WO2008153339A2

WO2008153339A2 - A vaccine for treating or preventing pasteurellosis using a yeast surface-expression system

Info

Publication number: WO2008153339A2
Application number: PCT/KR2008/003309
Authority: WO
Inventors: Joo Sung Yang; Young Hoon Jin; Sun Hwa Moon
Original assignee: Univ Sungkyunkwan Found; Joo Sung Yang; Young Hoon Jin; Sun Hwa Moon
Priority date: 2007-06-12
Filing date: 2008-06-12
Publication date: 2008-12-18
Also published as: KR101001023B1; WO2008153339A3; KR20080109673A

Abstract

The present invention relates to a vaccine composition for treating or preventing pasteurellosis using a yeast surface-expression system. In particular, the present invention relates to a recombinant nucleotide for the preparation of vaccine against pasteurellosis, comprising i) a nucleotide encoding an outer membrane peptide of Pasteurella multocida, ii) a nucleotide encoding an Aureobasidin A peptide, and iii) a nucleotide encoding an LTB (heat-labile enterotoxin subunit) peptide, a recombinant expression vector comprising the nucleotide, and a vaccine composition for treating or preventing pasteurellosis which is obtained by infecting a host cell with the vector. The vaccine composition prepared by the present invention can induce immune response by a simple immunization method including oral immunization, mucosal immunization, and intradermal immunization using a tattoo machine.

Description

[DESCRIPTION]

[Invention Title]

A VACCINE FOR TREATING OR PREVENTING PASTEURELLOSIS USING A YEAST SURFACE-EXPRESSION SYSTEM

[Technical Field]

The present invention relates to a vaccine for treating or preventing pasteurellosis using a yeast surface-expression system. In particular, the present invention relates to a recombinant nucleotide for the preparation of a vaccine against pasteurellosis, comprising i) a nucleotide encoding an outer membrane peptide of Pasteurella multocida, ii) a nucleotide encoding an Aureobasidin A peptide, and iii) a nucleotide encoding an LTB (heat-labile enterotoxin subunit) peptide, a recombinant expression vector comprising the nucleotide, and a vaccine composition for treating or preventing pasteurellosis, which is obtained by infecting a host cell with the vector.

[Background Art]

Pasteurellosis is caused by infection with bacteria of the Pasteurella genus, and common in a variety of animal species including cattle, buffaloes, sheep, swine, rabbits, fowls, dogs, rodents and cats, which is exemplified by shipping fever, calf enzootic pneumonia, mastitis, pleuropneumonia, snuffles, fowl cholera, epizootic hemorrhagic septicemia, atrophic rhinitis and abscess. There are three groups of strains that cause pasteurellosis, Pasteurella multocida (serotypes A, B, D, E, F), Pasteurella haemolytica (biotypes A and T), and Pasteurella pneumotropica. Pasteurella multocida type A is a cause of shipping fever, calf enzootic pneumonia, or mastitis in cattle, pleuropneumonia or mastitis in sheep, pneumonia by secondary infection in swine, pleuropneumonia in rabbits, and fowl cholera in fowls. Pasteurella multocida type B is a cause of epizootic/hemorrhagic septicemia in cattle and buffaloes, and Pasteurella multocida C is a cause of atrophic rhinitis in swine, hi addition, Pasteurella multocida type E is a cause of epizootic/hemorrhagic septicemia in cattle and buffaloes in Africa, and Pasteurella multocida type F causes infection in turkeys. Pasteurella haemolytica type A is a cause of shipping fever or pneumonia in cattle, and enzootic pneumonia, septicemia or gangrenous mastitis in sheep. Pasteurella haemolytica type T is a cause of septicemia in sheep. In addition, Pasteurella pneumotropica is a cause of pneumonia or abscess in rodents.

In particular, fowl cholera is acute contagious disease and remains a major disease of chickens, water fowls, and turkeys for over 200 years. Fowl cholera is caused by a common pathogenic bacterium known as Pasteurella multocida, which was first isolated by Pasteur about 100 years ago, and the world's first vaccine against it was developed. Fowl cholera is, categorized as a Communicable disease Class I in Korea, a bacterial contagious disease with clinical symptoms and lesions generated by the toxin of Pasteurella multocida, and its incident has been reported in Korea. As mentioned above, Pasteurella multocida is a major cause of fowl cholera, and is a bipolar staining Gram-negative coccobacillus, classified depending on the presence or absence of a capsule. The capsule has been implicated in virulence in Pasteurella multocida, and the virulence characteristics have been reported to be diverse among the strains. Pasteurella multocida grows well on blood or dextrose starch agar, but not on MacConkey agar. A gel diffusion precipitin test is used for serotyping Pasteurella multocida, and total 16 serotypes are recognized. As a result of analyzing the strains isolated from chickens, it is reported that the most common serotypes found in chickens are 1, 3, and 3x4.

