of other pathogens related to pigs.

[0017] The present invention is defined by the appended claims.

[0018] In the present application, an ApxIA, ApxIIA or ApxIIIA polypeptide of Actinobacillus pleuropneumoniae, a. i. The wild-type amino acid sequence of ApxIA set forth in SEQ ID NO: 1 is modified with at least one amino acid selected from the group consisting of K560 and K686, ii. The wild-type amino acid sequence of ApxIIA set forth in SEQ ID NO: 2 is modified with at least one amino acid selected from the group consisting of K557 and N687, iii. The wild-type amino acid sequence of ApxIIIA set forth in SEQ ID NO: 3 is modified with at least one amino acid selected from the group consisting of K571 and K702, and at least one amino acid is replaced with an amino acid that is not acylation-sensitive, or b. An analog or fragment of the modified amino acid sequence defined in a., which is at least 90% identical to the modified amino acid sequence defined in a., the fragment contains at least 50% of the contiguous amino acids of the modified amino acid sequence defined in a., and the analog or fragment contains an amino acid that is not acylation-sensitive as defined in a., an analog or fragment, or c. i. The wild-type amino acid sequence of ApxIA set forth in SEQ ID NO: 1 contains a deletion that includes at least one amino acid selected from the group consisting of K560 and K686, ii. The wild-type amino acid sequence of ApxIIA set forth in SEQ ID NO: 2 contains a deletion that includes at least one amino acid selected from the group consisting of K557 and N687, iii. The wild-type amino acid sequence of ApxIIIA set forth in SEQ ID NO: 3 contains a deletion that includes at least one amino acid selected from the group consisting of K571 and K702, the deletion does not delete more than 50% of the wild-type amino acid sequence, or d. An analog of the amino acid sequence containing the deletion defined in c., which is at least 90% identical to the amino acid sequence containing the deletion defined in c., an analog, ApxIA, ApxIIA or ApxIIIA polypeptides are disclosed.

Mode for Carrying Out the Invention

[0019] The following headings are merely for organizing the present disclosure and should not be considered as differentiating one section in the present disclosure from another. The features of each section can be combined with the features of another section.

[0020] General Definitions As used herein, the term "fragment or analog" is defined as follows: An "analog" can be regarded as an amino acid sequence that exhibits at least 60%, 70%, 80%, 85%, 90%, 95%, or 97% level of homology to the original amino acid sequence. Homology, as used herein, means identity. Thus, those sequences can differ from each other based on conservative substitutions, deletions, or insertions.

[0021] The degree of identity can be determined by the Protein BLAST program using the BLASTP algorithm with default parameters such as: Expect threshold 10, Word size: 3, Matrix: BLOMSUM 62, Gap Costs: Existence: 11, Extension: 1, and Compositional adjustments: Conditional compositional score matrix adjustment (BLAST is a registered trademark of the National Library of Medicine). The program can be used to search a protein database with a protein query. The exact matches between the aligned query and the database sequences are output as identity.

[0022] Preferably, a conservative amino acid "substitution" is the result of substituting one amino acid with another having similar structural and / or chemical properties, i.e., a conservative amino acid substitution. Amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the relevant residues. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine, polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine, positively charged (basic) amino acids include arginine, lysine and histidine, and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0023] "Insertions" or "deletions" are typically made in the range of about 1, 2 or 3 amino acids. Permissible modifications can be determined experimentally by systematically introducing amino acid insertions or deletions into the protein using recombinant DNA techniques and assaying the activity of the resulting recombinant variants. This is not outside the realm of routine experimentation for those skilled in the art.

[0024] A "fragment" of a polypeptide comprises at least 50%, 60%, 70%, 80%, 90%, 95% or 97% of the original polypeptide. These fragments can be used as the exclusive active ingredient in the vaccines according to the present invention.

[0025] The APP "toxin" is a polypeptide consisting of the amino acid sequence of ApxIA, ApxIIA or ApxIIIA (e.g., as shown in SEQ ID NOs: 1-3) and exhibiting cytotoxic and / or hemolytic activity. The "toxoid" in the present disclosure refers to a polypeptide that is a modified form of the "toxin", and this modification is carried out by substitution or deletion of amino acids having acylatable sensitivity in vivo in APP, thereby not exhibiting cytotoxic or hemolytic activity.

[0026] As used herein in the context of vaccine compositions, the term "comprising" means that additional active or immunogenic components may be present. "Consisting of" means that no additional components are present, and "essentially consisting of" means that certain additional components, i.e., components that do not substantially affect the essential characteristics of the vaccine (i.e., inert or non-immunogenic components), may be present.

[0027] The genus Actinobacillus includes gram-negative, non-spore-forming, mainly capsulated bacterial species that colonize the respiratory and urogenital mucosal surfaces. Veterinarily relevant species are, for example, A. pleuropneumoniae, A. suis, A. equuli, and A. lignieresii, which are preferred Actinobacillus species of the present disclosure. Actinobacillus species usually exhibit strong host specificity. Preferred serotypes of APP are serotypes 1, 5, 7, 8, 9, and 11.

[0028] Polypeptide The present disclosure relates to ApxIA, ApxIIA or ApxIIIA polypeptides (hereinafter, for the sake of brevity, interchangeably referred to as ApxA polypeptides), wherein the wild-type ApxIA, ApxIIA or ApxIIIA polypeptides are modified by amino acid substitution or deletion at amino acid positions having acylation sensitivity in the wild-type polypeptide. Amino acid substitution means introducing an amino acid that does not have acylation sensitivity. The ApxIA, ApxIIA or ApxIIIA polypeptides modified by the above method surprisingly no longer exhibit hemolytic activity and / or cytotoxic activity. Therefore, by using them, it is possible to confer immunoprotective ability against APP while suppressing side effects when vaccinating mammals. Therefore, they can be regarded as inactivated toxins, i.e., toxoids. Therefore, the polypeptides of the present disclosure can be safely used in vaccines for immunizing target animals, particularly mammals, against APP infection.

[0029] A polypeptide containing a substitution at an amino acid position having acylation sensitivity within the wild-type polypeptide In particular, the present disclosure relates to ApxIA, ApxIIA or ApxIIIA polypeptides of Actinobacillus pleuropneumoniae, wherein a. i. The wild-type amino acid sequence of ApxIA set forth in SEQ ID NO: 1 is modified with at least one amino acid selected from the group consisting of K560 and K686, ii. The wild-type amino acid sequence of ApxIIA set forth in SEQ ID NO: 2 is modified with at least one amino acid selected from the group consisting of K557 and N687, iii. The wild-type amino acid sequence of ApxIIIA set forth in SEQ ID NO: 3 is modified with at least one amino acid selected from the group consisting of K571 and K702, At least one amino acid is replaced with an amino acid that does not have acylation sensitivity.

[0030] The polypeptide can induce a humoral and / or cellular immune response against one or more serotypes of APP in mammals, particularly pigs, when administered to the mammal. The polypeptide can induce a humoral and / or cellular immunological response against one strain of APP, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 strains of APP in mammals, particularly pigs, when administered to the mammal. Preferably, the polypeptide induces a sterile immunity against APP, APP strain, or APP serotype.

[0031] Each of ApxIA, ApxIIA, or ApxIlIA contains two amino acids with acylating sensitivity. The modification can affect one or both of the amino acids.

[0032] The present disclosure also relates to analogs and / or fragments of the modified amino acid sequence defined above, which are at least 90% homologous to the modified amino acid sequence defined above, the fragment contains at least 50% of the contiguous amino acids of the modified amino acid sequence defined above, and contains amino acids that do not have the acylating sensitivity defined above.

