Vaccine containing glycosylated bacteria

Abstract

The present invention is directed to Gram-negative bacterial host cells for vaccine use, comprising a heterologous functional Actinobacillus pleuropneumoniae (APP) rfb gene cluster that binds to the lipid A core of the bacterial host cell and produces the APP O antigen located on the outer surface of the bacterial host, wherein the endogenous rfb gene cluster of the bacterial host cell is not functional. The invention further relates to compositions, particularly vaccines, comprising the host cells, and corresponding uses in the prevention and / or therapy of Actinobacillus pleuropneumoniae (APP) infection.

Description

[Technical field]

[0001] The present invention is directed to a Gram-negative bacterial host cell for vaccine use, comprising a heterologous functional Actinobacillus pleuropneumoniae (APP) rfb gene cluster that binds to the lipid A core of the bacterial host cell and produces the APP O antigen located on the bacterial host outer surface, where the endogenous rfb gene cluster of the bacterial host cell is not functional. The present invention further relates to compositions, particularly vaccines, comprising said host cells, and corresponding uses in the prevention and / or therapy of Actinobacillus pleuropneumoniae (APP) infections. [Background technology]

[0002] Actinobacillus pleuropneumoniae (APP) is the main cause of porcine pleuropneumonia, a highly contagious respiratory disease in pigs that causes major economic losses in the swine industry. Transmission occurs via aerosols or close contact with infected animals or asymptomatic carriers. To date, 18 serotypes of APP with different geographical distributions have been classified according to their surface capsular polysaccharides (Bosse et al., 2018, Vet Microbiol., Vol. 220, pp. 83-89). Although differences in pathogenicity exist, all serotypes are capable of causing disease. The existence of at least 18 serotypes makes the development of a broadly protective vaccine difficult. The economic importance of the disease has stimulated intensive research in the field of APP vaccination in the last few years. However, as antibiotics are used to control the disease and antibiotic resistance has reached alarming levels worldwide, alternative solutions are needed.

[0003] Currently available vaccines against APP consist mainly of inactivated whole cell bacterins (chemically inactivated bacterial cells) or subunit vaccines based on outer membrane proteins. Some vaccines are based on or supplemented with Apx toxins (ApxI-IV toxoids), a set of pore-forming cytolysins that play a central role in APP pathogenesis. To date, the protective effect induced by all commercially available vaccines is insufficient. Bacterin-based and subunit vaccines have been shown to confer limited protection against heterologous strains. Vaccines based on inactivated Apx toxins are effective in reducing infection-associated morbidity but are unable to prevent pulmonary colonization and their use poses a potential threat to the induction of infection by asymptomatic carriers (Andresen et al., 1997, Acta Vet Scand, Vol. 38, pp. 283-293; Antenucci et al., 2017, Vet Res., Vol. 48:74; Antenucci et al., 2018, Vet Res., 49:4; Haesebrouck et al., 2004, Vet Microbiol., Vol. 100, pp. 255-268; Loera-Muro and Angulo 2018, Vet Microbiol. Vol. 217, pp. 66-75; Ramjeet et al., 2008, Anim Health Res Rev, Vol. 9(1), pp. 25-45).

[0004] Potential antigenic structures other than surface proteins for bacterial vaccine development are the glycans displayed on the surface of pathogenic bacteria. These glycans are one of the first points of contact for the immune system of an infected host. Two prominent surface glycan structures of Gram-negative bacteria are extracellular capsular polysaccharides (CPS, in Gram-positive and -negative bacteria) and lipopolysaccharides (LPS, in Gram-negative bacteria). Development of glycan-based vaccines against bacterial diseases in the human field has mainly focused on CPS structures. Several bacterial diseases have been addressed by isolating CPS structures and chemically conjugating these glycans to immunostimulatory protein carriers. The H. influenza type b CPS conjugated to a tetanus toxoid from Sanofi Pasteur (product ActHIB), the CPS of four Neisseria meningitidis serotypes conjugated to a diphtheria toxoid from GSK Vaccines (Menveo), and the CPS of 13 Streptococcus pneumoniae serotypes conjugated to a diphtheria toxoid from Pfizer (Prevnar 13) represent three examples of successful development of vaccines based on this type of CPS.

[0005] However, for veterinary vaccine development, isolation of surface glycans and subsequent chemical conjugation to carrier structures results in uneconomical production costs. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Bosse et al., 2018, Vet Microbiol., Vol. 220, pp. 83-89 [Non-Patent Document 2] Andresen et al., 1997, Acta Vet Scand, Vol. 38, pp. 283-293 [Non-Patent Document 3] Antenucci et al., 2017, Vet Res., Vol. 48: 74

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[0007] The objective underlying the present invention is the provision of a safe and effective vaccine that confers protection against many, most and preferably essentially all serotypes of APP bacteria, which will allow a significant reduction in antibiotic treatments in food production and will reduce clinical outbreaks and losses during pig fattening. [Means for solving the problem]

[0008] The object is to provide a Gram-negative bacterial host cell for vaccine use, (a) a heterologous functional Actinobacillus pleuropneumoniae (APP) rfb gene cluster, wherein the heterologous functional APP rfb gene cluster binds to a lipid A core of a bacterial host cell to produce an APP O-antigen located on the bacterial host outer surface, and wherein the endogenous rfb gene cluster of the bacterial host cell is not functional; (b) optionally, a heterologous promoter for regulating transcription of the heterologous APP rfb gene cluster that is stronger than the endogenous promoter for the endogenous rfb gene cluster; (c) optionally, at least one additional gene for functionally expressing an enzyme that assists in the synthesis of the APP O antigen; (d) optionally at least one neutralizing epitope of an Apx toxin, optionally at least one neutralizing epitope of Apx toxins I, II and III, optionally located on the outer surface of a bacterial host cell and / or secreted from the cell. Including, Optionally, the solution is provided by providing a bacterial host cell, wherein at least one of (a), (c) and (d) is codon-optimized for the bacterial host cell.

[0009] It has surprisingly been found and clinically demonstrated that Gram-negative bacterial host cells that have a non-functional endogenous rfb gene cluster, i.e., no production of endogenous O antigen, but contain a heterologous functional Actinobacillus pleuropneumoniae (APP) rfb gene cluster and produce the APP O antigen bound to the lipid A core of the bacterial host cell and located on the bacterial host outer surface, will elicit a superior immune response in pigs and thereby be protected against APP infection.

[0010] As used herein, the adjective "heterologous" indicates that the thing so referred to, e.g., a cellular component such as a gene, protein, glycan, glycoprotein, metabolite, etc., is not naturally occurring in the cell in question, i.e., has been artificially introduced and is derived from a heterologous, i.e., genetically non-identical, organism.

[0011] As used herein, the adjective "endogenous" indicates that the thing so referred to, e.g., a cellular component such as a gene, protein, glycan, glycoprotein, metabolite, etc., is naturally present in the cell in question.

[0012] Those skilled in the art can routinely identify cellular components and compounds as heterologous or endogenous by known methods, for example, by comparative molecular genetics and biochemical analysis. For example, those skilled in the art can routinely identify the APP rfb gene cluster and / or APP O antigen in non-APP cells as heterologous. Similarly, it is routine to demonstrate that the rfb gene cluster is not functional, or its endogenous absence or non-functional genetic structure in Gram-negative bacteria, and / or the absence of the corresponding APP O antigen in or on the cell or secreted from the cell of interest.

[0013] As used herein, the term "non-functional", particularly in the context of the rfb gene cluster in a bacterial host cell, is intended to indicate a partial or complete absence, structural or functional change, or at least a malfunction of at least one gene, optionally all genes, of the cluster, resulting in the absence, aberrant expression and / or aberrant function of at least one protein, optionally all proteins, arising from the gene cluster, substantially eliminating the production of O-antigen from the gene cluster expression product. For example, one or more of the genes of the rfb gene cluster can be altered and / or deleted, and / or the gene regulation of the cluster can be altered, thereby rendering the expression of said genes dysfunctional, i.e., resulting in physiologically irrelevant or abolished expression of proteins for O-antigen synthesis. Analysis of the genes and proteins and evaluation of their functionality or absence can be achieved using conventional techniques in molecular biology and biochemistry.

[0014] To achieve a physiologically effective immune response, the bacterial host cell will bind the APP O antigen resulting from expression of the heterologous functional APP rfb gene cluster to the bacterial host lipid A core and translocate the conjugate to the outer surface of the bacterial host for presentation to and contact with appropriate immune-related components in the environment, e.g., live vaccinated pigs.

[0015] The bacterial host cell of the present invention can be broadly selected from Gram-negative bacterial cells, since all Gram-negative bacterial cells express and contain lipid A, which is necessary for binding to the heterologous O antigen of the bacterial host cell of the present invention. In certain embodiments, for example, the bacterial host cell is selected from the group consisting of Enterobacteriaceae, Burkholderiaceae, Pseudomonadaceae, Vibrionaceae, optionally Burkholderia thailandensis, Pseudomonas aeruginosa, Vibrio natriegens, Vibrio cholerae, Escherichia coli, optionally E. coli_5, Salmonella enterica, optionally Salmonella enterica subsp. enterica, optionally selected from the group consisting of serovar Typhimurium, Enteritidis, Heidelberg, Gallinarum, Hadar, Agona, Kentucky and Infantis, and Salmonella enterica subsp. enterica serovar Typhimurium SL1344.

[0016] It will, of course, be understood that the bacterial host cell selected must be suitable for pharmaceutical use, for example for vaccine use, either as a killed or live vaccine.

[0017] In a further embodiment, the bacterial host cell of the invention comprises a heterologous functional rfb gene cluster, which may be selected from all known APP rfb gene clusters, in particular the well-known APP1-18 rfb gene clusters. These gene clusters may be altered for use in the bacterial host cell of the invention, i.e., may differ from the naturally occurring gene clusters to the extent that the corresponding expressed heterologous APP O antigen is functional, i.e., capable of eliciting physiologically relevant immunity in pigs against APP challenge, and yet capable of binding to the lipid A core of the bacterial host cell.

[0018] In one embodiment, a heterologous rfb gene cluster for practicing the present invention comprises: (i) comprises or consists of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4; (ii) has at least 70, 80, 90, 95, or 98% nucleic acid sequence identity to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4, optionally over the entire sequence; (iii) hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4; and / or (iv) Degenerate with respect to any of the nucleic acid sequences of (i) to (iii); The APP2 or APP8 rfb gene cluster.

[0019] In the context of nucleic acids, as known to those of skill in the art, and as used herein, the term "% (percent) sequence identity" refers to the degree of relatedness between two or more nucleic acid molecules, as determined by the match between the sequences. The percentage of "sequence identity" is the result of the percentage of identical regions in two or more sequences, taking into account gaps and other sequence idiosyncrasies.

[0020] The identity of related nucleic acid molecules can be determined using known methods. Generally, specialized computer programs are used, with algorithms adapted to meet the specific requirements of this task. A preferred method for determining identity begins with generating the highest degree of identity between the sequences to be compared. Preferred computer programs for determining identity between two nucleic acid sequences include, but are not limited to, BLASTN (Altschul et al., 1990, J. Mol. Biol., Vol. 215, pp. 403-410) and LALIGN (Huang and Miller 1991, Adv. Appl. Math., Vol. 12, pp. 337-357). BLAST programs can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbook, Altschul et al., NCB NLM NIH Bethesda, MD 20894).

[0021] The identity of related nucleic acid molecules, eg, the APP rfb gene cluster, can also be determined by their ability to hybridize, optionally under stringent conditions, to specifically referenced nucleic acid sequences. Protocols common and / or standard in the art for determining the ability to hybridize to a specifically referenced nucleic acid sequence under stringent conditions (e.g., Sambrook and Russell, (2001) Molecular cloning: A laboratory manual (vol. 3)) may be used, for example, as mentioned above, for example, BLASTN (Altschul et al., 1990, J. Mol. Biol., Vol. 215, pp. 403-410), the LALIGN alignment tool and multiple alignment tools, such as CLUSTALW (Sievers et al., 2011, Mol. Sys. Bio., Vol. 7, pp. 539), MUSCLE (Edgar 2004, Nucl. Acids Res., Vol. 32, pp. 1792-7) or T-COFFEE (Notredame et al., 2000, J. of It is preferred to analyze and determine the ability to hybridize to a specific referenced nucleic acid sequence under stringent conditions by comparing nucleotide sequences that can be found in gene databases (e.g., http: / / www.ncbi.nlm.nih.gov / entrez / query.fcgi?db=nucleotide) using alignment tools such as Molecular Biology, Vol. 302(1), pp. 205-17.

[0022] Most preferably, the ability of the APP rfb gene cluster for use in the present invention to hybridize to a specifically referenced nucleic acid, such as any of those set forth in SEQ ID NOs: 1, 3 and 4, is confirmed in a Southern blot assay under the following conditions: 6x sodium chloride / sodium citrate (SSC), 45°C, followed by a wash at 0.2x SSC, 0.1% SDS, 65°C.

[0023] In a further embodiment, the bacterial host cell produces an APP1-18 O antigen, optionally an APP2 or APP8 O antigen, where the APP rfb gene cluster optionally expresses at least one protein comprising or consisting of any one of the amino acids of SEQ ID NO:2, 50-61, or SEQ ID NO:5, 62-72, or at least one protein having at least 70, 80, 90, 95 or 98% amino acid sequence identity to these sequences.

[0024] Percent identity of related amino acid molecules can be determined using known methods. Generally, specialized computer programs are used, with algorithms adapted to meet the specific requirements of this task. A preferred method for determining identity begins with generating the highest degree of identity between the sequences to be compared. Preferred computer programs for determining identity between two amino acid sequences include, but are not limited to, TBLASTN, BLASTP, BLASTX, TBLASTX (Altschul et al., 1990, J. Mol. Biol., Vol. 215, pp. 403-410), or ClustalW (Larkin et al., 2007, Bioinformatics, Vol. 23, pp. 2947-2948). BLAST programs can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbook, Altschul et al., NCB NLM NIH Bethesda, MD 20894). The ClustalW program can be obtained from http: / / www.clustal.org.

[0025] Heterologous APP rfb gene clusters for use in the present invention can be prepared synthetically by methods well known to those of skill in the art, but can also be isolated from suitable DNA libraries and other publicly available sources of nucleic acid, and then optionally mutated. Preparation of such libraries or mutations is well known to those of skill in the art.

[0026] In an alternative embodiment, the bacterial host cell of the invention is one in which the endogenous rfb gene cluster of the bacterial host cell is non-functional and at least partially, optionally completely deleted.

[0027] Introduction of a heterologous promoter to regulate transcription of the heterologous APP rfb gene cluster was found to improve APP O-antigen expression when the heterologous promoter was stronger than the endogenous promoter for the APP rfb gene cluster. The heterologous promoter for the heterologous APP rfb gene cluster is optionally selected from the group consisting of kanamycin promoter, proD promoter, j23101 promoter, proC promoter, STER_RS05525 promoter, STER_RS01225 promoter, STER_RS04515 promoter, STER_RS05020 promoter, STER_RS06870 promoter, STER_RS00780 promoter, P32 promoter, optionally consisting of kanamycin promoter, proD promoter, j23101 promoter, STER_RS04515 promoter and P32 promoter, optionally consisting of kanamycin promoter and proD (Datsenko et al., 2000, PNAS, Vol. 97(12), pp. 6640-6645; Davis et al., 2010, Nucleic acids research, Vol. 39(3), pp. 1121-1141; Kong et al., 2019, ACS Synthetic Biology, Vol. 8, pp. 1469-1472).

[0028] As used herein, in the context of comparing promoter efficiency, the term "stronger" is intended to indicate that a heterologous promoter will produce more transcription products, i.e., rfb gene cluster transcription and translation products, i.e., rfb cluster expression products (enzymes), and more APP O antigen, than the endogenous and functional APP rfb promoter naturally occurring in a host cell.

[0029] It has been found that some of the enzymatic activities resulting from the APP rfb gene cluster can limit the cellular production of APP O-antigen. In this regard, it has been demonstrated that the cellular production of APP O-antigen in the bacterial host cell of the present invention can be improved in efficiency by introducing at least one additional gene for functionally expressing an enzyme that supports, for example, participates in or converts an intermediate product of, APP O-antigen synthesis. Optionally, the at least one additional gene for functionally expressing an enzyme that supports APP O-antigen synthesis is selected from the group consisting of enzymes for nucleotide-activated glycan biosynthesis, undecaprenyl pyrophosphate glycosyltransferase, O-antigen glycosyltransferase, O-antigen polymerase, O-antigen chain length determinant protein, and N-glycan epimerase, and combinations thereof, and optionally selected from the group consisting of gne and wzy genes; i. wherein the gne gene encodes a UDP-galactose / UDP-N-acetylglucosamine epimerase, optionally from Campylobacter jejuni, and the gne gene optionally comprises or consists of SEQ ID NO:6, or has a nucleic acid sequence that is at least 70, 80, 90, 95 or 98% identical to SEQ ID NO:6, optionally over the entire sequence, and / or hybridizes to the nucleic acid sequence of SEQ ID NO:6 under stringent conditions; ii. wherein the wzy gene encodes an O-antigen polymerase of APP, optionally of APP2, and the wzy gene optionally comprises or consists of a codon-optimized wzy gene of SEQ ID NO:7 or SEQ ID NO:8, or has a nucleic acid sequence that is at least 70, 80, 90, 95 or 98% identical to SEQ ID NO:7 or SEQ ID NO:8, optionally over the entire sequence, and / or hybridizes to the nucleic acid sequence of SEQ ID NO:7 or SEQ ID NO:8 under stringent conditions.

[0030] Bacterial infections are often accompanied by the release of toxic compounds. APP bacteria typically secrete ApxI, ApxII, ApxIII and ApxIV toxins in various combinations depending on the APP serotype. To ameliorate symptoms of APP infection, the bacterial host cell of the invention can optionally comprise at least one neutralizing epitope of Apx toxin, optionally at least a neutralizing epitope of Apx toxin I-III, located on the bacterial host cell outer surface and optionally bound to a membrane protein selected from the group consisting of cytolysin A, trimeric autotransporter adhesins, preferably AIDA-I and EaeA, and an outer membrane protein (OMP), preferably OmpA of E. coli (Xu et al., 2018, PlosOne, Vol. 13(1); Rutherford et al., 2006, Vol. 188(11); Wentzel et al., 2001, J. Bacteriol., Vol. 183(24), pp. 7273-7284, 2001; Georgiou et al., 1996, Protein A, Vol. 183(24), pp. 7273-7284, 2001). Engineering, Vol. 9(2), pp. 239-247).