An acute outbreak of fowl cholera results in sudden death due to septicemia, accompanied by a mucous discharge from the mouth, and cyanotic combs and wattles, and commonly occurs in summer. A chronic outbreak of fowl cholera is prevalent in fall and winter, and may occur following an acute outbreak or by infection with low virulent strains. The chronic form occurs sporadically with a low mortality rate. A hyperacute form generally has no symptoms, the acute form is characterized by hepatomegaly with necrotic areas due to hemorrhages in the heart, forestomach, serous membranes, and abdominal fat, and follicle rapture and malformation. The chronic type is characterized by inflammatory exudates in the eye and nose, and cheesy exudates in the infected area. Recovered chickens acquire immunity against the same strain, but remain as carriers. Therefore, it is most important to find and remove all carriers for the control of fowl cholera. For the treatment of fowl cholera, a continuous medication program is needed, which has a drawback in that it requires higher costs than vaccination. Also, many sulfa drugs and antibiotics are effective for reduction in the mortality rate, but discontinuance of treatment increases the mortality rate. In addition, drugs such as sulfaquinoxaline are more effective, but cause a drop in egg production of layer chickens. Moreover, the drugs may exclude the egg-laying ability from layer chickens, and thus the drugs should be used with care.

A killed fowl cholera vaccine is not completely effective, but repeated vaccination of 2 week-old chicken is known to be enough to induce immune response. However, the killed vaccine does not effectively induce cross- protection between some serotypes. An oil vaccine is used for immunization of breeding hens. Since the oil vaccine causes a drop in egg production due to severe stress, the vaccination course has to be completed before initiation of egg laying. A live fowl cholera vaccine is administered by Wing web inoculation to 10-11 week-old layer chickens and breeding hens, followed by revaccination in 6-8 weeks. In the United States, a CU strain is used for the live vaccine. It is known that the live vaccine confers better protection than the killed vaccine against a wide range of serotypes.

Therefore, there is a need for effective vaccines, which is useful for inducing protective immunity for the prevention and treatment of fowl cholera. On the other hand, surface-displayed viral antigens on salmonella carrier vaccine were suggested by Lee, J. S., K. S. Shin et al. ((200O) Nat. Biotechnol. 18(6):645-8), but there are some differences between proteins expressed in the prokaryotic salmonella and human. Ln addition, for protein glycosylation to express a protein that is more similar to that in eukaryotes, yeast expressing surface antigen determinants on its surface and implications for a possible oral vaccine were suggested by Schreuder, M. P., C. Deen, et al. ((1996) Vaccine 14(5):383-8). However, the transformed yeast which expresses an antigen protein on its surface loses the selectivity during laboratory subcultures, since the gene of antigen protein is episomally maintained in the yeast, and a selection marker based on auxotrophy does not exhibit a strong selection power. In addition, since the gene encoding a yeast surface-displayed protein is not codon- optimized, the protein expression level is remarkably low. Upon being used for a vaccine, the yeast surface-displayed protein may be easily degraded by various factors to provide a relatively low immunity. Accordingly, in order to overcome the above problems, the improvement in immunization method and masking technology for surface-displayed protein stabilization are increasingly demanded.

[Disclosure]

[Technical Problem]

Accordingly, applying to the conventional yeast surface-expression system, the present inventors have selected only the antigenic epitope peptide of outer membrane protein from Pasteurella multocida type A, which is a cause of fowl cholera, as an antigen, and manufactured a construct fused with an LTB protein to enhance immunity. To increase the expression of the yeast surface- expression system and selectivity of the transformed yeast, they have improved the conventional expression vector, and then integrated the improved expression vector into a yeast genomic DNA to prepare a yeast which stably expresses the antigen protein on its surface with the aid of an antibiotic. They found that the yeast expression system can induce an excellent immune response by administration via various routes such as oral and intradermal immunization, and thus be applied to pasteurellosis including fowl cholera, thereby completing the present invention.

[Technical Solution]

It is an object of the present invention to provide a recombinant nucleotide for the preparation of vaccine against pasteurellosis, comprising i) a nucleotide encoding an outer membrane peptide of Pasteurella multocida, ii) a nucleotide containing an Aureobasidin A resistance gene, and iii) a nucleotide encoding an LTB (heat-labile enterotoxin subunit) peptide, an expression vector comprising the nucleotide, and a host cell transformed with the expression vector.

It is another object of the present invention to provide a method for preparing a vaccine for the prevention or treatment of pasteurellosis, comprising the step of transforming the host cell with the expression vector for its amplification, and a vaccine composition for the prevention or treatment of pasteurellosis, prepared by the method. [Advantageous Effects]

The vaccine composition prepared by the present invention can induce immune response by a simple immunization method including oral immunization, mucosal immunization, and intradermal immunization using a tattoo machine.