[0033] As defined above, the analog can differ from the wild type by deletion or conservative substitution. However, the analog always contains an amino acid that replaces an amino acid with acylating sensitivity.

[0034] The modified polypeptide does not contain amino acids with acylatable sensitivity, so the modified polypeptide cannot be acylated. The polypeptide modified in this way cannot be acylated by any endogenous or exogenous acyltransferase (for example, ApxC of Actinobacillus pleuropneumoniae). As shown experimentally below, these modified polypeptides no longer show substantial pathological effects and can therefore be safely used in vaccine compositions. Even if acyltransferase is provided exogenously in vivo from a naturally occurring APP strain or from any other source of acyltransferase, these modified polypeptides are safer for use in vaccine compositions than vaccines lacking acyltransferase ApxC because they exhibit pathological effects. Similar considerations apply to the following polypeptides in which one or two amino acids with acylatable sensitivity in APP are deleted.

[0035] A polypeptide containing a deletion at the position of an amino acid with acylatable sensitivity within the wild-type polypeptide Instead of substituting an amino acid with acylatable sensitivity in the wild-type polypeptide, the amino acid can be deleted to eliminate the pathological effect of the wild-type polypeptide.

[0036] Therefore, the present disclosure also relates to ApxIA, ApxIIA or ApxIIIA polypeptides, in which i. the wild-type amino acid sequence of ApxIA set forth in SEQ ID NO: 1 contains a deletion comprising at least one amino acid selected from the group consisting of K560 and K686, ii. the wild-type amino acid sequence of ApxIIA set forth in SEQ ID NO: 2 contains a deletion comprising at least one amino acid selected from the group consisting of K557 and N687, iii. the wild-type amino acid sequence of ApxIIIA set forth in SEQ ID NO: 3 contains a deletion comprising at least one amino acid selected from the group consisting of K571 and K702.

[0037] The polypeptide can induce a humoral and / or cellular immune response against one or more serotypes of APP in a mammal, particularly a pig, when administered to the mammal. The polypeptide can induce a humoral and / or cellular immunological response against one strain of APP, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 strains of APP in a mammal, particularly a pig, when administered to the mammal. Preferably, the polypeptide induces a sterile immunity against APP, APP serotype, APP strain, or APP serotype.

[0038] As described above, each of ApxIA, ApxIIA, or ApxIIIA contains two amino acids with acylation sensitivity. The deletion of the amino acids can include one or both of the amino acids.

[0039] Thus, the deletion can include a point deletion that deletes only one or two amino acids with acylation sensitivity in each wild-type sequence. Also disclosed as deletions are deletions in the amino acids of the region adjacent to one or two amino acids with acylation sensitivity. That is, as long as one of the two amino acids with acylation sensitivity is included in the deletion, each deletion can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 350, 400 amino acids. The size of the deletion in each amino acid with acylation sensitivity is independent and can also have the same size. Also disclosed are deletions that cover a continuous portion of the continuous amino acids between two amino acids with acylation sensitivity.

[0040] The deletion does not delete more than 50% of the wild-type amino acid sequence.

[0041] The present disclosure also relates to analogs of the modified amino acid sequences defined above that are at least 90% identical to the modified amino acid sequences defined above and that do not contain amino acids having the acylation sensitivity defined above. Such analogs can include conservative substitutions and deletions as defined in the section related to the general definition. Thus, the polypeptide may contain further deletions outside the deletion region containing one or two amino acids having acylation sensitivity, but these deletions will result in the deletion of only 1, 2, or 3 amino acids.

[0042] Amino acids having no acylation sensitivity Amino acids having acylation sensitivity are natural amino acids such as lysine and / or asparagine. Amino acids having no acylation sensitivity are known to those skilled in the art and can be used to substitute one or both of the amino acids having acylation sensitivity. For example, amino acids having no acylation sensitivity can independently be selected from the group consisting of alanine, glycine, isoleucine, leucine, methionine, valine, serine, threonine, asparagine, glutamine, aspartic acid, histidine, aspartic acid, cysteine, proline, phenylalanine, tyrosine, tryptophan, and glutamic acid. In particular, amino acids having no acylation sensitivity can independently be selected from the group consisting of alanine, glycine, serine, threonine, valine, isoleucine, and leucine. Furthermore, amino acids having no acylation sensitivity can independently be selected from the group consisting of alanine, glycine, and serine. The most preferred amino acid having no acylation sensitivity is alanine.

[0043] APXIA, APXIIA, or APXIIIA polypeptides having two modified amino acids Preferably, the two amino acids having acylation sensitivity are modified in each of ApxIA, ApxIIA, or ApxIIIA.

[0044] That is, the wild-type sequence of ApxIA set forth in SEQ ID NO: 1 is modified at K560 and K686. That is, the wild-type sequence of ApxIIA set forth in SEQ ID NO: 2 is modified at K557 and N687. That is, the wild-type sequence of ApxIIIA set forth in SEQ ID NO: 3 is modified at K571 and K702.

[0045] Also disclosed is that the two amino acids having acylating sensitivity in each of ApxIA, ApxIIA or ApxIIIA are modified to alanine.

[0046] That is, the disclosed modified polypeptide can have the sequence shown in SEQ ID NO: 4, 5 or 6, or can be an analog or fragment thereof as defined above.

[0047] Nucleic acid Also disclosed is a nucleic acid comprising a nucleic acid sequence capable of encoding the above polypeptide. The disclosed nucleic acid can be cDNA, DNA, RNA, cRNA or PNA (peptide nucleic acid). The term "nucleic acid sequence" refers to a heteropolymer of nucleotides having the sequence of these nucleotides. The nucleic acid can comprise the nucleic acid set forth in SEQ ID NO: 10, 11 or 12.

[0048] The nucleic acid can be included in a vector suitable for cloning or expressing the nucleic acid in the present disclosure. Exemplary vectors are pEX-A258 (SEQ ID NO: 19), pQE-80L (SEQ ID NO: 20) or pQE-60 (SEQ ID NO: 21). The nucleic acid or vector can include additional regulatory non-coding elements such as an inducible or non-inducible promoter, operator (e.g., lac-operator) or nucleic acid encoding other AP proteins. Further, the vaccine composition according to the present invention is preferably further characterized by a polynucleotide incorporated into the vector, and the polynucleotide is operably linked to the expression control region of the vector. That is, also disclosed is an expression vector that preferably contains one or more regulatory sequences in addition to the nucleic acid sequence encoding the modified form of ApxA in the expression vector. The term "expression vector" usually refers to a plasmid or phage or virus or vector for expressing a polypeptide from a DNA (RNA) sequence. An expression vector can include a transcription unit that includes an assembly of (1) one or more genetic elements that play a role in regulating gene expression, such as a promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into a protein, and (3) appropriate transcription start and termination sequences. The structural unit intended for use in a yeast or eukaryotic expression system preferably includes a leader sequence that allows extracellular secretion of the protein translated by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may contain an N-terminal methionine residue. This residue may then be cleaved from the expressed recombinant protein to become the final product, or it may not be.

[0049] Microorganism Also disclosed are microorganisms comprising the nucleic acids and / or vectors disclosed above. The microorganisms may be Escherichia coli (E. coli) strains, such as the E. coli Top10F’ strain, or Actinobacillus pleuropneumoniae strains, such as ST2 (e.g., APP23), ST2 (e.g., 07 / 07), ST5 (e.g., DZY47), ST7 (e.g., DZY33), or ST8 (e.g., DZY49). The microorganisms may also comprise nucleic acids and / or vectors encoding other genes.