[0031] Codon optimization by synonymous substitution is widely used for recombinant protein expression. Codon optimization refers to the adaptation of the codon composition of a recombinant gene to improve protein expression without changing the resulting amino acid sequence. This is possible because the majority of amino acids are coded for by more than one codon. Typically, codon optimization is adapted for a specific host organism (Burgess-Brown et al., 2008, Protein Expression & Purification Vol. 59, pp. 94-102; Elena et al., 2014, Frontiers in Microbiology, Vol. 5(21), pp. 1-8).

[0032] In an alternative embodiment, the bacterial host cell of the invention is one in which (a) the heterologous functional APP rfb gene cluster, (b) at least one additional gene for functionally expressing an enzyme assisting in APP O-antigen synthesis, and / or (c) at least one neutralizing epitope of the Apx toxin are codon-optimized for the bacterial host cell. Optionally, the heterologous functional APP rfb gene cluster is codon-optimized for the bacterial host cell.

[0033] The following specific, non-limiting embodiments of bacterial host cells of the invention are presented to further illustrate the general inventive concept.

[0034] In an alternative embodiment, the bacterial host cell of the invention is Escherichia coli, optionally E. coli_5, or Salmonella, optionally Salmonella enterica, optionally Salmonella enterica subsp. enterica serovar Typhimurium, optionally Salmonella enterica subsp. enterica serovar Typhimurium SL1344, wherein (a) the heterologous functional APP rfb gene cluster is selected from an APP1-18 rfb gene cluster, optionally an APP2 or APP8 rfb gene cluster; (b) the heterologous promoter for regulating the transcription of the heterologous APP rfb gene cluster is the kanamycin or proD promoter; (c) at least one further gene for functionally expressing an enzyme that assists in the synthesis of the APP O-antigen is the wzy gene, optionally codon-optimized wzy, and / or the gne gene, both genes optionally integrated into the genome of the bacterial host cell or located on a plasmid; (d) optionally comprising at least one neutralizing epitope of Apx toxins I, II and III, which optionally binds to a membrane protein, optionally binds to cytolysin A of E. coli, or is secreted from a host cell; wherein (i) the APP2 or APP8 rfb gene cluster, (ii) the gne gene and / or (iii) the wzy gene, optionally the APP2 rfb gene cluster and the wzy gene, are codon-optimized for the bacterial host cell Escherichia coli, optionally E. coli_5, or Salmonella, optionally Salmonella subsp. enterica, optionally Salmonella subsp. enterica serovar Typhimurium.

[0035] In another alternative embodiment, optionally as described above, the bacterial host cell of the invention is Salmonella enterica subsp. enterica serovar Typhimurium, optionally Salmonella enterica subsp. enterica serovar Typhimurium strain SL1344, wherein (a) the codon-optimized heterologous functional APP rfb gene cluster is an APP2 rfb gene cluster that optionally (i) comprises or consists of SEQ ID NO:3; (ii) has at least 70, 80, 90, 95 or 98% nucleic acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3, optionally over the entire sequence; (iii) hybridizes to the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions; and / or (iv) is degenerate with respect to any of the nucleic acid sequences of (i)-(iii), the endogenous rfb gene cluster of the bacterial host cell is at least partially or completely deleted; (b) the optional heterologous promoter for regulating transcription of the heterologous APP2 rfb gene cluster is the kanamycin promoter; (c) at least one additional gene for functionally expressing an enzyme that assists in the synthesis of the APP O-antigen, the gne gene and / or the wzy gene, optionally integrated into the genome of the bacterial host cell; i. wherein the gne gene, optionally the Campylobacter jejuni gne gene, optionally comprises or consists of SEQ ID NO:6, or optionally has a nucleic acid sequence that is at least 70, 80, 90, 95 or 98% identical to SEQ ID NO:6, optionally over the entire sequence, and / or hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:6; ii. wherein the wzy gene optionally comprises or consists of SEQ ID NO:7 or SEQ ID NO:8, or optionally has a nucleic acid sequence that is at least 70, 80, 90, 95 or 98% identical to SEQ ID NO:7 or SEQ ID NO:8, optionally over the entire sequence, and / or hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:7 or SEQ ID NO:8; (d) optionally comprising at least two neutralizing epitopes of Apx toxins I, II and III, optionally at least Apx toxins II and III, optionally bound to a membrane protein, optionally cytolysin A of E. coli.

[0036] In a further alternative embodiment, the bacterial host cell of the invention is Escherichia coli, optionally E. coli_5, wherein (a) A heterologous functional APP rfb gene cluster, optionally (i) comprising or consisting of SEQ ID NO:3; (ii) having at least 70, 80, 90, 95 or 98% nucleic acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3, optionally over the entire sequence; (iii) hybridizing to the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions; and / or (iv) being degenerate with respect to any of the nucleic acid sequences of (i) to (iii), the endogenous rfb gene cluster of the bacterial host cell is at least partially or completely deleted; (b) the heterologous promoter for regulating transcription of the heterologous APP2 rfb gene cluster is the kanamycin or proD promoter, optionally the kanamycin promoter; (c) at least one additional gene for functional expression of an enzyme that assists in the synthesis of the APP O antigen, the gne gene, optionally integrated into the genome of the bacterial host cell or located on a plasmid; wherein the gne gene, optionally the Campylobacter jejuni gne gene, optionally comprises or consists of SEQ ID NO:6, or optionally has a nucleic acid sequence that is at least 70, 80, 90, 95 or 98% identical to SEQ ID NO:6, optionally over the entire sequence, and / or hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:6; (d) optionally comprising at least one neutralizing epitope of Apx toxins I, II and III, optionally at least Apx toxins II and III, optionally bound to a membrane protein, optionally bound to cytolysin A of E. coli or secreted from a host cell; Here, the APP2 rfb gene cluster is optionally codon-optimized for E. coli.

[0037] In another embodiment of the invention, the bacterial host cell is Salmonella enterica subsp. enterica serovar Typhimurium, optionally Salmonella enterica subsp. enterica serovar Typhimurium strain SL1344 or Escherichia coli, optionally E. coli_5, wherein (a) the heterologous functional APP rfb gene cluster is an APP8 rfb gene cluster, which is optionally codon-optimized and optionally (i) comprises or consists of SEQ ID NO:4; (ii) has at least 70, 80, 90, 95 or 98% nucleic acid sequence identity to SEQ ID NO:4, optionally over the entire sequence; (iii) hybridizes to the nucleic acid sequence of SEQ ID NO:4 under stringent conditions; and / or (iv) is degenerate with respect to any of the nucleic acid sequences of (i)-(iii), (b) the optional heterologous promoter for regulating transcription of the heterologous APP2 rfb gene cluster is the kanamycin or proD promoter, optionally the kanamycin promoter; (c) at least one further gene for functionally expressing an enzyme of APP O-antigen synthesis is the wzy and / or gne gene, optionally codon-optimized, and optionally both genes are integrated into the genome of the bacterial host cell; i. wherein the gne gene, optionally the Campylobacter jejuni gne gene, optionally comprises or consists of SEQ ID NO:6, or optionally has a nucleic acid sequence that is at least 70, 80, 90, 95 or 98% identical to SEQ ID NO:6, optionally over the entire sequence, and / or hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:6; ii. wherein the wzy gene, optionally codon-optimized, optionally comprises or consists of SEQ ID NO:7 or SEQ ID NO:8, or has a nucleic acid sequence that is at least 70, 80, 90, 95 or 98% identical to SEQ ID NO:7 or SEQ ID NO:8, optionally over the entire sequence, and / or hybridizes to the nucleic acid sequence of SEQ ID NO:7 or SEQ ID NO:8 under stringent conditions; (d) optionally comprising at least two neutralizing epitopes of Apx toxins I, II and III, optionally at least Apx toxins II and III, optionally bound to a membrane protein, optionally cytolysin A of E. coli.

[0038] The bacterial host cells of the invention may be administered to pigs in viable or inactivated form.

[0039] The above bacterial host cells of the present invention are highly immunogenic and generate an immune response against APP infection. Moreover, once prepared, the bacterial host cells can be easily grown and mass-produced. The bacterial host cells can be administered live or inactivated, for example, as live or killed vaccines, where live vaccines allow long-term growth and sustained immune stimulation in the host, and a complete immune response without adjuvants.

[0040] The present invention therefore also relates to the medical use of a live or killed bacterial host cell of the invention, in particular for the preparation of a medicament, preferably a vaccine, for the prevention and / or therapy of APP infections.

[0041] Preferably, the medicament is useful for the prevention and / or treatment of an APP, in particular an APP1-18 infection, preferably an APP infection in pigs.

[0042] A further aspect of the invention relates to a composition, optionally a pharmaceutical composition, comprising at least one bacterial host cell of the invention as described herein, and a physiologically acceptable excipient.

[0043] In one embodiment, a composition or pharmaceutical composition of the invention comprises a bacterial host cell expressing at least two different O antigens, optionally from APP1-APP18, optionally selected from the group consisting of APP1, 2, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17 and 18, optionally a combination of O antigens selected from the group consisting of APP1, 2, 5, 7, 8 10, 12, 14 and 18.

[0044] In a further embodiment, the bacterial host cell or composition of the invention is for use in the prevention and / or treatment of Actinobacillus pleuropneumoniae (APP) infection, optionally in a mammal, and optionally in an APP2 infection in pigs (Sus) or domestic pigs (Sus scrofa domesticus).

[0045] In another embodiment, the present invention includes feed or drinking water for animals, particularly swine livestock, as well as physiologically acceptable excipients and / or foodstuffs. Such compositions, such as vaccines or feed, will significantly reduce APP colonization in swine livestock, thereby reducing the chances of infection spreading.

[0046] In another aspect, the present invention is directed to a therapeutic method for the treatment of APP infection, and optionally for the prevention of APP infection in pigs, comprising administering a physiologically effective amount of a bacterial host cell or composition, food or feed of the present invention to a mammalian subject in need thereof.

[0047] For therapeutic and / or prophylactic use, the compositions, pharmaceutical compositions, foods or feeds of the invention may be administered in any conventional manner and in any conventional dosage form.

[0048] Routes of administration include, but are not limited to, intranasal, oral, oronasal, transcutanous, conjunctive, intraembryo, subcutaneous, intradermal, intramuscular, sublingual, transdermal, or conjunctival. For example, application devices and methods include syringes, nebulizing and atomizing devices, sprays (coarse sprays, sprays on feed), and drinking water. Inhalation implies inhalation of liquids or powders. Application routes can be combined, for example, intranasal and oral application.

[0049] Preferred modes of administration are intranasal, oral, oronasal, sublingual, subcutaneous, intradermal, transdermal, conjunctival and intramuscular, with intranasal and oral being most preferred.

[0050] The bacterial host cells of the invention can be administered alone or in combination with adjuvants, including other active ingredients, that enhance the stability and / or immunogenicity of the bacteria, facilitate administration of pharmaceutical compositions containing the bacteria, provide increased solubility or dispersion, increase proliferative activity, provide adjunctive therapy, etc.

[0051] Pharmaceutical dosage forms of bacterial host cells, e.g., E. coli and Salmonella enterica, optionally serovar Typhimurium, as described herein, include pharma- ceutically acceptable carriers and / or adjuvants known to those skilled in the art. These carriers and adjuvants include, for example, water or buffer solutions with or without surfactants and / or salts, metal salts (e.g., aluminum-based), saponins, oils (mineral and non-mineral), oil emulsions, bacterial derivatives, cytokines, ISCOMs, liposomes, microparticles, vitamins (e.g., alpha tocopherol), dextrans, carbomers, microemulsions, synthetic oligodeoxynucleotides, and other immunostimulatory compounds, alone or in combination. Preferred dosage forms include solutions, suspensions, emulsions, powders, tablets, capsules, and transdermal patches. Methods for preparing dosage forms are well known, see for example Ansel et al., 1990, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th ed., ISBN: 978-0812112559 and, in particular, Pastoret 1999, Acad. Sci. Paris / Elsevier SAS, Vol. 322, pages 967-972.

[0052] For example, vaccination with the bacterial host cells and compositions of the invention can be performed as a single vaccination or with a booster vaccination, possibly followed by revaccination after a given period of time. Vaccines can consist of live bacteria, for example applied at a dose of 10E4-10E6 cfu (colony forming units), or can consist of inactivated bacteria, with or without adjuvant, for example applied at a dose of 10E6-10E11 cfu (colony forming units).

[0053] The following figures and examples serve to illustrate the invention and are not intended to limit the scope of the invention as set forth in the appended claims. [Brief description of the drawings]

