CA2593558C - Vaccine against clostridium perfringens - Google Patents

Abstract

There is provided a vaccine for controlling Clostridium perfringens in animals, and particularly necrotic enteritis in poultry?. The vaccine may comprise a C. perfringens antigenic polypeptide or variant thereof, a nucleic acid encoding the C. perfringens antigenic polypeptide or variant thereof, or a recombinant cell producing the C. perfringens antigenic polypeptide or variant thereof..

Description

June 22, 2007 Canadian Patent Application Vaccine against Clostridium perfringens Field of Invention The invention relates to the production of a vaccine. More specifically, the invention provides a vaccine for controlling Clostridium perfringens in animals.
Background of the Invention Clostridium are characterized as spore-forming, anaerobic, Gram positive bacilli. The species, Clostridium perfringens, can be subdivided into subspecies. Five subspecies have been described. These subspecies are generally known as "type" A-E. All subspecies produce several toxins, both major and minor toxins. The four major toxins are the alpha, beta, epsilon and iota toxin. All C. perfringens types produce the alpha-toxin. The beta-toxin is produced by C. perfringens types B and C. In addition, a range of minor toxins is produced by all C. perfringens types.
One or more of these various toxins can play a role in C. perfringens related pathogenesis. Type A is known to be pathogenic for various birds, man, cows and pigs. Type B is mainly pathogenic for lamb, sheep and goat, and causes "lamb dysentery"
and haemorrhagic enteritis. Type C is pathogenic for man, sheep, calf, lamb, pig, and bird. C.
perfringens can cause of "struck", haemorrhagic enteritis, necrotic enteritis and enterotoxemia.
Necrotic enteritis (NE) is an economically important enteric disease of birds, for example poultry, caused by Clostridium perfringens. The disease is usually controlled by antimicrobial drugs administered at prophylactic doses either in water or feed. However, there is concern about the routine prophylactic use of antimicrobial drugs in food animal production because of their contribution to resistance problems. If antimicrobial drugs were banned for such purposes in North America, there might be an increase in NE in poultry, for example chicken flocks, as has happened in Scandinavia (12).
Although vaccination offers an alternative approach to antimicrobial drugs in control of the disease, very little is known about immunity to NE. However, there has been considerable work on immunity to C. perfringens in other circumstances, since it is a cause of gas gangrene in people. This has identified the alpha-toxin, a phospholipase C

2 exoenzyme, both as a major virulence factor and as an important immunogen. For example, a genetically engineered vaccine inducing alpha-toxin (amino acids 247-370) serum antibodies was shown by Williamson and Titball (34) to neutralize hemolytic activity of the toxin and to provide protection against C. perfringens in mice. Bennett et al.
(5) showed that a recombinant Vaccinia virus vector expressing the non-toxic C-domain region of the alpha-toxin protein provided antibody-mediated protection against experimental toxin challenge.
More recently, Stevens et al. (30) showed significant prevention of gas gangrene in mice by immunization using the C-terminal domain of the alpha -toxin (amino acids 247-370). In addition, there is evidence based on naturally occurring antibodies or maternal vaccination that antibodies to alpha-toxin are involved in immunity to NE (10,19).
However, the importance of alpha-toxin or any other protein in immunity to NE in birds, for example chickens, remains to be demonstrated, and one study has shown the immunizing effects of alpha-toxin minus mutants (32). A recent study also demonstrated that an alpha-toxin minus mutant produced NE experimentally in chickens, demonstrating that factors other than alpha-toxin are important in the pathogenesis of NE (14). Other studies have shown that the immunizing ability to protect against NE was associated with virulent rather than with avirulent C. perfringens (32).
While the prior art has demonstrated some immunizing effect of whole-cell C.
perfringens in chickens, the basis of this immunity is poorly understood. NE
is usually controlled by antimicrobial drugs but, if these are unavailable or not used, there is currently no other simple way to control infection. Therefore, there is a need for novel vaccine for controlling Clostridium perfringens in birds.
An object of an aspect of the present invention is to provide a novel vaccine for controlling Clostridium perfringens in birds.
Summary of the Invention In an aspect, there is provided a vaccine for controlling C. perfringens in an animal comprising an isolated nucleic acid molecule which comprises a nucleic acid sequence that encodes a C. perfringens secreted antigenic polypeptide or a variant thereof.
In another aspect, there is provided a vaccine for controlling C. perfringens in an animal comprising an isolated C. perfringens secreted antigenic polypeptide or a variant thereof.
=

3 In yet another aspect, there is provided a vaccine for controlling C.
perfringens in an animal comprising a recombinant cell producing an isolated C. perfringens secreted antigenic polypeptide or a variant thereof.
In accordance with another aspect, there is provided a vaccine for controlling C.
perfringens in an animal comprising an isolated C. perfringens secreted antigenic polypeptide comprising an amino acid sequence of Figure 1 (SEQ ID NO:1) or a variant thereof conferring an immunoprotective effect against C. perfringens, wherein the variant comprises an amino acid sequence having at least 80 % identity with the amino acid sequence of Figure 1 (SEQ ID NO:1).
In accordance with another aspect, there is provided a vaccine for controlling C.
perfringens in an animal comprising a recombinant cell producing an isolated C.
perfringens secreted antigenic polypeptide comprising an amino acid sequence of Figure 1 (SEQ ID NO:1) or a variant thereof conferring an immunoprotective effect against C.
perfringens, wherein the variant comprises an amino acid sequence having at least 80 %
identity with the amino acid sequence of Figure 1 (SEQ ID NO:1).
In accordance with another aspect, there is provided a use of an isolated C.
perfringens secreted antigenic polypeptide comprising an amino acid sequence of Figure 1 (SEQ ID NO:1) or a variant thereof conferring an immunoprotective effect against C.
perfringens for preparation of a vaccine, wherein the variant comprises an amino acid sequence having at least 80 % identity with the amino acid sequence of Figure 1 (SEQ
ID NO:1).
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the invention are given by way of illustration only, since various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.
Brief Description of the Drawings The present invention will become more fully understood from the detailed description given herein and from the accompanying drawings, which are given by way of illustration only and do not limit the intended scope of the invention.
Figure 1 shows an amino acid sequence of Hypothetical Protein (HP) of C.
perfringens Strain 13, GenBank Accession # 18144943.

3a Figure 2 shows an amino acid sequence of Pyruvate ferredoxin oxidoreductase (PFOR) of C. perfringens Strain 13, GenBank Accession # 18311043. The underlined portion corresponds to the amino acid sequence of truncated PFOR (tPFOR).
Figure 3 shows an amino acid sequence of Elongation factor-G (EF-G) of C.
perfringens Strain 13, GenBank Accession # 18311390.
Figure 4 shows an amino acid sequence of Perfringolysin 0 of C. perfringens Strain 13, GenBank Accession # 18143820.
Figure 5 shows an amino acid sequence of Glyceraldehyde 3-phoshate dehydrogenase (GPD) of C. perfringens Strain 13, GenBank Accession # 18144966.
Figure 6 shows an amino acid sequence of Fructose bi-phosphate aldolase (FBA) of C. perfringens Strain 13, GenBank Accession # 18310332.

