WO2013034687A1

WO2013034687A1 - Avibacterium paragallinarum rtx toxin

Info

Publication number: WO2013034687A1
Application number: PCT/EP2012/067506
Authority: WO
Inventors: Joachim Frey; Eliane KÜNG
Original assignee: Universität Bern Verwaltungsdirektion
Priority date: 2011-09-09
Filing date: 2012-09-07
Publication date: 2013-03-14

Abstract

The present invention provides the polypeptide sequences of Avibacterium paragallinarum toxin, its coding nucleic acid sequence, as well as the use of the inventive sequences as a vaccine, antibodies and their use.

Description

AVIBACTERIUM PARAGALLINARUM RTX TOXIN

Background of the invention

The invention relates to a newly identified and characterized bacterial toxin of Avibacterium paragallinarum. The invention also encompasses methods for the production of the A. paragallinarum toxin, as well as its diagnostic, therapeutic, and preventive use, particularly as a traditional, recombinant or DNA vaccine.

Avibacterium (Haemophilus) paragallinarum is a Gram-negative bacterium that belongs to the family of Pasteurellaceae, which is the etiological agent of infectious coryza, a severe acute respiratory disease of chickens affecting mainly laying hens. A. paragallinarum was first described in 1969 as bacterium of the family of Pasteurellaceae requiring β-nicotine adenine di-nucleotide (β-NAD) as growth factor and allocated to the genus Haemophilus. Haemophilus paragallinarum was later reclassified as Avibacterium paragallinarum, which corresponds better with the phylogenetic classification. Currently, mostly β-NAD dependent and but also some β-NAD independent strains of A. paragallinarum are known. A. paragallinarum is non-haemolytic. It is classified into the three serovars A, B and C using a plate agglutination test and is further subdivided into various serotypes, currently recognized as the major Kume serotypes A1 , A2, A3, A4, B1 , C1 , C2, C3 and C4 that show particular geographical spread. Infections with A. paragallinarum generally result in nasal discharge, conjunctivitis, swelling of the sinuses and the face, lacrimation, anorexia and sometimes swelling of the wattles and diarrhoea. Chicken herds infected with A paragallinarum decrease their egg production by 10 to 40% and show mortality rates of about 5%, causing significant losses in the poultry industry.

Currently, little is known about the molecular nature of virulence factors of A. paragallinarum. Hemagglutinin is the antigen that was studied most extensively in A. paragallinarum; it is suggested to be important in its pathogenicity of the bacterium, showing considerable variability among serovars and serotypes. Serovar B has been reported to be devoid of hemagglutinin or has a hemagglutinin that differs strongly from serovars A and C. Further potential virulence factors that have been described are secreted metalloproteases, which are able to degrade chicken IgG, the capsular polysaccharides that are speculated to be involved in colonization of the host tissue, as well as a putative protein toxin that, however, never was purified or identified until present.

Vaccination against infectious coryza is possible by commercial vaccines consisting of whole formalinized bacteria. However, the particular antigens necessary to induce protective immunity are unknown and chickens have to be vaccinated separately against each of the three A. paragallinarum serovars, since there is no cross protection between the different serovars or serogroups. Thus, the use of autogenous vaccines appears to be more effective in control of infectious coryza if these vaccines contain serovars which are present in a country or locally. Design of novel vaccines, as well as of methods for the assessment of their in vitro potency and of diagnostic methods, is mainly hampered by a lack of basic knowledge about the molecular mechanisms of pathogenicity, particularly the main virulence attributes of A. paragallinarum.

The objective of the present invention to overcome or at least mitigate the disadvantages of the prior art. This objective is attained by the subject-matter of the independent claims.

Summary of the invention

The present invention focuses on a hitherto unpublished polypeptide, which represents a major cytotoxic activity of A. paragallinarum. This polypeptide, herein referred to as Avibacterium RTX toxin A (AvxA), in its naturally occurring toxic form confers A. paragallinarum its strong cytotoxic activity against the avian macrophage like cell line HD11.

The toxin, AvxA, is a secreted bivalent serine-protease - RTX-porin toxin. Anti-AvxA antibodies fully neutralize secreted cytotoxin in cell free supernatants of liquid cultures of serotypes A, B and C of A. paragallinarum.

The polypeptide of the invention is defined by its amino-acid sequence SEQ ID NO 1 and the subsequence containing the major protective antigen SEQ ID NO 2 which is part of SEQ ID NO 1 and by the nucleotide (nt) sequence of the gene encoding this polypeptide SEQ ID NO 3 or of the subsequence SEQ ID NO 4. The polypeptide of the invention binds to polyclonal antibodies raised against the protein according to SEQ ID NO 2, defined as the AvxA-RTX moiety. Polyclonal antibodies directed against the AvxA-RTX moiety of the polypeptide of the invention neutralize the cytotoxic activity of polypeptide AvxA or of supernatants of bacterial cultures of virulent A. paragallinarum against avian cells of the avian macrophage like cell line HD11. Anti-AvxA antibodies are specific to AvxA and do not cross-react with ApxIA and conversely anti-ApxIA antibodies do not cross-react with AvxA, showing that this novel protein, AvxA, or its sub-domain AvxA-RTX is different from the predicted RTX protein of A. paragallinarum as postulated previously (Ramon Rocha M.O. et al., 2006; FEMS Microbiol Lett. 257, 63-68).. Hence the polypeptide of the invention enables the design of a novel vaccine to treat an infection with A. paragallinarum and adequate potency tests and diagnostic tests to detect A. paragallinarum infections.