[Description of Drawings]

Fig. 1 shows the sequences of PCR primers for pYDl-ompH(A:3);

Fig. 2 shows the cloned product of pYDl-ompH(A:3), which was cleaved by restriction enzymes;

Fig. 3 shows the sequencing result of pYDl-ompH(A:3);

Fig. 4 shows a strategy for cloning p AUR YD;

Fig. 5 shows the sequences of PCR primers for pAURYD;

Fig. 6 shows the sequencing result of p AURYD (fragment 1);

Fig. 7 shows the sequencing result of p AUR YD (fragment 2);

Fig. 8 shows the sequences of pAURYD of Fig. 6;

Fig. 9 shows a construct map of pAURYD vector;

Figs. 10 to 12 show antigenic epitope prediction of ompH(A:3) for pAURYD-peptide construction, in which Fig. 10 is the result showing the structure of transmembrane protein predicted from the amino acid sequence of outer membrane, Fig. 11 is a reference drawing for the selection of highly antigenic regions, disclosed in a paper of analyzing the sequences of major outer membrane proteins from various serotypes of Pasteurella multocida, and Fig. 12 shows the selected highly antigenic regions, resulting from analyzing the sequences of the outer membrane proteins in Figs. 10 and 11 ; Fig. 13 shows the sequences of PCR primers for pAURYD-peptide;

Fig. 14 shows the cloned product of pAURYD-peptide, which was cleaved by restriction enzymes;

Fig. 15 shows the sequencing result of pAURYD-peptide and its sequence;

Fig. 16 shows the sequences of PCR primers for p AUR YD-LTB;

Fig. 17 shows the cloned product of pAUR YD-LTB, which was cleaved by restriction enzymes;

Fig. 18 shows the sequencing result of pAUR YD-LTB and its sequence;

Fig. 19 shows the cloned product of pAURYD-fusion, which was cleaved by restriction enzymes;

Figs. 20 and 21 show the sequencing result of pAURYD-fusion;

Fig. 22 shows primers for cloning the LTB (heat-labile enterotoxin subunit B) gene into the pAURYD vector;

Figs. 23 and 24 show the sequencing result of the PCR product, resulting from PCR analysis using the primers prepared for cloning into the pAURYD vector;

Fig. 25 shows the primers which are prepared for confirmation of the clones;

Fig. 26 shows the band size of each DNA isolated from the transformed yeast, resulting from PCR;

Fig. 27 shows the band size of each PCR product, resulting from colony PCR analysis of the transformed single colony;

Fig. 28 shows the IFA result of pAURYD clones of the transformed yeast; Fig. 29 shows confocal microscopic images of pAURYD clones of the transformed yeast;

Fig. 30 shows the result of GMl binding assay of the surface expressed yeast;

Figs. 31 to 33 are the results of showing the antigen-specific antibody reaction, upon administration via various routes (oral, intranasal, intradermal);

Figs. 34 to 36 are the results of serum IgG ELISA upon oral and intranasal administration;

Fig. 37 is the result of serum IgG ELISA upon intradermal administration;

Fig. 38 is the result of serum IgG and IgA ELISA;

Fig. 39 is a schematic diagram showing the p AUR YD constructs; and

Fig. 40 is a schematic diagram showing the OmpH peptide-LTB fusion construct in the yeast surface-expression system.

[Best Mode]

To achieve the object, the present invention provides a recombinant nucleotide for the preparation of a vaccine against pasteurellosis, comprising i) a nucleotide encoding an outer membrane peptide of Pasteurella multocida, ii) a nucleotide containing an Aureobasidin A resistance gene, and iii) a nucleotide encoding an LTB (heat-labile enterotoxin subunit) peptide.

As used herein, the term "pasteurellosis" refers to a disease which is caused by infection with bacteria of the Pasteurella genus, and common in a variety of animal species including cattle, buffaloes, sheep, swine, rabbits, fowls, dogs, rodents and cats, and encompasses all diseases caused by Pasteurella multocida (serotypes A, B, D, E, F), Pasteurella haemolytica (biotypes A and T), and Pasteurella pneumotropica. The diseases are exemplified by shipping fever, calf enzootic pneumonia, mastitis, pleuropneumonia, snuffles, fowl cholera, epizootic hemorrhagic septicemia, atrophic rhinitis and abscess. In one preferred embodiment, the vaccine according to the present invention is provided for the prevention or treatment of fowl cholera.

In the present invention, a nucleotide encoding the outer membrane peptide of Pasteurella multocida type A is used as an antigen. It is preferable that only epitope of the outer membrane protein is used to increase its surface expression level, since the outer membrane protein of Pasteurella multocida type A which is a cause of fowl cholera is a macromolecule possessing high hydrophobicity, and thus hardly expressed on the surface of yeast. The nucleotide has preferably a length of 140 to 180 bp, and more preferably a length of 169 bp. In one preferred embodiment of the present invention, Pasteurella multocida type A is used, hi one embodiment of the present invention, used were L2 and L5 regions, which were predicted to have high hydrophilicity and antigenicity, among regions of the outer membrane H gene of Pasteurella multocida type A:3 obtained by antigenic epitope prediction.