4 Figure 7 shows recombinant C. perfringens histidine-tagged proteins purified from Escherichia coli cells. (A) Coomassie stained purified proteins (B) Reactivity of purified proteins to immune serum from chickens immune to necrotic enteritis. In each panel, Lane 1- Alpha-toxin (45 kDa), Lane 2- GPD of Figure 5 (40 kDa), Lane 3- FBA of Figure 6 (35 kDa), Lane 4- tPFOR of Figure 2 (67 kDa), Lane 5- HP of Figure 1 (90-100 kDa) and Lane M-Molecular mass standards.
Figure 8 shows a summary of mean lesion scores of immunized broiler chicken groups challenged with C. perfringens infected feed, together with the concurrent unimmunized controls. VC-vehicle-only controls, A-tox- alpha-toxin, FBA-Fructose 1,6-biphosphate aldolase, GPD- Glyceraldehyde 3-phosphate dehydrogenase, tPFOR-Truncated pyruvate: ferredoxin oxidoreductase, HP- Hypothetical protein, Sup- culture supernatant of C. perfringens, G+H- combination of GPD and HP, Exp- Experiment. + -birds in this group were challenged for 3 days and autopsied on day 6. ++ -birds in this group were given a severe challenge. * -immunized group that had significantly fewer chickens with lesions compared to unimmunized vehicle-only controls; Fisher's exact test, p< 0.05.
Figure 9 shows serum IgY ELISA titres of broiler chickens immunized intramuscularly with C. perfringens purified proteins. Serum collected at three time-points;
Day 0- Pre-immunization titre, Day 10- Mid-experiment, Day 20- Pre-challenge titre. FBA-Fructose 1,6-biphosphate aldolase, GPD- Glyceraldehyde 3-phosphate dehydrogenase, tPFOR-Truncated pyruvate: ferredoxin oxidoreductase, HP-Hypothetical protein, Exp- Experiment.
* Significant titre values when compared to pre-immunization titres, p < 0.05.
Figure 10 shows intestinal IgY and IgA ELISA titres of broiler chickens immunized intramuscularly with C. perfringens purified proteins. Samples analyzed were from pooled intestines collected from at least 10 chickens in each group. FBA-Fructose 1,6-biphosphate aldolase, GPD- Glyceraldehyde 3-phosphate dehydrogenase, tPFOR- Truncated pyruvate:
ferredoxin oxidoreductase, HP- Hypothetical protein, Exp-Experiment..
Detailed Description of the Preferred Embodiments A vaccine is provided for controlling Clostridium perfringens in animals. The vaccine may comprise a C. perfringens antigenic polypeptide or variant thereof, a nucleic acid molecule encoding the C. perfringens antigenic polypeptide or variant thereof, or a recombinant cell producing the C. perfringens antigenic polypeptide or variant thereof.

Administration of the vaccine to a subject can confer an immunoprotective effect to the subject against C. perfringens. The vaccine may be for prophylactic, therapeutic, or both prophylactic and therapeutic treatment.
The vaccine will typically comprise an isolated C. perfringens secreted antigenic

5 polypeptide or variant thereof, an isolated nucleic acid molecule encoding the C. perfringens secreted antigenic polypeptide or variant thereof, or a recombinant cell producing the C.
perfringens secreted antigenic polypeptide or variant thereof.
An antigenic polypeptide may be provided by any source or method, for example, natural isolate or recombinant or synthetic origin or suitable combinations thereof.
Administration of the antigenic polypeptide to a subject can confer an immunoprotective effect to the subject against C. perfringens. The antigenic polypeptide may be of any length provided that the immunoprotective activity is maintained. The sequence of the antigenic polypeptide may be based on a complete or partial naturally occurring amino acid sequence of a polypeptide that naturally occurs in virulent C. perfringens type A. An antigenic polypeptide may be used either singly or in combination with other polypeptides, antigenic or otherwise, in the preparation of a vaccine. A polypeptide refers to a chain of amino acids, for example peptides, oligopeptides, or proteins, having a biological function, and does not refer to a specific length of the chain.
An isolated C. perfringens antigenic polypeptide is a polypeptide that has been identified and separated and/or recovered from at least one component of its natural environment. The isolated polypeptide will typically have been purified by at least one purification step, and, in some embodiments purification may be achieved (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the C.
perfringens antigenic polypeptide natural environment will not be present. An isolated polypeptide may be produced by synthetic or recombinant techniques, for example as described in J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press. An isolated polypeptide produced as a result of recombinant techniques may be referred to as a recombinant polypeptide.
A nucleic acid encoding an antigenic polypeptide may be any nucleic acid molecule of, for example. cDNA, genomic DNA, synthetic DNA or RNA origin or suitable combinations thereof. Administration of the nucleic acid encoding an antigenic polypeptide to a subject

6 can confer an immunoprotective effect to the subject against C. perfringens.
The nucleic acid may be of any length provided that the immunoprotective activity is maintained by the encoded antigenic polypeptide. The sequence of the nucleic acid encoding an antigenic polypeptide may be based on a complete or partial naturally occurring nucleic acid sequence found in virulent C. perfringens type A. A nucleic acid sequence encoding an antigenic polypeptide may be used either singly or in combination with other nucleic acid sequences, encoding antigenic polypeptides or encoding any other desired polypeptide, in the preparation of a vaccine.
An isolated nucleic acid molecule encoding a C. perfringens antigenic polypeptide is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid.
Such an isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. An isolated nucleic acid molecule encoding a C.
perfringens antigenic polypeptide includes nucleic acid molecule encoding a C.
perfringens antigenic polypeptide contained in cells that ordinarily express the C.
perfringens antigenic polypeptide where, for example, the nucleic acid molecule is in a chromosomal or extrachromosomal location different from that of natural cells. The isolated nucleic acid molecule may be referred to as a recombinant nucleic acid molecule where the isolated nucleic acid molecule has been manipulated using recombinant techniques, for example, as described in 3. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press.
Variants include, without limitation, analogs, derivatives, fragments, truncations, mutants, deletions, substitutions, insertions, fusions and the like.
An antigenic polypeptide or a nucleic acid encoding an antigenic polypeptide may be mutated or changed or derivatised in any manner desired (for example, any number or combination of deletions, insertions, or substitutions) to produce a corresponding variant.
Use of such variants in producing vaccines and in vaccinating a subject is contemplated, and such a variant nucleic acid or variant polypeptide may be mutated or changed or derivatised in any manner in comparison to a naturally occurring nucleic acid or polypeptide sequence, respectively, found in virulent C. perfringens (type A), provided that the capability of conferring an immunoprotective effect against C. perfringens is maintained.
Similarly, nucleic acids or polypeptides having varying degrees of sequence identity to a corresponding naturally occurring nucleic acid or polypeptide sequence found in virulent C.
perfringens (type A) may be tolerated without eliminating an immunoprotective activity against C.

7 perfringens. For example, a vaccine may comprise an antigenic polypeptide having a sequence that is identical to a naturally-occurring form of the antigenic polypeptide or a variant thereof that has a sequence that is at least 80% identical to a naturally-occurring form of the antigenic polypeptide. As another example, a vaccine may comprise a nucleic acid molecule having a coding sequence that is identical to a naturally-occurring form of the coding sequence or a variant thereof that has a sequence that is at least 70%
identical to a naturally-occurring form of the coding sequence. Determination of sequence identity of proteins and nucleic acids by computer based methods, as well as nucleic acid hybridization techniques using high stringency conditions for determining or identifying nucleic acid sequences that share high (eg., at least 70%) sequence identity are well known to the skilled person.
Stringency of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.
Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of sequence identity between the probe and hybridizable sequence, the higher the relative temperature which can be used. High stringency conditions may be identified by those that:
(1) employ low ionic strength and high temperature for washing, for example 0.015 M
sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50 C.;
(2) employ during hybridization a denaturing agent, such as formamide, for example, 50%
(v/v) formamide with 0.1% bovine serum albumin/0.1% Fico11/0.1%
polyvinylpyrrolidone/50 mM
sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 C.; or (3) employ 50% formamide, 5xSSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM
sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 pg/ml), 0.1% SDS, and 10% dextran sulfate at 42 C., with washes at 42 C. in 0.2xSSC (sodium chloride/sodium citrate) and 50% formamide at 55 C., followed by a high-stringency wash consisting of 0.1xSSC containing EDTA at 55 C.
Hybridization and wash times should be sufficient for achieving equilibrium.
Percent (%) sequence identity of amino acid or nucleic acid sequences with respect to antigenic polypeptides as, for example in Figures 1 to 6, and nucleic acid sequences encoding antigen polypeptides is the percentage of residues in a candidate sequence that are identical with the antigenic polypeptide amino acid sequence or the antigenic polypeptide-encoding nucleic acid sequence, as the case may be, after aligning the