[0050] Vaccine composition Also disclosed is a vaccine composition comprising at least one of the modified ApxIA, ApxIIA or ApxIIIA polypeptides as defined above, the nucleic acid molecule as defined above, or the vector as defined above, or the microorganism as defined above. The vaccine composition may comprise at least a pharmaceutical carrier, diluent, and / or adjuvant.

[0051] The preparation of the vaccine composition according to the present invention is known in the art and is described in handbooks known to those skilled in the art. When manufacturing the vaccine composition according to the present invention, examples of pharmaceutically acceptable carriers, diluents or adjuvants include mineral salt adjuvants (e.g., aluminum, calcium, iron, zirconium-based), tensioactive adjuvants (e.g., Quil A, QS-21, other saponins), bacterium-derived adjuvants (e.g., N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), lipopolysaccharide (LPS), monophosphoryl lipid A, trehalose dimycolate (TDM), DNA, CpG, bacterial toxins), adjuvant emulsions (e.g., FIA, Montanide, adjuvant 65, Lipovant), liposome adjuvants, polymer adjuvants and carriers, cytokines (e.g., granulocyte-macrophage colony-stimulating factor), carbohydrate-based adjuvants, live antigen delivery systems (e.g., bacteria, particularly modified APP). Furthermore, carriers also include dry preparations such as coating patches made of titanium or polymer. Also, the techniques for formulating and administering the vaccines of the present application are described in "Remington, The Science and Practice of Pharmacy" (22nd Edition).

[0052] The vaccine composition as a unit dosage composition may contain 0.01 to 2.0 mg of protein, 0.01 to 2.0 mg of nucleic acid, or 0.5 to 200 mg (or 1×10 4 CFU~1×10 10 CFU, colony-forming unit) of microorganisms. The required active amount of the protein, nucleic acid or microorganism can be determined by those skilled in the art through routine tests on, for example, pigs or piglets.

[0053] The vaccine composition containing at least one of the above ApxIA, ApxIIA or ApxIIIA polypeptides may be a subunit vaccine. The subunit vaccine may further contain additional APP polypeptides.

[0054] Also contemplated is a vaccine that contains substantially only nucleic acid or a vector, such as a DNA vaccine. A DNA vaccine is a third-generation vaccine. The nucleic acid or DNA vaccine contains DNA / nucleic acid encoding a specific protein (ApxA) derived from APP. The DNA / nucleic acid is injected into a mammalian body, taken up by cells, and in the normal metabolic process of the cells, proteins are synthesized based on the genetic code within the nucleic acid they have taken up. Since these proteins contain regions of the amino acid sequence characteristic of APP, when they are recognized as foreign substances, processed by host cells, and presented on their surface, the immune system is alerted and then an immune response is induced.

[0055] Alternatively, the nucleic acid may be encapsulated in a protein to facilitate entry into cells. When this capsid protein is included in the nucleic acid, the resulting vaccine can combine the efficacy of live vaccines without the risk of toxicity reversion.

[0056] The above microorganisms in the vaccine composition may be, for example, species of Actinobacillus such as Actinobacillus pleuropneumoniae, Actinobacillus suis, strains of species of Actinobacillus such as Actinobacillus pleuropneumoniae, Actinobacillus suis, or serotypes of strains of a specific species of Actinobacillus. The vaccine composition may contain at least one, for example, 1, 2, 3, 4, 5, 6, or 7 strains or serotypes of species of Actinobacillus. Each of the species, strains, or serotypes of Actinobacillus may encode at least one polypeptide as defined above.

[0057] The above-mentioned microorganism in the vaccine composition may not contain an APP nucleic acid encoding an APP protein different from the disclosed modified ApxIA, ApxIIA or ApxIIIA polypeptide. For example, the microorganism may contain a deletion of the apxIV gene and / or the sxy gene.

[0058] In addition, at least one of Actinobacillus pleuropneumoniae in the vaccine composition may contain one or at least two (e.g., single or multiple deletions) of the following modifications: ΔtbpA, ΔtonB2, ΔsodC, ΔdsbA, Δfur, ΔmlcA, ΔmglA, ΔexbB, ΔureC, ΔexbB ΔureC double mutant, ΔfhuA, ΔhlyX, ΔapxIC and ΔapxIIC double mutant, ΔapxIC ΔapxIIC Δorf1 triple mutant, ΔapxIIA ΔureC ΔdmsA ΔhybB ΔaspA Δfur hexamutant, ΔapxIIIB ΔapxIIID double mutant, ΔcIpP and ΔapxIIC double mutant, ΔznuA, ΔapfA, ΔapxIIA and ΔureC double mutant, ΔapxIC ΔapxIIC Δorf1 ΔcpxAR ΔarcA pentamutant, ΔapxIC ΔompP2 double mutant, ΔapxIIC ΔapxIVA double mutant, inactive apxIIC, inactive apxIC, Δlip40, ΔcpxA / cpxR, ΔpotD2, ΔtolC2, ΔsapA or ΔPdxS / PdxT.

[0059] Vaccine compositions containing the toxoid form of ApxIA as disclosed above and at least one ApxIA, ApxIIA or ApxIIIA polypeptide exhibit significantly enhanced immunogenicity and protective effects. Vaccine compositions containing the toxoid form of ApxIA as disclosed above and at least one ApxIA, ApxIIA or ApxIIIA polypeptide do not exhibit cytotoxic effects, do not exhibit hemolytic activity, and do not cause significant post-immunization fever that can occur after conventional immunization.