In addition, the nucleotide containing an Aureobasidin A resistance gene of the present invention is used for introduction of Aureobasidin A as a selection marker, in order to improve the selectivity of the yeast cell capable of surface- expressing the antigen. The preferred one is an AURl gene. In one embodiment of the present invention, an AURC-I selection marker gene, which is the Aureobasidin A resistance gene, was cleaved with restriction enzymes, and then used as a linear plasmid to transform the yeast.

In addition, the nucleotide encoding an LTB (heat-labile enterotoxin subunit) peptide of the present invention is used to transfer the antigen protein to a cell expressing a GMl receptor with selectivity and target specificity and to enhance immune response, which is achieved by the strong GMl ganglioside binding activity of LTB protein. The nucleotide has preferably a size of 300 to 350 bp, and more preferably a size of 321 bp. hi one embodiment of the present invention, used was a heat-labile enterotoxin subunit B (LTB) gene isolated from enterotoxigenic Escherichia coli (ETCT). In one preferred embodiment, the nucleotide of the present invention is a nucleotide having a nucleic acid sequence represented by SEQ ID NO. 1.

In still another embodiment, the present invention provides an expression vector comprising the nucleotide.

As used herein, the term "vector" means any vehicle to allow insertion of a desirable gene into a host cell, and includes all of the typical vectors such as a plasmid vector, a cosmid vector, a bacteriophage vector, and adenoviral and retroviral vectors, preferably a plasmid vector. The vector capable of effectively expressing a foreign protein in a host cell, in particular, yeast system includes a promoter, an extracellular secretion signal sequence, a cytokine gene and a transcription termination signal sequence. In a preferred embodiment, the expression vector of the present invention is integrated into a genomic DNA of host cell to transform the host cell. In a specific embodiment of the present invention, each of ompH(A:3) gene or LTB gene is cloned into pAURYD having the constitution shown in Fig. 7 to prepare a pAURYD-peptide vector and a pAURYD-LTB vector. The pAURYD-LTB vector was cleaved with restriction enzymes, and then cloned into the pAURYD-peptide vector to prepare a pAURYD-fusion expression vector.

When the expression vector of the present invention is introduced into the host cell, it does not exist as a plasmid in the host cell, but is integrated into a genomic DNA of host cell, and thus a LTB-fused recombinant outer membrane protein of Pasteurella multocida is stably expressed as the antigenic peptide on the surface of host cell.

In still another embodiment, the present invention provides a host cell transformed with the expression vector. The host cell is preferably yeast, and more preferably Saccharomyces cerevisiae EBY 100.

The host cell of the present invention expresses the LTB-fused recombinant outer membrane protein of Pasteurella multocida type A on its surface by the expression vector. In particular, the host cell can express the recombinant protein, as shown in Fig. 23.

In still another embodiment, the present invention provides a method for preparing a vaccine for the prevention or treatment of pasteurellosis, comprising the step of transforming the host cell with the expression vector to express the LTB (heat-labile enterotoxin subunit)-fused recombinant outer membrane protein of Pasteurella multocida.

In still another embodiment, the present invention provides a vaccine composition for the prevention or treatment of pasteurellosis, comprising the LTB (heat-labile enterotoxin subunit)-fused recombinant outer membrane protein of Pasteurella multocida which is prepared by the above method.

The vaccine composition according to the present invention may be formulated into a pharmaceutically or veterinary acceptable suspension, solution or emulsion well known to those skilled in the art. The preferred pharmaceutically acceptable carrier may include a sterilized aqueous or nonaqueous solution, a suspension, and an emulsion, which is suitable for ingestible or inhalable formulation, or rectal or vaginal suppository. Examples of the nonaqueous solution may include propylene glycol, polyethylene glycol, vegetable oil (e.g., olive oil), and organic ester (e.g., ethyl oleate). Examples of the aqueous carrier may include water, alcoholic/aqueous solution, emulsion or suspension (saline and buffered media). In addition, the pharmaceutically acceptable carrier may include a preserving agent and other additives such as an antimicrobial agent, an antioxidant, a chelating agent and inert gas.

In addition, the vaccine composition according to the present invention may be administered via any route selected in the medical or veterinary field. For example, the vaccine composition may be administered by oral, inhalable, intrarectal, intranasal, intradermal or intravenous route, which varies depending on administration dose, tissue to which vaccine is administered, titer of vaccine, treatment requirement for immunization subject, and cost. In particular, the vaccine composition according to the present invention exhibited immunization effect by oral, intranasal or intradermal administration, as shown in Figs. 21a and 21b, thereby being intradermally administered using a tattoo machine or being orally administered as a feed additive along with feed.

In addition, the vaccine composition according to the present invention may be provided singly or as an ingredient of multivalent vaccine, that is, in combination with other vaccine. Examples of the other vaccine may include a known live or killed vaccine against pasteurellosis, preferably fowl cholera.