8 sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity or percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over a desired length of sequence, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 residues or even the full-length of the sequences being compared.
When considering an antigenic polypeptide or variant thereof, the variant antigenic polypeptide will typically have an amino acid sequence that is at least 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98 percent identical to the corresponding antigenic polypeptide.
When considering a nucleic acid sequence encoding an antigenic polypeptide or variant thereof, the variant nucleic acid sequence will typically be at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98 percent identical to the corresponding nucleic acid encoding the antigenic polypeptide.
Techniques and strategies for producing variants are well known in the art. In one example, with regard to polypeptides, an antigenic polypeptide may be modified in vivo or in vitro by, glycosylation, amidation, phosphorylation, carboxylation, truncation, fragmentation, substitution, and the like without eliminating an immunoprotective activity against C.
perfringens. In another example, with regard to nucleic acids, substitution mutations can be made in a nucleic acid encoding an antigenic polypeptide such that a particular codon is changed to a codon which codes for a different amino acid. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e. by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. Groupings of amino acids are known to the skilled person. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine,

9 tyrosine, asparagine, and glutamine. The positively charges (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any number of such substitutions or any other type of alteration (eg., deletion or insertion) or modification may be tolerated provided that the immunoprotective effect of the antigenic polypeptide is not eliminated.
Recombinant cells, comprising an antigenic polypeptide or a nucleic acid sequence that encodes an antigenic polypeptide may be used as vaccines for controlling C.
perfringens. Recombinant cell types may include any cell type that is compatible with the physiology of an intended vaccination subject. Cells of eukaryotic or prokaryotic origin may be used. Prokaryotic cells that can survive within the gastrointestinal system of an intended vaccination subject may be particularly useful for preparation of oral or enteral vaccines. For example, cells that form part of the intestinal flora of an intended vaccination subject (such as Eschercichia coli or Lactobacillus species) may be used. In another example, aviru lent Salmonella or Listeria or other attenuated invasive bacterial cells may be used.
A cell may be altered or modified to comprise a nucleic acid sequence that does not naturally occur in the cell, and as such the cell will be considered recombinant. In other examples, a cell may be altered or modified to comprise an additional copy of a nucleic acid sequence that naturally occurs in the cell, and such cells will also be considered recombinant. As is understood by one of skill in the art, a nucleic acid encoding an antigenic polypeptide may be introduced into a cell using any known technique, for example, microinjection, electroporation, viral transfection, lipofectamine transfection, calcium phosphate precipitation and the like. In certain non-limiting examples, a bacterial cell may be modified by introduction of a nucleic acid molecule encoding an antigenic polypeptide, and then the modified cells may be administered to a subject. In certain other examples, a nucleic acid molecule encoding an antigenic polypeptide may be incorporated into an appropriate construct or vehicle, for example a viral construct, and administered to a subject such that the nucleic acid molecule encoding the antigenic polypeptide is introduced and expressed in at least a portion of the cells of the subject.
A nucleic acid encoding an antigenic polypeptide may be operably linked to control sequences, typically in the context of a suitable vector. A useful control sequence may be any nucleic acid element that is necessary or advantageous for expression of the coding sequence of the nucleic acid sequence. Each control sequence may be native or foreign to the nucleic acid sequence encoding the antigenic polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, or a transcription terminator. Alternatives for incorporating control sequences are readily available to the skilled person. For example, a nucleic acid encoding an antigenic polypeptide may be under the control of an endogenous upstream promoter, or it may be put under control of a heterologous upstream promoter.
Examples of suitable promoters for directing the transcription of the modified nucleotide sequence, such 5 as PS4 nucleic acids, in a bacterial host include the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis alpha-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the promoters of the Bacillus subtilis xylA and xylB
genes, the

10 promoter of the Bacillus subtilis aprE gene and a promoter derived from a Lactococcus sp.--derived promoter including the P170 promoter. When the gene encoding the PS4 variant polypeptide is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter.
For transcription in a fungal species, examples of useful promoters are those derived from the genes encoding the, Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral .alpha.-amylase, A. niger acid stable .alpha.-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans acetamidase.
Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris A0X1 or A0X2 promoters.
Still further suitable promoters are available to the skilled person, for example, cytomegalovirus, Rous Sarcoma Virus, synthetic pox viral promoter, pox synthetic late promoter 1, pox synthetic late promoter 2 early promoter 2, pox 01L promoter, pox 14L
promoter, pox 13L promoter; pox 12L promoter, pox IlL promoter, pox DIOR
promoter, PRV
gX, HSV-1 alpha 4, chicken beta-actin promoter, HCMV immediate early, MDV gA, MDV gB, MDV gD, ILT gB, BHV-1.1 VP8 and ILT gD and internal ribosomal entry site promoter.
A suitable vector may be any vector (for example, a plasmid or virus) which can incorporate a nucleic acid sequence encoding an antigenic polypeptide and any desired control sequences and can bring about the expression of the nucleic acid sequence . The choice of the vector will typically depend on the compatibility of the vector with a host cell into which the vector is to be introduced. In certain examples, the vector may exist as an extrachromosomal entity, with replication being independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome, or an artificial

11 chromosome. In other examples, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Still other examples of vectors and techniques for manipulating vectors will be known and apparent to the skilled person.
Recombinant cells may comprise an antigenic polypeptide or a nucleic acid sequence encoding an antigenic polypeptide, either singly or in combination, with other desired polypeptide or nucleic acid molecules, respectively, to optimize vaccination efficacy.
Furthermore, a nucleic acid sequence may be mutated or altered prior to introduction into the cells as desired, for example for codon optimization for expression in a particular cell .. type. In addition, a nucleic acid sequence may be altered to encoded a fusion of an antigenic polypeptide with one or more other polypeptide as desired in an application, for example fusion with a targeting polypeptide or a carrier polypeptide.
As is understood by the skilled person, administration of a vaccine can be done in a variety of manners. For example, administration may be done intramuscularly, subcutaneously, intravenously, intranasally, intradermaly, intrabursally, in ovo, ocularly, orally, intra-tracheally or intra-bronchially, as well as combinations of such modalities. The dose of the vaccine may vary with the size of the intended vaccination subject. Methods of administration are known to the skilled person, for example, U.S. Pat. Nos.
5,693,622;
5,589,466; 5,580,859; and 5,566,064. The amounts of polypeptide, nucleic acid sequence, or recombinant cell needed for preparation of a vaccine is well understood by one of skill in the art.
An antigenic polypeptide, a nucleic acid encoding an antigenic polypeptide, or a recombinant cell, may be used in combination with a pharmaceutically acceptable carrier for preparation of a vaccine. Pharmaceutically acceptable carriers for vaccines are well known .. to those skilled in the art and include but are not limited to proteins, sugars, and the like.
One example of such a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as hydrolyzed proteins, lactose, and the like.
Another example of an acceptable carrier is 0.01-0.1M, and preferably 0.05M, phosphate buffer or 0.8% saline. Acceptable carriers may be aqueous or non-aqueous solutions, .. suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Examples of aqueous carriers are water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Preservatives and other additives for vaccines are also well know to the skilled person, for example antimicrobials, antioxidants, chelating agents, inert gases, organic acids and the like.