[0060] The above modifications are described below: Baltes, Nina; Hennig-Pauka, Isabel; Gerlach, Gerald-F (2002): “Both transferrin binding proteins are virulence factors in Actinobacillus pleuropneumoniae serotype 7 infection.” In FEMS microbiology letters 209(2), pp. 283-287. Sheehan, B.J.; Bosse, J.T.; Beddek, A.J.; Rycroft, A.N.; Kroll, J.S.; Langford, P.R. (2003): “Identification of Actinobacillus pleuropneumoniae Genes Important for Survival during Infection in Its Natural Host.” In Infection and immunity 71(7), pp. 3960-3970. DOI:10.1128 / IAI.71.7.3960-3970.2003. Baltes, N.; Tonpitak, W.; Gerlach, G.F.; Hennig-Pauka, I.; Hoffmann-Moujahid, A.; Ganter, M.; Rothkotter, H.J. (2001): “Actinobacillus pleuropneumoniae iron transport and urease activity. Effects on bacterial virulence and host immune response.” In Infection and immunity 69(1), pp. 472-478. DOI:10.1128 / IAI.69.1.472-478.2001. Jacques, Mario (2004): “Surface polysaccharides and iron-uptake systems of Actinobacillus pleuropneumoniae.” In Canadian journal of veterinary research = Revue canadienne de recherche veterinaire 68(2), pp. 81-85. Baltes, Nina; N’diaye, Mohamed; Jacobsen, Ilse D.; Maas, Alexander; Buettner, Falk F.R.; Gerlach, Gerald-F (2005): “Deletion of the anaerobic regulator HlyX causes reduced colonization and persistence of Actinobacillus pleuropneumoniae in the porcine respiratory tract.” In Infection and immunity 73(8), pp. 4614-4619. DOI:10.1128 / IAI.73.8.4614-4619.2005. Lin, Liwen; Bei, Weicheng; Sha, Yonggang; Liu, Jinlin; Guo, Yi; Liu, Weihong et al. (2007): “Construction and immunogencity of a DeltaapxIC / DeltaapxIIC double mutant of Actinobacillus pleuropneumoniae serovar 1.” In FEMS microbiology letters 274(1), pp. 55-62. DOI:10.1111 / j.1574-6968.2007.00813.x. Yuan, Fangyan; Liu, Jinlin; Guo, Yi; Tan, Chen; Fu, Shulin; Zhao, Jin et al. (2011): “Influences of ORF1 on the virulence and immunogenicity of Actinobacillus pleuropneumoniae.” In Current microbiology 63(6), pp. 574 - 580. DOI: 10.1007 / s00284-011-0016-0、 Maas, Alexander; Jacobsen, Ilse D.; Meens, Jochen; Gerlach, Gerald-F (2006): “Use of an Actinobacillus pleuropneumoniae multiple mutant as a vaccine that allows differentiation of vaccinated and infected animals.” In Infection and immunity 74(7), pp. 4124 - 4132. DOI: 10.1128 / IAI.00133-06、 Park, Changbo; Ha, Yooncheol; Kim, Soohee; Chae, Chanhee; Ryu, Doug-Young (2009): “Construction and characterization of an Actinobacillus pleuropneumoniae serotype 2 mutant lacking the Apx toxin secretion protein genes apxIIIB and apxIIID.” In The Journal of veterinary medical science 71(10), pp. 1317 - 1323、 Xie, Fang; Li, Gang; Zhou, Long; Zhang, Yanhe; Cui, Ning; Liu, Siguo; Wang, Chunlai (2017): “Attenuated Actinobacillus pleuropneumoniae double - deletion mutant S - 8ΔclpP / apxIIC confers protection against homologous or heterologous strain challenge.” In BMC veterinary research 13(1), p. 14. DOI: 10.1186 / s12917 - 016 - 0928 - 9、 Yuan, Fangyan; Liao, Yonghong; You, Wujin; Liu, Zewen; Tan, Yongqiang; Zheng, Chengkun et al. (2014): “Deletion of the znuA virulence factor attenuates Actinobacillus pleuropneumoniae and confers protection against homologous or heterologous strain challenge.” In Veterinary microbiology 174(3 - 4), pp. 531 - 539. DOI: 10.1016 / j.vetmic.2014.10.016、 Zhou, Yang; Li, Lu; Chen, Zhaohui; Yuan, Hong; Chen, Huanchun; Zhou, Rui (2013): “Adhesion protein ApfA of Actinobacillus pleuropneumoniae is required for pathogenesis and is a potential target for vaccine development.” In Clinical and vaccine immunology: CVI 20(2), pp. 287 - 294. DOI: 10.1128 / CVI.00616 - 12、 Tonpitak, Walaiporn; Baltes, Nina; Hennig-Pauka, Isabel; Gerlach, Gerald F. (2002): “Construction of an Actinobacillus pleuropneumoniae serotype 2 prototype live negative-marker vaccine.” In Infection and immunity 70(12), pp. 7120-7125. Yuan, Fangyan; Liu, Jinlin; You, Wujin; Bei, Weicheng; Wang, Chunlai; Zhao, Jin et al. (2018): “Generation, safety and immunogenicity of an Actinobacillus pleuropneumoniae quintuple deletion mutant SLW07(ΔapxIC ΔapxIIC Δorf1 ΔcpxAR ΔarcA).” In Vaccine 36(14), pp. 1830-1836. DOI:10.1016 / j.vaccine.2018.02.083. Liu, Qiong; Gong, Yuheng; Cao, Yuqin; Wen, Xintian; Huang, Xiaobo; Yan, Qigui et al. (2013): “Construction and immunogenicity of a ΔapxIC / ompP2 mutant of Actinobacillus pleuropneumoniae and Haemophilus parasuis.” The Onderstepoort journal of veterinary research 80(1), p. 519. DOI:10.4102 / ojvr.v80i1.519. Liu, Jinlin; Chen, Xia; Lin, Liwen; Tan, Chen; Chen, Yan; Guo, Yi et al. (2007): “Potential use an Actinobacillus pleuropneumoniae double mutant strain DeltaapxIICDeltaapxIVA as live vaccine that allows serological differentiation between vaccinated and infected animals.” Vaccine 25(44), pp. 7696 - 7705. DOI: 10.1016 / j.vaccine.2007.07.053、 Bei, Weicheng; He, Qigai; Yan, Lin; Fang, Liurong; Tan, Yadi; Xiao, Shaobo et al. (2005): “Construction and characterization of a live, attenuated apxIICA inactivation mutant of Actinobacillus pleuropneumoniae lacking a drug resistance marker.” In FEMS microbiology letters 243(1), pp. 21 - 27. DOI: 10.1016 / j.femsle.2004.11.033、 Xu, Fu-Zhou; Shi, Ai-Hua; Chen, Xiao-Ling; Yang, Bing; Wang, Jin-Luo(2007): “Construction and immunogenicity of an attenuated mutant of Actinobacillus pleuropneumoniae by insertional inactivation of apxIC.” Wei Sheng wu xue bao = Acta microbiologica Sinica 47(5), pp. 923 - 927、 Prideaux, C.T.; Lenghaus, C.; Krywult, J.; Hodgson, A.L. (1999): “Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site - specific mutagenesis of the ApxII operon.” In Infection and Immunity 67(4), pp. 1962 - 1966. Liu, 2018 “Outer Membrane Lipoprotein Lip40 Modulates Adherence, Colonization, and Virulence of Actinobacillus pleuropneumoniae.” Front Microbiol. 2018 Jul 3; 9: 1472. doi: 10.3389 / fmicb.2018.01472. Li, 2018 “The CpxA / CpxR Two - Component System Affects Biofilm Formation and Virulence in Actinobacillus pleuropneumoniae.” Front Cell Infect Microbiol. 2018 Mar 20; 8: 72. doi: 10.3389 / fcimb.2018.00072. Zhu 2017 “Polyamine - binding protein PotD2 is required for stress tolerance and virulence in Actinobacillus pleuropneumoniae.” Antonie Van Leeuwenhoek. 2017 Dec; 110(12): 1647 - 1657. doi: 10.1007 / s10482 - 017 - 0914 - 7. Li 2017 "TolC2 is required for the resistance, colonization and virulence of Actinobacillus pleuropneumoniae." J Med Microbiol. 2017 Jul 31. doi:10.1099 / jmm.0.000544. Xie 2017 "The SapA Protein Is Involved in Resistance to Antimicrobial Peptide PR-39 and Virulence of Actinobacillus pleuropneumoniae." Front Microbiol. 2017 May 10;8:811. doi:10.3389 / fmicb.2017.00811, and Xie 2017 "Pyridoxal phosphate synthases PdxS / PdxT are required for Actinobacillus pleuropneumoniae viability, stress tolerance and virulence." PLoS One. 2017 Apr 27;12(4):e0176374. doi:10.1371 / journal.pone.0176374.

[0061] Combinations of the modified polypeptides disclosed herein with the additional known modifications listed above are expected to result in synergistic attenuation of Actinobacillus pleuropneumoniae.

[0062] Medical uses or methods The disclosed vaccine composition can be used in prophylactic, metaphylactic or therapeutic treatment of pneumonia, pleurisy or pleuropneumonia in a subject, particularly pneumonia, pleurisy or pleuropneumonia caused by Actinobacillus pleuropneumoniae. The subject to be treated can be a mammal, particularly a pig. The vaccine composition can be administered by intramuscular, intradermal, intravenous, subcutaneous, or mucosal administration (e.g., intranasal administration).

[0063] The vaccine composition can be administered at least once, e.g., once or twice, using the above unit dosage composition. Specifically, the composition can be administered for the first time on the subject's birth day, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks or 12 weeks from birth. Thus, the vaccine can be effective when administered at an early point in the mammal's life. However, it is also disclosed that the vaccine can be administered at any point during the mammal's life.

[0064] The vaccine composition can also be administered at a second time, and the period between the two administrations can be 1 - 4 weeks, 1 - 3 weeks or 1 - 2 weeks. Preferably, the vaccine composition containing the microorganism is administered only once.