In still another embodiment, the present invention provides a yeast surface- expression system, comprising the step of (a) preparing a recombinant vector which comprises a recombinant nucleotide including i) a nucleotide encoding an outer membrane peptide of Pasteurella multocida, ii) a nucleotide containing an Aureobasidin A resistance gene, and iii) a nucleotide encoding an LTB (heat- labile enterotoxin subunit) peptide;

(b) transforming a yeast with the recombinant vector; and

(c) culturing the transformed yeast to express the recombinant protein on its surface.

The yeast surface-expression system of the present invention can be used for the development of effective vaccines against pathogens infectious to animals and fowls, which requires low costs, and is easily handled by a user. Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

[Mode for Invention]

Example 1. Construction of plasmid DNA

An 0mpH(A:3) gene was obtained by isolating an outer membrane H gene of Pasteurella multocida serotype A: 3. To clone the gene into pYDl, a set of PCR primer pairs was prepared, as shown in Fig. 1. After performing PCR, the product was cloned into pYDl. Then, the cloned vector was digested with restriction enzymes, BamHI and Notl. As a result, the ompH(A:3) was found to have a size of 1210 bp (Fig. 2), and sequence analysis thereof was requested (Core-bio). The sequencing results are shown in Fig. 3. The obtained clone was designated as "pYDl-ompH", which is a clone not integrated into the yeast chromosome.

In order to solve the problem that the pYDl vector is expressed in the yeast as a plasmid to lose stability during yeast division, and in order to compensate for the low selectivity of tryptophan auxotrophic marker, a method of integrating an antibiotic selection marker gene into the yeast genomic DNA was employed. The strategy is as follows. The fragment from the GALl promoter region to the MATα transcription termination region in the pYDl vector was divided into two fragments, and then the PCR primers therefor were prepared. At this time, to provide the new vector with multiple cloning site (MCS), new restriction enzyme target sites (Smal, Sail, SacII, Ndel) were introduced into the primers. After performing PCR, two fragments were obtained by using a TA cloning kit (Invitrogen™), and subjected to sequencing analysis. From the TA clone, the fragments were inserted between Sad and Sphl restriction sites of pAURlOl (Takara Bion, Otsu, Japan), which is a chromosomal integrating vector for yeast, using restriction enzymes and ligase (see Figs. 4 and 5). The pAURlOl vector does not replicate autonomously in yeast, and is stably maintained only when integrated into a chromosome. An Aureobasidin A resistance gene, AURl-C selection marker gene was cleaved with restriction enzymes, and used in a linear plasmid form. Thus, it can be integrated into the aurl locus on the yeast chromosome with high efficiency.

The prepared pAURYD plasmid is integrated into the yeast genomic DNA to have a resistance to Aureobasidin A in YPD media, and expresses a desirable protein on the surface of the yeast in 2% galactose media. Sequence analysis thereof was requested (Core-bio), and the results are shown in Figs. 6 and 7. The obtained clone was designated as "pAURYD", and further used as a vector to clone an antigen gene.

To increase the surface expression level, only epitope was selected from the 0mpH(A:3) gene. PCR primers were prepared in order to clone L2 and L5 regions, which were predicted to have high hydrophilicity and antigenicity, among regions obtained by antigenic epitope prediction (see Figs. 10 to 13). PCR was performed using ompH(A:3) as a template and a primer set of set #1 and #2 under the conditions of denaturation at 94 ^°C for 1 min, annealing at 54 ^°C for 30 sec and extension at 72 ^°C for 1 min. Each PCR product was purified using a purification kit, and then overlapping PCR was performed using two products as the template under the conditions of denaturation at 94 ^°C for 1 min, annealing at 54 ^°C for 30 sec and extension at 72 ^°C for 1 min. The obtained PCR product was cloned into pAURYD, and then enzyme restriction was performed to confirm the clones. As a result, the peptide was found to have a size of 169 bp (Fig. 14), and sequence analysis thereof was requested (Core-bio). Then, the result was aligned with the original sequence to confirm the sequencing results (Fig. 15). The obtained clone was designated as "pAURYD-peptide".

To clone the gene of heat-labile enterotoxin subunit B (LTB) isolated from enterotoxigenic Escherichia coli (ETEC) into the pAURYD vector, primers were prepared. In this connection, when LTB was fused with the antigen gene whihc had been cloned into pAURYD, a glycine-proline-glycine-proline linker was formed by introducing the corresponding sequence G-P-G-P(ggc-ccg-ggc-ccg) into the sense primer (Fig. 16), and PCR was performed using the primer under the conditions of denaturation at 94 ^°C for 1 min, annealing at 58 ^°C for 30 sec and extension at 72 ^°C for 1 min. The PCR product was cloned into Xhol and Bstl restriction sites of pAURYD, and then enzyme restriction was performed to confirm the clones. As a result, the LTB was found to have a size of 321 bp (Fig. 17), and sequence analysis thereof was requested (Core-bio). The results are shown in Fig. 18. The obtained clone was designated as "pAURYD-LTB". To express the fusion protein of peptide and LTB on the surface of yeast, the fusion protein was cleaved from the pAUR YD-LTB vector with restriction enzymes, and then cloned into the pAURYD-peptide vector. Then, enzyme restriction was performed to confirm the clones (see Figs. 19 to 21). The clone obtained by fusion of OmpH peptide and LTB was designated as "pAURYD- fusion". Accordingly, a construct was prepared by linking the cloned peptide with LTB via the GPGP linker.