In a construction, the polypeptide of the invention may be expressed recombinantly, i.e. expressed as a result of genetic engineering, which engineering leads to a combination of genes not present in naturally occurring organisms. This way, the polypeptide of the invention can be designed in a dedicated fashion, thus enabling the provision of a constitution that comprises in essence only the polypeptide of the invention in its inactive from of the present invention or at least no other polypeptides that correspond to A. paragallinarum polypeptides.

The present invention also pertains to DNA that encodes the polypeptide as described above. The DNA may be purified as a separate entity from the organism or synthesised and placed in the genome of different species using genetic engineering techniques to arrive at recombinant DNA at recombinant DNA. In an application, the DNA comprises the DNA according to SEQ ID NO 3 or SEQ ID NO 4, or even restricted to DNA according to those sequence identifiers.

Detailed description of the invention

According to a first aspect of the invention, an isolated AvxA toxin polypeptide is provided. This polypeptide comprises an amino acid sequence at least 85% identical to SEQ ID 2, which is the protein sequence of Avibacterium paragallinarum toxin AvxA-RTX domain

Identity in the context of the present invention is a single quantitative parameter representing the result of a sequence comparison position by position. Methods of sequence comparison are known in the art; the BLAST algorithm available publicly is an example.

In one embodiment, the isolated AvxA toxin polypeptide comprises an amino acid sequence at least 85% identical to SEQ ID 1 , which is the protein sequence of Avibacterium

paragallinarum toxin AvxA.

In one embodiment, the isolated polypeptide comprises a sequence with at least 90%, 91 %, 92%, 93%, or 94% sequence identity, preferably more than 95% 96% or 97% sequence identity, most preferably 98% or more sequence identity to SEQ ID. 1 or SEQ. ID 2.

According to a second aspect of the invention, the isolated AvxA toxin polypeptide sequence of the invention is provided for use as a vaccine against infection by Avibacterium

paragallinarum, particularly in poultry.

In one embodiment, the isolated AvxA toxin polypeptide is provided as a recombinant protein vaccine, optionally adsorbed on alumina and/or formulated with other acceptable excipients and adjuvants.

In one embodiment, the isolated AvxA toxin polypeptide vaccine may be provided as a recombinant transgenic vaccine, such as, by way of non-limiting example, a recombinant salmonella bacterium or a recombinant Escherichia coli bacterium expressing a polypeptide comprising a sequence tract at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID 1 or SEQ ID 2. Similarly, a yeast cell such as Pichia pastoris secreting recombinant AvxA fused to an adjuvant peptide is considered.

In one embodiment, the isolated AvxA toxin polypeptide vaccine comprises an AvxA polypeptide according to the instant invention that further comprises, or is covalently or non- covalently associated to, a polypeptide sequence active as an immune stimulant or vaccine carrier protein, such as a tetanus toxoid or diphtheria toxin -particularly CRM 197 (cross- reactive material), a nontoxic mutant diphtheria toxin-, an outer membrane protein complex derived from Neisseria meningitidis serogroup B strain B1 1 , H. influenzae-der^'wed protein D or Escherichia coli heat labile LT-toxin-B-subunit.

According to yet another aspect of the invention, an isolated bacterial cell wall is provided that comprises a recombinant AvxA toxin. Such system is referred to as a "bacterial ghost" and described in detail in US patents 6,951 ,756, 7,541 ,039, and 7,858,357, all of which are incorporated herein by reference.

According to another aspect of the invention, a ligand such as, by way of non-limiting example, an antibody or antibody fragment selectively reactive to an AvxA toxin polypeptide sequence is provided. According to one embodiment, the ligand, for example the antibody or antibody fragment, binds to and neutralizes the polypeptide of SEQ ID 2.

In some embodiments, the antibody or antibody fragment is a monoclonal antibody, particularly preferred therein an IgG molecule, binding to or raised against an AvxA toxin polypeptide as provided herein. According to another embodiment, a preferred ligand is a single-domain antibody, for instance a single-chain variable fragment (scFv) antibody selected by phage display methods, or a camelide antibody, specifically reactive to AvxA toxin polypeptide.