[0054] [Figure 1] Schematic diagram of the APP rfb cluster identified from sequenced APP2 strains. The rfb cluster of APP2, with a length of 12928 bp, consists of 13 genes encoding putative glycosyltransferases, glycan-modifying enzymes, chain-length determinant proteins, O-antigen transporters, acetyltransferases and a hypothetical protein (with homology to the O-antigen polymerase wzy). Gene positions and cluster sizes are given in base pairs. hP-hypothetical protein. [Diagram 2] Schematic representation of the O-antigen biosynthetic cluster of APP2, including the adjacent gene erpA, and additional downstream sequences used for integration into SL1344 and E. coli_5. The complete fragment has a length of 13758 bp. Gene positions and cluster sizes are given in base pairs. hP-hypothetical protein. [Diagram 3] Schematic diagram of the APP2 O-antigen biosynthesis cluster located on a pDOC plasmid with the flanking homologous host cell regions of the rfb cluster. The 13758 bp fragment containing the APP2 rfb cluster and its flanking regions (see FIG. 2) was engineered for integration into SL1344 and E. coli_5 in an antisense orientation relative to the endogenous rfb cluster. To achieve this, a kanamycin resistance cassette flanked by FRT sites was fused downstream. The fusion construct is flanked upstream and downstream by homologous regions flanking the rfb cluster (including the galF gene in the antisense orientation) of either SL1344 or E. coli_5. The entire construct is flanked by I-SceI restriction endonuclease recognition sites. [Figure 4]Schematic diagram of the codon-optimized APP2 O-antigen biosynthesis cluster located on a pDOC plasmid with flanking homologous regions of the endogenous rfb cluster. For integration of the codon-optimized rfb cluster of APP2 into SL1344 and E. coli_5, the original integration site in the endogenous rfb cluster was kept the same. A codon-optimized (for E. coli expression) APP2 rfb cluster was synthesized that encoded a kanamycin resistance cassette (flanked by FRT sites) in its 5' region, resulting in transcription regulated by the promoter of the kanamycin gene. The entire construct is flanked by I-SceI restriction endonuclease recognition sites. [Diagram 5]Figure 1 shows photographs of Western blots (A,C) and silver staining (B,D) of gels against APP2 LPS for confirmation of extrachromosomal expression of wzy and gne in SL1344 expressing the APP2 O-antigen biosynthesis cluster (A,B) and of gne in E.coli_5 expressing the APP2 O-antigen biosynthesis cluster (C,D). wzy and / or gne were encoded on a plasmid under the control of an arabinose-inducible promoter in SL1344 and E.coli_5 cells expressing the APP2 rfb cluster (with or without codon optimization) downstream of a kanamycin resistance cassette. Cells were induced with 0.1% (SL1344 cells) or 0.2% (E.coli_5) arabinose and overnight cultures were analyzed for APP2 LPS expression by proteinase K treatment of cells and analysis of digested extracts via SDS-PAGE. On the left, molecular marker bands are indicated in kDa. Lane 1: APP2 P1875, lane 2: SL1344, lane 3: SL1344Δrfb, lane 4: SL1344Δrfb::kanR-APP2.LPS pMLBAD-gne pEC415-wzy, lane 5: SL1344Δrfb::kanR-APP2.LPS(cod.opt.) pMLBAD pEC415, lane 6: SL1344Δrfb::kanR-APP2.LPS(cod.opt.) pMLBAD-gne, lane 7: SL1344Δrfb::kanR-APP2.LPS(cod.opt.) pEC415-wzy, lane 8: SL1344Δrfb::kanR-APP2.LPS(cod.opt.) pMLBAD-gne pEC415-wzy, lane 9: E. coli_5, lane 10: E. coli_5Δrfb, lane 11: E. coli_5Δrfb::kanR-APP2.LPS(cod.opt.), lane 12: E. coli_5Δrfb::kanR-APP2.LPS(cod.opt.)pMLBAD, lane 13: E. coli_5Δrfb::kanR-APP2.LPS(cod.opt.)pMLBAD-gne. [Figure 6]Schematic representation of overlap PCR to generate fragments rfaL-Ωgne / cat (A) and rfaL-Ωgne-wzy(cod.opt.) / cat (B) to be integrated into E.coli_5 and SL1344 derivatives. (A) To generate rfaL-Ωgne / cat, individual fragments for gne and cat with overlapping regions were amplified. Overlap PCR was used to fuse the two fragments, extending the 5' and 3' ends and encoding homologous regions with the target integration sites in the genomes of E.coli_5 and SL1344. The same principle was followed to generate the integration construct rfaL-Ωgne-wzy(cod.opt.) / cat (B), except that an additional fragment encoding codon-optimized wzy was generated and the three fragments were then combined by overlap PCR and integration of homologous recombination sequences at the 5' and 3' ends. [Figure 7] Schematic representation of the cat / kP integration construct. To integrate the kanamycin promoter (kP) upstream of the codon-optimized APP2 rfb cluster, a 1414 bp construct was synthesized containing a chloramphenicol resistance cassette with flanking FRT sites followed by the 373 bp promoter region of the kanamycin resistance cassette (encoded on pKD4). [Figure 8] Figure 8 shows a comparison of APP2 O-antigen expression levels in SL1344 and E. coli_5 expressing the APP2 O-antigen biosynthetic cluster with or without additional glycosylation modifications (KanR and Kp control of APP2 rfb cluster expression, wzy and / or gne integration). Saturated overnight cultures of the cells listed in Figure 8A were analyzed for APP2 O-antigen expression by proteinase K treatment and analysis of digested cell extracts via SDS-PAGE. Figure 8B is a photograph of a silver stain for APP2 LPS and Figure 8C is a Western blot with rabbit serum reactive with APP2 LPS of the gel. On the left, molecular marker bands are indicated in kDa. [Figure 9]Schematic diagram of the APP rfb cluster identified from sequenced APP8 strains. The rfb cluster of APP8, with a length of 13598 bp, consists of 13 genes (potential functions of individual genes are listed in the table). Gene positions and cluster size are given in base pairs. [Figure 10] Schematic diagram of the codon-optimized APP8 O-antigen biosynthesis cluster located on pDOC_SL1344_Δrfb::cat-kP-APP8.LPS(cod.opt.) with flanking homologous regions of the endogenous rfb cluster (including galF of SL1344). Upstream of the codon-optimized rfb cluster of APP8 is a chloramphenicol resistance cassette (flanked by FRT sites) followed by the kP (kanamycin) promoter. The entire construct is flanked by I-SceI restriction endonuclease recognition sites. [Figure 11] Figure 1 shows APP8 O antigen expression levels in SL1344 encoding the codon-optimized APP8 rfb cluster under the control of the kP promoter. Saturated overnight cultures of SL1344 (lane 1), SL1344Δrfb (lane 2), APP8 (lane 3), APP3 (lane 4) and SL1344Δrfb::cat-kP-APP8.LPS(cod.opt.) (lane 5) were analyzed for APP8 O antigen expression by proteinase K treatment and analysis of digested cell extracts via SDS-PAGE. (A) is a photograph of a silver stain for LPS and (B) is a Western blot with pig serum reactive with APP3 LPS of the gel. Molecular marker bands (M) are indicated in kDa. [Figure 12]FIG. 10: Demonstration of expression and purification of HIS10-ApxII(439-801aa). HIS10-ApxII(439-801aa) was expressed in BL21 cells encoding pMLBAD-HIS10-ApxII(439-801aa) after arabinose induction. After 4 h incubation, cells were harvested and disrupted via lysozyme treatment followed by sonication-freeze-thaw cycles. Protein was purified under denaturing conditions by Ni-NTA binding and gravity flow. Elution from Ni-NTA beads (E) was achieved by five times 1 ml denaturing buffer containing 0.5 M imidazole. 7.5 μl of each elution fraction (lane 1: E1, lane 2: E2, lane 3: E3, lane 4: E4, lane 5: E5) was loaded for SDS-PAGE and analyzed via Coomassie staining (A) and Western blot against HIS epitopes (B). On the left and right, molecular marker bands are indicated in kDa. [Figure 13] Figure 1: Demonstration of expression and purification of ApxIII(27-245aa)-HIS9. ApxIII(27-245aa)-HIS9 in BL21 cells encoding pMLBAD-ApxIII(27-245aa)-HIS9 after arabinose induction. After 4 hours of incubation, cells were harvested and disrupted via lysozyme treatment followed by sonication-freeze-thaw cycles. Protein was purified by Ni-NTA binding and gravity flow. In pooled elution fractions, buffer was exchanged into PBS. 7.5 μl of pooled and dialyzed samples were used for SDS-PAGE and analyzed via Coomassie staining (A) and Western blot against HIS epitopes (B). On the left, molecular marker bands are indicated in kDa. [Figure 14] Schematic representation of the synthesized ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6 with flanking homologous recombination sites, a synthetic promoter and a 3' chloramphenicol resistance cassette. [Figure 15]Figure 1 demonstrates expression of ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII-(aa626-860)-HIS6 in SL1344 with improved APP2 O antigen expression. The analyzed strains are shown with their loading patterns shown in (A). Overnight cultures in liquid medium were diluted to 0.05 OD600 and grown to logarithmic growth (OD600 approx. 1). Cells were harvested, boiled in 1x Laemmli, loaded onto gradient BisTris gels for SDS-PAGE and analyzed via Coomassie staining (A) and Western blot against the HIS epitope (B). On the left and right, molecular marker bands are indicated in kDa. [Figure 16] Figure 1 shows an overview of the immunization schedule. Pigs were immunized twice with inactivated test material on study day (SD) 0 and SD 14 and euthanized 2 weeks after the last immunization (SD 28). Blood was sampled weekly for serum collection. Animals were monitored daily for clinical signs. [Figure 17] Figure 1 shows serum IgG-mediated absorbance at SD0 and SD28 for all animals tested against purified LPS of APP serotypes 2 (APP2) and 7 (APP7). Individual animals are indicated according to their ear tags. OD at 450 nm was recorded in a single measurement. Individual figures are grouped with respect to the antigen applied and its application (oral & intranasal vs. instillation). [Figure 18] Figure 1 shows BALF IgA-mediated absorbance at SD28 for all animals tested against purified LPS of APP serotypes 2 (APP2), 1 (APP1), 5 (APP5) and 7 (APP7). Individual animals are indicated according to their ear tags. OD at 450 nm was recorded in duplicate (error bars are shown). Individual figures are grouped with respect to the antigen applied and its application (oral & intranasal vs. instillation). [Figure 19]Figure 1 shows serum IgG-mediated absorbance at SD0 and SD28 and BALF IgA-mediated absorbance at SD28 for all animals in groups 2 and 7, two animals in groups 3 and 10, and one animal in group 11 tested against purified ApxII(439-801aa) and AcrA-HIS6 protein (as a negative control). Individual animals are shown according to their ear tags. OD at 450 nm was recorded in a single measurement. Individual figures are classified with respect to the antigen applied and its application (oral & intranasal vs. instillation). [Figure 20] Figure 1 shows a schematic overview of the immunization schedule. Pigs were immunized twice with live recombinant bacteria on study day (SD) 0 and SD 14, and euthanized 2 weeks after the last immunization (SD 28). Blood was sampled once a week for serum collection. Animals were monitored daily for clinical signs. [Figure 21] Figure 1. Overview of vaccination and challenge schedule. Pigs were vaccinated twice, SD0 and SD21, and then challenged with APP2 bacteria on SD42. Pigs were euthanized on day 48 (SD48). Serum was collected at the indicated time points. Animals were monitored regularly (at least twice daily for 6 days post-challenge) for clinical signs. [Figure 22] Lesion scoring according to Hannan et al., 1982. Group 1: live SL1344Δrfb::kanR-APP2.LPS-(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat, purified HIS10-APXII(439-801aa), purified APXIII(27-245aa)-HIS9. Group 2: inactivated SL1344Δrfb::kanR-APP2.LPS pEC415-wzy pMLBAD-gne, purified HIS10-APXII(439-801aa), purified APXIII(27-245aa)-HIS9. Group 3: purified HIS10-APXII(439-801aa), purified APXIII(27-245aa)-HIS9. Group 4: non-vaccinated control group. Statistical analysis was performed by Dunnett's multiple comparison test. Statistical significance is indicated as (*) P value 0.01. [Figure 23]Figure 1 shows the % survival over 6 days post-challenge stratified by groups, with day 0 being the day of challenge and day 6 being the end of the study. Group 1: inactivated SL1344Δrfb::kanR-APP2.LPS pEC415-wzy pMLBAD-gne, purified HIS10-APXII(439-801aa), purified APXIII(27-245aa)-HIS9; Group 2: commercial vaccine Porcilis® APP. Group 3: non-vaccinated control group. [Figure 24] Lesion scoring according to Hannan et al., 1982. Group 1: inactivated SL1344Δrfb::kanR-APP2.LPS pEC415-wzy pMLBAD-gne, purified HIS10-APXII(439-801aa), purified APXIII(27-245aa)-HIS9. Group 2: commercial vaccine Porcilis® APP. Group 3: non-vaccinated control group. Statistical analysis was performed by Dunnett's multiple comparison test. Statistical significance is indicated as (**) P value < 0.01. [Diagram 25] Figure 1 shows APP2 reisolation assessed for all animals and categorized by group. Bacteriology score values ​​are set as follows: 0 = no APP2 bacteria, 1 = <20 CFU (colony forming units) APP2 bacteria, 2 = <200 CFU APP2 bacteria, 3 = >200 CFU APP2 bacteria reisolation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] The present invention is described below by way of exemplary examples, none of which should be construed as limiting the scope of the claims as appended hereto. EXAMPLES

[0056] Materials and Methods The bacterial strains and their sources used in the examples are listed below:

[0057] [Table 1A]

[0058] [Table 1B]

[0059] Plasmids used in the examples and their sources are listed below:

[0060] [Table 2]

[0061] The oligonucleotides and their nucleic acid sequences of the plasmids used in the experimental examples are listed below:

[0062] [Table 3A]

[0063] [Table 3B]

[0064] Methods – Growth of bacterial strains Bacterial strains and plasmids listed in Tables 1 and 2 above were grown in Luria-Bertani (LB) medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl), TB medium (12 g / L tryptone, 24 g / L yeast extract, 0.017 M KH2PO4, 0.072 M K2HPO4) or BHI+NAD (37 g / L Brain Heart Infusion medium (BHI) supplemented with 2 mg / L β-nicotinamide adenine dinucleotide (NAD); for exact composition see Sigma Aldrich cat.nr.53286)).

[0065] Agar plates were supplemented with 1.5% (w / v) agar. Antibiotics were used at the following final concentrations: ampicillin (Amp) 100 μg / mL, kanamycin (Kan) 50 μg / mL, chloramphenicol (Cm) 25 μg / mL, streptomycin (Strep), trimethoprim (Tmp) 10 μg / mL, and gentamicin (gent) 15 μg / mL.

[0066] Example 1 1) Generation of a vaccine strain expressing the APP2 O-antigen biosynthesis cluster 1.1) Integration of the APP2 O-antigen biosynthetic cluster in E. coli and SL1344 In a first step, Actinobacillus pleuropneumoniae serotype 2 (APP2) strain (P1875) was sequenced and the O-antigen biosynthesis cluster (rfb cluster) was identified according to Xu et al., 2010, J. Bacteriol., Vol. 192(21), pp. 5625-5636. It was shown that the rfb cluster of all APP serotypes used in this publication is located between the erpA and rpsU genes. Figure 1, Table 4 and Seq. 1 show the gene organization and annotated genes / proteins for the sequenced APP2 strains of this study.

[0067] [Table 4]

[0068] As vaccine strains, two vector bacteria were selected, derived from Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli (see strain list). The Salmonella enterica subsp. enterica serovar Typhimurium SL1344 parental strain was isolated from an infected cow (Hoiseth et al., 1981, Nature, Vol. 291(5812), pp. 238-239). The SL1344 strain is a gene-marked version of the parental strain. The second strain, Escherichia coli strain 5 (E. coli_5), was isolated from the tonsils of a healthy pig in Switzerland. It was shown to be PCR negative for the virulence factors stx, eae, LT and ST. Both strains were sequenced and the endogenous O-antigen biosynthesis cluster (rfb cluster) was identified between the genes galF and gnd. Furthermore, both strains were genetically modified so that they did not express endogenous O antigens but instead expressed a gene cluster encoding the O antigen biosynthetic pathway of APP2 inserted at this position (Seq. 42, Fig. 2 ).

[0069] The 13758 bp fragment containing the APP2 rfb cluster and its flanking regions (Figure 2) was engineered for integration into SL1344 and E. coli_5 in an antisense orientation relative to the endogenous rfb cluster. For this, a kanamycin resistance cassette flanked by FRT sites was fused downstream. The fusion construct was flanked upstream and downstream by homologous regions flanking the rfb cluster of SL1344 or E. coli_5 (containing the galF gene in the antisense orientation).

[0070] In detail, shuttle vector plasmids pDOC_E.coli_5_Δrfb::APP2.LPS / kanR (for E.coli_5) and pDOC_SL1344_Δrfb::APP2.LPS / kanR (for SL1344) based on the pDOC system of Lee et al., 2009 were generated / synthesized (by CRO) to integrate the APP2 rfb cluster (Seq.1) into E.coli_5 and SL1344. The identified APP2 rfb operon (genes wzzB to rfbC) was extended at the 5' and 3' ends to include the adjacent upstream promoter region (also containing the erpA gene) and downstream terminator region (containing the 3' region of the rpsU gene) of SEQ ID NO:1 (see FIG. 2). This sequence was fused at the 3' end to a kanamycin resistance marker gene (kanR) with 5' and 3' flanking FRT recognition sites (palindromic flippase recognition site). From the sequencing results of E. coli_5 and SL1344, the flanking regions of the endogenous rfb cluster were recovered and used as flanking regions for the APP2 rfb cluster and the kanR resistance cassette as follows: An 800 bp fragment immediately following the stop of the last gene of the endogenous rfb cluster in E. coli_5 and SL1344 was chosen as the upstream homologous recombination sequence located in front of the APP2 rfb cluster. A region of 1252 bp (E. coli_5) or 1399 bp (SL1344) was chosen as the downstream homologous recombination sequence of kanR. These regions contain the entire galF gene and all nucleotides up to the +1 position before the start codon of the first gene of the cluster. The entire integration cluster was flanked by I-SecI restriction endonuclease cleavage sites. The integration cassette was designed to integrate in the antisense orientation of the endogenous rfb cluster. An overall schematic representation of the construct is shown in Figure 3. For integration of the APP2 rfb operon on the shuttle vector, additional genes were required. As plasmid selection marker, an ampicillin resistance cassette was used. The sucB gene (involved in sucrose conversion, which produces toxic metabolites in the presence of sucrose) was integrated for counterselection of the vector plasmid.

[0071] The shuttle vector plasmid (pDOC_E.coli_5_Δrfb::APP2.LPS / kanR in E.coli_5 and pDOC_SL1344_Δrfb::APP2.LPS / kanR in SL1344) was transformed into the target cells together with the helper plasmid pACBSCE, which encodes the arabinose-inducible λ-recombination system and carries the I-SceI restriction enzyme as well as an I-SceI cleavage site. After transformation, the expressed I-SceI enzyme cut the shuttle vector plasmid at the I-SceI restriction endonuclease cleavage site, linearizing the above-mentioned modified APP2 rfb operon carrying the kanamycin resistance gene flanked by endogenous homologous sequences. The λ-recombination system recognized the homologous flanking regions at the respective sites in the genome of the recipient cells and exchanged the endogenous DNA with the donor DNA, resulting in the integration of the APP2 O-antigen biosynthetic cluster with the kanamycin resistance cassette in either E. coli_5 or SL1344 (resulting in strains E. coli_5Δrfb::APP2.LPS / kanR and SL1344Δrfb::APP2.LPS / kanR). Cells selected on kanamycin- and sucrose-containing medium were positively tested by PCR for the presence of foreign DNA and for the absence of shuttle vector and helper plasmid by lack of growth on the selective medium.

[0072] To "recombine out" the kanamycin resistance marker, the cells were introduced with the temperature-sensitive plasmid pCP20 (Cherepanov et al., 1995, Gene, Vol. 158, pp. 9-14) for SL1344 derivatives or pCP20-Gent (in which a gentamicin resistance gene was integrated downstream of a chloramphenicol resistance gene) for E. coli_5 derivatives, encoding a flippase that recognizes a palindromic FRT site. After the "flip-out" event, only one FRT site remained in the genome. The incubation temperature was increased to counterselect positive clones against the flippase-encoding plasmid. A final PCR confirmed the absence of all helper plasmids, the absence of the kanamycin resistance marker and the introduction of the APP2 rfb cluster construct (resulting in strains E. coli_5Δrfb::APP2.LPS and SL1344Δrfb::APP2.LPS). The expression of APP2 O antigen displayed on the surface of E. coli_5 and SL1344 was confirmed by SDS-PAGE and immunoblotting (Figure 8). The analysis procedures and results are described under 1.6.

[0073] As a control, the endogenous rfb cluster was deleted in wild-type cells E. coli_5 and SL1344. For this, knockout cassettes were generated by PCR using pKD4 as template (Datsenko et al., 2000, PNAS, Vol. 97(12), pp. 6640-6645) and oligonucleotides Ec_5_Δrfb fw / Ec_5_Δrfb rev for the E. coli_5 rfb cluster deletion and SL1344_Δrfb fw / SL1344_Δrfb fw for the SL1344 rfb cluster deletion. The resulting PCR product contains a 21-24 bp overlap sequence encoding a kanamycin resistance cassette flanked by FRT sites and, in addition, the endogenous 5' (before the start of the first gene of the rfb cluster → wfgD (E. coli_5) or rfbB (SL1344)) and 3' (after the stop codon of the last gene of the rfb cluster → pglH (E. coli_5) or rfbP (SL1344)) sequences required for homologous recombination. The DNA fragments (E. coli_5 1759 bp, SL1344 1623 bp) and temperature-sensitive helper plasmids encoding the λ-recombination system of pKD46 (Datsenko et al., 2000, PNAS, Vol. 97(12), pp. 6640-6645) for SL1344 and pLAMBDA46 (in which the β-lactamase gene was replaced with a gentamicin resistance gene) for E. coli_5 were introduced. The λ-recombination system recognized the homologous flanking regions at the respective sites before the start of the first gene and after the stop codon of the last gene of the rfb cluster, resulting in the deletion of the endogenous rfb gene cluster by integrating a kanamycin resistance cassette flanked by FRT sites. The resulting strains E. coli_5Δrfb::kanR and SL1344Δrfb::kanR were further engineered to lose the antibiotic marker. To "recombine out" the kanamycin resistance marker, the flip-out technique established by Cherepanov et al., 1995, Gene, Vol. 158, pp. 9-14, was used as described above. The final E. coli_5Δrfb and SL1344Δrfb strains were confirmed by PCR and analyzed for the lack of O-antigen expression by immunoblotting (Figure 8). The analytical procedures and results are described under 1.6.

[0074] 1.2) Improved expression of APP2 O antigen in E. coli_5 and SL1344 by introducing the kanR resistance marker upstream of the APP2 rfb cluster To improve the presentation of the APP2 O antigen in glycoengineered E. coli_5 and SL1344 strains, the promoter region of the rfb cluster was exchanged with a kanamycin resistance gene, of which the kanamycin promoter is utilized to drive the transcription of downstream genes. For this, a PCR fragment was generated using oligonucleotides Ec_SL / Kan_fw and Ec_SL / Kan_rev and pKD4 as template to amplify the resistance gene with the encoded kanamycin promoter and the 5' and 3' encoded FRT sites. Furthermore, oligonucleotides introduced homologous recombination sequences homologous to the erpA gene before the start codon and after the stop codon. The resulting product with a size of 1644 bp was used for a second PCR reaction together with oligonucleotides Ec_SL / Kan_fw_elo and Ec_SL / Kan_rev_elo to extend the homologous recombination sequence in order to increase the integration efficiency. This DNA fragment and a temperature-sensitive helper plasmid encoding the λ-recombination system of pKD46 (Datsenko et al., 2000, PNAS, Vol. 97(12), pp. 6640-6645) for SL1344Δrfb::APP2.LPS and pLAMBDA46 for E. coli_5Δrfb::APP2LPS strain were introduced. The λ-recombination system recognized the homologous flanking regions at the respective sites at the start and stop codons of erpA, "recombined out" the erpA gene, and integrated the kanamycin promoter and resistance gene flanked by FRT sites into the respective sites. The introduced kanamycin resistance gene allowed the selection of positive clones (successful integration). These positive candidates were confirmed by PCR for the deletion of erpA and the integration of the PCR fragment. The temperature-sensitive helper plasmid was lost from the cells by increasing the growth temperature over several rounds of incubation. O antigen presentation in the final strains E. coli_5Δrfb::kanR-APP2.LPS and SL1344Δrfb::kanR-APP2.LPS was examined by immunoblotting (FIG. 8). The analytical procedures and results are described under 1.6.