To clone the gene of heat-labile enterotoxin subunit B (LTB) isolated from enterotoxigenic Escherichia coli (ETEC) into the pAURYD vector, primers were prepared (Fig. 22). PCR was performed using the primer under the conditions of denaturation at 94 ^°C for 1 min, annealing at 54 ^°C for 30 sec and extension at 72 ^°C for 1 min. The PCR product was cloned into Xhol and BstBI restriction sites of pAURYD, and then enzyme restriction was performed to confirm the clones. As a result, the LTB was found to have a size of 309 bp, and sequence analysis thereof was requested (Core-bio). The results are shown in Figs. 23 and 24. The obtained clone was designated as "ρAURYD-LTB(NoGP)".

Example 2. Transformation of yeast

Saccharomyces cerevisiae EBYlOO strain was transformed with each of the pAURYD vector clones. Single colonies of EBYlOO were inoculated in 2 ml of YPD broth, and cultured at 250 rpm and 30^°C for 16 hrs. Then, 500 /d of the cells were inoculated in 50 ml of YPD broth, and cultured at 250 rpm and 30^°C until reaching OD600 of 1.2. The cells were centrifuged at 1000xg, and then the pellet was resuspended in 10 ml of a solution A (LiAc, Tris-HCl, EDTA). Then, the cells were centrifuged again, the pellet was resuspended in 1 ml of the solution A, and aliquoted into 100 #ϋ, followed by incubation at 30^°C for 1 hrs. Then, 5 μg of Stul cut DNA, 150 μg of salmon testes DNA treated at 100^°C for 10 min, and 850 μJL of solution B (solution A, 40% PEG) were added thereto, followed by incubation for 30 min under the same conditions. Then, the cells were immediately heat shocked at 42 ^°C for 15 min, and then left at room temperature for 10 min, followed by centrifugation at 5000 rpm. Then, 5 ml of YPD broth was added thereto, and incubated at 30^°C overnight. After centrifugation, the cell pellet was resuspended in 1 ml of YPD broth, and 0.5 mg/ml of YPD plate was smeared with 200 μA of the resuspended cells. After 3 days, colonies resistant to Aureobasidin A were obtained.

Example 2-1. Confirmation of transformed yeast by PCR

The transformed yeast was inoculated in 2 ml of YPD, and then cultured at 250 rpm and 30 ^°C for 24 hrs. Then, the cells in E-tube were centrifuged, and the pellet was washed with 1 ml of distilled water once. 200 ul of lysis buffer (2% (v/v) Triton X-100), 1% (v/v) SDS, 100 mM NaCl, 10 mM Tris base pH 8.0, 1 mM EDTA pH 8.0) was added thereto to resuspend the pellet. 200ul of acid- washed glass beads (sigma, Cat No. G8772) and 200ul of phenol: chloroform: isoamyl alcohol (25:24:1) (Bioneer Cat. No. C-9017) were added thereto, followed by vortexing for 1 min. Then, 200 ul of TE buffer (10 mM Tris base pH 8.0, 1 mM EDTA pH 8.0) was added thereto, and mixed well by inversion, followed by centrifugation at 13200 rpm for 5 min. The obtained supernatant was mixed with 1 ml of 100% ethanol, and centrifuged at 13200 rpm for 5 min. The obtained pellet was washed with 70% ethanol, and dried well. The dried pellet was dissolved in 50 ul of distilled water to obtain the yeast DNA. The DNA was quantified, and PCR was performed using 50 ng of DNA as a template under the conditions of denaturation at 94 ^°C for 1 min, annealing at 54 ^°C for 1 min and extension at 72 "C for 1 min. Primers to be annealed to both ends of multiple cloning site (MCS) of pYDl vector were prepared to confirm the clones (Fig. 25). PCR was performed to confirm each band size corresponding to DNA from the transformed yeast (Fig. 26). In addition, colony PCR was performed using the transformed single colonies and the primers. As a result, a band size corresponding to genomic DNA PCR was observed to confirm transformation of the yeast (Fig. 27).

Example 3. Induction of transformed yeast

Single colony was inoculated in 2 ml of YPD containing 0.5 /«j/ml of Aureobasidin A, and then cultured at 250 rpm and 30^°C until reaching OD600 of 2 to 5. Then, 1 ml of the cells in E-tube was centrifuged, and the pellet was resuspended in 10 ml of induction medium (10 ml of 0.67% YNB (with ammonium sulfate, without amino acids), 0.5% Casamino acids, 2% glucose or galactose, 0.01% tryptophan), followed by incubation at 250 rpm and 30^°C for 48 hrs.