Alternatively, a ligand employed in practising the invention may be a molecule engineered or selected to demonstrate a high specificity and avidity for an AvxA toxin polypeptide. Such engineered or selected molecule may be a antibody-like polypeptide or synthetic antibody selected for by phage-display (see Pini et al., J.Biol. Chem. (1998) 273, 21769-21776; United States Patent Nos. 5,223,409; 5,403,484; 5,571 ,698; 5,837,500; 5,849,500; 5,985,588; 6,127, 132; 6,387,627, and 6,730,483, all of which are incorporated by reference herein), or by other evolutionary selection methods. Alternatively, the ligand may be a polynucleotide or spiegelmer with high selectivity for an AvxA toxin polypeptide (e.g. SELEX, see Ellington & Szostak, Nature 346, 818-822; United States Patent Nos.5,270, 163; 5,475,096; 5,496938; 5,567,588; 5,580,737, all of which are incorporated by reference herein).

According to yet another aspect of the invention, the antibody, antibody fragment or ligand of the invention is provided for prevention or therapy of infection by Avibacterium paragallinarum, particularly in poultry. Such compound may be administered to a patient in need thereof in a form or dosage as required by the indication, wherein administration by injection is preferred.

According to yet another aspect of the invention, a nucleic acid sequence encoding an isolated AvxA toxin polypeptide sequence of SEQ ID 1 or SEQ ID2 is provided. One embodiment of this aspect of the invention is exemplified by the sequences of SEQ ID 3 and SEQ ID 4. Other sequences encoding the AvxA toxin fall within the scope of the present invention; particularly sequences optimized for codon usage in the respective intended host, for example a bacterium for recombinant production of isolated protein as a vaccine, or for recombinant expression by a mammalian or bacterial host for use as a live or attenuated vaccine.

According to another aspect of the invention, a vaccine against infection by Avibacterium paragallinarum is provided, comprising a nucleic acid sequence of the invention, as defined in the previous paragraph, under control of a promoter sequence operable in a mammalian cell. DNA is particularly preferred in practicing this aspect of the invention. In one

embodiment, the promoter is a CMV promoter.

The art of DNA vaccine development is well known. Any DNA expression cassette comprising the vaccine DNA sequence as defined above or exemplified by the sequences ID 3 and ID 4 of the present invention under control of a promoter sequence operable in a mammalian cell, optionally having an eukaryotic termination sequence on its 3' terminus, will suffice. "Classic" examples are plasmid DNA molecules containing the described elements; other designs such as linear sequence tracts are known also (see US7074772, incorporated by reference herein).

According to yet another aspect of the invention, an isolated cell is provided that comprises a transgene expression construct for the expression of AvxA toxin. Such expression construct comprises a nucleic acid sequence of the invention, according to the above definition. In one embodiment, the cell is a bacterial cell such as an attenuated salmonella cell. US

applications or granted patents US2008220022 (A1), 8062645, 7935355 and 8062645, all of which are incorporated herein by reference, exemplify the art of making salmonella-based vaccines.

The use of such isolated cell or bacterial ghost comprising a transgene expression construct for the expression of AvxA toxin as a vaccine against infection by Avibacterium

paragallinarum, particularly in poultry, is another aspect of the invention.

According to another aspect of the invention, a method for manufacturing of an isolated AvxA toxin polypeptide is provided, comprising expressing a nucleic acid sequence of the invention in a transgenic host under control of a suitable promoter. Expression systems and suitable vectors are known in the art and include, without limitation, prokaryotic systems such as E. coli, Bacillus sp., particularly B. subitilis, yeast expression systems such as pichia, or mammalian expression systems. Prokaryotic systems are preferred in practicing the present invention.

According to yet another aspect of the invention, a method for the detection of Avibacterium paragallinarum in a sample is provided, comprising contacting the sample with an antibody, antibody fragment or ligand of the invention that recognizes an AvxA toxin polypeptide. The binding of the antibody, antibody fragment or ligand of the invention may then be determined by reaction of a second antibody or other ligand, optionally attached to a fluorescence marker or reporter enzyme or a macroscopically detectable ligand such as a latex bead. Binding of the antibody, antibody fragment or ligand of the invention to a toxin may similarly be detected directly by, for example, surface plasmon resonance.

Similarly, a kit for detection of Avibacterium paragallinarum toxin is provided, comprising an antibody, antibody fragment or ligand of the invention of the invention.

Short description of the figures

Fig. 1 shows an overview of the genetic structure of the avx operon (upper part; C:

Putative acetyltransferase AvxC, A: AvxA structural toxin gene, B: AvxB protein, D: AvxD, protein with bridge function to outer membrane for type I secretion; Π: Transcription attenuator (between A ad B) and transcription stop (downstream D); a detail of the avx promoter (lower part, left; P: promoter; SD: Shine Dalgarno sequence) and details of AvxA protein (lower part, right).

Fig. 2 shows an SDS PAGE gel (left), and immunoblots of secreted proteins (middle and right; AH: amphiphatic helix; GGXGXD: gly-rich repeats; SeSi: secretion signal; numbers refer to base pairs).

Fig. 3 shows the cytotoxicity determination using chicken macrophage-like cells

HD11.