Brief Description of the Drawings

[0065]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Example

[0066] The following examples provide experimental data for explaining the present invention and should be understood only as illustrations of the present invention. In particular, it is contemplated that each specific feature disclosed in the examples can be combined with the features described in the preceding detailed description section.

[0067] Example 1: Cloning of wild-type and modified APXIA, APXIIA, or APXIIIA in PEX-A258 The protein sequences of wild-type ApxIA - ApxIIIA were obtained from the database Uniprot, namely ApxIA App ST5b (Uniprot: A3N292), ApxIIA APP ST3 (Uniprot B0BPP0, with modified S148G), and ApxIIIA ST8 (Uniprot: P55131).

[0068] In addition, protein sequences containing the following modifications were provided.

Table 1

[0069] The nucleic acids were derived from the protein sequences, and these nucleic acid sequences were codon-optimized and manufactured synthetically (Eurofins Genomics) without including the start codon. Each nucleic acid sequence was inserted into plasmid pEX-A258 (SEQ ID NO: 19) so as to be flanked by the restriction site BamHI at the 5'-end and SacI at the 3'-end.

[0070] Example 2: Cloning and Expression of Wild-Type and Modified ApxIA - ApxIIIA in PQE-80L (SEQ ID NO: 20) 1 μg of pEX-A258 containing each of ApxIA - ApxIIIA was restricted with BamHI FD (ThermoFisher) and Sacl FD (ThermoFisher) according to the manufacturer's instructions and separated on a 1% agarose gel. Nucleic acid fragments of appropriate sizes were extracted from the agarose gel (QUIAEX-II). pQE-80L was restricted with BamHI FD (ThermoFisher) and Sacl FD (ThermoFisher) according to the manufacturer's instructions. Using a 1:5 ratio of pQE-80L and each nucleic acid fragment, ligation was performed overnight at room temperature using T4-DNA ligase (ThermoFisher). The ligated DNA was electroporated into E. coli Topf10F', and the success of cloning of wild-type and modified forms of ApxIA - ApxIIIA was confirmed by restriction analysis and sequencing. pQE-80L contains ampicillin resistance.

[0071] Example 3: Cloning of ApxIC, ApxIIC, and ApxIIIC Genes in PQE-60 Using the primers shown in Table 2, the apxIC - IIIC genes were amplified from APP-DNA by polymerase chain reaction (PCR) and then purified.

[0072] According to the instructions of each manufacturer, the PCR fragments containing each of apxIC - IIIC nucleic acids and pQE-60 were restricted with the restriction enzymes shown in Table 2 below and separated on a 1% agarose gel.

Table 2

[0073] Nucleic acid fragments of appropriate sizes were extracted from an agarose gel (QUIAEX-II). Using a 1:5 ratio of pQE-60 and each nucleic acid fragment, ligation was performed overnight at room temperature using T4-DNA ligase (ThermoFisher).

[0074] The ligated DNA was electroporated into E. coli Topf10F‘, and the success of cloning was confirmed by restriction analysis and sequencing.

[0075] Example 4: Cloning and Expression of the APXIC, APXIIC, and APXIIIC Genes in PACYC184 The apxIC, apxIIC, and apxIIIC genes were amplified from each pQE-60 vector of Example 2 using the primers shown in Table 3 below, respectively.

Table 3

[0076] In this way, the promoter / lac-operator and ribosome binding site were further included in the amplicon (SEQ ID NOs: 16, 17, and 18).

[0077] Each PCR product was restricted using the restriction enzymes shown in Table 3 and cloned into pACYC184 using the same method and conditions as above with the restriction enzyme sites XbaI and NruI (apxIC and apxIIIC) or ClaI / BscI and NruI (apxIIC). pACYC184 contains chloramphenicol resistance.

[0078] The ligated DNA was electroporated into E. coli Topf10F‘, and the success of cloning was confirmed by restriction analysis and sequencing.

[0079] Example 5: Co - transformation of pACYC184 containing the ApxIC, ApxIIC, or ApxIIIC gene and pQE - 80L containing ApxIA, ApxIIA, or ApxIIIA Activation of the ApxA toxin requires ApxC. Therefore, pACYC184 containing the apxIC, apxIIC, or apxIIIC gene obtained in Example 4 was co - transformed with E. coli Top10F' together with pQE - 80L containing the apxIA, apxIIA, or apxIIIA gene, respectively, and those that were successfully co - transformed were selected on LB agar plates containing 50 μg / ml of ampicillin and 15 μg / ml of chloramphenicol. The success or failure of the transformation was confirmed by restriction analysis.

[0080] Example 6: Expression and isolation of ApxA, especially ApxIA, toxin and toxoid Using the E. coli colonies obtained in Example 5, which contain pACYC184 and pQE - 80L encoding the ApxA and ApxC genes, respectively, they were inoculated into 40 ml of LB medium (100 μg / ml of ampicillin and 30 μg / ml of chloramphenicol). The culture was incubated with shaking at 30 °C overnight. The next day, the overnight culture was inoculated into 400 ml of LB medium (100 μg / ml ampicillin and 30 μg / ml chloramphenicol) to an optical density of 0.1 (optical density at 600 nm = OD600). The resulting culture was incubated with shaking until the OD600 reached 0.5. To induce the expression of the recombinant protein, 1 mM of IPTG (isopropyl β - D - 1 - thiogalactopyranoside) was added. The culture was incubated with shaking at 30 °C for an additional 4 hours. Next, the culture was centrifuged at 7000 g for 10 minutes. After removing the supernatant, the bacterial pellet was resuspended in LEW buffer (50 mM NaH2PO4, 300 mM NaCl, in distilled water, pH 8.0) to a concentration of 5 ml per 1 g of bacterial pellet and stored at - 20 °C.

[0081] 1 ml of 10× Bug Buster (Novagen), 5 μl of Benzoase (25 U / μl, Novagen) and 10 mg of lysozyme were added to the 10 ml of resuspended bacterial pellet. The resulting suspension was gently rotated at room temperature for 20 minutes. After centrifugation at 4°C and 15,000 g for 60 minutes, the pellet was washed with LEW buffer (10 mL of LEW buffer per 1 mg of pellet) and centrifuged (4°C, 15,000 g, 60 minutes). The washed pellet was resuspended in 2 mL of denaturing solubilization buffer (8 M urea in LEW buffer, pH = 8.0) per 1 g of pellet, and then 1 mL of Bug Buster (Novagen) per 10 mL of resuspended pellet was added. The suspension was incubated at 4°C for 1 hour and then centrifuged (4°C, 18,000 g, 15 minutes). The supernatant containing the protein was used for the next step of protein purification by column.

[0082] Following the manufacturer's instructions, the column (Protino Ni-IDA 2000 column, Machery-Nagel) was equilibrated with 4 mL of equilibration buffer (8 M urea in LEW buffer, pH = 8.0). Next, half of the supernatant containing the protein was loaded onto the column and washed twice with 4 ml of equilibration buffer (a total of 8 ml of buffer). The protein was eluted twice with 3 ml of elution buffer (equilibration buffer containing 250 mM imidazole, a total of 6 ml of buffer). This procedure was repeated with the remaining half of the supernatant containing the protein. The protein-containing eluates were pooled and dialyzed overnight at 4°C in PBS (pH 8.0) containing 1 M urea. The dialysis buffer was exchanged with PBS (pH 8.0) containing 0.5 M urea and dialysis was continued for an additional 4 hours. Next, the buffer was exchanged with PBS (pH 8.0) without urea and dialysis was continued for an additional 4 hours. The protein concentration was measured using the Pierce BCA Protein Assay Kit (ThermoFisher) according to the manufacturer's instructions. The protein was stored at -80°C until use.