[0075] 1.3) Improved expression of APP2 O antigen in E. coli_5 and SL1344 by codon optimization of the APP2 rfb cluster To improve APP2 O-antigen cluster protein / enzyme expression and ultimately increase APP2 O-antigen presentation on lipid A, the nucleotide triplet codons of the gene coding sequence could be optimized for SL1344 and E. coli_5. This would reduce the use of undesired triplet codons that may be preferred in A. pleuropneumoniae but not in SL1344 and E. coli_5. In this study, codon optimization was performed by CRO and on E. coli expression levels. Whether the same was true for Salmonella enterica subsp. enterica serovar Typhimurium SL1344 remained to be shown by integration of the same codon-optimized APP2 rfb cluster into the SL1344 endogenous rfb cluster.

[0076] To integrate the codon-optimized rfb cluster of APP2 into SL1344 and E. coli_5, the original integration site at the endogenous rfb cluster was kept the same. A codon-optimized (for E. coli expression) APP2 rfb cluster was synthesized, which encoded a kanamycin resistance cassette (flanked by FRT sites) at its 5' end. This results in the transcription of the APP2 rfb cluster (codon-optimized) regulated by the promoter of the kanamycin gene. In detail, a new integration cassette was designed combining the kanamycin resistance gene integration (Seq. 1 and 7) upstream of the APP2 rfb cluster and the codon optimization of the APP2 rfb cluster (the CRO that performed the synthesis applied the codon optimization for E. coli expression of the plasmid) (Figure 4). The resulting plasmid (Seq. 43; pDOC_E.coli_5_Δrfb::kanR-APP2.LPS(cod.opt.)) was used to generate the plasmid pDOC_SL1344_Δrfb::kanR-APP2.LPS(cod.opt.) (Seq. 44) that was used for engineering E.coli_5 and to genetically engineer SL1344 cells. The oligonucleotides BamHI-Fw KanR-Fw / XhoI 3'rspU-Rev were used to amplify a 14972 bp fragment from the plasmid pDOC_E.coli_5_Δrfb::Kan-APP2.LPS(cod.opt.) containing the codon-optimized APP2 rfb cluster with an upstream integrated kanamycin resistance cassette, flanked by FRT sites. In addition, new restriction cleavage sites were incorporated at the 5' (BamHI) and 3' (XhoI) ends by PCR. A second PCR was performed to generate a fragment containing the pDOC backbone encoding the homologous recombination sequences for integration of the cassette in the rfb cluster of SL1344. Using pDOC_SL1344_Δrfb::APP2.LPS / kanR as template, a 8113 bp fragment with BamHI and XhoI cleavage sites was generated using primers BamHI-SL-gnd Fw ext / XhoI-SL-GalF Rev ext. After restriction digestion of the PCR product with BamHI and XhoI, both fragments were ligated.The sequence was confirmed by sequencing (pDOC_SL1344_Δrfb::kanR-APP2.LPS(cod.opt.)) and this plasmid was further used to replace the rfb cluster of SL1344 with the codon-optimized APP2 rfb cluster downstream of the kanamycin resistance cassette. To incorporate the kanamycin resistance cassette and the codon-optimized APP2 rfb cluster into E. coli_5 and SL1344, the plasmids pDOC_E.coli_5_Δrfb::kanR-APP2.LPS(cod.opt.) and pDOC_SL1344_Δrfb::kanR-APP2.LPS(cod.opt.) were transformed into E. coli_5 or SL1344, respectively, as described above and according to the procedure by Lee et al., 2009. The final strains E. coli_5Δrfb::kanR-APP2.LPS (cod.opt.) and SL1344Δrfb::kanR-APP2.LPS (codon optimized) were confirmed by PCR and O antigen expression was tested by immunoblotting (Figure 8). The analytical procedures and results are described under 1.6.

[0077] 1.4) Improved expression of APP2 O antigen in SL1344 expressing the APP2 rfb cluster by introducing gne and wzy As can be seen in the constructed strains expressing the APP2 rfb cluster, the typical "ladder" pattern of lipopolysaccharides was only weakly present or not detectable (Figure 8). Instead, a strong accumulation of O antigen as single subunit oligosaccharides was observed on lipid A. This indicated that the O antigen polymerase (Wzy) could not efficiently assemble LPS glycans. The APP2 O antigen-specific wzy was chosen to express Wzy encoded on a plasmid. wzy is annotated by Xu et al., 2010, J. Bacteriol., Vol. 192(21), pp. 5625-5636 as the sixth gene in the APP2 cluster and corresponds to a hypothetical protein located between setA and rfbX in Seq. 1 and Figure 1. Blast analysis of the hypothetical protein sequence against the NCBI protein database identified Wzy (locus AZY91860) from Lactococcus lactis with 29% identity, polysaccharide polymerase from Oenococcus oeni (locus WP_071436899) ​​with 22% identity, and polysaccharide polymerase from Streptococcus sp. DD12 (locus KXR76217) with 24% identity. Based on these findings, it was postulated that the hypothetical protein may represent the O-antigen polymerase Wzy of the APP2 rfb cluster. Oligonucleotides 5'NdeI_wzy / 3'EcoRI_wzy were used to amplify the potential wzy gene from the plasmid pDOC_E.coli_5_Δrfb::APP2.LPS / kanR with 5'NdeI and 3'EcoRI restriction cleavage sites (fragment size of PCR product was 1138 bp). For expression of wzy, vector pEC415 with an arabinose-inducible promoter was chosen. To clone wzy into pEC415, the vector was linearized, amplified and modified by PCR with oligonucleotides 3'EcoRI_pEC415fw / 5'NdeI_pEC415rev (fragment of 5419 bp). Again, EcoRI and NdeI cleavage sites were introduced at the ends of the fragment.The PCR-generated EcoRI-wzy-NdeI and EcoRI-pEC415-NdeI fragments were digested with the respective restriction endonucleases and ligated. After sequence verification (pEC415-wzy), the plasmid was further used for testing in cells expressing the APP2 rfb cluster.

[0078] A second potential limiting factor for O-antigen biosynthesis could be glycan availability and / or grafting. LPS structural data for specific APP serotypes was published by Perry et al., 1990, which showed that the APP2 O-antigen pentasaccharide [→2)-α-D-Galp-(1→3)-β-D-Glcp-(1→4)-α-D-Glcp(6-(Ac) 0-65 )-(1→4)-β-D-GalpNAc-(1→2)-α-L-Rhap-(1→] n Galactose was identified at the reducing end of gne. One key enzyme in the production of galactose in bacterial cells is the conversion of galactose from glucose as a substrate. This requires the action of an epimerase. The UDP-GlcNAc / Glc 4-epimerase gne of Campylobacter jejuni was identified to provide galactose and N-acetylgalactosamine in bacterial cells to cell surface carbohydrates (Bernatchez et al., 2005). The sequence of gne (locus C8J_1070) of C. jejuni 81116 was fused with a C-terminal HA (hemagglutinin) tag and 5'EcoRI and 3'XbaI restriction sites. This fragment was synthesized and then cloned into the EcoRI / XbaI sites of pMLBAD under the control of an arabinose-inducible promoter (pMLBAD-gne-HA).

[0079] Plasmid-based expression of wzy and / or gne was tested in SL1344 and E. coli_5, which express the APP2 O antigen on their surface. SL1344 and its derivatives (Δrfb, Δrfb::kanR-APP2.LPS pMLBAD-gne pEC415-wzy, Δrfb::kanR-APP2.LPS(cod.opt.)pMLBAD pEC415, Δrfb::kanR-APP2.LPS(cod.opt.)pMLBAD-gne, Δrfb::kanR-APP2.LPS(cod.opt.)pEC415-wzy and Δrfb::kanR-APP2.LPS(cod.opt.)pMLBAD-gne) pEC415-wzy) and E. coli_5 and its derivatives (Δrfb, Δrfb::kanR-APP2.LPS(cod.opt.), Δrfb::kanR-APP2.LPS(cod.opt.)pMLBAD, Δrfb::kanR-APP2.LPS(cod.opt.)pMLBAD-gne) were cultured at 37°C with shaking to an OD of 0.6. 600 Cultures were grown in LB medium supplemented with antibiotics (according to Tables 1 and 2) until stationary phase. Arabinose was added to a final concentration of 0.1% (for SL1344 and its derivatives) or 0.2% (for E. coli_5 and its derivatives) to induce wzy and / or gne expression. After a further 5 h of incubation at 37° C. (shaking), arabinose was added again to 0.1% (for SL1344 and its derivatives) or 0.2% (for E. coli_5 and its derivatives) and the cultures were incubated at 37° C. (shaking) for approximately 16 h. Stationary phase cells were harvested and further processed. As a control for APP2 O antigen presentation on lipid A, the APP2 P1875 strain was grown in BHI+NAD at 37° C. with gentle shaking (110 rpm) until stationary phase, after which the cells were harvested. For APP2 O antigen analysis, cells were resuspended in 1× Laemmli buffer (1OD 600 cells / 100 μl 1× Laemmli buffer). Samples were incubated at 95°C for 5 min. OD 60012 μg proteinase K (stock 20 mg / mL in 10 mM Tris-HCl pH 7.5, 20 mM CaCl2, 50% glycerol) was added per equivalent amount of cells and the samples were incubated at 60 °C for 1 h. Proteinase K-treated samples (0.1 OD 600A total of 1000 APP2 LPS-binding proteins (1000 x 1000 cells) were loaded onto a 4-12% Bis-Tris gel and the molecules were separated by size in MES buffer. The gel was further processed for immunoblotting and silver staining. To analyze LPS synthesis via immunoblotting, LPS was transferred from the gel onto a PVDF membrane. The membrane was incubated in blocking solution (PBS pH 7.5 / 0.05% Tween / 0.1% casein) for 2 h at room temperature with shaking. The membrane was then incubated overnight at 4°C with shaking in antibody binding solution (PBS pH 7.5 / 0.05% Tween / 0.05% casein) containing rabbit serum reactive against APP2 LPS (rabbit immunized with proteinase K-treated extract of APP2 P1875) at a 1:2000 dilution. The immunoblot was washed three times for 5 min with excess PBS 0.05% Tween buffer pH 7.5. The membrane was then incubated in antibody binding solution with secondary goat anti-rabbit IgG-HRP antibody (BETHYL Cat#A120-401P) at 1:2000 dilution for 1 hour with shaking at room temperature. The membrane was washed 4 times for 5 minutes with excess PBS 0.05% Tween buffer pH 7.5. Antibody binding was visualized by overlaying ECL solution (GE healthcare #RPN2105) on the membrane and detecting the light signal using Stella8300 (Raytest). For silver staining, the protocol described by Tsai et al., 1982, Anal.Biochem., Vol.119(1), pp.115-9 was used. Briefly, the gel was fixed overnight in 40% EtOH / 5% acetic acid at room temperature. The gel was then treated with 0.7% periodic acid in 40% EtOH / 5% acetic acid for 10 min, followed by washing three times with ddH2O for 15 min. The gel was stained with staining solution (0.187 N sodium hydroxide, 0.2 N ammonium hydroxide, 0.667% silver nitrate) and washed extensively again three times with ddH2O for 10 min. LPS on the gel was visualized by using a developing solution (0.25 mg / ml citric acid monohydrate, 0.0185% formaldehyde solution).

[0080] Analysis of immunoblots of membranes treated with rabbit serum against APP2 LPS demonstrated strong staining of the lipid A fraction at around 10 kDa in APP2 P1875 strain as well as a ladder pattern (O antigen polymerization on lipid A) migrating between approximately 12.5 and 190 kDa (Fig. 5A, C, lane 1). No recognition by rabbit serum was observed for SL1344 and SL1344Δrfb (Fig. 5A, lanes 2, 3) or E. coli_5 and E. coli_5Δrfb. By silver staining, the LPS of SL1344 could be detected and was absent in SL1344Δrfb, indicating successful deletion of endogenous O antigen biosynthesis (Fig. 5B, lanes 2, 3). The LPS of E. coli_5 was visible only at a lower molecular weight and was absent in E. coli_5Δrfb cells (Fig. 5D, lanes 9 and 10). Integration of the codon-optimized APP2 rfb cluster downstream of the kanamycin resistance cassette in the rfb cluster of SL1344 (Figure 5A, lane 5) resulted in a 10-15 kDa band that appeared to correspond to lipid A linked to a single O antigen. When gne or wzy were overexpressed in these cells (Figure 5A, lanes 6, 7), the ladder pattern described above appeared, which could be further enhanced when overexpressing both proteins in SL1344Δrfb::kanR-APP2.LPS(cod.opt.) (Figure 5A,B, lane 8). These cells also show stronger presentation than cells without the codon-optimized APP2 rfb cluster using the same expression settings (Figure 5A, lane 4). Thus, it is demonstrated that gne and wzy expression enhances APP2 LPS expression in SL1344Δrfb::kanR-APP2.LPS(cod.opt.) cells. Moreover, for E. coli_5 cells expressing the codon-optimized APP2 rfb cluster, the ladder pattern against the APP2 O antigen could be further improved by overexpression of gne (Fig. 5C and D , compare lanes 12 and 13).

[0081] 1.5) Integration of gne and codon-optimized wzy into SL1344 expressing APP2 O antigen Since an improvement in APP2 O-antigen synthesis was observed after introducing gne and / or wzy on a plasmid into the genetically modified SL1344, these genes were integrated into the genome of a strain expressing the APP2 rfb cluster. To compare the expression levels of APP2 LPS, gne alone and gne fused to codon-optimized wzy (wzy(cod.opt.) of APP2, Seq.8) were integrated. As an integration site, the downstream region of the O-antigen ligase rfaL was chosen in order to use the rfaL promoter to transcribe integrated gne and wzy(cod.opt.) in a similar manner. The general design (Figure 6) is based on the generation of two (Figure 6A) to three (Figure 6B) separate PCR fragments flanked by FRT sites, each encoding the two enzymes (gne, wzy-cod.opt.) and the antibiotic resistance cassette cat (chloramphenicol resistance cassette).

[0082] To generate rfaL-Ωgne / cat (Figure 6A), separate fragments for gne and cat with overlapping regions were amplified. Using overlapping PCR, the two fragments were fused and the 5' and 3' ends were extended to encode a homologous region with a targeted integration site in the genome of SL1344. The same principle was followed to generate the integration construct rfaL-Ωgne-wzy(cod.opt.) / cat (Figure 6B), except that an additional fragment encoding codon-optimized wzy was generated, followed by overlapping PCR to combine the three fragments and add homologous recombination sequences at the 5' and 3' ends. The 5' and 3' ends of the fusion construct were further extended to include a homologous region that can recombine with the targeted genomic integration site downstream of the rfaL stop codon (method adapted from Bryskin et al., 2010, Biotechniques, Vol. 48(6), pp. 463-465). Template and oligonucleotide information for the individual and overlap PCRs to generate substrates for engineering SL1344 is listed in Table 5.

[0083] [Table 5]

[0084] The fusion construct resulting from overlap PCR was transformed with the temperature-sensitive helper plasmid pLAMBDA46 (modified from Datsenko et al., 2000, PNAS, Vol. 97(12), pp. 6640-6645) encoding the λ-recombination system for SL1344Δrfb::kanR-APP2.LPS, SL1344Δrfb::kanR-APP2.LPS(cod.opt.). The λ-recombination system recognized the homologous flanking regions of the fusion construct and recombined them into the genome downstream after the stop codon of rfaL. The introduced chloramphenicol resistance gene (cat) allowed the selection of positive clones (successful integration). These positive candidates were confirmed by PCR for integration of the PCR fragment. The temperature-sensitive helper plasmid was lost from the cells by increasing the growth temperature over several rounds of incubation. The O antigen presentation in the final strains SL1344Δrfb::kanR-APP2.LPS rfaL-Ωgne / cat, SL1344Δrfb::kanR-APP2.LPS rfaL-Ωgne-wzy(cod.opt.) / cat, SL1344Δrfb::kanR-APP2.LPS(cod.opt.)rfaL-Ωgne / cat and SL1344Δrfb::kanR-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat was examined by immunoblotting (FIG. 8). The analytical procedure and results are described under 1.6.

[0085] 1.6) Replacement of the kanamycin resistance cassette with only the kanamycin promoter "driving" expression of the APP2 rfb cluster For whole-cell bacteria-based vaccines, the absence of antibiotic resistance is recommended. Therefore, the kanamycin resistance introduced to enhance APP2 rfb expression had to be deleted from the genome. However, the advantage of increased transcription by the kanamycin promoter may be used in future bacterial vaccine strains. In a first step, the kanamycin and chloramphenicol resistance cassettes were "flipped out" by introducing the temperature-sensitive plasmid pCP20 (Cherepanov et al., 1995, Gene, Vol. 158, pp. 9-14) into SL1344Δrfb::kanR-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat cells. After the "flip-out" event of the kanamycin and chloramphenicol resistance cassettes at each position, only one FRT site remained in the genome. The incubation temperature was increased to counterselect positive clones against the flippase-encoding plasmid. The absence of pCP20 and the absence of kanamycin and chloramphenicol resistance markers was confirmed by PCR. To integrate the kanamycin promoter (kP) upstream of the codon-optimized APP2 rfb cluster, a 1414 bp construct was synthesized containing a 373 bp promoter region of a chloramphenicol resistance cassette followed by a kanamycin resistance cassette with flanking FRT sites (Figure 7, Seq. 45). To introduce sequence 22 upstream of the APP2 rfb cluster, a PCR fragment was generated using oligonucleotides Ec_SL / Kan_fw and Ec_SL / Kan_rev and sequence 24 as a template, thereby amplifying the kP integration cassette and adding a homologous recombination sequence to the upstream region of the rfb cluster. The resulting product was used for a second PCR reaction together with oligonucleotides Ec_SL / Kan_fw_elo and Ec_SL / Kan_rev_elo to extend the homologous recombination sequence for increased integration efficiency (fragment size 1671 bp).This DNA fragment and the temperature-sensitive helper plasmid pKD46 (Datsenko et al., 2000, PNAS, Vol. 97(12), pp. 6640-6645) encoding the λ-recombination system for SL1344Δrfb::APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) were introduced. The λ-recombination system recognized the homologous flanking regions upstream of the rfb cluster and integrated into the respective site a chloramphenicol resistance gene with flanking FRT sites fused to the kanamycin promoter. The introduced chloramphenicol resistance gene allowed the selection of positive clones (successful integration). These positive candidates were confirmed for integration of the PCR fragment. The temperature-sensitive helper plasmid was lost from the cells by increasing the growth temperature over several rounds of incubation. To remove the chloramphenicol resistance cassette, the resulting strain SL1344Δrfb::cat / kP-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) was transformed with pCP20 and the marker flipped out as described above. The resulting strain SL1344Δrfb::kP-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) was analyzed for its APP2 O-antigen production and compared to its parental strain (Figure 8).