Fig. 4 shows cytotoxicity or neutralization of cytotoxicity of culture supernatants

(secreted cytotoxins) of A. paragallinarum serovar A, B and C using chicken macrophage-like cells HD11. HD1 1 cells were incubated with supernatant of cultures from A. paragallinarum strains JF4211 serovar A ("A"); JF4999 serovar B ("B"); JF5000 serovar C ("C") or with supernatants that were treated antecedent with IgG from hyper-immune anti-rAvxA rabbit serum or with IgG from pre-serum as control. The cells had been stained 24h post inoculation using vital stain (see Examples) to determine the amount of live cells (dark) and dead cells (bright). A, B, C: no serum; AS, BS, CS: Anti-rAvxA serum; R: rabbit pre-immunization serum; K: positive control. Examples

Bacterial strains and culture conditions

Avibacterium paragallinarum type strain ATCC29545^T (= NCTC11296) and a field strain of A. paragallinarum serovar A strain JF421 1 , serovar B strain JF4999 and serovar C strain JF5000 isolated from chicken with coryza, were used. A. paragallinarum was grown either on Chocolate agar medium + IsoVitaleX (BD diagnostic systems, Sparks, MD, USA) or AVG agar medium (1 % Bacto Trypton, 0.5% Bacto Yeast Extract, 0.5% NaCI, 0.25% Dextrose, 0.25% K₂HP0₄ 1.5% Bacto Agar w/v, ^g/ml β-NAD) incubated at 37°C with 5% C0₂ for 36-48. Liquid cultures were grown in AVG medium (1 % Bacto Trypton, 0.5% Bacto Yeast Extract, 0.5% NaCI, 0.25% Dextrose w/v, 0.25% K₂HP0₄, 10 μg/ml β-NAD) at 37°C with slow shaking.

Escherichia coli strain DH5a (endA1 , hsdRM (rk^",mk⁺), supEAA, thi-1 , recA^ , gyrA (NaIR) rem , A(lacZYA-argF), strain BL21 (DE3) (F, ompT, hsdSB, (r_B-,m_B-), DE3 [ DE3 i21 l /ac/ /acL/\/5 /acZ T7-RNA-pol, int^"]), strain 5K (thi, thr, leuBQ, lacY^ , supE, tonA2 , hsdR) and strain JF522 (thi, thr, leuBQ, /acY1 , supE, tonAT.†, /7sdfi7pLG575 hlyBD) were grown on Luria-Bertani (LB) broth or LB agar complemented with the corresponding antibiotics for selection of recombinant plasmids (Ampicillin 100 μg/ml, Kanamycin 50 μg/ml and Chloramphenicol 25 μg/ml) at 37°C.

Isolation and sequence analysis of genomic DNA from A. paragallinarum

Genomic DNA from A. paragallinarum was isolated from bacteria directly taken from the Chocolate agar plates using chaotrophic guanidium-thiocyanate buffer. The isolated DNA was stored at -20°C.

Genome sequencing (75bp paired-ends, insert size 600bp) was performed using an lllumina Genome Analyzer II using standard protocols. Reads were first controlled for quality and filtered using the Fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) to remove any read with undetermined nucleotides "N". Finally the reads were trimmed at position 70. The cleaned data set was used for de novo assembly using concurrently Velvet, ABySS and

SOAPdenovo with varying parameters. The three best assemblies (one for each tool) were combined using MUMmer. The draft was iteratively improved with a combination of tools like

GapCloser, resulting in 2475 contigs with a total length of 2.8 x 10⁶ base pairs We annotated this draft genome sequence automatically using a pipeline developed previously. The data were screened for potential virulence genes.

Gene cloning and expression of cloned genes on plasmid vectors

Gene cloning, restriction enzyme digestion, ligation, transformation, plasmid extraction, agarose gel electrophoresis and isolation of DNA fragments were performed using standard methods. Expression of cloned genes was induced by 100μηι IPTG in E. coli host strains DH5a or BL21 (DE3).

Plasmid constructs were sequenced to control the correct construction using BigDye v.3.1 with T3, T7 and internal primers on an ABI prism 3130 Gene Sequencer. Sequencer 4.6 (Gene Codes Corporation, Ann Arbor, Ml, USA) was used for sequence analysis.

Cloning expression and purification of recombinant AvxA-RTX part and AvxC

The part of the avxA gene containing the RTX domain was amplified by PCR using the following oligonucleotide primers: AvxA_RTX_L (tatgcggccg caGATTTCGA TGATGTGCTA GTGGGG) and AvxA_RTXR (gagctcCJAJ TTCCAATTCGC TGCC) (nucleotides in lower cases added to create Not\ and Sacl restriction sites, respectively [in italics]) (Microsynth, Balgach, Switzerland), Pwo DNA-polymerase (Roche) and genomic DNA of A. paragallinarum JF4211 as template at an annealing temperature of 53°C. The PCR fragment was purified, digested with restriction enzymes Not\ and Sad and cloned into the Not\ and Sad restriction sites of expression vector pBluescript KS (Stratagene), resulting in plasmid pAvxA-MP7 that expresses the RTX domain of AvxA (AvxA-RTX) from the vector's IPTG inducible lac-promoter pi_ac.