Example 4. Immunofluorescence assay of transformed yeast The transformed yeast was induced in galactose media, and then subjected to IFA. At this time, since a V5 tag was fused at 3 '-end of antigen gene, a monoclonal anti V5 antibody (Invitrogen™) (room temperature, 2 hrs) and an Alexa Fluor 488 (room temperature, 2 hrs) were used as primary and secondary antibodies, respectively. Unlike a negative control group EBYlOO, round signal was observed around the surface of the transformed yeast (Figs. 28 and 29).

Example 5. GMl binding assay of transformed yeast

The GMl ganglioside binding property of LTB protein was employed to measure activity of the proteins which were expressed on the surface of yeast. The LTB protein was known not to bind to GMl ganglioside when it is a monomer. The assay method is similar to ELISA, and the wells were coated with GMl ganglioside (Calbiochem cat. No. 345724) in an amount of 0.15 ug/well at 4^°C for 16 hrs. Then, the numbers of yeasts expressing LTB and fusion were equalized to the number of yeast expressing pAURYD only as a negative control group, and the cells were added to each well by serial dilution. To analyze the reaction with lysate, glass beads and the pellet (1 :1) were added to E-tube, and 400 ul of Tris-Cl (pH 8.0) was added thereto, followed by vortexing for 30 sec three times. After centrifugation, the supernatant was separated to obtain the lysate. The lysate was also diluted, and added to the well coated with GMl, followed by incubation at 37°C for 1 hr. As in ELISA, the well was incubated at 37^°C for 1 hr with poly anti LTB antibody as a primary antibody, and then incubated at 37^°C for 1 hr with anti rabbit IgG antibody conjugated to HRP as a secondary antibody. The extent of color development by a TMB solution was measured using a spectrophotometer to determine the amount of yeast. It was found that the yeast expressing a fusion exhibited a stronger affinity than the yeast expressing LTB only (Fig. 30).

Example 6. Immunization and ELISA

The transformed yeast was induced, and the expression was confirmed by IFA. Then, mouse immunization was performed. 4-week old BALB/C mouse was used, and administered via oral, intrarectal, intranasal, sublingual, and intradermal routes. Oral immunization (Fig. 31) was performed using a stainless- steel animal needle (zonde) in the same dose. Intrarectal immunization (Fig. 33), subligual immunization (Fig. 32), and intranasal immunization (Figs. 31 and 33) were performed by lightly anesthetizing the mice. Intradermal immunization (Fig. 32) was performed using a tattoo machine. Immunization was performed following the schedule which is shown in Fig. 19, and according to the schedule, blood samples were collected from the orbital cavity vein of each mouse. Sera were separated from the blood samples, and then IgG ELISA was performed to detect the antigen-specific antibody reaction. In addition, fecal samples were collected, and IgA was isolated from the fecal samples using an extraction buffer (PBS, 0.01% sodium azide, 5% FBS, protease inhibitor cocktail). Then, IgA ELISA was performed to detect the antigen-specific antibody reaction in intestinal mucosa. At this time, the ompH(A:3) protein purified from bacteria was used (Figs. 34 to 36). At 6 weeks after oral administration of a low dose, the level of specific antibody was slightly increased, as compared to the negative group, preimmune sera (Figs. 34 and 35). At 6 weeks after intranasal administration, the level of specific antibody was also slightly increased, as compared to the negative group and preimmune sera (Fig. 36). At 5 weeks after intradermal administration, the highest increase in the level of specific antibody was observed in the group immunized with the ompH(A:3) peptide-LTB fusion construct, and an increase in the level of specific antibody was also observed in the group immunized with ompH(A:3) (Fig. 37). The result that the highest increase in the level of specific antibody was observed in the group immunized with the ompH(A:3) peptide-LTB fusion construct is thought to be correlated with the result of GMl binding assay in which a stronger affinity was observed in the yeast expressing a fusion construct (Fig. 30). That is, it is inferred that the sample showing the highest level in the GMl binding assay was specifically targeted to GMl ganglioside distributed on cells in vivo. Upon intrarectal and intranasal immunization of pYD-ompH with or without the purified LTB protein, the immune adjuvant effect of LTB was not observed. However, the serum IgG antibody reaction being specific to the OmpH antigen was found to increase in a time-dependent manner (Fig. 38). With respect to the level of fecal IgA for evaluation of efficacy of mucosal vaccine, the immune adjuvant effect of LTB was not observed in the groups immunized via intrarectal and intranasal routes. However, the fecal IgA antibody reaction being specific to the OmpH antigen was found to increase in a time-dependent manner (Fig. 38).

[Industrial Applicability]

The present invention relates to a vaccine composition for treating or preventing pasteurellosis using a yeast surface-expression system. In particular, the present invention relates to a recombinant nucleotide for the preparation of a vaccine against pasteurellosis, comprising i) a nucleotide encoding an outer membrane peptide of Pasteurella multocida type A, ii) a nucleotide encoding an Aureobasidin A peptide, and iii) a nucleotide encoding an LTB (heat-labile enterotoxin subunit) peptide, a recombinant expression vector comprising the nucleotide, and a vaccine composition for treating or preventing pasteurellosis which is obtained by infecting a host cell with the vector.