[0086] Wild-type SL1344 and E. coli_5 and strains deficient in endogenous O-antigen biosynthesis (E. coli_5Δrfb and SL1344Δrfb) or expressing the APP2 rfb cluster (E. coli_5Δrfb::APP2.LPS, Δrfb::kanR-APP2.LPS, Δrfb::kanR-APP2.LPS(cod.opt.) and SL1344Δrfb::APP2.LPS, Δrfb::kanR-APP2.LPS, Δrfb::kanR-APP2.LPS(cod.opt.), Δrfb::kanR-APP2.LPS rfaL-Ωgne / cat, Δrfb::kanR-APP2.LPS rfaL-Ωgne-wzy(cod.opt.) / cat, Δrfb::kanR-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat, Δrfb::kP-APP2-.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.), Δrfb rfaK-ΩClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-rfaL, Δrfb::kP-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.)pliC-ΩClyA-ApxI(aa626-845)-ApxII-(aa612-801)-ApxIII(aa626-860)-HIS6-pagC) genetically engineered cells were grown to saturation (OD ) in LB medium with shaking at 37 °C. 600 The cells were grown to stationary phase at 37°C with gentle shaking (110 rpm) in BHI+NAD. Cells were harvested and used for further processing. For APP2 O antigen analysis, cells were resuspended in 1x Laemmli buffer (1OD 600 cells / 100 μl 1× Laemmli buffer). Samples were incubated at 95°C for 5 min. OD 60012 μg proteinase K (stock 20 mg / ml, in 10 mM Tris-HCl pH 7.5, 20 mM CaCl2, 50% glycerol) was added per equivalent amount of cells and the samples were incubated for 1 h at 60°C. The proteinase K-treated samples (0. 1OD 600A total of 1000 APP2 LPS-binding proteins (1000 x 1000 cell equivalents) were loaded onto a 4-12% Bis-Tris gel and the molecules were separated by size in MES buffer. The gel was further processed for immunoblotting and silver staining. To analyze LPS synthesis via immunoblotting, LPS was transferred from the gel onto a PVDF membrane. The membrane was incubated in blocking solution (PBS pH 7.5 / 0.05% Tween / 0.1% casein) for 2 h at room temperature with shaking. The membrane was then incubated overnight at 4°C with shaking in antibody binding solution (PBS pH 7.5 / 0.05% Tween / 0.05% casein) containing rabbit serum reactive against APP2 LPS at a 1:2000 dilution. The immunoblot was washed three times for 5 min with excess PBS 0.05% Tween buffer pH 7.5. The membrane was then incubated in antibody binding solution with secondary goat anti-rabbit IgG-HRP antibody (BETHYL Cat#A120-401P) at 1:2000 dilution for 1 h at room temperature with shaking. The membrane was washed 4 times for 5 min with excess PBS 0.05% Tween buffer pH 7.5. Specific antibody binding was then visualized by adding ECL solution (GE healthcare #RPN2105) to the membrane and recording the light signal detected with a Stella8300 (Raytest). For silver staining, the protocol described by Tsai et al., 1982, Anal. Biochem., Vol. 119(1), pp. 115-9 was used. Briefly, the gel was fixed overnight in 40% EtOH / 5% acetic acid at room temperature. The gel was then treated with 0.7% periodic acid in 40% EtOH / 5% acetic acid for 10 min, followed by washing three times for 15 min with ddH2O. The gel was stained with staining solution (0.187 N sodium hydroxide, 0.2 N ammonium hydroxide, 0.667% silver nitrate) and washed extensively again three times for 10 min with ddH2O. LPS on the gel was visualized by adding a developing solution (0.25 mg / ml citric acid monohydrate, 0.0185% formaldehyde solution).

[0087] Immunoblotting with rabbit serum against LPS of APP2 showed strong staining of the lipid A fraction at around 10 kDa in APP2 P1875 strain as well as a ladder pattern (O antigen polymerization on lipid A) migrating between approximately 12.5 and 190 kDa (Fig. 8C, lane 1). No recognition by the rabbit serum was observed for SL1344 and SL1344Δrfb (Fig. 8C, lanes 2 and 3), SL1344Δrfb rfaK-ΩClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-rfaL (Fig. 8C, lane 11), or E. coli_5 and E. coli_5Δrfb (Fig. 8C, lanes 13 and 14). Silver staining could detect LPS in SL1344 (range 10 kDa to >115 kDa) and E. coli_5 (10 kDa to 15 kDa) and was absent in SL1344Δrfb and E. coli_5Δrfb, indicating successful deletion of endogenous O-antigen biosynthesis (Fig. 8C, lanes 2, 3, 13, 14). Integration of the APP2 rfb cluster led to the appearance of a very faint band between 10 and 15 kDa, which appeared to correspond to lipid A linked to a single O-antigen (Fig. 8C, lanes 4, 15). This signal could be enhanced when APP2 O-antigen biosynthesis was located downstream of a kanamycin resistance cassette (Fig. 8C, lanes 5, 16). Codon optimization of the APP2 rfb cluster in combination with an upstream kanamycin resistance cassette further improved APP2 O-antigen biosynthesis (Fig. 8C, lanes 6, 17). Specifically for E. coli_5, this resulted in the appearance of polymerized O antigen chains (ladder pattern, FIG. 8C, lane 17). A ladder pattern at lower molecular weights could also be detected by silver staining analysis (FIG. 8B, lane 17). After introduction of gne or gne and wzy into SL1344Δrfb::kanR-APP2.LPS (FIG. 8C, lanes 7, 8), a stepwise increase in APP2 O antigen expression was observed with bands appearing at higher molecular weights, indicating polymerization of O antigen on lipid A.Genomic expression of gne and codon-optimized wzy in SL1344 expressing the codon-optimized APP2 rfb cluster downstream of either a kanamycin resistance cassette (Fig. 8C, lane 9) or the kanamycin promoter alone (Fig. 8C, lane 10) increased O-antigen polymerization and lipid A presentation to the point where glycan structures were detectable even at high molecular weights (>190 kDa). In both cell lines, lower molecular weight bands could also be detected by silver staining analysis (Fig. 8B, lanes 9, 10). The final glycoengineered SL1344, which additionally displays ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6 on the cell surface ( Figure 8 C, lane 12), shows the same intensity of APP2 O antigen expression as its parent strain SL1344Δrfb::kP-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.).

[0088] 1.7) Integration of the codon-optimized APP8 O-antigen biosynthetic cluster controlled by the kP promoter in SL1344 To demonstrate that the described transplantation of heterologous O antigens into a host bacterium can be applied to various O antigen types, the rfb cluster of Actinobacillus pleuropneumoniae serotype 8 (AAP8) strain (MIDG2331) was genomic integrated into SL1344 and tested for AAP8 O antigen presentation on lipid A.

[0089] A codon-optimized (codon-optimized for E. coli expression, shown for the APP2 rfb cluster to function similarly for SL1344) 13598 bp fragment (Figure 9, Seq. 4) containing the APP8 rfb cluster was engineered for integration in the antisense orientation relative to the endogenous rfb cluster into SL1344. Upstream of the APP8 rfb cluster was fused a 373 bp promoter region of a chloramphenicol resistance cassette flanked by FRT sites followed by a kanamycin resistance cassette. The construct was flanked upstream and downstream by homologous regions flanking either the SL1344 rfb cluster (containing the galF gene in the antisense orientation) flanked by I-SceI restriction endonuclease recognition sites (Seq. 46, Figure 10). To incorporate the kP-controlled codon-optimized APP8 rfb cluster into SL1344, a shuttle vector plasmid pDOC_SL1344_Δrfb::cat-kP-APP8.LPS(cod.opt.) based on the pDOC system of Lee et al., 2009 was generated / synthesized (by CRO).

[0090] The shuttle vector plasmid (pDOC_SL1344_Δrfb::cat-kP-APP8.LPS(cod.opt.)) was transformed into SL1344 together with the helper plasmid pACBSCE. The procedure was as described above and in Lee et al., 2019. The final strain SL1344Δrfb::cat-kP-APP8.LPS(cod.opt.) was confirmed by PCR and O antigen expression was tested by immunoblotting (Figure 11).

[0091] Wild-type SL1344 and genetically modified cells deficient in endogenous O-antigen biosynthesis (SL1344Δrfb) or expressing the codon-optimized APP8 rfb cluster under the control of the kP promoter (SL1344Δrfb::cat-kP-APP8.LPS(cod.opt.)) were grown to saturation (OD ) in LB medium with shaking at 37°C. 600>2). Cells were harvested for further processing. As a control for APP8 O antigen presentation on lipid A, APP8 (MIDG2331) and APP3 (ORG1224) strains were grown in BHI+NAD at 37°C with gentle shaking (110 rpm) to stationary phase. Cells were harvested and used for further processing. For APP8 O antigen analysis, cells were resuspended in 1x Laemmli buffer (1OD 600 cells / 100 μl 1× Laemmli buffer). Samples were incubated at 95°C for 5 min. OD 600 12 μg proteinase K (stock 20 mg / ml in 10 mM Tris-HCl pH 7.5, 20 mM CaCl2, 50% glycerol) was added per equivalent amount of cells and the samples were incubated at 60°C for 1 h. Proteinase K-treated samples (0.1 OD 600A total of 1000 APP3 LPS-binding proteins (1000 x 1000 cell equivalents) were loaded onto a 4-12% Bis-Tris gel and the molecules were separated by size in MES buffer. The gel was further processed for immunoblotting and silver staining. To analyze LPS synthesis via immunoblotting, LPS was transferred from the gel onto a PVDF membrane. The membrane was incubated in blocking solution (PBS pH 7.5 / 0.05% Tween / 0.1% casein) for 2 h at room temperature with shaking. The membrane was subsequently incubated overnight at 4 °C with shaking in antibody binding solution (PBS pH 7.5 / 0.05% Tween / 0.05% casein) containing porcine serum reactive against APP3 LPS at a 1:500 dilution. The immunoblot was washed three times for 5 min with excess PBS 0.05% Tween buffer pH 7.5. The membrane was then incubated in antibody binding solution with secondary swine anti-IgG-HRP antibody (BETHYL Cat#A100-105P) at 1:2000 dilution for 1 h at room temperature with shaking. The membrane was washed 4 times for 5 min with excess PBS 0.05% Tween buffer pH 7.5. Specific antibody binding was then visualized by adding ECL solution (GE healthcare #RPN2105) to the membrane and recording the light signal detected with a Stella8300 (Raytest). For silver staining, the protocol described by Tsai et al., 1982, Anal. Biochem., Vol. 119(1), pp. 115-9 was used. Briefly, the gel was fixed overnight in 40% EtOH / 5% acetic acid at room temperature. The gel was then treated with 0.7% periodic acid in 40% EtOH / 5% acetic acid for 10 min, followed by washing three times for 15 min with ddH2O. The gel was stained with staining solution (0.187 N sodium hydroxide, 0.2 N ammonium hydroxide, 0.667% silver nitrate) and washed extensively again three times for 10 min with ddH2O. LPS on the gel was visualized by adding a developing solution (0.25 mg / ml citric acid monohydrate, 0.0185% formaldehyde solution).

[0092] Since the O antigen structures of APP3 and APP8 are identical, rabbit serum reactive against serotype 3 was used (Perry et al., 1990, Serodiagnosis and Immunotherapy in Infectious Disease, Vol. 4(4), pp. 299-308).

[0093] Immunoblotting with rabbit serum against LPS of APP3 showed strong staining of lipid A fraction around 10 kDa for APP8 and 3 strains as well as a ladder pattern (O antigen polymerization on lipid A) migrating between about 12.5 and 190 kDa (Fig. 11B, lanes 3, 4), demonstrating cross-reactivity of pig serum with both serotype O antigens. For SL1344 and SL1344Δrfb (Fig. 11B, lanes 1, 2), no recognition by rabbit serum was visible. Silver staining could detect LPS (range 10 kDa to >115 kDa) for SL1344, which was abolished for SL1344Δrfb (Fig. 11A, lanes 1, 2). Integration of the codon-optimized APP8 rfb cluster under the control of the kP promoter resulted in the appearance of a strong band at 10-15 kDa, which appeared to correspond to lipid A linked to a single O antigen (Fig. 11B, lane 5). Furthermore, it was reactive with APP3-reactive pig serum, and a faint ladder pattern between 15 and 190 kDa, indicating O antigen polymerization, was detected.

[0094] It can be concluded that the heterologous APP8 rfb cluster (codon optimized and under the control of the kP promoter) could be successfully grafted into the heterologous host SL1344, resulting in the presentation of the APP8 O antigen on lipid A.

[0095] Example 2 2) Cloning of neutralizing epitopes of Apx toxins of APP as potential vaccination candidates 2.1) Construction of plasmids encoding soluble neutralizing epitopes of ApxII and ApxIII For an effective vaccine against APP infection, inactivated toxins (Apx toxoids) are expected to be present in the final vaccine formulation. These pore-forming Apx toxins belong to the main virulence factors of APP and strongly contribute to the pathogenesis of the infection (Bosse et al., 2002, Microbes Infect., Vol. 4(2), pp. 225-235). The four identified Apx toxins (ApxI, II, III and IV) are encoded in their pretoxin structural form in the apx operon, which further encodes the activator gene and the secretion apparatus-encoding genes. The four toxins are expressed in various combinations in the different APP serotypes. APP serotype 2 encodes ApxII, III and IV (Beck et al., 1994, J. Clin. Microbiol., Vol. 32(11), pp. 2749-2754). In this paper, truncated forms of ApxII and III were generated, expressed in E. coli and purified. In recent publications, neutralizing epitopes of ApxII and III were identified that could induce immune responses, and antibodies generated against these epitopes were protective in vitro. Kim et al., 2010 described an N-terminally HIS10-tagged truncated ApxII from APP2, containing amino acids 439-801aa. The sequence of RTX toxin IIA (ApxII) was retrieved from a database (GenBank: AF363362.1), synthesized, and used to generate a truncated HIS10-tagged ApxII(439-801aa) (Seq. 47) encoded on the pMLBAD vector pMLBAD-HIS10-ApxII(439-801aa). The sequence of ApxIII was retrieved from a database (GenBank: AF363363.1) and used as a template for synthesis. Previous studies have shown that the N-terminal domain of ApxIII is recognized by convalescent pig serum, has cytotoxic neutralizing activity, and prevents ApxIII-induced neutrophil apoptosis in vitro (Seah et al., 2004, Vaccine, Vol. 22(11-12), pp. 1494-1497).The N-terminal domain of ApxIII (amino acids 27–245) was synthesized including a C-terminal HIS10 tag (Seq. 48) and used as a template to introduce a start codon and a 5′XmaI / 3′HindIII restriction endonuclease cleavage site by using primers 5′XmaI-XhoI-APXIIIne-HIS-fw / 3′APXIIIne-HIS-HindIII-rv in a PCR reaction. The resulting fragment was digested with the respective enzymes and ligated into XmaI / HindIII treated pMLBAD vector (pMLBAD-ApxIII(27-245aa)-HIS9; one HIS epitope was lost during the course of cloning. Both plasmids were introduced into E. coli BL21 for purification for protein expression studies and pig vaccination studies. To purify HIS10-ApxII(439-801aa), an overnight culture of BL21 pMLBAD-HIS10-ApxII(439-801aa) grown at 37°C with shaking in LB+Tmp (10 μg / ml) was diluted to 0.1 OD in 1 L LB+Tmp. 600 Dilute to OD 600The cells were incubated at 37°C with shaking until the β-Kα was approximately 0.8-0.9. Arabinose (0.2% final concentration) was added to induce protein expression. The cells were incubated for another 4 hours at 37°C on a shaker and subsequently harvested by centrifugation (5000×g / 4°C / 10 minutes and the supernatant discarded). For cell lysis, 20 ml of 0.1 mg / ml lysozyme (in 1×PBS) was added. The cells were disrupted by sonication and freeze-thaw rounds. Briefly, the cell pellet was sonicated at 85% amplitude, frozen in liquid nitrogen for 1 minute and thawed at 37°C (shaking) for 10 minutes. This procedure was repeated three times. The cell suspension was centrifuged at 10000×g for 5 minutes at 4°C. During the above procedure, it was found that HIS10-ApxII(439-801aa) aggregated, whereby the protein was found in the pellet fraction after centrifugation. Approximately 20 ml denaturing buffer (6 M guanidine hydrochloride, 0.1 M Tris-HCl pH 8.0) was added to the pellet and the material was centrifuged again at 10000×g for 20 min at 4° C. 4 ml of equilibrated nickel-NTA resin was added to the supernatant and the material was incubated for 1 h at 4° C. on a rotating wheel. The suspension was then loaded onto a gravity-flow column. The resin was washed three times with 10 ml 1×PBS and the protein was eluted with five 1 ml denaturing buffer containing 0.5 M imidazole. To analyze the recovered material, 60 μl of each elution fraction was mixed with 20 μl of 4× Laemmli buffer. Samples were heated at 95° C. for 5 min and 10 μl was loaded onto a 4-12% Bis-Tris gel to separate molecules by size in MES buffer. The gel was further processed for Coomassie staining (FIG. 12A) and immunoblotting with a HIS-specific antibody (FIG. 12B). For Coomassie staining, the gel was layered with Coomassie staining solution (3 mM Coomassie Brilliant Blue R 250, 40% ethanol, 10% acetic acid) and incubated overnight at room temperature with shaking. The gel was repeatedly placed in Coomassie destaining solution (30% ethanol, 10% acetic acid) until the desired destaining of the gel and staining of the protein was observed. Proteins were transferred from the gel onto a PVDF membrane to detect purified HIS10-ApxII(439-801aa) via immunoblotting.The membrane was incubated in blocking solution (PBS pH 7.5 / 0.05% Tween / 0.1% casein) for 2 hours at room temperature with shaking. The membrane was then incubated in antibody binding solution (PBS pH 7.5 / 0.05% Tween / 0.05% casein) containing Tetra-HIS antibody (Qiagen #34670) at 1:2000 dilution with shaking at 4°C overnight. The membrane was washed three times for 5 minutes with an excess of PBS 0.05% Tween buffer pH 7.5. The membrane was then incubated in antibody binding solution with secondary goat anti-mouse IgG-HRP antibody (BETHYL Cat#A90-116P) at 1:2000 dilution with shaking at room temperature for 1 hour. The membrane was washed four times for 5 minutes with an excess of PBS 0.05% Tween buffer pH 7.5. Specific antibody binding was then visualized by adding ECL solution (GE healthcare #RPN2105) to the membrane and recording the optical signal detected using a Stella8300 (Raytest).