In order to produce a recombinant, poly-histidine tailed RTX domain of AvxA (AvxA- RTX-10xHis) peptide the following oligonucleotide primers: AvxA_RTX-His_L atccafggGATTTCGATGATGTGCTAGTGGGG and AvxA_RTX-His-R ATGGATCC atggafccTTTCCAATTCGCTGCCAA (nucleotides in lower cases added to create Nco\ and SamHI restriction sites, respectively [in italics]; Microsynth, Balgach, Switzerland), Pwo DNA- polymerase and genomic DNA of A. paragallinarum JF421 1 as template at an annealing temperature of 54°C. The PCR fragment was purified, digested with restriction enzymes Nco\ and SamHI and cloned in the Nco\ and SamHI restriction sites of a His-tail expression vector (pETHIS-1) resulting in plasmid pAVX2 that expresses the 10xHis-tailed RTX domain of AvxA (AvxA-RTX-10xHis) from the vector's T7 promoter in E. coli strain BL21 (DE3) after induction with 0.1 mM IPTG. The poly-His tagged proteins AvxA-RTX-10xHis were pruified on a nickel affinity column (Qiagen, Basel, Switzerland) from guanidium thiocyanate lysed bacterial cultures with a pH gradient for elution, followed by extensive dialysis of the corresponding fractions against PBS buffer (50 mM Na-phosphate, pH 7.5, 144 mM NaCI). The purified protein appears as a single 80 kDa peptide on SDS PAGE gels.

The avxC gene encodes protein AvxC, the putative activator of AvxA. For recombinant expression of AvxC, an IncQ vector based on the replicon of RSF1010 was constructed. The plasmid is compatible with the above expression systems (based on plasmid pETHIS-1 and E. coli expression strain BL21 (DE-3) carrying the avxA or hlyBD genes). For this purpose the 1.5 kb SamHI - Sma\ fragment with the chloramphenicol resistance gene and part of the polylinker of vector pJFF224NX (Frey J., 1992; Res. Microbiol. 143, 263-269) was replaced by the 1.2 kb SamHI - Stu\ kanamycin resistance gene fragment (aph(3')-la from Tn903) from plasmid pSSVI 186-in (Viret, 1993) to result in vector pJFF225Km. The avxC gene was amplified by PCR using the primers AVXCupLSac (gggagcTCTG ATAGTGGGAG AATTAGATAA C) and AVXCmidRPst (ttctgcagAJ AGTCAAATTC CATTATTTTC TC) (nucleotides in lower cases added to create Sacl and Pst\ restriction sites, respectively), Pwo DNA-polymerase with proofreading activity and genomic DNA of A. paragallinarum JF4211 as template at an annealing temperature of 62°C. The PCR fragment was purified, cut with restriction enzymes Sad and Pst\, and ligated into the Sacl, Pstl site of vector pJFF225Km to obtain plasmid pJFF225-AvxC.

Expression and secretion of inactive recombinant AvxA-RTX was obtained with E. coli strain JF4814 (containing the specific recombinant genes avxA-RJX and hlyBD), which was constructed by transforming E. coli strain JF522 (Maier, E. et al., 1996; Infect. Immun. 64, 4415-4423) containing the HlyBD type I secretion system with plasmid pAvxA-MP7. Expression and secretion of toxically active AvxA-RTX was obtained from strain JF4965 (containing the specific recombinant genes avxA-RTX, avxC and hlyBD), which was constructed by transformation of strain JF4814 with plasmid pJFF225-AvxC. Both strains JF4814 and JF4965 secrete a 80 kDa protein that is found the culture supernatant as analyzed on SDS PAGE control gels and that reacts with rabbit anti AvxA-RTX antibodies on immunoblots (Figure 2).

Figure 2 shows an SDS PAGE gel of secreted proteins as culture supernatant (Su) and total proteins (To) of strain JF4814 (E. coli 5K hlyBD, avxA) after IPTG induction. Middle: Immunoblot of secreted proteins as culture supernatant (Su) and total proteins (To) of strain JF4814 (E. coli 5K hlyBD, avxA) after IPTG induction reacted with IgG anti-AvxA-RTX antibodies. Right: Immunoblot of supernatants of A. paragallinarum JF421 1 (1 and 2) and ATCC29545 (3) and washed bacteria A. paragallinarum JF4211 (4) and ATCC29545 (5) reacted with IgG anti-AvxA-RTX antibodies. St: protein molecular mass standard, masses indicated on the left border. Note that endogenous AvxA secreted by A. paragallinarum has a molecular mass of 95 kDa while recombinant AvxA-RTX is 80 kDa on the immunoblot and 75.4 as calculated from the amino-acid sequence(see arrows).

Production of rabbit anti-ApxA antiserum

Aliquots of 500 μΙ_ containing 200 μg of highly purified recombinant protein AvxA- RTX-10xHis were mixed with the same quantity of Gerbu adjuvant for immunizing rabbits three times at intervals of two weeks. Rabbits were bled two weeks after the last immunization and all serum samples were then incubated for 30 min at 56°C to inactivate complement. Immunoglobulin G (IgG) was purified using a Sephadex Protein A column.

Immuno blot analysis of recombinant peptides and A. paragallinarum culture supernatant.