12 Acceptable adjuvants, for example as described in US Patent No. 6,908,620, may be used to enhance the immune response to an antigenic polypeptide. Acceptable adjuvants include, without limitation: polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers; immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one ore more non-methylated CpG
units (W098/16247); oil in water emulsion; cation lipids containing a quaternary ammonium salt;
cytokines; aluminum hydroxide or aluminum phosphate; or any combinations or mixtures thereof.
An oil in water emulsion adjuvant can be based on, for example, light liquid paraffin oil (European pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil resulting from the oligomerization of alkenes, e.g. isobutene or decene, esters of acids or alcohols having a straight-chain alkyl group, such as vegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate), glycerol tri(caprylate/caprate) and propylene glycol dioleate, or esters of branched, fatty alcohols or acids, especially isostearic acid esters. The oil is used in combination with emulsifiers to form an emulsion. The emulsifiers may be nonionic surfactants, such as: esters of on the one hand sorbitan, mannide (e.g.
anhydromannitol oleate), glycerol, polyglycerol or propylene glycol and on the other hand oleic, isostearic, ricinoleic or hydroxystearic acids, said esters being optionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymer blocks, such as Pluronic, e.g., L121.
Examples of adjuvant polymers crosslinked acrylic or nnethacrylic acid, especially crosslinked by polyalkenyl ethers of sugars or polyalcohols. U.S. Pat. No.
2,909,462, provides examples of such acrylic polymers crosslinked by a polyhydroxyl compound having at least three hydroxyl groups, preferably no more than eight such groups, the hydrogen atoms of at least three hydroxyl groups being replaced by unsaturated, aliphatic radicals having at least two carbon atoms.
An example of a cationic lipid adjuvant is DMRIE (N-(2-hydroxyethyl)-N,N-dimethy1-2,3-bis(tetradecyloxy)-1-propane ammonium; W096/34109), either alone or associated with a neutral lipid, for example, DOPE (dioleoyl-phosphatidyl-ethanol amine), to form DMRIE-DOPE.
Examples of cytokine adjuvants are granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), interferon alpha (IFN alpha), interferon beta (TEN beta), interferon gamma, (IFN gamma), interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12),

13 tumor necrosis factor alpha (TNF alpha), tumor necrosis factor beta (TNF
beta), and transforming growth factor beta (TGF beta). A cytokine adjuvant can be in form of a cytokine polypeptide or a nucleic acid sequence encoding a cytokine polypeptide.
Still further adjuvants known to the skilled person include, without limitation, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, keyhole limpet hemocyanins, dinitrophenol, and the like.
Adjuvants may be co-administered or sequentially administered with a vaccine.
The vaccine described herein can be useful for controlling C. perfringens in an animal, for example a bird, a cow or a pig. The vaccine may be useful in any bird, wild, domesticated or commercially farmed, for example, chicken, turkey, goose, duck, pheasant, quail, pigeon and ostrich.
When introducing elements disclosed herein, the articles "a", "an", "the", and "said"
are intended to mean that there are one or more of the elements unless the context dictates otherwise. For example, the term "a compound" and "at least one compound" may include a plurality of compounds, including mixtures thereof. The terms "comprising", "having", "including" are intended to be open-ended and mean that there may be additional elements other than the listed elements.
The above disclosure generally describes preferred embodiments. A more complete understanding can be obtained by reference to the following specific Examples.
These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
Examples Example 1: Clostridium perfringens Antigens Recognized by Broiler Chickens Immune to Necrotis Enteritis Four strains of C. perfringens (CP1, CP4, CP5, and CP6) used in this study are clinical isolates from field cases of NE. Strains CP1 and CP4 are virulent, and CP5 and CP6 avirulent, isolates, as assessed by their abilities to cause NE (32).
Clostridium perfringens cells were grown anaerobically in an empirically formulated medium (tryptic soy broth [Difco] 50%, nutrient broth [Difco] 25% and peptone water [Difco] 25%) for 24 h at 37 C, and the cells and culture supernatant were collected thereafter. The cells were lysed by ,

14 eight freeze-thaw cycles with liquid nitrogen to obtain whole-cell proteins.
The culture supernatant was dialyzed and concentrated by use of 10-kDa cutoff AmiconTM
filters (Millipore Inc., Billerica, MA) to obtain secreted proteins. The protein concentration was determined using a PlusOneTM 2-D QuantTM kit (Amersham Biosciences, San Francisco, CA). The protein contents of concentrated secreted and whole-cell protein samples were 3 to 4 mg/ml. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis under reducing conditions, 100 pg of protein sample was applied.
The secreted and whole-cell proteins were separated by one-dimensional SDS-PAGE in a 12.5 /0 acrylamide gel under reducing and nonreducing conditions (16).
The gels were visualized by Coomassie R-250 staining. The proteins from the gel were transferred to a nitrocellulose membrane of 0.45-pm pore size (Bio-Rad Laboratories) by use of a Hoefer tank buffer system (Amersham Biosciences) followed by reaction with primary antibodies (serum or intestinal washing) at 1:1,000 and 1:500 dilutions, respectively. Serum (source of immunoglobulin Y [IgY]) used in this study was pooled from broiler chickens immune to virulent C. perfringens challenge in infection-immunization experiments (32). The pooled small intestinal washings made from these birds by use of phosphate-buffered saline were dialyzed, concentrated, and used as the source of primary antibody (IgA and IgY) in Western blotting and neutralization experiments. Anti-chicken IgY (heavy plus light chains) and anti-chicken IgA
were used as secondary antibodies at 1:2,000 and 1:1,000 dilutions, respectively.
Specific immunoreactive protein bands were visualized using an alkaline phosphatise-conjugated substrate kit (Bio-Rad Laboratories).
Several protein bands from strain CP4 showed reactivity to immune serum, but similar reactivity was not observed for secreted proteins from CP5. This lack of reactivity was also observed when secreted proteins from avirulent strain CP6 were reacted with immune serum. Secreted proteins from another virulent strain, CP1, showed reactivity similar to that seen for CP4. The secreted protein bands of CP4 that showed reactivity to immune serum were consistently reactive in multiple gels run at different times.
Although there was little reactivity of CP4- and CP5-secreted proteins to intestinal IgA, the reactivity of these secreted proteins to intestinal IgY was similar to that of Western blots done with immune serum. Therefore, it seems that both intestinal and serum IgY
antibodies are important in immunity to this infection. No differences in the whole-cell protein reactivities to serum or intestinal washings between virulent and avirulent strains were observed, suggesting that the trait of immune protection against NE lies in the secreted components of virulent C. perfringens.

Six immunoreactive secreted proteins unique to virulent strains, of which five were highly antigenic, were identified in the parallel-run Coomassie-stained gels by use of the coordinates of molecular-weight-marker hands and the distance of migration.
The gels from the centers of these bands were excised, in-gel digested, and identified by 5 mass spectrometric techniques, namely, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS/MS).
The peptide masses and sequence data from MS analysis were searched against the National Center for Biotechnology Information (NCBI) protein database by MS-Fit and Matrix-Science Mascot search to identify the protein that had the highest homology 10 percentage match. Of the six antigenic secreted proteins unique to the two virulent, innmunoprotective strains identified by MS (Figures 1 to 6), three (perfringolysin 0, fructose 1,6 biphosphate aldolase [FBA], and elongation factor G [EF-G]) are regulated by the VirR-VirS virulence regulon of C. perfringens (3). In addition to virulence genes, this regulon controls genes involved in energy metabolism, such as those encoding FBA

15 and NAD-dependent 13-hydroxybutyryl coenzyme A dehydrogenase, as well as others that may be indirectly involved in bacterial virulence (3,13, 28). It therefore seems possible that the marked difference in the innmunoreactivities of the secreted proteins between the two virulent and the two avirulent strains is the result of a mutation in this regulatory region in the avirulent strains and that the quantity of these proteins produced is too low to be detected in SDS-PAGE and Western blotting experiments. The avirulent strains do produce alpha-toxin; however, the amount produced was not quantified.
Example 2: Antigenic Epitopes of alpha-Toxin Purified C. perfringens alpha-toxin (Sigma Laboratories) was separated by SDS
PAGE and Western blotting performed using chicken immune serum and intestinal antibodies, as well as alpha-toxin antiserum (I G. Songer, University of Arizona) raised in goats. As expected, alpha-toxin antiserum detected purified alpha toxin (43 kDa).
Interestingly, in the Western immunoblot no antibodies to alpha-toxin were detected either in serum or intestinal washings of immune birds. However, further study showed antibodies to alpha toxin were detected in serum at titres of 5000 when native (and not denatured) alpha toxin was used in a lecithinase inhibition assay. In addition, there was immunoreactivity when a Western immunoblot was run against alpha toxin electrophoresed in a non-denaturing gel. This suggests that neutralizing antibodies to alpha toxin are present in immune birds but these may be to conformational rather than linear protein epitopes.