[0096] Using the applied method of protein expression and purification, HIS10-ApxII(439-801aa) could be purified in large quantities via binding to nickel-NTA resin and elution with gravity flow (Figure 12). The highest protein amount was found in elution fractions 3-5. In addition to the purified HIS10-ApxII(439-801aa) at around 41 kDa, some impurities could be detected at higher and lower molecular weights (Figure 12A), which were also recognized by the HIS antibody (Figure 12B). Using this experiment, we were able to demonstrate the successful expression and purification of truncated HIS10-ApxII(439-801aa).

[0097] To purify ApxIII(27-245aa)-HIS9, an overnight culture of BL21 pMLBAD-ApxIII(27-245aa)-HIS9 grown at 37°C with shaking in TB+Tmp (10 μg / ml) was diluted to 0.1 OD 600 Dilute to OD 600The cells were incubated with shaking at 37°C until the NA was approximately 0.6-0.8. Arabinose (0.2% final concentration) was added to induce protein expression. The cells were incubated for another 4 h at 37°C on a shaker and subsequently harvested by centrifugation. A similar procedure was followed as described for HIS10-ApxII(439-801aa). Briefly, cell lysis was performed in a final volume of 70 ml (30 mM Tris HCl pH 7.5, 300 mM NaCl, 20% sucrose, 1 mM EDTA, 1 g / l lysozyme, 1x protease inhibitor cocktail). Cells were lysed by three rounds of sonication-freeze-thaw rounds (as above). The cell suspension was centrifuged at 5525 x g for 15 min at 4°C. The supernatant was centrifuged again at 20000 x g for 1 h at 4°C. 0.3 ml of nickel-NTA resin equilibrated in binding buffer (30 mM Tris HCl pH 7.5, 300 mM NaCl) was added to the supernatant and the material was incubated for 1 hour at 4° C. on a rotating wheel. The suspension was then loaded onto a gravity-flow column. The resin was washed twice with 3 ml binding buffer and once with 3 ml wash buffer (30 mM Tris HCl pH 7.5, 300 mM NaCl, 10 mM imidazole). Elution was performed by adding 4 times 0.3 ml elution buffer 1 (30 mM Tris HCl pH 7.5, 300 mM NaCl, 200 mM imidazole), 2 times 0.5 ml elution buffer 2 (30 mM Tris HCl pH 7.5, 300 mM NaCl, 500 mM imidazole) and 3 times 0.5 ml elution buffer 3 (30 mM Tris HCl pH 7.5, 300 mM NaCl, 1 M imidazole). To analyze the cell preparation and protein purification, all fractions were tested by immunoblotting and Coomassie staining against the HIS epitope and it was decided to pool elution fractions 4-8 and to exchange the buffer by dialysis in PBS. To analyze the pooled and dialyzed samples, 60 μl of material was mixed with 20 μl 4× Laemmli buffer. Samples were heated at 95°C for 5 min, and 10 μl was loaded onto a 4-12% Bis-Tris gel to separate molecules by size in MES buffer.Immunoblotting and Coomassie staining with HIS-specific antibodies were performed as described above (Figure 13). A HIS-specific signal was identified around 25 kDa, which is consistent with the calculated molecular weight of ApxIII(27-245aa)-HIS9 (Figure 13B), and could also be detected on a Coomassie-stained gel (Figure 13A). Some impurities at lower and higher molecular weights are also detected. Successful expression and purification of truncated ApxIII(27-245aa)-HIS9 was demonstrated (Figure 10).

[0098] 2.2) Genomic integration of ApxI, II and III neutralizing epitopes as fusion constructs to be expressed on the cell surface of either E. coli_5 or SL1344, both of which express the APP2 O antigen. To generate a live Salmonella vaccine strain that displays neutralizing epitopes of APP2 LPS and Apx toxin on its surface, the corresponding genes were integrated into the genome, resulting in surface display of neutralizing epitopes. Xu et al., 2018, PlosOne, Vol. 13(1) described a fusion construct consisting of ClyA, a pore-forming hemolytic protein expressed on the cell surface, linked to truncated ApxI (628-845aa), truncated ApxII (612-801aa) and truncated ApxIII (626-860aa) with a C-terminal HIS6 tag, which induces a specific immune response. In a vaccination and challenge study of mice, the groups showed increased immunoglobulin and cytokine levels as well as increased survival after challenge with different APP serotypes. The sequences of the constructs are publicly available and were modified as follows: The codon usage of ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6 was optimized for the E. coli expression system using the algorithm provided by the synthesis company. For strong transcription rates, the sequence of the synthetic promoter proD (Davis et al., 2010, Nucleic acids research, Vol. 39(3), pp. 1121-1141) was incorporated before the start codon. To select for successful integration into the genome, a chloramphenicol resistance cassette (cat) flanked by FRT sites (based on pKD3; Datsenko et al., 2000, PNAS, Vol. 97(12), pp. 6640-6645) was added after the stop codon of the fusion construct. For homologous integration into the genome, sequences homologous to the intergenic rfaK-rfaL region were chosen upstream and downstream of the construct (213 bp 5' of the proD promoter and an additional 250 bp after the chloramphenicol resistance cassette). The complete synthetic construct is represented diagrammatically in Figure 14 (Seq. 49).To integrate the fusion construct proD-ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6 / cat into SL1344Δrfb::kP-APP2.LPS rfaL-Ωgne-wzy(cod.opt.), the flanking regions of sequence 32 were modified by PCR with oligonucleotides FW_pliCint / REV_pagCint followed by extension of the 5' and 3' ends with oligonucleotides eloFW_pliCint / eloREV_pagCint. This procedure created an 83 bp homologous region with the downstream region of pliC in SL1344 and an 87 bp homologous region with the downstream region of pagC in SL1344. This modification allowed the integration of the fusion construct proD-ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6 / cat into the intergenic region between pliC and pagC. The final construct was transformed into SL1344Δrfb::kP-APP2.LPS rfaL-Ωgne-wzy(cod.opt.) together with the temperature-sensitive helper plasmid pKD46 (Datsenko et al., 2000, PNAS, Vol. 97(12), pp. 6640-6645), which encodes the γ-recombination system. The λ-recombination system recognized the homologous flanking regions at each genomic site and recombined the fusion construct proD-ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6 / cat into the integration site described above (SL1344Δrfb::kP-APP2.LPS rfaL-Ωgne-wzy(cod.opt.)pliC-ΩproD-ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-cat / pagC).To generate a control strain lacking the APP2 rfb cluster but expressing the proD-ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6 fusion construct, Seq.32 was amplified with oligonucleotides FW-rfaK_rfaL / REV_rfaL_rfaK to generate a 4513 bp fragment containing the integration construct with flanking homology regions for integration at the intergenic region between rfaK and rfaL of SL1344. This fragment was transformed with pKD46 into SL1344Δrfb and the procedure was followed as described above. The construct proD-ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6 / cat was integrated into the intergenic region between genes rfaK and rfaL (SL1344Δrfb rfaK-ΩproD-ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-rfaL / cat). In both strains, the introduced antibiotic resistance cassette allowed the selection of positive clones (successful integration). These positive candidates were confirmed by PCR for integration of the PCR fragment. The temperature-sensitive helper plasmid was lost from the cells by increasing the growth temperature over several rounds of incubation. To remove the chloramphenicol resistance cassette, the resulting strains SL1344Δrfb::kP-APP2.LPS rfaL-Ωgne-wzy(cod.opt.)pliC-ΩproD ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-cat / pagC and SL1344Δrfb rfaK-ΩproD-ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-cat / rfaL were transformed with the temperature-sensitive plasmid pCP20 (Cherepanov et al., 1995, Gene, Vol. 158, pp. 9-14), which encodes a flippase recognizing a palindromic FRT site. After the "flip-out" event, one FRT remained in the genome.Again, the incubation temperature was increased and positive clones were counterselected against the flippase-encoding plasmid. A final PCR confirmed the absence of all helper plasmids, the absence of the chloramphenicol resistance marker and the integration of proD-ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6. The resulting strains SL1344Δrfb::kP-APP2.LPS rfaL-Ωgne-wzy(cod.opt.)pliC-ΩproD-ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-pagC and SL1344Δrfb rfaK-ΩproD-ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-rfaL were analyzed for their APP2 O antigen production as well as ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6 expression. To confirm the expression of ClyA-ApxI(628-845aa)-ApxII(612-801aa)-ApxIII(626-860aa)-HIS6, SL1344Δrfb::kP-APP2.LPS rfaL-Ωgne-wzy(cod.opt.) and SL1344Δrfb whole cell extracts were analyzed by immunoblotting and Coomassie staining.

[0099] Wild-type SL1344, deficient in endogenous O-antigen biosynthesis (SL1344Δrfb), or expressing the APP2 rfb cluster (SL1344Δrfb::APP2.LPS, Δrfb::KanR-APP2.LPS, Δrfb::KanR-APP2.LPS(cod.opt.), Δrfb::KanR-APP2.LPS rfaL-Ωgne / cat, Δrfb::KanR-APP2.LPS rfaL-Ωgne-wzy(cod.opt.) / cat, Δrfb::KanR-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat, Δrfb::kP-APP2.LPS-(cod.opt.)rfaL-Ωgne-wzy(cod.opt.)) genetically modified cells or cells expressing ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-pagC with or without the improved APP2 rfb cluster integration (SL1344Δrfb Overnight cultures of rfaK-ΩproD-ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-rfaL, Δrfb::kP-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.)pliC-ΩproD-ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6-pagC) were incubated at an OD of 0.05 in LB medium. 600 Dilute to 1 ml / mL and place at logarithmic growth phase (OD 600 The SL1344 derivatives and APP2 cells were grown at 37°C on a shaker until stationary phase (approximately 1 OD). In addition, the APP2 P1875 strain was grown in BHI+NAD at 37°C with gentle shaking (110 rpm) until stationary phase. SL1344 derivatives and APP2 cells were harvested and incubated with 1× Laemmli (1OD) at 95°C for 5 min. 600 Cell equivalent / 100 μl 1× Laemmli) was heated at 0.1 OD 600Cell equivalents were loaded onto 4-12% Bis-Tris gels. As controls, 2.5 μg purified HIS10-APXII(439-801aa) and 5 μg APXIII(27-245aa)-HIS9 in 1× Laemmli were loaded onto the gels. Proteins and whole cell extracts were size-separated with MOPS buffer. (Loading scheme in Figure 15A). The gel was further processed for Coomassie staining (Figure 15B) and Western blotting to detect HIS epitopes (Figure 15C). For Coomassie staining, the gel was layered with Coomassie staining solution (3 mM Coomassie Brilliant Blue R 250, 40% ethanol, 10% acetic acid) and incubated overnight at room temperature with shaking. The gel was repeatedly placed in Coomassie destaining solution (30% ethanol, 10% acetic acid) until the desired destaining of the gel and staining of the proteins was observed. Proteins were transferred from the gel onto a PVDF membrane for detection of ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6, HIS10-APXII(439-801aa) and APXIII(27-245aa)-HIS9 via immunoblot. The membrane was incubated in blocking solution (PBS pH 7.5 / 0.05% Tween / 0.1% casein) for 2 hours at room temperature with shaking. The membrane was then incubated overnight at 4°C with shaking in antibody binding solution (PBS pH 7.5 / 0.05% Tween / 0.05% casein) containing Tetra-HIS antibody (Qiagen #34670) at a 1:2000 dilution. The immunoblots were washed three times for 5 min with excess PBS 0.05% Tween buffer pH 7.5. The membranes were then incubated in antibody binding solution with secondary goat anti-mouse IgG-HRP antibody (BETHYL Cat#A90-116P) at 1:2000 dilution for 1 h at room temperature with shaking. The membranes were washed four times for 5 min with excess PBS 0.05% Tween buffer pH 7.5. Specific antibody binding was then visualized by adding ECL solution (GE healthcare #RPN2105) to the membranes and recording the light signal detected using Stella8300 (Raytest).When comparing SL1344 and its derivatives on a Coomassie stained gel, the same amount of cell equivalents were loaded (Figure 15B, lanes 4-14), except for SL1344Δrfb::Kan-APP2.LPS rfaL-Ωgne-wzy(cod.opt.) / cat (potential loading step error, Figure 15B, lane 10). Coomassie analysis of APP2 whole cell extracts revealed a reduction in overall protein staining, also distinct from SL1344 cells. Purified HIS10-APXII(439-801aa) was visible as a faint band around 50 kDa (Figure 15B, lane 1), which was recognized by the HIS antibody used in the Western blot analysis (Figure 15C, lane 1). Also, APXIII(27-245aa)-HIS9 was detectable around 25 kDa (Figure 15B, lane 2). In the HIS immunoblot, a very strong signal was monitored for this protein (Figure 15C, lane 2). Cells expressing ClyA-ApxI(aa626-845)-ApxII(aa612-801)-ApxIII(aa626-860)-HIS6 (Figure 15C, lanes 13, 14) showed a signal in the HIS immunoblot over 115 kDa, higher than the calculated molecular weight around 104 kDa. This may be due to SDS-PAGE parameters resulting in an altered migration pattern for this transmembrane protein. Also, an additional band below 115 kDa was detected, which may be due to proteolytic cleavage of the fusion construct. The detected signal was absent in all other tested control cells lacking the fusion construct.

[0100] Example 3 Clinical Trials 3.1) Immunization studies in piglets using inactivated vaccine strains presenting recombinant APP2 O antigen and inactivated APP2 bacteria The goal of this initial immunization study was to test the safety and immunogenicity of APP2 LPS following intranasal / oral or intramuscular immunization of pigs with inactivated E. coli_5 and SL1344 encoding the APP2 rfb cluster (see Table 6 for study layout).

[0101] Bacterial whole cell vaccines were prepared as follows: Groups 1 and 6 contained SL1344Δrfb::kanR-APP2.LPS pMLBAD-gne pEC415-wzy, which required induction of wzy and gne expression (both genes are under the control of an arabinose-inducible promoter) before preparing the strain for immunization. Therefore, cells were cultured at an OD of approximately 0.6. 600 The cells were cultured in LB with the respective antibiotics (see Table 2) and 0.01% arabinose at 37°C with shaking at 180 rpm until OD 2000. The cells were induced with 0.1% arabinose. After 6 hours of incubation, the cells were harvested and resuspended in PBS buffer. The OD 2000 of these cell suspensions was 2.2 mg / mL. 600 It was determined that (1OD 600 Groups 4 and 8 cells consisted of SL1344Δrfb and E. coli_5Δrfb, which were used in a 1:1 mixture in individual immunizations. These cells were cultured until the culture reached an OD of >2. 600 The APP serotype 2 cells (groups 5 and 9) were inoculated in BHI+NAD at 37°C with gentle shaking (110 rpm) until stationary phase, harvested, and resuspended in PBS buffer. The OD of these cell suspensions was 600 Due to the culture conditions, the OD 600 The conversion rate between OD and cfu differs from that mentioned above (1OD for APP2). 600 (Equivalent to 4.1 x 10E5 cfu). Glycoengineered SL1344 as well as SL1344Δrfb, E. coli_5Δrfb and APP2 cells (in PBS buffer) were heat inactivated by incubating the materials at 80°C for 90 min with shaking at 600 rpm. Before applying the materials, the cells were tested for complete inactivation.

[0102] Protein-based material was prepared as follows: N-terminal HIS10-tagged neutralizing epitope of ApxII from APP2 (439-801aa) was expressed and purified from E. coli (BL21 pMLBAD-HIS10-ApxII(439-801aa)) via Ni-NTA Sepharose binding and imidazole elution on FPLC. Purified protein was dialyzed in PBS. Protein concentration was determined.

[0103] To prepare material for intranasal and oral immunizations (groups 1-5), two doses of 1 mL per immunization were prepared per animal, each dose containing 10E11 (groups 1, 4) or 10E8 (group 5) cfu, 400 μg HIS10-ApxII(439-801aa) (group 2) or PBS only (group 3) in PBS, mixed with Montanide IMS1313 (Seppic) according to the supplier's information to reach a concentration of 25%.

[0104] For intramuscular immunization, a total volume of 0.5 ml per animal was prepared. Each dose contained 0.375 ml of inactivated cells (in PBS, groups 6, 8 had 10E8 cfu and group 9 had 10E5 cfu) or 400 μg HIS10-ApxII(439-801aa) (group 7) and 0.125 ml Montanide ISA25 (Seppic) to reach a final concentration of 25% adjuvant. The mixture was homogenized according to the supplier's recommendations. Group 10 was the adjuvant control (0.375 ml PBS buffer mixed with 0.125 ml Montanide ISA25 and homogenized) and group 11 was kept as an untreated control and did not receive any antigen.

[0105] Table 6 provides an overview of immunization groups 1-11 with applied inactivated cells, proteins, adjuvant only or none for intranasal and oral or intramuscular applications. Antigen amounts and dose volumes are given. For intranasal or oral vaccination, three applications were prepared for each nostril (0.5 ml per nostril) and oral (1 ml).

[0106] [Table 6]

[0107] For the study, commercially bred pigs (Swiss Landrace x Large White) were used, weaned at approximately 4 weeks of age on study day (SD) 0. The pigs were raised in approved APP-free housing facilities and all animals were negative for antibodies to APP by ELISA on SD-7. Three animals per group (randomized) were immunized twice on SD0 and SD14 (Figure 16).

[0108] Intranasal administration was performed using a MAD Nasal intranasal mucosal spray device MAD100 (Teleflex) with a 3 ml syringe. For each animal, two syringes / MAD devices were filled with 1 ml air and 0.5 ml of the relevant antigen / adjuvant mixture, respectively. Each pig was administered 0.5 ml per nostril.

[0109] Oral dosing was performed using a MAD Nasal intranasal mucosal spray device MAD100OS (Teleflex) with a 3 ml syringe. For each animal, one syringe / MAD device was filled with 1 ml of air and 1 ml of the relevant antigen / adjuvant mixture. The material was administered above the tonsils.

[0110] For intramuscular administration, each animal received 0.5 ml of the antigen / adjuvant mixture by intramuscular injection into the right side of the neck.