Culture supernatant and sedimented bacteria of A. paragallinarum grown in AVG medium for 24h, were obtained by centrifugation at 10Ό00 x g for 30 min. Aliquots of 15 μΙ of suspended and washed bacteria or culture supernatants (containing secreted proteins from A. paragallinarum) or recombinant proteins obtained from E. coli culture supernatants or after purification by Ni-NTA chelating chromatography were analyzed by sodiumdodecyl sulfate- polyacrylamide gel electrophoresis (PAGE) on 12% polyacrylamide gels. The proteins separated by PAGE were transferred to nitrocellulose membranes (BioRad Laboratories, Hercules, USA). The membranes were blocked by incubation for 12h in 1 % blocking buffer, washed and then incubated with the corresponding rabbit polyclonal anti antibodies or purified IgG, diluted 1 : 1000 in blocking buffer followed by incubation with phosphate labelled conjugate goat anti-rabbit IgG heavy and light chains (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) diluted 1 :2000 in blocking buffer for 90 min and then reacted with the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) (Sigma Aldrich, St. Louis, MO, USA) according to the suppliers instructions.

Cell lines, and cytotoxicity assay

HD1 1 macrophage-like cells, derived from MC29 transformation of chicken bone marrow cells ( Beug H. et al., 1979; Cell 18, 375-390) were grown in 24-well microtiter plates containing 0.5 ml RPMI1640 medium + GlutaMAX™-! + 25 mM HEPES (Gibco, Carlsbad, USA) supplemented with 10% foetal bovine serum (FBS) in a humidified environment of 5% C0₂ at 37°C.

To test for cytotoxicity, cells were cultured over night to approximately 20% confluence reading corresponding to 2x10⁵ cells per well. Polymyxin was added at a concentration of 10μg/ml in order to protect cells against LPS 3 h prior adding the potential protein-toxins to be tested. Subsequently, 100 μΙ of proteins obtained from culture supernatants of A. paragallinarum strain ATCC29545 or JF421 1 , adjusted to a concentration of 5 μς/ΓπΙ, or from recombinant E. coli strains secreting non activated AvxA-RTX (strain JF4814) or activated AvxA-RTX (strain JF4965) and adjusted to a concentration of 50 μg/ml, were added to the HD1 1 cells and incubated for 24 h or 48 h. Cytotoxicity was determined by vital cell staining using the kit Cytotox neutral red version 2.3 according to the producer's instructions. Live and dead cells were determined photospectrometrically using an ELISA reader at 540 nm.

For neutralization experiments, recombinant AvxA-RTX protein-toxin or native AvxA toxin from culture supernatant of exponential of stationary phase liquid cultures of A. paragallinarum was incubated with rabbit anti AvxA-RTX antibodies 1 : 10 (vol./vol.) or with monospecific anti-AvxA-RTX IgG (3 μg IgG per 1 μg recombinant activated AvxA-RTX) at 37°C for 10 min prior application to HD11 cells. Rabbit pre-immunization serum or IgG purified from pre-immunization serum were used as controls.

The avx operon in A. paragallinarum expresses AvxA, a composite protease-RTX toxin

Fig. 1 shows an overview of the genetic structure of the avx operon (upper part), details of the avx promoter (left, lower part) and details of the AvxA protein showing the serine-peptidase domain (fair grey) and the RTX domain (dark grey) with the 11 glycine rich repeats that have Ca2+ binding capacity (right side, lower part). C: Putative acetyltransferase AvxC, A: AvxA structural toxin gene, B: AvxB protein, an ATPase activity and selectivity type I secretion system D: AvxD, protein with bridge function to outer membrane for type I secretion; Π: Transcription attenuator (between A ad B) and transcription stop (downstream D).

A. paragallinarum type strain ATCC29545 and the serovar A field strain JF421 1 both secrete a heat labile cytotoxic product that is sensitive to proteinase K as revealed by incubating 100 μΙ aliquots of supernatants of A. paragallinarum cultures in AVG medium (corresponding 0.5 μg protein) of standard cultures in AVG medium of these strains on chicken cells HD1 1 , incubating for 24 h and subsequent vital staining of the cells. The HD11 cells showed rounding and detached from the surface after exposure to A. paragallinarum supernatants. This indicates that A. paragallinarum produces or secretes one or several major protein toxins. Supernatants of A. paragallinarum strains ATCC29545 and JF4211 show no haemolytic activity towards sheep or chicken erythrocytes.