The vaccine composition prepared by the present invention can induce immune response by a simple immunization method including oral immunization, mucosal immunization, and intradermal immunization using a tattoo machine, and can be utilized against pasteurellosis in a variety of animal species including cattle, swine, rabbits, and fowls.

Claims

[CLAIMS]

[Claim 1]

A recombinant nucleotide for the preparation of a vaccine against pasteurellosis, comprising i) a nucleotide encoding an outer membrane peptide of Pasteurella multocida, ii) a nucleotide containing an Aureobasidin A resistance gene, and iii) a nucleotide encoding an LTB (heat-labile enterotoxin subunit) peptide.

[Claim 2]

The nucleotide according to claim 1, wherein the nucleotide is a nucleotide having a nucleic acid sequence of SEQ ID NO. 1.

[Claim 3]

An expression vector comprising the nucleotide of claim 1 or 2.

[Claim 4]

A host cell which is transformed with the expression vector of claim 3.

[Claim 5]

The host cell according to claim 4, wherein the host cell is a yeast cell.

[Claim 6]

The host cell according to claim 4 or 5, wherein the recombinant peptide is expressed on the surface of the host cell.

[Claim 7]

The host cell according to claim 6, wherein the recombinant peptide is a fusion peptide of an outer membrane peptide of Pasteurella multocida type A and LTB (heat-labile enterotoxin subunit).

[Claim 8]

The host cell according to claim 6, wherein the recombinant peptide is a fusion peptide of an outer membrane peptide of enterotoxigenic Escherichia coli (ETEC) and LTB (heat-labile enterotoxin subunit).

[Claim 9]

The host cell according to claim 6, wherein the recombinant peptide is prepared by linking an antigen peptide with LTB via a linker.

[Claim 10]

The host cell according to claim 8, wherein the linker is a GPGP linker.

[Claim 11]

A method for preparing a vaccine for the prevention or treatment of pasteurellosis, comprising the step of transforming a host cell with the expression vector of claim 3 to express a recombinant peptide.

[Claim 12] A vaccine composition for the prevention or treatment of pasteurellosis, comprising the host cell of claim 4 or 5.

[Claim 13]

The vaccine composition according to claim 8, wherein the composition is formulated for oral or intradermal administration.

[Claim 14]

A method for expressing a recombinant peptide for the prevention or treatment of pasteurellosis on the surface of yeast, comprising the step of

(a) transforming a yeast with a recombinant nucleotide including i) a nucleotide encoding an outer membrane peptide of Pasteurella multocida, ii) a nucleotide containing an Aureobasidin A resistance gene, and iii) a nucleotide encoding an LTB (heat-labile enterotoxin subunit) peptide; and

(b) culturing the transformed yeast.

[Claim 15]

A method for transferring an antigen protein to a GMl receptor-expressing cell using the host cell of claim 4 or 5.

[Claim 16]

A pAURYD-fusion construct illustrated in Fig. 22.

[Claim 17] A vaccine composition for the prevention or treatment of pasteurellosis, comprising the recombinant peptide expressed on the surface of yeast according to the method of claim 13.

[Claim 18]

The vaccine composition according to claim 12, wherein the intradermal administration is performed using a tattoo machine.

[Claim 19]

A pAURYD-LTB(NoGP) construct illustrated in Fig. 16c.

[Claim 20]

The nucleotide according to claim 1, wherein the pasteurellosis is a disease caused by a stain selected from the group consisting of Pasteurella multocida types A, B, D, E and F.

[Claim 21]

The nucleotide according to claim 20, wherein the pasteurellosis is fowl cholera.

[Claim 22]

The method according to claim 11 , wherein the pasteurellosis is a disease caused by a stain selected from the group consisting of Pasteurella multocida types A, B, D, E and F.

[Claim 23]

The method according to claim 22, wherein the pasteurellosis is fowl cholera.

[Claim 24]

The vaccine composition according to claim 12, wherein the pasteurellosis is a disease caused by a stain selected from the group consisting of Pasteurella multocida types A, B, D, E and F.

[Claim 25]

The vaccine composition according to claim 24, wherein the pasteurellosis is fowl cholera.

[Claim 26]

The method according to claim 14, wherein the pasteurellosis is a disease caused by a stain selected from the group consisting of Pasteurella multocida types A, B, D, E and F.

[Claim 27]

The method according to claim 26, wherein the pasteurellosis is fowl cholera.

[Claim 28] The vaccine composition according to claim 17, wherein the pasteurellosis is a disease caused by a stain selected from the group consisting of Pasteurella multocida types A, B, D, E and F.

[Claim 29]

The vaccine composition according to claim 28, wherein the pasteurellosis is fowl cholera.