[0111] Blood was collected weekly for isolation of serum, and animals were examined at least daily for clinical signs.A transient increase in rectal temperature up to 41.0°C was observed 4-10 hours after intramuscular injection of the adjuvant as well as the adjuvanted vaccine, compared with untreated controls, suggesting a nonspecific increase in body temperature in response to the adjuvant.

[0112] After euthanasia at SD28, lungs were removed and BALF (bronchoalveolar lavage fluid) was collected. For each set of lungs, a 500 mL volume of PBS was flushed into the trachea, the lungs were gently inverted for 5-10 seconds, and the fluid was collected.

[0113] IgG and IgA responses to LPS in serum and BALF were analyzed by ELISA. Phenol / chloroform extracted LPS (10 OD per reaction according to the instruction manual of Intron Biotechnology #17141) of APP2 and 7 for serum analysis and APP1, 2, 5a and 7 for BALF analysis was used. 600 Bacteria were treated with 0.05OD 600Equivalent volume, 100 μl coating buffer (PBS buffer pH 7.5) / well). Plates were incubated overnight at 4° C. on a shaker. Plates were washed once with 200 μl washing solution (PBS pH 7.5 / 0.05% Tween / 0.05% casein) per well and 150 μl blocking buffer (PBS pH 7.5 / 0.05% Tween / 0.1% casein) per well was added. Plates were incubated for 1 hour at room temperature with shaking. Blocking buffer was removed and pig sera from SD0 (pre-immune) and 28 (2 weeks after the second immunization / euthanasia day) were added at 1:500 dilution (in washing solution). BALF from SD28 was added at 1:2 dilution (in washing solution). After 1 hour incubation time at room temperature with shaking, the plates were washed 3 times with 200 μl washing solution per well and secondary swine anti-IgG-HRP (BETHYL Cat#A100-105P) for serum or swine anti-IgA-HRP (BETHYL Cat#A100-102P) for BALF antibodies were added at 1:1000 dilution in washing solution (total 100 μl per well). After 1 hour incubation time at room temperature on a shaker, the plates were washed 4 times with 200 μl washing solution per well. Plates were developed by adding 110 μl developer solution per well (1.5 mg 3,3',5,5'-tetramethylbenzidine dihydrochloride dissolved in 1.5 ml 100% DMSO per plate followed by 13.5 ml 0.05 M phosphate-citrate buffer; 2 μl fresh 30% H2O2 added before use). After adequate color development was observed, the reaction was stopped by adding 110 μl stop reagent BioFX. Absorbance was measured at 450 nm using a Tecan Infinite M Nano reader.

[0114] To analyze the immune response against ApxII, purified N-terminal HIS10-tagged neutralizing epitope of ApxII expressed in BL21 pMLBAD-HIS10-ApxII(439-801aa) and purified AcrA-HIS6 protein purified from E. coli (HIS6-tagged C. jejuni used as negative control) were used. 500 ng of each protein was coated per well (in 100 μl coating buffer) in 96-well plates (TPP, 92096) and probed against porcine serum and BALF. The procedure was performed as described above. For ELISA to detect specific ApxII antibody production, all animals from groups 2 and 7, two animals from groups 3 and 10 and one animal from group 11 were tested.

[0115] Serum IgG analysis (Figure 17) revealed specific anti-APP2 LPS responses at SD28 in both animals immunized with heat-inactivated APP2 (mucosally and intramuscularly immunized animals, groups 5 and 9) and in pigs immunized with glycoengineered SL1344 (SL1344Δrfb::kanR-APP2.LPS pMLBAD-gne pEC415-wzy, mucosally and intramuscularly immunized animals, groups 1 and 6). No IgG responses were detectable before immunization at SD0. IgG levels in the serum of animals receiving SL1344Δrfb and E.coli_5Δrfb (groups 4, 8), adjuvant (groups 3, 10) or negative control (group 11) were at background levels in the ELISA performed.

[0116] For the analysis of IgA responses to APP2 LPS, BALF was used at a 1:2 dilution. The control, adjuvant and non-immunized groups immunized with SL1344Δrfb and E.coli_5Δrfb showed strong background levels (Figure 18). In addition, cross-reactivity to APP1, APP5 and APP7 LPS was detected in all animals. In all animals immunized with inactivated APP2, the response to APP2 clearly exceeded those to APP1, 5 and 7, both by mucosal and intramuscular routes, indicating a specific IgA response to APP2 LPS. When analyzing groups 1 and 6 (application of glycoengineered SL1344-SL1344Δrfb::kanR-APP2.LPS pMLBAD-gne pEC415-wzy), it becomes clear that mucosally immunized animals showed higher IgA titers to APP2 LPS than intramuscularly immunized animals. Furthermore, one individual animal (pig 5062) demonstrated similar levels of recognition of all four tested APP LPS serotypes.

[0117] Animals immunized with purified HIS10-ApxII(439-801aa) (FIG. 19) developed a serum IgG rise against ApxII at SD28 after intramuscular immunization but not after mucosal immunization.

[0118] Specific IgA against ApxII was not detectable in the BALF of pigs immunized with purified HIS10-ApxII-(439-801aa). Animal 5136 also showed elevated BALF IgA against AcrA-HIS6, suggesting the development of HIS antibodies rather than against ApxII (both modified proteins AcrA and ApxII have only the HIS tag in common, without further homology). In addition, animals in the control groups (adjuvant group and non-immunized control group) nonspecifically recognized HIS10-ApxII(429-801aa) and AcrA-HIS6.

[0119] In conclusion, the inactivated antigen was safe in pigs. The observed transient increase in body temperature was most likely due mainly to an adjuvant reaction. Glycoengineered SL1344 (SL1344Δrfb::kanR-APP2.LPS pMLBAD-gne pEC415-wzy) was immunogenic, specific serum IgG responses against APP2 LPS were detectable in all six pigs, and specific IgA against APP2 LPS was found in four of the six pigs. Immunization with purified HIS10-ApxII(439-801aa) initiated the formation of specific IgG in the serum of intramuscularly immunized pigs (but not after mucosal immunization). Specific IgA against ApxII was not detectable in BALF.

[0120] 3.2) Immunization studies with improved live recombinant APP vaccine strains in piglets In a second immunization study, the safety and immunogenicity of live SL1344 encoding the APP2 rfb cluster with improved APP2 O antigen presentation on the cell surface was tested in pigs (see Table 7 for study layout). Group 1 used SL1344Δrfb::kanR-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat and group 2 was kept as untreated control (no antigen applied).

[0121] For this experiment, viable bacteria were prepared as follows: cultures reached an OD of >2. 600 Bacteria were grown in LB medium with shaking at 37°C until an OD of 0.5 was reached and harvested. 600 was determined and cfu / ml was estimated (1OD 600 (Equivalent to 4.1 x 10E8 cfu). Cells were washed with sterile PBS and resuspended in PBS to a cell concentration of 1 x 10E8 cfu / ml. Cells were kept on ice until applied to animals.

[0122] Table 7 provides an overview of immunization group 1 with applied live cells (intranasal and oral) or untreated control group 2. Antigen amount and dose volume are given. For intranasal and oral vaccination, two applications (1 ml, 2 times) were prepared and given into each nostril (0.5 ml each nostril) and into the oral cavity (1 ml).

[0123] [Table 7]

[0124] For the study, Danebreed (Landrace and Large White commercial cross) pigs with Duroc sires, approximately 4-5 weeks old on SD0 were used. Six animals per group (randomized) were immunized twice on SD0 and SD14 (Figure 20). The pigs were raised in approved APP-free housing facilities and all animals were negative for antibodies to APP by ELISA on SD-7. Intranasal administration of live bacteria in PBS was performed using a MAD Nasal intranasal mucosal spray device MAD100 (Teleflex) with a 3 ml syringe. For each animal, two syringes / MAD devices were filled with 1 ml air and 0.5 ml of the relevant live cell / PBS mixture, respectively. Each pig was administered 0.5 ml per nostril. Oral administration was performed using a MAD Nasal intranasal mucosal spray device MAD100OS (Teleflex) with a 3 ml syringe. For each animal, one syringe / MAD device was filled with 1 ml of air and 1 ml of the relevant viable cell / PBS mixture. The material was administered above the tonsils. Blood was collected weekly for serum isolation and animals were examined for clinical signs.

[0125] After euthanasia at SD28, lungs were removed and BALF was collected. For each set of lungs, a volume of 500 ml PBS was flushed into the trachea, the lungs were gently inverted for 5-10 seconds, and the fluid was collected (BALF). The persistence of the bacterial strain used for immunization was tested after euthanasia. Swab samples were collected from the tonsils, lungs (left and right diaphragmatic lobes), and tracheobronchial lymph nodes of each animal in groups 1 and 2. Each swab was aseptically streaked onto a sterile LB agar plate containing kanamycin and incubated at 37°C for 16-24 hours.

[0126] All animals remained in good health throughout the study. No abnormal clinical signs were observed in any of the animals. No pulmonary abnormalities were observed after necropsy. No lesions or other pathology were noted in the lungs of any of the animals.

[0127] Bacterial colonies were recovered from two animals in Group 1. One animal (#341609) had a total of 366 colonies recovered from the tonsils but no other tissues. The second animal (#341618) had single colonies present in the tonsils and tracheobronchial lymph nodes but no other tissues. No bacteria could be isolated from any animal in the non-immunized control group.

[0128] Serum and BALF immune responses to APP2 LPS were analyzed by immunoblot. APP2 P1875 strain was grown in BHI+NAD to stationary phase at 37°C with gentle shaking (110 rpm). Cells were harvested and used for further processing. For APP2 O antigen analysis, cells were resuspended in 1x Laemmli buffer (1OD 600 cells / 100 μl 1× Laemmli buffer). Samples were incubated at 95°C for 5 min. OD 600 12 μg proteinase K (stock 20 mg / ml in 10 mM Tris-HCl pH 7.5, 20 mM CaCl2, 50% glycerol) was added per equivalent amount of cells and the samples were incubated at 60°C for 1 h. Proteinase K-treated samples (0.1 OD 600A total of 1000 cells (cell equivalents) were loaded onto a 4-12% Bis-Tris gel and the molecules were separated by size in MES buffer. For immunoblotting, the gel was further processed. LPS was transferred from the gel onto a PVDF membrane. The membrane was incubated in blocking solution (PBS pH 7.5 / 0.05% Tween / 0.1% casein) for 2 h at room temperature with shaking. The membrane was then incubated overnight at 4 °C with shaking in antibody binding solution (PBS pH 7.5 / 0.05% Tween / 0.05% casein) containing pig serum at 1:500 dilution from 6 animals in group 1 and 2 animals in group 2 (untreated control) immunized with SL1344Δrfb::kanR-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat at SD0 and SD28, and BALF at SD28 at 1:2 dilution. The immunoblots were washed three times for 5 min with excess PBS 0.05% Tween buffer pH 7.5. The membranes were then incubated in antibody binding solution with secondary swine anti-IgG-HRP antibody (BETHYL Cat#A100-105P) or swine anti-IgA-HRP (BETHYL Cat#A100-102P) at 1:2000 dilution with shaking for 1 h at room temperature. The membranes were washed four times for 5 min with excess PBS 0.05% Tween buffer pH 7.5. Specific antibody binding was then visualized by adding ECL solution (GE healthcare #RPN2105) to the membranes and recording the light signal detected using a Stella8300 (Raytest).

[0129] [Table 8]

[0130] Evaluation of the specific immune response against APP2 LPS (Table 8) showed clear APP2 LPS recognition by serum IgG at SD28 for 6 out of 6 animals (group 1) immunized with live SL1344Δrfb::kanR-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat, which was absent at SD0. The two tested animals in group 2 (untreated control) did not show any significant increase in serum IgG specifically against APP2 LPS.

[0131] Analysis of IgA against APP LPS in BALF shows an increase in specific recognition of APP2 O antigen in 5 out of 6 animals in Group 1. Both tested animals in the untreated control Group 1 showed no IgA response against APP2.

[0132] In conclusion, live glycoengineered SL1344 was safe and colonization could be confirmed in two pigs after euthanasia. In addition, the recombinant APP2 O antigen was immunogenic, inducing both systemic IgG and mucosal IgA responses.

[0133] 3.3) Efficacy of glycosylated APP vaccine candidates against APP serotype 2 in piglets The aim of this study was to investigate the efficacy of preventing APP2 infection in pigs immunized with the developed glycoengineered SL1344 vaccine strain, which displays the APP2 O antigen on its surface (see Table 9 for an overview of the groups).

[0134] The vaccine strain SL1344Δrfb::kanR-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat was used as live vaccine (group 1) and a second strain (SL1344Δrfb::kanR-APP2.LPS pEC415-wzy pMLBAD-gne with induced wzy and gne expression) was used as inactivated vaccine (group 2). Both were applied in combination with ApxII and III neutralizing epitopes. On SD0 and 21, the vaccine was administered by oral and intranasal routes and the neutralizing epitope of Apx toxin by intramuscular application (FIG. 21). These groups were compared with animals vaccinated with either inactivated APP2 in combination with injection of two Apx neutralizing epitopes (group 3), Apx neutralizing epitope alone (group 4) or with non-vaccinated animals (group 5). Pigs were challenged with an infectious dose of APP serotype 2 (HK361, NCTC10976) on SD42 and euthanized on SD48. Animals were monitored regularly for clinical signs (at least twice daily for 6 days after challenge) and rectal temperature measurements were taken once or twice daily. Online telemetry of body temperature (AniPill, Body Cap) was used in 50% of animals by subcutaneously placed temperature probe.

[0135] For this experiment, bacteria and proteins were prepared as follows: To prepare SL1344Δrfb::kanR-APP2.LPS(cod.opt.)rfaL-Ωgne-wzy(cod.opt.) / cat for live vaccination (group 1), cells were inoculated in LB medium at 37° C. with shaking for approximately 24 hours. On the day of vaccination, cells were cultured in stationary phase (OD 600 Bacterial cultures grown to an optical density (OD) of 100 nm (OD) were cooled on ice for approximately 10 minutes. The cultures were centrifuged at 4100×g for 15 minutes at 4° C. The pelleted cells were carefully resuspended in sterile pre-chilled PBS buffer. The suspension was centrifuged again at 4100×g for 15 minutes at 4° C., the supernatant was removed, and the resulting cell pellet was resuspended in pre-chilled PBS. The optical density at 600 nm (OD 600 ) was determined (1OD 600(Equivalent to 4.1 x 10E8 cfu). Each vaccine dose applied to pigs contained 1 x 10E8 cfu in a 1 ml volume.

[0136] To prepare SL1344Δrfb::kanR-APP2.LPS pEC415-wzy pMLBAD-gne for inactivation and vaccination (group 2), cells were inoculated into LB medium overnight at 37° C. with shaking. The following day, OD 600 was determined, and the cells were grown in LB medium supplemented with Amp, Tmp, and 0.01% arabinose at 0.075 OD 600 The cultures were incubated at 37°C with shaking. An OD of 0.6 was obtained. 600 Arabinose was added to a final concentration of 0.1% at 20° C. to induce expression of the plasmid-encoded proteins (gne and wzy). The culture was further incubated at 37° C. with shaking. Arabinose was added again to 0.1% after 6 hours. The culture was further incubated at 37° C. with shaking for 10-12 hours. The next day, OD 600 The OD was determined and the cultures were centrifuged at 7,000×g for 10 min at 4° C. The supernatant was removed and the pelleted cells were then carefully resuspended with PBS. 600 The optical density at 1 OD 600 (Equivalent to 4.1 x 10E8 cfu). For heat inactivation, the cell suspension was incubated at 80°C in a water bath for 90 min. Afterwards, the suspension was frozen and stored at -80°C until the day of vaccination. Before applying the material, the cells were tested for complete inactivation.

[0137] Protein-based vaccines were prepared as follows: N-terminal HIS10-tagged neutralizing epitopes of ApxII (439-801aa) (groups 1-4) were expressed and purified from E. coli (BL21 pMLBAD-HIS10-ApxII (439-801aa)) via Ni-NTA Sepharose binding and imidazole elution on FPLC. Purified proteins were dialyzed in PBS. Protein concentrations were determined. C-terminal HIS9-tagged neutralizing epitopes of ApxIII (27-245aa) (groups 1-4) were expressed and purified from E. coli (BL21 pMLBAD-APXIII (27-245aa)-HIS9) via Ni-NTA Sepharose binding and imidazole elution. Purified proteins were dialyzed in PBS. Protein concentrations were determined.

[0138] To prepare the live vaccine for intranasal and oral application (Group 1), two doses of 1 ml were prepared per animal. Each vaccine dose applied contained 1 x 10E8 cfu in a 1 ml volume.

[0139] To prepare the inactivated vaccine for intranasal and oral vaccination (groups 2 and 3), two doses of 1 ml per animal were prepared, each containing 10E11 (group 2) and 10E8 (group 3) cfu in PBS, mixed with the adjuvant Montanide IMS1313 (Seppic) according to the supplier's information to reach a concentration of 25%.

[0140] For intramuscular vaccinations with HIS10-APXII(439-801aa) and APXIII(27-245aa)-HIS9 (groups 1-3), a total volume of 0.5 ml per antigen and per animal was prepared. Each dose contained 0.375 ml 400 μg protein antigen and 0.125 mL Montanide ISA28 (Seppic) to reach a final concentration of 25%. The mixture was homogenized according to the supplier's recommendations. Group 4 was kept as an untreated control (no antigen application).

[0141] The challenge material was prepared as follows: Two days prior to challenge infection, APP2 strain HK361 was cultured on HIS+V culture plates and grown overnight at 37°C and 5% CO2. The following day, cultures were prepared by transferring one colony to 10 new HIS+V culture plates and incubated for 6 hours. All colonies from each plate were then transferred to PBS-filled test tubes and stored overnight in the refrigerator. The cfu cells / PBS solution was determined by diluting the cells and seeding them on HIS+V culture plates. The following morning, the cfu was determined and the solution was diluted to obtain a challenge concentration of 10E6 cfu / ml.

[0142] [Table 9]

[0143] For the study, commercial breeding pigs (TOPIGS-Norsvin, TN70, Z line) of approximately 5 weeks of age were used. The pigs were raised in approved APP-free housing facilities and all animals were negative for antibodies to APP by ELISA on SD-7. Seven to eight animals per group (randomized) were vaccinated twice with an interval of 3 weeks (SD0, SD21; Figure 21). Groups 1 and 2 contained only seven animals due to two animals that died before the start of the study (group 1) or at SD5 (group 2), respectively, without any detectable lesions at necropsy.