[0083] Example 7: Isolation of ApxA Toxin from APP Solution APVX contains 1 g of D(+)-glucose, monohydrate, 0.1 g of L-glutamine, 0.362 g of L-cysteine hydrochloride monohydrate, and 10 mL of nuclease-free H2O. The APP isolate was seeded onto chocolate agar containing IsovitaleX and incubated overnight at 37°C. Three APP colonies were combined and used to inoculate 3 mL of Columbia medium (10 mM Ca 2+ , 0.2 mg / mL of NAD, 1% of APPVX), and the resulting culture was incubated with shaking at 37°C for 5 hours. Next, the culture was centrifuged at 3200 g and 4°C for 20 minutes. The clear supernatant was transferred to a new tube, 44.05 g of ammonium sulfate was added gradually, and the mixture was stirred until the ammonium sulfate was completely dissolved. Next, the solution was incubated overnight at 4°C. The precipitated protein was centrifuged at 3200 g and 4°C for 3 hours, and the supernatant was removed. The protein pellet was air-dried and then suspended in 1 mL of PBS containing 6 M urea. The resulting protein solution was purified by gel filtration using a PDMidiTrap G25 column (GE Healthcare) to remove the residual buffer. After equilibrating 1 ml of the protein solution in PBS containing 6 M urea, it was loaded onto the gel filtration column and then eluted with 1.5 ml of PBS containing 6 M urea. Next, the eluted protein was stored at -20°C. The identity of the eluted protein was confirmed by SDS-PAGE.

[0084] Example 8: Establishment of a Cytotoxicity Assay To analyze the cytotoxic activity of recombinant ApxA proteins (wild-type and toxoid forms), a cytotoxicity assay is performed using BL3 cells (BL3.1 CRL-2306) as target cells. BL3 cells are obtained from bovine B cells and are known to be lysed by ApxA toxin (Tu, A.H.; Hausler, C.; Young, R.; Struck, D.K. (1994): “Differential expression of the cytotoxic and hemolytic activities of the ApxIIA toxin from Actinobacillus pleuropneumoniae.” In Infection and immunity 62 (5), pp. 2119-2121). The ApxA toxin obtained in Example 7 was used as a positive control, and PBS containing 6M urea was used as a negative control.

[0085] BL3 cells were cultured in medium (DMEM containing 100 U / mL penicillin, 100 μg / mL streptomycin, 10% FCS and 1× non-essential amino acids) at 37 °C and 5% CO2. T75 flasks with 25 ml of medium were used for culturing. Using new flasks, 50,000 cells / cm 2 of BL3 cells were seeded and subcultured weekly.

[0086] For use in the cytotoxicity assay, 50 μl of BL3 cells / media was transferred to the wells of a 96-well plate. The recombinant proteins and controls were diluted in DMEM. 50 μl of the diluted recombinant proteins and controls were added to each well containing cells. Next, the plate was incubated at 37 °C and 5% CO2 for 24 hours. After evaluating the cell morphology by microscopy, 10 μl of WST-1 (Sigma Aldrich) was added to each well and the plate was incubated at 37 °C and 5% CO2 for an additional 24 hours.

[0087] Next, the absorbance is measured using a 450 nm ELISA reader. WST-1 is cleaved by complex cellular mechanisms on the cell surface of living cells, generating soluble formazan. Therefore, the amount of formazan dye formed and the extinction measured by the ELISA reader are directly correlated with the number of metabolically active cells in the culture medium.

[0088] Example 9: Establishment of Hemolysis Assay To analyze the hemolytic activity of recombinant ApxA proteins (wild-type and toxoid-type), a hemolysis assay was established. A cell culture plate containing Columbia agar with 5% sheep blood (Bection Dickinson) was used. The cell culture plate was punched with holes 3 mm in diameter. 10 μL of recombinant protein and control samples were filled into each hole, and the plate was incubated at 37 °C overnight. The ApxA toxin obtained in Example 7 was used as a positive control, and PBS containing 6 M urea was used as a negative control.

[0089] When the sheep blood agar decolorizes and appears white, it is considered that hemolytic activity is present.

[0090] Example 10: Isolation of ApxA Toxin from APP Using the ST2, ST5, ST7, and ST8 strains of APP, and the expression and isolation methods described in Example 7, the supernatant containing ApxA toxin was isolated. As a result of SDS-PAGE of the supernatant, bands appeared at the predicted molecular weight positions corresponding to ApxIA, ApxIIA, ApxIIIA, and ApxIVA (data not shown). Therefore, the supernatant of each of the obtained ST2, ST5, ST7, and ST8 strains contains a mixture of ApxIA, ApxIIA, ApxIIIA, and ApxIVA.

[0091] Example 11: Cytotoxicity by ApxA Derived from APP in BL-3 Cells Using the method of Example 8, 50 μl of the ApxA toxin from Example 7 obtained from APP (ST2, ST5, ST7, and ST8 strains of APP) was serially diluted (1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048, 1:4096) in DMEM and tested.

[0092] The cytotoxic effect of the supernatant of APP was observed and decreased with the increase in the dilution rate (see Figure 1). The toxin isolated from ST8 showed the highest cytotoxicity, and the toxin isolated from ST7 showed the lowest toxicity. No correlation was observed between LPS in the ApxA preparation and cytotoxicity (data not shown).

[0093] Example 12: Cytotoxicity of Recombinant ApxIA and ApxIIIA in BL-3 Cells Using the method described in Example 8, the following samples were tested. [Table 4]

[0094] 50 μl of each sample was tested in serial dilutions (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512) in DMEM.

[0095] Using a control group with cells diluted in PBS, the effect of PBS was evaluated. No effect was observed (see Figure 2). The results are shown in Figure 2.

[0096] As shown in Figure 2A, in the cytotoxicity assay, recombinant wild-type ApxIA lysed BL3 cells, thus demonstrating its activity. After dilution of 1:64 (13.1 μg / ml of wild-type ApxIA toxin), the cell survival rate values were equivalent to those of the PBS control. The survival rate of BL3 cells incubated in the ApxIA toxoid form was equivalent to that of the PBS control. This was also the case for cell morphology (data not shown).

[0097] The toxoid form of ApxIA had effects similar to the PBS control with respect to cell viability and morphology, while the wild-type ApxIA form had a strong cytotoxic effect on BL3 cells. Therefore, it can be concluded that the toxoid form of ApxIA has no cytotoxic effect. Similar results were also obtained for the toxoid forms of ApxIIA (data not shown) and ApxIIIA (Figure 2B).

[0098] Example 13: Hemolytic activity of ApxA derived from APP Using the method of Example 9, 10 μl of the ApxA toxin of Example 7 obtained from APP (ST2, ST5, and ST8 strains of APP) was tested.

[0099] The results are shown in Figure 3. The ST8 strain showed strong hemolysis, the ST5 strain showed moderate hemolysis, and the ST2 strain showed no hemolysis as can be inferred from the decreasing white color in Figure 3 from left to right. It is known that ApxIA, ApxIIA, and ApxIIIA each cannot lyse red blood cells. Due to strain differences, the secretion levels of each ApxA are different, so the supernatants from each strain containing a mixture of different ApxA forms can show different hemolytic activities.

[0100] In summary, it became clear that the hemolytic activity of the ApxA toxin can be demonstrated by the method using Columbia agar containing 5% sheep blood described in Example 9.

[0101] Example 14: Hemolytic activity of recombinant ApxIA Using the method described in Example 9, the following samples were tested.