Screening of the annotated genomic sequence of A. paragallinarum strain JF421 1 revealed a 1 1 '385 nucleotide polycystronic operon, representing a typical operon structure of a pore forming RTX cytolysin with 4 genes encoding the activator protein AvxC, the structural toxin AvxA and the two type I secretion proteins AvxB and AvxD (Figure 1). The avxCABD operon is preceded by a transcription stop signal from the operon upstream avxCABD followed by a prokaryotic promoter sequence. This is followed by a consensus ribosome binding site (Shine-Dalgarno sequence, RBS) 6 bp upstream the start codon AGT of avxC. The gene avxC has a length of 432 bp encoding a 144 amino acid (aa) protein of 15.8 kDa (pi 6.25) and is terminated by a TGA stop codon. Gene avxC is followed by a 601 nt intergenic sequence and then followed by avxA of 6'582 nt, which encodes the structural AvxA toxin of 2194 aa and a calculated molecular mass of 238.147 kDa and a pi of 4.6. Gene avxA is preceded by an RBS 6 bp upstream the ATG start codon and terminated by a TAG stop codon. Gene avxA is followed by a transcriptional attenuator sequence and then by genes encoding the type I secretion system, avxB and avxD both containing their proper RBS (Figure 1). Interestingly, the calculated molecular mass of AvxA of 238 kDa is more than twice as that of typical RTX toxins, which is in the order of 110 kDa. The 703 aa C- terminal domain of AvxA shows characteristics of RTX toxins with 11 glycin rich nona-peptide repeats, preceded by a potential acylation site Gly-Lys and reveals highest similarity to the calcium binding haemolysin of Chromobacteirum violaceum (22% identical aa and 44% similar aa). The 1491 N-terminal aa domain of AvxA shows characteristics to a serine protease of the subtilase family and reveals highest similarity to the serine protease of Pseudomonas putida KT2440 (38% identical aa and 55% similar aa).

Southern blot DNA: DNA hybridization analysis of genomic DNA from A. paragallinarum strain ATCC29545 using DNA probes made from sequences of avxA and avxB and PCR amplification of the individual genes avxCABD from A. paragallinarum strain ATCC29545 and subsequent DNA sequence analysis revealed that the same avxCABD operon is present in both the A. paragallinarum field strain JF4211 and type ATCC29545.

Activation of cytotoxicity of recombinant RTX moiety of AvxA by AvxC

In order to analyze whether the RTX moiety of AvxA is a cytotoxin, recombinant

AvxA-RTX was expressed from cloned avxA-RTX gene on IPTG inducible cloning vector pBluescript KS in E. coli 5K harbouring the E. co//-haemolysin HlyBD type I secretion system (strain JF4814). A recombinant peptide of 80 kDa was found in the culture supernatant after IPTG induction, which was absent in the parent strain JF522 harbouring the empty cloning vector only. When chicken HD11 macrophage-like cells were incubated with this 80 kDa recombinant AvxA-RTX peptide, only minor cell rounding and detachment was shown and vital staining revealed 70% surviving cells after 24 h or 48 h. However, when the same recombinant protein was produced in E. coli 5K from the same vector but in the presence of AvxC, expressed in trans constitutively from an IncQ cloning vector (strain JF4965) the protein showed distinct toxicity towards HD11 cells after 24 h, with 0% surviving cells as measured by vital staining (Figure 3). These experiments show that the AvxA-RTX moiety has characteristic cytotoxic activity after activation with its corresponding AvxC activator protein.

Figure 3 shows the cytotoxicity determination using chicken macrophage-like cells HD11. The cells had been stained with vital stain (see material and methods) to determine the amount of live cells (dark) and dead cells (bright). The following substances were used to inoculate the cell cultures:

A1 , B1 , supernatant of Escherichia coli 5K JF522 (control)

A2, B2, non-activated recombinant AvxA-RTX

A3, B3, recombinant AvxA-RTX activated by AvxC in the E. coli host

A4, B4, recombinant activated AvxA-RTX preincubated with anti-AvxA-RTX rabbit serum

A5, B5, activated AvxA-RTX pre-incubated with purified anti-AvxA-RTX rabbit IgG A6, B6, no substance added (control)

C1 , D1 , activated AvxA-RTX pre-incubated with anti-AvxA-RTX preserum.

C2, D2, activated AvxA RTX pre-incubated with anti-AvxA-RTX preserum IgG

C3, D3, supernatant of A. paragallinarum ATCC29545T

C4, D4, supernatant of A. paragallinarum JF421 1

C5, D5, supernatant of A. paragallinarum JF4211 pre-incubated with anti-AvxA-RTX preserum IgG

C6, D6, supernatant of A. paragallinarum JF421 1 pre-incubated with anti-AvxA-RTX IgG The cytotoxic activity of activated Avx-RTX was inhibited with monospecific polyclonal antibodies directed against non-activated AvxC-RTX or with IgG from monospecific polyclonal antibodies directed against highly purified AvxA-RTX-10xHis.