16 Achieving conformational but non-toxic alpha-toxin epitopes in a vaccine may prove challenging. A structure-function analysis of alpha-toxin suggests that the toxicity is associated with the N-terminus whereas the immunogenicity is associated with the C-terminal domain (1, 7, 33). However, the hemolytic activity was found to result from an interaction of both the domains (1). Although many studies have emphasized the importance of non-toxic C-terminal domain in protection against experimental gas gangrene (5, 30, 34), some have shown the neutralizing epitopes to be on the N-terminus (18). It seems likely that the positioning of protective, neutralizing, conformational epitopes of alpha-toxin is subtle and that these epitopes are shared upon folding between both domains, which play a key role in toxicity and also in protective innmunogenicity, thus making them hard to access by antibodies.
ExaMple 3: Cloning, Expression and Purification of Secreted Antigenic Polypeptides from virulent C. Derfrincens:
The chromosomal DNA of the virulent, protective, C. perfringens strain CP4 was used as the source of DNA for cloning of secreted antigens: alpha-toxin, glyceraldehyde 3-phosphate dehydrogenase (GPD), pyruvate: ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase (FBA) and a Hypothetical Protein (HP). PCR was performed using the proofreading DNA polymerase (Qiagen, Mississauga, ON) and primers designed to specifically amplify DNA fragments in the genes. The primers used to amplify the genes are given in Table 1.

17 Table 1. List of primers used to amplify genes encoding proteins used in immunization experiments Gene Sequences 5'-3' Amplicon size (bp) Alpha-toxin Forward-ccgctcgagttgggatggaaaaattgat 1100 Reverse-ccggaattctttatattataagttgaattt Hypothetical protein Forward- 5400 ccgctcgaggaataagagaaaaatagcag Reverse-ccgggtaccacgttaaataaatagaacat Glyceraldehyde 3- Forward- 1000 phosphate ccgctcgagggtaaaagtagctattaacgg dehydrogenase Reverse-ccgggtaccttagaaactaagcattttaaa Fructose 1,6- Forward- 900 biphosphate aldolase ccgcggatccatggcattagttaacgcaaa Reverse-ccgcctcgagagctctgtttactgaaccga Truncated pyruvate: Forward- 1600 ferredoxin ccgcctcgagcacttcattagaaccagttg oxidoreductase Reverse-ccgcggatcctagctaagtagtcttggtct After purification (PCR Purification Kit, QiagenTM, Mississauga, ON), the PCR
products were cloned into plasmid expression vectors so as to generate proteins fused with histidine residues (6-His) either at the N-terminus or at the C-terminus of the protein sequence. Two plasmid vectors, pBAD (for cloning alpha-toxin, GPD and HP) and pET-28 (for cloning FBA and tPFOR), were used in this study.
The resulting plasmids were introduced into E. coil LMG 194 or BL21 Star (DE3) (Invitrogen, Carlsbad, CA), following the manufacturer's instructions. For protein expression, overnight cultures were used to inoculate a fresh Luria broth (LB) medium supplemented with ampicillin or kanamycin (100 pginn1). Bacteria were grown at under aerobic conditions and L-arabinose (St. Louis, MO) (0.2% final) or isopropyl-beta-D-thiogalactopyranoside (IPTG) (Qbiogene, Vista, CA) was added (1 mM) to the bacterial culture in exponential growth phase (0D500-0.5). After a further incubation for 4 h, cells were harvested by centrifugation.
Purification of recombinant proteins was performed by affinity chromatography on nickel-nitrilotriacetic acid (Ni-NTA) agarose following the manufacturer's instructions

18 (Qiagen). Briefly, when the proteins were expressed as soluble proteins, bacterial pellets were resuspended in a buffer (50mM NaH2PO4, 300 mM NaCI) containing lyzozyme (1 mg/ml) and incubated for 60 min in ice. The bacterial cells were lysed using a French pressure cell (3-4 cycles of 1000 psi). The supernatant was collected by centrifugation and added to Ni-NTA agarose. The washing and elution steps were performed using buffers containing increasing concentration of imidazole. Finally, imidazole was removed from the eluted material by dialysis against phosphate buffered saline, pH 7.2, (PBS) and the recombinant proteins were concentrated using Amicon filter -10kD (Millipore, Billerica, MA) and the protein concentration was determined using PlusOne' 2-D Quant kit (Amersham Biosciences, San Francisco, CA).
Purified recombinant proteins were separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a 12.5% acrylamide gel under denaturing conditions as described by Laemmli (16). Proteins were transferred to nitrocellulose membrane of 0.45 pm pore size using a mini-gel transfer assembly (Bio-Rad Laboratories, Hercules, CA). After completion of transfer, non-specific binding sites on the membranes were blocked with blocking buffer containing 1% casein (Bio-Rad Laboratories) and incubated with primary antibodies (immune serum collected from infection-immunized birds in a previous study (32) at 1:1000 dilution. Anti-chicken IgY (H+L) (Cedarlane Laboratories, Hornby, ON) was used as secondary antibody at 1:2000 dilution.
The blots were developed and specific irnmunoreactive protein bands were visualized using an alkaline phosphatase-conjugated substrate kit (Bio-Rad Laboratories) (see Figure 7).
All five genes selected for the immunization study were successfully cloned, expressed and purified to homogeneity.
Cloning of the full length pfor gene was not successful, possibly because of homologous recombination. However, a portion of the gene that encoded a truncated protein (tPFOR) of 67 kDa size (see underlined portion of Figure 2) that contained the Iron- Sulphur (Fe-S) active sites of this enzyme was successfully cloned and purified in large quantities.
Although alpha-toxin was successfully cloned, expressed and purified, the quantity obtained was insufficient for immunization, possibly because of toxicity for the E. coil host.
Hence, commercially available purified alpha-toxin (Sigma Laboratories, St.
Louis, MO) was used for immunization experiments.
Hypothetical Protein (190 kDa) was found to be cleaved into two bands of 90-kDa size upon expression. Attempts to express the entire protein by changing E. coil expression hosts were unsuccessful. Since these two bands reacted strongly to anti-histidine antibodies as well as to immune serum collected from infection-immunized birds (Figure 7)

19 from a previous study (32) these two bands were further purified in large quantities and used in immunization experiments.
Example 4: Immunization Experiments in Chickens Experiments with chickens and conditions for their use were approved by the University of Guelph Animal Care Committee in accordance to the Canadian Council on Animal Care's Guidelines. Commercial 1-day-old male White Plymouth Rock broiler chickens (Bonnie's Chick Hatchery, Elmira, Ont., Canada) were fed an antibiotic-free chicken starter containing 20% protein for 13 days followed by a formulated wheat-based grower feed, containing 28% protein (Arkell Research Station, University of Guelph). Birds were immunized intramuscularly in the pectoral muscle in a volume of 0.2 ml per bird with purified recombinant proteins at different concentrations and frequencies.
In all experiments, the number of birds in each group was between 10 and 20 and all birds were identified individually. Blood was collected from the wing vein from all the groups at three times: pre-immunization, mid-experiment and pre-challenge. Intestinal washings were collected using PBS at autopsy.
For the experimental infection of birds, virulent C. perfringens CP4 was grown in cooked meat medium (Difco) (CMM) for 24 h at 37 C. Fluid thioglycolate medium (FTG, Difco) was then inoculated with a 3% (v/v) inoculum from the C. perfringens infected CMM
and incubated at 37 C for 24 h. The growth at 24 h was log,o 8.24 0.09 C.
perfringens CFU/ml. The inoculated FTG was then mixed with feed at a ratio of 2:1 (v/w).
Inoculated feed was prepared freshly twice per day and fed to chickens for 3-5 days as described.
Autopsy Chickens were euthanized with carbon dioxide gas and their small intestines (duodenum to ileum) examined for grossly visible lesions. Any chickens that had reached a pre-determined severity of clinical illness prior to autopsy were euthanized and later autopsied. Intestinal lesions in the small intestine (duodenum to ileum) were scored as follows: 0 = no gross lesions; 1 = thin or friable wall; 2 = focal necrosis or ulceration; 3 =
large patches of necrosis; 4 = severe extensive necrosis; 5 = chickens that died during experiment, having 4+ lesions (25). Blind scoring was employed to avoid scorer bias. A
pathological change of mild generalized and superficial intestinal necrosis, not previously included in this scoring system (25), was assigned a score of 1+.