Table 5

[0102] The results are shown in Figure 4. Wild-type ApxIA lyses red blood cells, while the toxoid form of ApxIA does not. Wild-type ApxIIIA showed no hemolytic activity as also described in the literature.

[0103] Since the toxoid form of ApxIA, like the PBS control, did not show hemolytic activity, while the wild-type ApxIA form had strong hemolytic activity against sheep red blood cells, it can be concluded that the ApxIA toxoid has no hemolytic activity. Similar results were obtained for the toxoid form of ApxIIA (data not shown).

[0104] Example 15: Immunogenicity of the ApxIA, ApxIIA, and ApxIIIA polypeptides of the present invention in pigs (1) Preparation of antigen and vaccine formulations Nucleic acids encoding the modified ApxIA (SEQ ID NO: 4), ApxIIA (SEQ ID NO: 5), and ApxIIIA (SEQ ID NO: 6) codons optimized for expression in E. coli were synthetically produced (Eurofins Genomics) and introduced into the plasmid pEX-A258 (SEQ ID NO: 19) such that the sequences were flanked by the restriction enzyme site BamHI at the 5' end and SacI at the 3' end. According to the instructions, 1 μg of pEX-A258 containing ApxIA and ApxIIIA respectively was restricted with BamHI FD (ThermoFisher) and Sacl FD (ThermoFisher). The nucleotides encoding the amino acids 439 - 801 of ApxIIA, the pEX-A258 (SEQ ID NO: 19) containing the ApxIIA gene, and a primer introducing a BamHI restriction site at the 5' end and a SacI restriction site at the 3' end (forward:

Chemical formula

Chemical formula

[0105] Montanide ISA 201 VG (lot 36075M, Seppic) was used according to the manufacturer's instructions to prepare test batches using modified ApxIA, ApxIIA (cleaved form), and ApxIIIA (Table 6). Briefly, the adjuvant and antigen were pre-warmed to 31°C in a water bath. The volume of the antigen(s) was made equal to that of the adjuvant (50:50, w / v) by adding sterile PBS and maintained at 31°C. Montanide ISA 201 VG was gently stirred in a beaker. Next, the antigen was added to the adjuvant and stirred for an additional 5 minutes at 31°C. The formulation was stored at 20°C for 1 hour, filled into glass vials, and further stored at 4°C until use.

Table 6

[0106] (2) Animal studies (immunogenicity) The immunogenicity of the vaccine formulation was determined by quantification of ApxIA-, ApxIIA-, and ApxIIIA-specific IgG antibodies in ELISA and evaluation of ApxIA neutralizing properties in serum after vaccination of 8-week-old pigs.

[0107] Animal Male and female pigs (castrated males and females) were obtained from a general pig breeder (BHZP, 21337 Luneburg, Zuchtbetrieb Garlitz (DE-LWL 1051)). Each pig was identified with a unique ear tag number and subjected to a veterinary examination for compliance with the selection / exclusion criteria specified in the test protocol. All animals showed no clinical signs of APP infection, and the animals had no clinical signs (dyspnea, cough, diarrhea, lameness) at the start of the test. Before the animals were used in the test, it was confirmed that the serum was negative by a commercially available APP-ApxIV ELISA (IDEXX). The pigs were isolated and managed in an animal facility of IDT Biologika GmbH, Am Pharmapark, D-06861 Dessau-Rosslau (Germany) against contaminants. Two independent rooms / pens were provided at equal intervals on a hard floor within the unit. Straw was used as bedding and replaced daily. A manually filled dry feeder was used to allow the pigs to freely ingest (commercially available mixed pellets). Water of tap water quality was supplied by faucets and nipple drinkers. All facilities were cleaned daily.

[0108] Test Design A total of 25 eight-week-old pigs were randomly assigned to five groups, with five animals per group. In three groups, 1.5 mL of either "Vaccine 1", "Vaccine 2" or "Vaccine 3" was used and administered intramuscularly twice at an interval of 14 days. Five animals in one group were administered 2 mL of a commercially available vaccine against Actinobacillus Pleuropneumoniae (Porcilis® APP, MSD Animal Health, batch: A784A01) twice at an interval of 14 days as a positive control. Another group was administered a placebo vaccine as a negative control. The summary of the groups and treatments is shown in Table 7.

Table 7

[0109] On the 0th day of the test (the first vaccination day), 14th day (the second vaccination day), and 28th day (the end of the test), serum specimens were collected from each animal.

[0110] Regarding the local and systemic reactions related to vaccination, evaluations were performed once a day from 1 day before to 3 days after each of the two vaccinations. In the evaluation of local reactions, the redness and swelling at the injection site were evaluated.

[0111] From 1 day before to 3 days after each of the two vaccinations, the rectal temperature was measured once a day. On the vaccination days, the temperature was measured immediately before vaccination and 6 hours later. The results of the temperature are shown in Table 8 and Figure 5.

[0112] Results:

Table 8

[0113] (4) Serological data (neutralization assay) The neutralization assay enables quantification of the recombinant WT ApxIA neutralizing ability of each porcine serum.

[0114] BL3 cells (BL3.1 CRL-2306) were used as target cells. BL3 cells were obtained from bovine B cells and the lysis of this cell line by ApxIA toxin has been reported (Tu, A.H.; Hausler, C.; Young, R.; Struck, D.K. (1994)). BL3 cells were cultured in medium (DMEM containing 100 U / mL penicillin, 100 μg / mL streptomycin, 10% FCS and 1× non-essential amino acids) at 37 °C and 5% CO2. T75 flasks with 25 ml of medium were used for culturing. New flasks were used and BL3 cells at 50,000 cells / cm2 were seeded weekly and subcultured. For use in the neutralization assay, 50 μl of BL3 cells / media were transferred to the wells of a 96-well plate.

[0115] Porcine sera were heat-inactivated at 56 °C for 30 minutes. Next, the sera were diluted and prepared as a 2-fold dilution series in DMEM. Recombinant WT ApxIA (active toxin) was prepared according to Example 7 and diluted to a final concentration of 240 μg / mL. 70 μl of each serum dilution was mixed with 30 μl of the WT ApxIA dilution (final amount of ApxIA per well: 6 μg) on a 96-well plate (serum dilution of 1:2 to 1:256) and incubated at 37 °C for 1 hour. 50 μl of the contents of each well was added to 50,000 BL3 cells / cm in 50 μL of DMEM 2Transfer to a round-bottom 96-well plate containing [the relevant substance], and make the final serum dilution 1:4 to 1:512. Incubate the plate at 37 °C for 24 hours in an atmosphere of 5% CO₂ and 100% humidity. After 24 hours, add the WST-1 substrate (Sigma) to each well of the plate according to the manufacturer's instructions. Incubate the plate at 37 °C for an additional 24 hours in an atmosphere containing 5% CO₂ and 100% humidity. Next, measure the absorbance using an ELISA reader at 450 nm. WST-1 is cleaved by the complex cellular machinery on the cell surface of live cells, generating soluble formazan. Therefore, the amount of formazan dye formed and the extinction measured by the ELISA reader are directly correlated with the number of metabolically active cells in the culture medium. In the serological tests of the animals in groups 1, 2, and 3, a neutralization assay using the serum obtained on day 28 was used. The results are shown in Figure 7.

[0116] Example 16: Efficacy of the ApxIA, ApxIIA, and ApxIIIA polypeptides of the present invention in pigs (1) Preparation of antigen and vaccine formulations The preparation of the antigen and formulations was carried out as described in Example 15. Table 9 summarizes the formulations in each vaccine test batch.