Autocleavage of AvxA by Avibacterium paragallinarum

Supernatants of exponential growth or stationary phase liquid cultures of A. paragallinarum strains ATCC29545^T and JF4211 (serovar A) show on immunoblots, reacted with monospecific anti AvxA-RTX IgG, a distinct protein of 95 kDa size, however, at low concentration which is invisible on simple SDS PAGE stained with coomassie blue. This indicates that the RTX moiety of AvxA is cleaved and secreted separately from the serine- peptidase moiety of AvxA (Figure 2). No larger bands corresponding to the full sized 240 kDa AvxA peptide could be detected, even in early exponentially phase and after addition of exogenous proteinase inhibitors, indicating that cleavage off the RTX moiety of AvxA must occur before or concomitantly with secretion during release to the growth medium. Antibodies directed to the Actinobacillus pleuropneumoniae toxin Apxl did not detect any peptides on these immunoblots and did not cross react with recombinant AvxA-RTX. Neutralization of total cytotoxic activity of secreted Avibacterium. paraaallinarum proteins by anti - AvxA-RTX-10xHis antibodies as proof of concept

Since purified IgG from monospecific rabbit antiserum directed against AvxA-RTX- 10xHis was able to neutralize the cytotoxic activity of recombinant, activated AvxA-RTX, we tested whether these IgG also were able to neutralize native AvxA produced and secreted by A. paragallinarum serovars A, B and C. Secreted proteins of A. paragallinarum serovars A, B and C from exponentially and stationary phase liquid cultures revealed toxicity to chicken HD11 cells as shown by vital staining of the chicken cells after incubation with supernatants for 24 h (Figure 3, 4). However, when supernatants of these cultures were pre-incubated with rabbit anti-Avx-RTX serum or with monospecific, polyclonal rabbit anti-Avx-RTX IgG, fully neutralized the cytotoxic effect of all serovars towards HD1 1 cells even after extended incubation for 48 h leaving > 90% of the cells alive (Figure 3, 4). Serum or IgG from the same rabbits taken before immunization had no neutralizing effect. Also anti-Apxl antibodies did not neutralize the cytotoxic activity of recombinant AvxA-RTX or native AvxA (Figure 3). Conclusions

The examples illustrate a novel 240 kDa toxin AvxA-RTX that is secreted by Avibacterium paragallinarum. This toxin belongs to the composite RTX toxins having two main domains in the structural toxin, the N-terminal domain representing a serine protease and the C-terminal domain representing a RTX cytotoxin. During secretion, or prior to secretion, AvxA is proteolytically cleaved into a 95 kDa peptide that is recognized by antibodies directed against recombinant AvxA-RTX domain peptide. Since monospecific polyclonal rabbit antibodies and IgG directed against recombinant non-activated AvxA-RTX domain neutralize the entire cytotoxic activity found in supernatant of cultures of any of the serotypes A, B and C of A. paragallinarum, the RTX domain of AvxA appears to represent the major cytotoxic activity of A. paragallinarum. Recombinant non activated AvxA-RTX that is produced e.g. in a recombinant Escherichia coli strain containing a gene encoding for the structural AvxA-RTX protein but not the activator gene avxC or consequentially native, inactivated AvxA, produced in an Avibacterium paragallinarum strain in which the avxC gene has been deleted, can induce neutralizing antibodies against the cytotoxic effect of A. paragallinarum. Hence AvxA- RTX or native AvxA or a genetically engineered strain of A. paragallinarum with a deleted avxC gene can serve as a vaccine or vaccine componens, inducing neutralizing antibodies in vaccinated animals that protect against disease.

Claims

1. An isolated polypeptide comprising an amino acid sequence at least 85% identical to SEQ ID 2, for use as a vaccine against infection by Avibacterium paragallinarum.

2. An isolated polypeptide according to claim 1 , comprising an amino acid sequence at least 85% identical to SEQ ID 1 , for use as a vaccine against infection by

Avibacterium paragallinarum.

3. An antibody, antibody fragment or ligand selectively reactive to an isolated

polypeptide sequence as defined in any of claims 1 or 2, for use in a method for prevention or therapy of infection by Avibacterium paragallinarum.

4. A nucleic acid sequence encoding an isolated polypeptide sequence as defined in any of claims 1 or 2, for use in a method for prevention or therapy of infection by Avibacterium paragallinarum.

5. A nucleic acid sequence according to claim 4, comprising the sequence of SEQ ID 3 or SEQ ID 4.

6. A vaccine against infection by Avibacterium paragallinarum, comprising a nucleic acid sequence according to claim 4 or 5.

7. An isolated cell comprising a transgene expression construct comprising a nucleic acid sequence according to claim 4 or 5.

8. A method for manufacturing an isolated polypeptide sequence according to claim 1 or 2, comprising expressing a nucleic acid sequence of claim 4 or 5 in a transgenic host under control of a suitable promoter.

9. An isolated polypeptide comprising an amino acid sequence at least 85% identical to SEQ ID 2.

10. An isolated polypeptide according to claim 1 , comprising an amino acid sequence at least 85% identical to SEQ ID 1.

1 1. An antibody, antibody fragment or ligand selectively reactive to an isolated

polypeptide sequence as defined in any of claims 9 or 10.

12. A nucleic acid sequence encoding an isolated polypeptide sequence according to any of claims 9 or 10.

13. A nucleic acid sequence according to claim 12, comprising the sequence of SEQ ID 3 or SEQ ID 4.

14. A method for the detection of Avibacterium paragallinarum in a sample, comprising contacting the sample with an antibody according to claim 1 1.

15. A kit for the detection of Avibactenum paragallinarum in a sample, comprising an antibody according to claim 11.