A visual summary of mean lesion scores of birds from all immunized groups that received different doses of antigens and challenge across different experiments, together with the concurrent unimmunized controls, is shown in Figure 8.
5 Measurement of the antibody titers in chicken sera and intestinal washings The specific antibody titers were determined by the end point dilution method using TM
an enzyme-linked immunosorbent assay (ELISA). Microtiter plates (Immulon-2, Chantilly, VA) were coated with recombinant proteins (5pg/m1 in 0.1 M carbonate buffer pH
9.6) for 60 min at 37 C followed by an overnight incubation at 4 C. After blocking coated plates for 60 10 min at 37 C with PBS containing 3% of bovine serum albumin (BSA Sigma), immune sera were serially diluted in PBS containing BSA 1% and incubated for 2 h with recombinant protein-coated plates at room temperature. After washing with PBS containing Tween 20 0.1% (PBST), alkaline phosphatase (AP)- coupled anti-chicken IgY (H+L) (1:5,000 in PBS-Tween 20 0.1%-BSA 1%) was added to the microplates and incubated for 60 min at room 15 temperature. After extensive washing with PBST, color reaction was developed using alkaline phosphate substrate kit (Bio-Rad Laboratories) following the manufacturer's instructions. The reaction was stopped by adding 0.4 M NaOH. The absorbance was measured at 405 nm in an ELISA spectrophotometer. The specific antibody titer of immune serum was expressed as the reciprocal of the serum dilution (log2 OD) that gave an A405 value above the cut-off, defined

20 as twice the absorbance value of the un-immunized and mock control wells.
Intestinal antibody response was also measured by ELISA following the procedure described above for serum. Intestinal washings from at least 10 chickens per group were pooled and the total protein content was measured using PlusOneTM 2-D Quant kit (Amersham Biosciences) and used as source of primary antibody after keeping the protein content of the initial dilution (1:10) constant across all the groups.
Alkaline phosphatase conjugated anti-chicken IgY and IgA were used as secondary antibodies at dilutions of 1:4000 and 1:2000 respectively. The end-point titres were determined as described above for serum ELISA.
Although purification of histidine tagged recombinant proteins through Ni-NTA
agarose column should yield eluted protein free of LPS, using mock-immunized (as well as non-immunized control bird) serum to determine the cut-off value further strengthened the interpretation of results. All the proteins used to immunize birds in the immunization experiments described produced significant antigen-specific serum antibody titres (Figure 9) compared to their pre-immunization titres. Alpha-toxoid immunized but non-protected birds

21 as shown in Experiment-1 below had higher titres than toxoid-active toxin immunized birds that were highly protected in Experiment-3 below.
Intestinal antibody responses to all the immunized proteins showed higher IgY
than IgA titers (Figure 10). However, unlike serum, such a difference was not observed between alpha-toxoid (Experiment-1) and toxoid-active toxin boosted (Experiment-3) birds.
Statistical analysis Statistical analysis was performed to determine whether there was a significant difference between the number of birds with lesions from immunized groups and birds from the unimmunized, vehicle-only controls. A two-tailed Fisher's exact test determined whether the two groups differed in the proportion with which they fell into the two classifications of either lesions or no lesions, under the null hypothesis that the proportions were the same.
Data was analyzed in a 2 x 2 contingency table with the unimmunized control group in one column and immunized groups in the other column. Lesion scores were ranked from 0 to 5+, however, a "protective" response was given to birds with <1+ lesions. The null hypothesis was rejected at a = 0.05. For serum ELISA, a one-way ANOVA (Mini-tab 14 software) was used to determine significant differences the antibody titres between pre-immunized and immunized time points across all the groups. Statistical analysis could not be made on intestinal ELISA, since the washings collected were pooled per group.
A summary of the immunization experiments is shown in Table 2.
Table 2. Summary of experimental design Experiment Immunization Dosage of Frequency of Oral Groups vaccine/ administration challenge Bird 1 VC, Supa, Alpha- 20 pg Three times; day 3 days toxoid, GPD, HP, 7, 14 and 21 (mild) FBA and tPFOR
2 VC, MCb, GPD, HP, 40 pg Two times; day 7 5 days FBA and tPFOR and 14 (moderate) 3 VC, Alpha-toxoid/ 20 pg Three times; day 5 days toxin`, GPD, HP, 7, 14 and 21 (severe) tPFOR, combination of GPD and HP
4A VC and FBA 20 pg Three times; day 3 days 7, 14 and 21 (mild-moderate)

22 VC, Alpha-toxin d and 20 pg Three times; day 5 days FBA 7, 14 and 21 (severe) VC- Vehicle-only controls, GPD- glyceraldehyde 3-phosphate dehydrogenase, tPFOR-truncated pyruvate: ferredoxin oxidoreductase, FBA- fructose 1,6-biphosphate aldolase, HP-Hypothetical protein, Sup- crude culture supernatent of virulent C.
perfringens and MC-mock-immunized controls a Birds received 60 pg/inj of culture supernatant that was processed and concentrated following a protocol described earlier (15).
b Birds were mock immunized with an unrelated protein that was cloned, expressed and purified from E. coli in the same manner as C. perfringens related proteins.
C Birds received alpha-toxoid in the first two injections followed by active alpha-toxin in the third.
d Birds in this group received three injections of alpha-toxin where in the first and the third injections were with 20 pg and the second was reduced to 10 pg.
In Experiment-1, birds were immunized with alpha-toxoid, HP, GPD, FBA and tPFOR.
Each bird received three injections of 20 pg of recombinant protein and 50 pg of Quil-A
adjuvant (Superfos Biosector, Vedbaek, Denmark) on days 7, 14 and 21 of age. A
week later, following a 20 h fast, birds were orally challenged 2-3 times a day with virulent C.
perfringens. This challenge was considered 'mild' since the duration of challenge was 3 days and lesions produced in non-immunized birds were relatively mild. Post mortem examination was performed on all birds on day 31, or on birds that died earlier, and the intestinal lesions were scored. A group of birds also received crude culture supernatant of CP4 obtained, processed and concentrated following a protocol described earlier (15) at a dose of 60 pg/
bird/ injection. The unimmunized controls received only Quil-A adjuvant followed by a challenge similar to immunized groups. Purified alpha-toxin was toxoided following the protocol described (11) and used for immunization.
HP, GPD, tPFOR and FBA showed significant protection against a mild infection challenge (Table 3). Immunization with crude culture supernatant that contained all secreted proteins including those purified also showed significant protection. Alpha-toxoid did not protect birds against challenge. Hypothetical Protein showed the greatest protection.

23 Table 3. Intestinal lesion scores of birds immunized with three injections intramuscularly, then infected with a mild challenge by C. perfringens No. of Lesion scores Protein Mean chickens + + + + +
Vehicle-only controls 10 1 3 4 1 1 0 1.55 Culture 8 1 1 0 0 , 0 0.4 supernatant*
Alpha-toxoid 12 3 4 3 1 1 0 1.41 HP* 12 10 2 0 0 0I 0 0.16 GPD* 10 7 2 1 0 0 0 0.4 tPFOR* 10 4 5 1 1 0 0 0 ____________ 01 I
FBA* 10 4 6 010 010 I 0.6 * Immunized groups that had significantly fewer chickens with lesions compared 5 to unimmunized vehicle-only controls; Fisher's exact test, p< 0.05.
In Experiment-2, birds were immunized with HP, GPD and tPFOR. Each bird received two injections of 40 pg of recombinant protein and 50 pg of QuilATM
adjuvant on days 14 and 21 of age. A week later, following 20 h fasting birds were orally 10 challenged with virulent C. perfringens twice a day for five days, from days 28-32 of age.
Since feeding was restricted to twice daily and birds ran out of feed on some occasions, and in light of the moderate lesion scores in unimmunized birds, this challenge was considered moderate'. Post mortem examination was performed on day 33 and intestinal lesions were scored. The unimmunized controls received only QuilATM
adjuvant followed by a challenge similar to immunized groups. A group of birds were mock immunized with an unrelated protein that was cloned, expressed and purified from E. coli in the same manner as C. perfringens related proteins.
HP alone showed significant protection against a moderate challenge, whereas GPD, tPFOR and FBA did not (Table 4). Immunization (mock) with an unrelated purified recombinant fusion protein showed a mean lesion score similar to that of unimmunized controls. The increased mean lesion score of controls compared to Experiment-1 indicated the effect of increased length of challenge.