Table 9

[0117] (2) Animal tests (efficacy) The purpose of this test is to evaluate the protective effect obtained by vaccination with the recombinantly expressed modified ApxIA, ApxIIA, and ApxIIIA vaccine formulations against exposure to serotype 9 of Actinobacillus pleuropneumoniae (APP) in pigs.

[0118] Animals Male and female pigs (castrated males and females) were obtained from a general pig breeder (Dalmandi Mezogazdasagi Zrt., 7211 Dalmand, Felszabadulas u. 42). Each pig was identified with a unique ear tag number and subjected to veterinary examination for compliance with the selection / exclusion criteria specified in the test protocol.

[0119] Neither APP nor Mycoplasma was present in the origin (herd). Piglets derived from sows were not vaccinated against APP to avoid interference by maternally derived antibodies with vaccination. The animals had no clinical signs (dyspnea, cough, diarrhea, lameness). Furthermore, before the animals were used in the test, it was confirmed that the sera were negative by a commercially available APP-ApxIV ELISA (IDEXX). The pigs were fully grown, had no functional disorders, and no congenital malformations.

[0120] The animals were isolated and managed for contaminants in the animal facility of PROPHYL Ltd, Top. No.: 045 / 7, Bar H-7711, Hungary. A manual filling type dry feeder was used to allow the pigs to freely consume (commercially available mixed pulverized feed). Water of tap water quality was supplied by faucets and nipple drinkers. All facilities were cleaned daily.

[0121] Test Design The efficacy induced by a vaccine containing 25 μg of each modified ApxIA, ApxIIA (cleaved), and ApxIIIA formulated with Montanide ISA 201 VG (lot 36075M, Seppic) against intranasal exposure to APP serotype 9 was compared with a registered APP vaccine (Porcilis® APP, MSD Animal Health, lot: A739A01) group and a placebo group that received PBS. An overview of the groups and treatments is shown in Table 10.

Table 10

[0122] For all pigs at 42 - 45 days of age, for each treatment group, 2.0 mL of each vaccine was used to inoculate the vaccine intramuscularly (day 0 of the test). Twenty - eight days later, the pigs were given a second vaccination with each vaccine via the intramuscular route (day 28 of the test). Two weeks after the second vaccination, all animals were subjected to intranasal exposure with APP serotype 9 (5×10 7 CFU / pig) (day 42 of the test).

[0123] Clinical tests were performed daily for 7 days from the exposure day. In each test, scores were obtained according to Table 11 based on clinical observations. The clinical scores for each animal over time were accumulated, and the average score per group over the entire observation period was calculated. For statistical analysis, the results of the clinical scores were analyzed using the Kruskal - Wallis test and Dunn's multiple comparison test. The data for each group were compared with the corresponding data of the placebo group (group 3). A p - value of 0.05 or less was considered significant. The results are shown in Figure 8. The results of the survival rate of the animals after exposure are shown in Figure 9.

Table 11

[0124] Seven days after exposure, all surviving animals were humanely euthanized and necropsied. Autopsies were performed on the lungs of all animals to observe the lesions caused by APP. Lung evaluation was performed according to the scoring system shown in Table 12. The lung lesions were scored from 0 to 5 for each lobe according to the percentage of solidified lung tumors. The results of the lung scoring were presented as the cumulative lung score (in the range of 0 - 35) for each pig. The cumulative lung scores were analyzed using the Kruskal - Wallis test and Dunn's multiple comparison test. The data for each group were compared with the corresponding data of the placebo group (group 3). A p - value of 0.05 or less was considered significant. The results are shown in Figure 10.

Table 12

Claims

1. A vaccine composition comprising ApxIA, ApxIIA and ApxIIIA polypeptides of Actinobacillus pleuropneumoniae, wherein: a. i. The wild-type amino acid sequence of ApxIA set forth in SEQ ID NO: 1 is modified with at least one amino acid selected from the group consisting of K560 and K686; ii. The wild-type amino acid sequence of ApxIIA set forth in SEQ ID NO: 2 is modified with at least one amino acid selected from the group consisting of K557 and N687; iii. The wild-type amino acid sequence of ApxIIIA set forth in SEQ ID NO: 3 is modified with at least one amino acid selected from the group consisting of K571 and K702; and the at least one amino acid is deleted or replaced with an amino acid that is not acylation-sensitive, ApxIA, ApxIIA and ApxIIIA polypeptides, or b. An analog or fragment that induces a humoral and / or cellular immunological response of the amino acid sequence defined in a., which is at least 90% identical to the full length of the amino acid sequence defined in a., and the analog or fragment contains an amino acid that is not acylation-sensitive or at least an amino acid deletion defined in a., an analog or fragment, at least a pharmaceutical carrier, diluent, and / or adjuvant, A vaccine composition comprising.

2. The vaccine composition according to claim 1, wherein the amino acid that is not acylation-sensitive is independently selected from the group consisting of alanine, glycine, isoleucine, leucine, methionine, valine, serine, threonine, asparagine, glutamine, aspartic acid, histidine, aspartic acid, cysteine, proline, phenylalanine, tyrosine, tryptophan, and glutamic acid.

3. a. The wild-type amino acid sequence of ApxIA set forth in SEQ ID NO: 1 is modified at K560 and K686, b. The wild-type amino acid sequence of ApxIIA set forth in SEQ ID NO: 2 is modified at K557 and N687, c. The wild-type amino acid sequence of ApxIIIA set forth in SEQ ID NO: 3 is modified at K571 and K702, the vaccine composition according to claim 1 or 2.

4. a. The wild-type amino acid sequence of the modified ApxIA is SEQ ID NO: 4, b. The wild-type amino acid sequence of the modified ApxIIA is SEQ ID NO: 5, c. The wild-type amino acid sequence of the modified ApxIIIA is SEQ ID NO: 6, the vaccine composition according to any one of claims 1 to 3.

5. A nucleic acid molecule or combination of nucleic acid molecules encoding the ApxIA, ApxIIA and ApxIIIA polypeptides according to claim 1.

6. The nucleic acid molecule or combination of nucleic acid molecules according to claim 5, wherein the nucleic acid molecule is contained in a vector.

7. A microorganism expressing the ApxIA, ApxIIA and ApxIIIA polypeptides according to claim 1.

8. The microorganism according to claim 7, which is an Escherichia coli strain or an Actinobacillus strain.

9. The microorganism according to claim 8, which is an Actinobacillus pleuropneumoniae strain.

10. The vaccine composition according to any one of claims 1 to 4, wherein the vaccine composition is a subunit vaccine.

11. The vaccine composition according to claim 10, wherein the subunit vaccine comprises at least one additional polypeptide of Actinobacillus pleuropneumoniae.

12. The vaccine composition according to claim 1, wherein the vaccine composition is a live vaccine comprising an Actinobacillus pleuropneumoniae strain that expresses the ApxIA, ApxIIA, and ApxIIIA polypeptides.

13. The vaccine composition according to claim 12, comprising at least two different Actinobacillus pleuropneumoniae strains.

14. The vaccine composition according to any one of claims 12 or 13, wherein the Actinobacillus pleuropneumoniae comprises a deletion of the ApxIV gene and / or the sxy gene.

15. The vaccine composition according to any one of claims 1 to 4 and 10 to 14, which is used for prophylactic, metaphylactic or therapeutic treatment of pneumonia, pleurisy or pleuropneumonia.

16. The vaccine composition according to any one of claims 1 to 4 and 10 to 14, which is used for prophylactic, metaphylactic or therapeutic treatment of pneumonia, pleurisy or pleuropneumonia caused by Actinobacillus pleuropneumoniae.

17. The vaccine composition according to any one of claims 1 to 4 and 10 to 14, which is administered by application intramuscularly, intradermally, intravenously, subcutaneously or mucosally.

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