[0144] Intranasal administration was performed using a MADgic laryngotracheal mucosal spray device MAD600 (Teleflex) with a 3 ml syringe. For each animal, two syringes / MAD devices were filled with 1 ml air and 0.5 ml of the relevant antigen / adjuvant mixture, respectively. Each pig was administered 0.5 ml per nostril.

[0145] Oral dosing was performed using a Nasal intranasal mucosal spray device MAD100OS (Teleflex) with a 3 ml syringe. For each animal, one syringe / MAD device was filled with 1 ml of air and 1 ml of the relevant antigen / adjuvant mixture. The material was administered above the tonsils.

[0146] For intramuscular administration, each animal received 0.5 ml of each antigen / adjuvant mixture by intramuscular injection in the neck.

[0147] Challenge was performed on day 42 by intranasal inoculation of 2 ml with 10E6 cfu / ml of APP2 strain HK361 using a MAD Nasal™ intramucosal spray device (Teleflex® Corporation).

[0148] Blood was collected on SD-6, 7, 14, 21, 28, 35, 41 and 48 (FIG. 21) for isolation of serum and animals were examined for clinical signs.

[0149] Animals were euthanized at 48 days SD and dissected. Lung and pleural lesions were scored according to Hannan et al., 1982, Res. Vet. Sci., Vol. 33(1), pp. 76-88. Scoring was based on the percentage of each lobe (7 sites) affected by typical APP lesions (Table 10). Both the dorsal and ventral surfaces of each lung were palpated and visually inspected, but a single value was arrived at for each lobe based on the average of the total surface area. Tukey-Kramer mean difference all-pairwise confidence intervals and P values ​​were used as statistical evaluation methods.

[0150] [Table 10]

[0151] Tissue specimens from lungs were sampled from all groups for bacteriological analysis to test for the presence or absence of challenge APP2 strains. For reisolation of APP2 bacteria, lung tissue samples were immersed in treated water for 7 seconds, then disrupted in a stomacher to give a suspension, of which 100 μl was plated on HIS+V plates and incubated overnight at 37° C. and 5% CO2. Colonies were identified by MALDI-TOF.

[0152] None of the pigs showed any abnormal local or systemic reactions after vaccination. Mild elevations in body temperature were observed in all vaccinated pigs 0.5 days after both vaccinations, which returned to normal values ​​1 day after vaccination.

[0153] Body temperatures were measured twice daily after challenge. An increase in rectal temperature was observed beginning one day after challenge. In general, the temperature increase was lower in the vaccinated groups compared to the non-vaccinated controls. This difference was most evident four days after challenge, at which point the mean temperatures of all vaccinated groups were statistically significantly lower compared to the non-vaccinated controls (Table 11).

[0154] [Table 11]

[0155] When lung lesions were assessed (see FIG. 22, Table 12), lung scoring of individual animals revealed animals in the negative control group (Group 4) exhibiting lung damage from Hannan scores of 0 to 7 (mean of 3.75). Animals vaccinated with Apx toxin alone (Group 3) did not show a significant reduction in lung scores compared to the non-vaccinated control group, although the mean value in this group was reduced to 2.25.

[0156] Six of seven animals in group 1 vaccinated with inactivated APP2 and Apx neutralizing epitopes had no detectable lung lesions and one animal showed a lung lesion with a score of 2. Lung lesions occurred significantly (P=0.00369) less frequently than in control animals (group 4), with a mean lung lesion score of 0.286.

[0157] [Table 12]

[0158] The semiquantitative results of the reisolation of the challenge strain (Table 13) showed that in all animals of group 4 it was possible to reisolate high numbers of APP2 bacteria. Although a statistical evaluation could not be performed with the recorded data, a tendency was observed that the lung lesion score positively correlated with the number of reisolated bacteria: in group 1, only one pig with detectable lesions revealed a high number of reisolated challenge bacteria. In two further animals of this group, low numbers of APP2 could be reisolated from lung areas without visible lesions. Groups 1 and 2 did not differ with regard to their lung lesion scores (6 out of 7 pigs in both groups without lesions), whereas in group 2, one pig with detectable lesions and 5 pigs without lesions were positive for reisolation of low to high numbers of challenge bacteria (6 out of 7 animals positive for APP2 challenge strain). For the four animals with lung lesions in Group 3, high amounts of APP2 could be isolated from the lungs either from areas with lesions or from areas without visible lesions.

[0159] [Table 13]

[0160] In summary, the glycoengineered candidate vaccines were highly effective and significantly reduced lung pathology in vaccinated animals compared to untreated animals. The experiments demonstrated that recombinant bacterial vaccines presenting heterologous APP O antigens on their surface, optionally in combination with ApxII and III neutralizing epitopes, can almost completely prevent the development of lung pathology and strongly reduce lung colonization by APP-loaded bacteria.

[0161] 3.4) Efficacy of glycoengineered APP vaccine candidates against APP serotype 2 in piglets administered by oral and nasal routes The aim of this study was to reproduce the efficacy of an inactivated APP2 vaccine strain (SL1344Δrfb::kanR-APP2.LPS pEC415-wzy pMLBAD-gne with induced wzy and gne expression) when applied orally and intranasally in combination with neutralizing epitopes of ApxII and III injected intramuscularly (group 1). Furthermore, efficacy was compared with animals treated with the commercial vaccine Porcilis® APP (group 2). The procedure was comparable to the experiment described in chapter 3.3. The vaccine was administered on SD0 and 21. Both groups were compared with non-vaccinated animals (group 3). Pigs were challenged with an infectious dose of APP serotype 2 (HK361, NCTC10976) on SD42 and euthanized on SD48. Animals were monitored regularly for clinical signs (at least twice daily for 6 days after challenge) and rectal temperature measurements were performed once or twice daily.

[0162] For this experiment, bacteria and protein as well as loading materials were prepared as described in section 3.3 above.

[0163] [Table 14]

[0164] For the study, commercial breeding pigs (TOPIGS-Norsvin, TN70, Z line) of approximately 5 weeks of age were used. Pigs were raised in approved APP-free housing facilities and all animals were negative for antibodies against APP prior to study day 1. Eight animals per group (randomized) were vaccinated twice with an interval of 3 weeks (SD0, SD21). Intranasal and oral administration of the glycomodified vaccine and intramuscular injection of Apx antigen (group 1) were performed as described in section 3.3 above. 2 ml of Porcilis® APP was injected intramuscularly in group 2 animals.

[0165] Challenge was performed on day 42 with a 2 ml intranasal inoculation containing 10E6 cfu / ml of APP2 strain HK361 via a MAD Nasal™ intramucosal spray device (Teleflex® Corporation).

[0166] Blood was collected on SD-1, 7, 14, 20, 28, 35, 41 and 48 for serum isolation and animals were examined for clinical signs.

[0167] Animals were euthanized and dissected on SD48. Lung and pleural lesions were scored according to Hannan et al., 1982, Res. Vet. Sci., Vol. 33(1), pp. 76-88. Scoring was based on the percentage of each lobe (7 sites) affected by typical APP lesions (Table 10). Both the dorsal and ventral surfaces of each lung were palpated and visually inspected, but a single value was arrived at for each lobe based on the average of the total surface area. Dunnett's multiple comparison test and P value were used as the statistical evaluation method.

[0168] Tissue specimens from lungs were sampled from all groups for bacteriological analysis to test for the presence or absence of challenge APP2 strains. For reisolation of APP2 bacteria, lung tissue samples were immersed in treated water for 7 seconds, then disrupted in a stomacher to give a suspension, of which 100 μl was plated on HIS+V plates and incubated overnight at 37° C. and 5% CO2. Colonies were confirmed by MALDI-TOF. Bacterial score values ​​were interpreted as 0=no APP2 bacteria isolated, 1=<20 CFU (colony forming units) APP2 bacteria isolated, 2=<200 CFU APP2 bacteria isolated, and 3=>200 CFU APP2 bacteria isolated.

[0169] None of the pigs showed any abnormal local or systemic reactions after vaccination. Mild elevations in body temperature were observed in all vaccinated pigs 0.5 days after both vaccinations, which returned to normal values ​​1 day after vaccination.

[0170] Body temperature was measured twice daily after challenge. A mild increase in rectal temperature was observed in control group 3 starting one day after challenge. In general, the temperature increase was lower in the vaccinated groups compared to the non-vaccinated control group.

[0171] Animals reaching the human endpoint criteria due to symptoms caused by infection were euthanized and clinically evaluated. Figure 23 shows a graph of the probability of survival for animals in all three groups. On day 2 post-challenge, five of the eight animals in control group 4 had to be euthanized due to increased morbidity. On day 4, one animal in commercial vaccine group 2 had to be euthanized. None of the animals in glycomodified vaccine group 1 showed clinical signs, and all animals survived to the end of the study on day 6 post-challenge.

[0172] Lung lesions were assessed (see FIG. 24, Table 15), and lung scoring of individual animals revealed animals in the negative control group (Group 3) exhibiting lung damage from Hannan scores of 0 to 8 (mean of 3.75). Animals vaccinated with the commercial vaccine (Group 2) showed a trend toward a decrease in lung scores compared to the non-vaccinated control group, although not significant, with a P value slightly above 0.05.

[0173] None of the animals in Group 1 vaccinated with inactivated APP2 and Apx neutralizing epitopes had detectable lung lesions. Vaccinated Group 1 was significantly different from control Group 3 (P=0.007).

[0174] [Table 15]

[0175] Semi-quantitative results of challenge strain reisolation (Figure 25) showed that high numbers of APP2 bacteria could be reisolated in all but one animal in group 3. In the commercial vaccine group 2, APP2 bacteria could be reisolated from four of eight animals, with two animals having high numbers of bacteria. In contrast, none of the animals vaccinated with the glycoengineered vaccine in group 1 were positive for APP2 bacterial reisolation.

[0176] In summary, the results of previous studies could be confirmed: the glycoengineered candidate vaccine was highly effective, did not result in APP2 bacterial reisolation 6 days after challenge, and did not result in the development of lung lesions in vaccinated animals compared to untreated animals. Moreover, efficacy was higher than that observed for animals treated with the commercial APP vaccine.

Claims

1. 1. A Gram-negative bacterial host cell for vaccine use comprising: (a) a heterologous functional Actinobacillus pleuropneumoniae (APP) rfb gene cluster, wherein the heterologous functional APP rfb gene cluster binds to a lipid A core of a bacterial host cell to produce an APP O-antigen located on the bacterial host outer surface, and wherein the endogenous rfb gene cluster of the bacterial host cell is not functional. Including, (b) a heterologous promoter for regulating transcription of the heterologous APP rfb gene cluster, which is stronger than the endogenous promoter for the endogenous rfb gene cluster; (c) at least one further gene for the functional expression of an enzyme that aids in the synthesis of said APP O antigen; and / or (d) containing or not containing at least one neutralizing epitope of the Apx toxin; The heterologous rfb gene cluster (i) comprises or consists of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4; (ii) has at least 90% nucleic acid sequence identity to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4; and / or (iii) is degenerate with respect to the nucleic acid sequence of (i) or (ii); Selected from the APP1-18 rfb gene cluster, The heterologous functional APP rfb gene cluster produces O antigens of APP1-18. Bacterial host cell.

2. 2. The bacterial host cell of claim 1, wherein the bacterial host cell is selected from the group consisting of Enterobacteriaceae, Burkholderiaceae, Pseudomonadaceae, Vibrionaceae, Escherichia coli, and Salmonella.

3. 3. The bacterial host cell of claim 1 or 2, wherein the heterologous functional APP rfb gene cluster expresses at least one protein comprising or consisting of any one of the amino acids of SEQ ID NO: 2, 50-61, or SEQ ID NO: 5, 62-72, or at least one protein having at least 70, 80, 90, 95 or 98% amino acid sequence identity to these sequences.

4. 4. A bacterial host cell according to any one of claims 1 to 3, wherein the endogenous rfb gene cluster of said bacterial host cell is at least partially or completely deleted.

5. 5. The bacterial host cell of claim 1, wherein the heterologous promoter for regulating transcription of the heterologous APP rfb gene cluster is a promoter selected from the group consisting of kanamycin promoter, proD promoter, j23101 promoter, proC promoter, STER_RS05525 promoter, STER_RS01225 promoter, STER_RS04515 promoter, STER_RS05020 promoter, STER_RS06870 promoter, STER_RS00780 promoter, P32 promoter.

6. at least one additional gene for functionally expressing an enzyme that assists in the synthesis of the APP O-antigen is selected from the group consisting of enzymes for nucleotide-activated glycan biosynthesis, undecaprenyl pyrophosphate glycosyltransferase, O-antigen glycosyltransferase, O-antigen polymerase, O-antigen chain length determinant protein, and N-glycan epimerase, and combinations thereof; i. the gne gene encodes a UDP-galactose / UDP-N-acetylglucosamine epimerase, and the gne gene may comprise or consist of SEQ ID NO:6, or may be at least 90% identical to SEQ ID NO:6; and ii. the wzy gene encodes an APP O-antigen polymerase, and the wzy gene may comprise or consist of the codon-optimized wzy of SEQ ID NO:7 or SEQ ID NO:8, or may be at least 90% identical to SEQ ID NO:7 or SEQ ID NO:8; 6. A bacterial host cell according to any one of claims 1 to 5.

7. 7. The bacterial host cell according to claim 1, wherein at least one neutralizing epitope of the Apx toxin is located on the outer surface of the bacterial host cell and is bound to a membrane protein.

8. (a) said heterologous functional APP rfb gene cluster; (b) at least one further gene for the functional expression of an enzyme that aids in the synthesis of said APP O antigen, and / or (c) at least one neutralizing epitope of the Apx toxin; The bacterial host cell according to any one of claims 1 to 7, wherein said gene is codon-optimized for said bacterial host cell.

9. The bacterial host cell for use according to claim 8, wherein said heterologous functional APP rfb gene cluster (a) is codon-optimized for said bacterial host cell.

10. the bacterial host is Escherichia coli or Salmonella; (a) the heterologous functional APP rfb gene cluster is selected from an APP1-18 rfb gene cluster; (b) the heterologous promoter for regulating the transcription of the heterologous APP rfb gene cluster is a kanamycin or proD promoter; and (c) the at least one additional gene for functionally expressing an enzyme that assists in the synthesis of the APP O-antigen is a wzy gene and / or a gne gene, both genes being integrated into the genome of the bacterial host cell or located on a plasmid; (d) containing or not containing at least one of the neutralizing epitopes of Apx toxins I, II and III; 10. A bacterial host cell according to any one of claims 1 to 9.

11. the bacterial host is Salmonella enterica subsp. enterica serovar Typhimurium; (a) the codon-optimized heterologous functional APP rfb gene cluster, (i) comprises or consists of SEQ ID NO:3; (ii) has at least 90% nucleic acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3; and / or (iii) is degenerate with respect to the nucleic acid sequence of (i) or (ii); APP2 rfb gene cluster, the endogenous rfb gene cluster of said bacterial host cell is at least partially or completely deleted; (b) the heterologous promoter for regulating the transcription of the heterologous APP2 rfb gene cluster is the kanamycin promoter; and (c) the at least one additional gene for functionally expressing an enzyme that assists in the synthesis of the APP O antigen is a gne gene and / or a wzy gene; i. the gne gene, comprising or consisting of SEQ ID NO:6, or having a nucleic acid sequence at least 90% identical to SEQ ID NO:6; and ii. the wzy gene comprises or consists of SEQ ID NO:7 or SEQ ID NO:8, or has a nucleic acid sequence that is at least 90% identical to SEQ ID NO:7 or SEQ ID NO:8; (d) containing or not containing at least two neutralizing epitopes of at least Apx toxins II and III; 11. The bacterial host cell of claim 10.

12. the bacterial host is Escherichia coli; (a) the heterologous functional APP rfb gene cluster (i) comprises or consists of SEQ ID NO:3; (ii) has at least 90% nucleic acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3; and / or (iii) is degenerate with respect to the nucleic acid sequence of (i) or (ii); APP2 rfb gene cluster, the endogenous rfb gene cluster of said bacterial host cell is at least partially or completely deleted; (b) the heterologous promoter for regulating the transcription of the heterologous APP2 rfb gene cluster is a kanamycin or proD promoter; and (c) the at least one additional gene for functionally expressing an enzyme that assists in the synthesis of the APP O antigen is a gne gene; and the gne gene comprises or consists of SEQ ID NO:6, or has a nucleic acid sequence at least 90% identical to SEQ ID NO:6; (d) containing or not containing at least one of the neutralizing epitopes of Apx toxins I, II and III; 11. The bacterial host cell of claim 10.

13. the bacterial host is Salmonella enterica subsp. enterica serovar Typhimurium or Escherichia coli; (a) the heterologous functional APP rfb gene cluster is an APP8 rfb gene cluster that (i) comprises or consists of SEQ ID NO:4; (ii) has at least 90% nucleic acid sequence identity to SEQ ID NO:4; and / or (iii) is degenerate with respect to the nucleic acid sequence of (i) or (ii), (b) the heterologous promoter for regulating the transcription of the heterologous APP2 rfb gene cluster is a kanamycin or proD promoter; and (c) said at least one further gene for functionally expressing an enzyme of the synthesis of said APP O antigen is a wzy and / or gne gene; i. the gne gene comprises or consists of SEQ ID NO:6, or has a nucleic acid sequence at least 90% identical to SEQ ID NO:6; and ii. the wzy gene comprises or consists of SEQ ID NO:7 or SEQ ID NO:8, or has a nucleic acid sequence that is at least 90% identical to SEQ ID NO:7 or SEQ ID NO:8; (d) containing or not containing at least two neutralizing epitopes of at least Apx toxins II and III; 11. The bacterial host cell of claim 10.

14. 14. The bacterial host cell of claim 1, wherein the bacterial host is live or inactivated.

15. 15. A composition comprising at least one bacterial host cell according to any one of claims 1 to 14.

16. 15. The composition or pharmaceutical composition of claim 14, comprising a bacterial host cell expressing at least two different O antigens from APP.

17. A bacterial host cell according to any one of claims 1 to 14 or a composition according to claim 15 or 16 for use in the prevention and / or therapy of Actinobacillus pleuropneumoniae (APP) infection.

18. 18. The bacterial host cell for use according to claim 17, wherein said bacterial host is administered intranasally, orally, oronasally, sublingually, subcutaneously, intradermally, transdermally, conjunctivally or intramuscularly.

19. A composition comprising a bacterial host cell described in any one of claims 1 to 14, or a composition described in claim 15 or 16, for use in a therapeutic method comprising administering a physiologically effective amount of a bacterial host cell described in any one of claims 1 to 14, or a composition described in claim 15 or 16, to a mammalian subject in need thereof for the treatment and / or prevention of APP infection.

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