24 Table 4. Intestinal lesion scores of birds immunized with two injections intramuscularly, then infected with a moderate challenge by C. perfringens 1 ___________________________________________________________________ No. of Lesion scores Protein Mean chickens 3. 2 3 1 4 5 + + + + +
Vehicle-only controls , 18 1 4 9 2 1 1 2.05 Mock controls 10 0 1 6 3 0 0 2.20 HP* 17 10 3 4 0 0 0 0.64 GPD 17 3 8 6 0 0 0 1.17 tPFOR 18 7 4 3 3 0 1 1.33 FBA 18 2 7 6 I 2 0 1 1.66 , *Immunized group had significantly fewer chickens with lesions compared to unimmunized vehicle-only controls; Fisher's exact test, p< 0.05.
In Experiment-3, the immunization schedule was same as in the Experiment-1 but birds were challenged orally for 5 days. This challenge was considered 'severe' since the birds were fed 2 to 3 times a day to ensure that they always had infected feed available.
Birds in this experiment were immunized with alpha-toxoid/toxin, HP, GPD, and tPFOR. A
group of birds received alpha-toxoid in the first two injections followed by active, non-toxoided, alpha-toxin in the third. This modification was made since injections with alpha-toxoid alone in the Experiment-1 did not show protection. The unimmunized controls received only Quil-A adjuvant followed by a challenge similar to immunized groups. A group of birds were immunized with a combination GPD and HP (20 pg each) along with Quil-A and challenged similarly as other groups.
This infection challenge was characterized as severe, since lesions scores in non-immunized chickens were greater than in Experiments- 1 and 2, an enhancement of severity attributed to constant challenge with infected feed. Alpha-toxin immunized birds, which received two initial injections of alpha-toxoid but a third injection with native, non-toxoided, alpha-toxin showed the greatest protection against heavy challenge (Table 5).
Birds immunized with either HP or tPFOR also showed significant protection against severe =

challenge. Although birds immunized with GPD, FBA and the combination of GPD
and HP had mean lesion scores lower than non-immunized controls, no statistical significance was observed.
Table 5. Intestinal lesion scores of birds immunized with three injections intramuscularly, 5 then infected with a severe challenge by C. perfringens.
No. of Lesion scores Protein Mean chickens ++ + + +
Vehicle-only controls 22 0 5 5 6 4 2 2.68 Alpha toxoid/toxin*
a 19 10 8 1 0 0 0 0.53 HP* 20 8 6 4 2 0 0 1.0 GPD 18 4 4 6 1 1 1 , 1.64 tPFOR* 19 9 2 6 2 0 0 1.05 GPD + HP 19 5 5 7 1 I 1 0 I 1.36 *Immunized groups that have significantly fewer chickens with lesions compared to unimmunized vehicle-only controls; Fisher's exact test, p< 0.05.
a Birds in this group received alpha-toxoid in the first two injection and toxin in the third.
In Experiment-4, the immunization schedule was same as in the Experiment-1 and 3, but birds were challenged orally for either 3 ("mild-moderate") or 5 days ("severe"). One group of birds was immunized with FBA and challenged for 3 days and autopsied on the day 6 together with the 3-day challenged unimmunized controls. Two other groups were immunized with either FBA or active (non-toxoided) alpha-toxin, followed by a severe challenge for 5 days. In the active alpha-toxin group, birds received three injections of active alpha-toxin where in the first and the third injections were with 20 pg and the second was reduced to 10 pg since 20 pg dose in the first injection was toxic, causing the death of some birds.
The mean lesion scores in birds of either immunized or unimmunized groups that were challenged for 3 days and autopsied on day 6 (Table 6) were higher than observed in birds of Experiment-1 that were challenged for 3 days and autopsied on day 4 (Table 2). The FBA immunized birds showed significant protection compared to unimmunized controls. In the 5-day-challenged (heavy dose) groups, neither FBA nor the alpha-toxin immunized birds were protected (Table 6). Mean lesion scores of unimmunized controls were comparable to the scores of unimmunized controls in Experiment-3 that also received a severe challenge.
Table 6. Intestinal lesion scores of birds immunized with three injections intramuscularly, then infected with mild-moderate or severe challenge by C. perfringens Protein =No. of Lesion scores Mean chickens + + + + +
A ¨
Groups infected with mild-moderate challenge Vehicle-only controls 10 0 5 2 0 1 2 2.3 FBA* 13 4 6 1 1 0 1 1.23 Groups infected with severe challenge Vehicle-only controls 11 0 3 2 3 1 2 2.72 Alpha-toxina 10 0 2 4 4 0 0 2.2 FBA 14 1 4 9 0 0 0 1.57 *Immunized group that had significantly fewer chickens with lesions compared to unimmunized vehicle-only controls; Fisher's exact test, p< 0.05.
a- Birds in this group received three injections of alpha-toxin where in the first and the third injections were with 20 pg and the second was reduced to 10 pg.
Experiments 1 to 4 show that a degree of immunity to C. perfringens infection in broiler chickens can be produced by immunization with several different secreted C.
perfringens proteins. All the proteins used in immunization, including alpha-toxin, showed significant protection depending on the severity of challenge. Of the five secreted proteins used for immunization experiments, three proteins namely alpha-toxin, HP and tPFOR
significantly protected chickens against a heavy challenge, whereas the other two proteins, GPD and FBA, significantly protected against mild challenge. Nevertheless a degree of protection was apparent with these latter proteins even against severe challenge.
Priming with alpha-toxoid and boosting with active toxin showed significant protection, whereas immunization with three injections of either alpha-toxoid or of active .. toxin did not protect (Tables 3, 6). The failure of active toxin to protect birds against a heavy challenge may have resulted from the toxin's activity on immune system cells.
The failure of alpha-toxoid to protect in the Experiment-1 may be the result of a degradation effect of toxoiding on the protein that was observed on SDS-PAGE gel (data not shown).
However, it is clear from Figure 9 that toxoiding was adequate to induce antibodies sufficient for the .. birds to tolerate the active toxin given as a booster in the Experiment-3.
The findings suggest that antibodies to conformational (rather than linear) epitopes of alpha-toxin are needed in protection against NE.
Perfringolysin 0 (also known as theta toxin), a 52 kDa protein is a potent hemolytic cytolysin that mediates necrosis in the pathogenesis of clostridial gas gangrene (29) and is an important protective immunogen in mouse and guinea-pig gas gangrene models (8). The purified alpha-toxin (Sigma Laboratories) used to immunize birds had traces of perfringolysin 0, identified using mass spectrometry (data not shown).
However, the relative amounts in the otherwise apparently pure alpha-toxin preparation (assessed by SDS-PAGE) were not quantified. It is possible that the protection observed in alpha-toxoid/toxin immunized birds (Table 5), can be partly attributed to perfringolysin 0 and that a synergistic effect on the induction of neutralizing antibodies against both the toxins may have contributed to better protection. A synergistic effect of alpha-toxin and perfringolysin 0 in the pathogenesis of C. perfringens- mediated gas gangrene has been observed (2). Proteins identified as important in immunity to NE, may also be involved in the pathogenesis of, and immunity to, gas gangrene.
Among proteins other than alpha-toxin used for immunization, HP showed significant protection against all severities of challenge (Tables 3 - 5). The truncated PFOR protein also produced significant protection against a heavy challenge in Experiment-3. The tPFOR did not produce significant protection when administered only twice (Experiment-2), possibly because this dosage produced a lower antibody response than observed in birds immunized three times, albeit with a lower dose (Figure 9). Because of the inconsistency in the protective effect observed with FBA in Experiments 1 and 2, a group of birds in Experiment-4 was immunized with FBA and challenged for 3 or 5 days, then necropsied on day 6 together with the concurrent unimmunized controls. The study confirmed that immunization with FBA
provided some protection against mild-moderate challenge. The mean lesion scores in 3-day- challenged birds of either immunized or unimmunized groups (Experiment-4A) were higher than the birds of Experiment-1 that were challenged for 3 days and necropsied on day 4, suggesting that delay to necropsy of birds following 3 day challenge was associated with higher lesion scores.
Experiments 1 to 4 shows that immunization with secreted C. perfingens proteins provides some immunity to birds against C. perfringens infection. Both alpha-toxin and perfringolysin 0 are regulated in C. perfringens by the VirR-VirS two-component regulon. (4, 26) a regulon that also controls genes involved in energy metabolism such as FBA and NAD-dependent 8-hydroxybutyryl co-enzyme-A dehydrogenase, as well as others that may be indirectly involved in bacterial virulence (3, 13, 28). There is growing evidence that certain enzymes, such as GPD and FBA, that are conventionally regarded as metabolic or "house-keeping" enzymes, may have a 'dual role' in both the pathogenesis of, and immunity to, other infections (17, 23). For example, recent studies of the virulence of Group A
streptococci (GAS) indicate that GPD assists in attachment of GAS to host cell plasmin and fibronectin receptors (6, 20, 35) and also plays an important role in cellular communication by activating host protein phosphorylation mechanisms (24). Furthermore, GPD
has been suggested to be a putative virulence factor in staphylococcal and neisserial infections (9, 22). Interestingly, a recent study showed that antibodies to FBA and GPD of Streptococcus pneumoniae showed age-dependent increased serum titers in children of different ages.
Immunization of mice with recombinant GPD and FBA showed significant protection against respiratory challenge with virulent S. pneumoniae (17). A role for FBA in immunity to Onchocerca volvulus, a filarid nematode causing River Blindness in humans, has also been suggested (21). Similarly, PFOR, an enzyme crucial for anaerobic energy metabolism, has been suggested to have a role in immunity to invasive amoebiasis (31).
Hypothetical Protein is a novel protein of C. perfringens of unknown function identified in its genome (27) that may have protease activity (zinc- nnetallopeptidase) based on the analysis of its protein structure (15).
Serum ELISA responses suggested that protection against NE is antibody mediated, since failure of protection in immunized groups in Experiment- 2 was associated with low antibody titers (Figure 8). However, alpha-toxoid/ toxin immunized- protected birds had lower antibody titers than toxoid- immunized birds that were not protected, suggesting the importance of conformational epitope- specific neutralizing antibodies, despite in low titers, in mounting a protective immune response. The intestinal antibody response, as expected, was biased towards IgY, since systemic immunization results in more antigen-specific IgY
than IgA (Figure 9). Immunization with HP, that significantly protected birds against all severities of challenge doses, produced higher IgA titers in all three experiments compared to other immunized groups. However, this association of IgA titers to protection was not evident in either alpha-toxoid/ toxin or tPFOR immunized groups that were also significantly protected birds against a heavy challenge in Experiment- 3.
Experiment 1 to 4 have demonstrated the immunizing ability of C. perfringens secreted proteins in protecting against C. perfringens in broiler chickens.

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The above-described embodiments are intended to be examples and alterations and modifications may be effected thereto, by those of skill in the art, without departing from the scope of the invention.

Claims (33)

We Claim:

1. A vaccine for controlling C. perfringens in an animal comprising an isolated C.
perfringens secreted antigenic polypeptide comprising an amino acid sequence of SEQ ID
NO:1 or a variant thereof conferring an immunoprotective effect against C.
perfringens, wherein the variant comprises an amino acid sequence having at least 80 %
identity with the amino acid sequence of SEQ ID NO:1.

2. The vaccine of claim 1, wherein the variant comprises an amino acid sequence having at least 90 % identity with the amino acid sequence of SEQ ID NO:1.

3. The vaccine of claim 2, wherein the antigenic polypeptide comprises an amino acid sequence of SEQ ID NO:1.

4. The vaccine of any one of claims 1 to 3, wherein the animal is selected from a bird, cow and pig.

5. The vaccine of any one of claims 1 to 3, wherein the animal is a bird selected from the group consisting of chicken, turkey, goose, duck, pheasant, quail, pigeon and ostrich.

6. The vaccine of claim 5, wherein the bird is a chicken.

7. The vaccine of claim 4, wherein said animal is a cow.

8. The vaccine of claim 4, wherein said animal is a pig.

9. A vaccine for controlling C. perfringens in an animal comprising a recombinant cell producing an isolated C. perfringens secreted antigenic polypeptide comprising an amino acid sequence of SEQ ID NO:1 or a variant thereof conferring an immunoprotective effect against C. perfringens, wherein the variant comprises an amino acid sequence having at least 80 % identity with the amino acid sequence of SEQ ID NO:1.

10. The vaccine of claim 9, wherein the variant comprises an amino acid sequence having at least 90 % identity with the amino acid sequence of SEQ ID NO:1.

11. The vaccine of claim 9, wherein the antigenic polypeptide comprises an amino acid sequence of SEQ ID NO:1.

12. The vaccine of any one of claims 9 to 11, wherein the animal is selected from a bird, cow or pig.

13. The vaccine of any one of claims 9 to 11, wherein the animal is a bird selected from the group consisting of chicken, turkey, goose, duck, pheasant, quail, pigeon and ostrich.

14. The vaccine of claim 13, wherein the bird is a chicken.

15. The vaccine of claim 12, wherein the animal is a cow.

16. The vaccine of claim 12, wherein the animal is a pig.

17. The vaccine of any one of claims 9 to 16, wherein the recombinant cell is a bacterial cell.

18. The vaccine of claim 17, wherein the recombinant cell is selected from Escherichia coli, Lactobacillus species, Salmonella species, and Listeria species.

19. The vaccine of any one of claims 9 to 17, wherein the recombinant cell is not C. perfringens.

20. The vaccine of any one of claims 1 to 19, further comprising a pharmaceutically acceptable carrier.

21. The vaccine of any one of claims 1 to 20, further comprising an adjuvant.

22. The vaccine of any one of claims 1 to 21, further comprising a preservative.

23. The vaccine of any one of claims 1 to 22, formulated for intramuscular, subcutaneous, intravenous, intranasal, intradermal, intrabursal, in ovo, ocular, oral, intra-tracheal, or intra-bronchial delivery.

24. The vaccine of any one of claims 1 to 23, further comprising a second antigenic polypeptide.

25. The vaccine of claim 24, wherein the second antigenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ
ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

26. Use of an effective amount of the vaccine of any one of claims 1 to 25 in a subject for immunization of the subject against a C. perfringens induced condition selected from the group consisting of necrotic enteritis, haemorrhagic enteritis, and enterotoxemia.

27. Use of an effective amount of the vaccine of any one of claims 1 to 25 for treatment of a C. perfringens induced condition selected from the group consisting of necrotic enteritis, haemorrhagic enteritis, and enterotoxemia in a subject.

28. Use of an isolated C. perfringens secreted antigenic polypeptide comprising an amino acid sequence of SEQ ID NO:1 or a variant thereof conferring an immunoprotective effect against C. perfringens for preparation of a vaccine, wherein the variant comprises an amino acid sequence having at least 80 % identity with the amino acid sequence of SEQ ID
NO:1.

29. The use of claim 28, wherein the vaccine is for immunization of a subject against a C. perfringens induced condition selected from the group consisting of necrotic enteritis, haemorrhagic enteritis, and enterotoxemia.

30. The use of claim 28, wherein the vaccine is for treatment of a C.
perfringens induced condition selected from the group consisting of necrotic enteritis, haemorrhagic enteritis, and enterotoxemia in a subject.

31. The use of any one of claims 26, 27, 29, and 30, wherein the C.
perfringens induced condition is necrotic enteritis.

32. A feed additive comprising the vaccine of any one of claims 1 to 25.

33. A kit comprising the vaccine of any one of claims 1 to 25, and instructions for use of the vaccine in a subject.