WO2009035160A1 - Candidate molecule for falciparum malaria vaccine - Google Patents

Abstract

It is intended to provide a malaria vaccine which contains one member selected from among the following substances (1) to (3): (1) a polypeptide containing the amino acid sequence of malaria parasite GPI8P transamidase; (2) a polypeptide containing a partial amino acid sequence of malaria parasite GPI8P transamidase, wherein the partial amino acid sequence consists of six or more amino acids, and having an immunogenicity; and (3) an expression vector which is capable of expressing the polypeptide (1) or (2).

Description

 Details

 P. falciparum vaccine

 The present invention relates to a vaccine against malaria. Specifically, the present invention relates to the use of the Plasmodium G P I 8 P transamidase gene as a malaria vaccine. Background art

 Malaria is a protozoal infection distributed in more than 70 countries in the tropics and subtropics. It is reported that 300 to 500 million people worldwide are infected with malaria, and a total of about 800 million people are infected with malaria, and 1 to 1.5 million people die from malaria.

 Malaria pathogens are unicellular organisms, malaria parasites, which are mediated by anopheles force (Anopheles s pp.). There are four species: P. falciparum, P. vivax, P. malariae, and P. ovale. Malaria caused by Plasmodium falciparum is particularly severe. The highest protective effect of a malaria vaccine is confirmed by a vaccine in which sporozoites, which are infectious malarial parasites, are inactivated by irradiation. However, because there are advanced facilities for production, it is not suitable for dissemination in developing regions. Therefore, the development of a new vaccine that is powerful and easy to prepare is expected. However, human malaria is basically a primate and does not force infection, so the effectiveness of the vaccine can only be confirmed by experimental ability using monkeys and simulated experiments that infect mice with mouse malaria parasites. I can't. Therefore, the development of a new vaccine against human malaria is extremely difficult.

It is believed that it is more practical to purify a vaccine protein having the same effect as the above vaccine and produce it as a subunit vaccine (peptide preparation). To date, several malaria vaccine scavenger molecules have been discovered, and some have started clinical trials, but no reports have been made that their effectiveness has been confirmed. In addition, DNA vaccines are known as vaccines that are very simple to handle, such as productivity and storage, compared to live vaccines. Since a DNA vaccine can be easily produced, it is possible to provide an improved vaccine quickly against a virus with a quick mutation. However, an effective DNA vaccine against P. falciparum has not been developed yet.

 On the other hand, analysis of the genome sequence of the malaria parasite reveals the amino acid sequence and nucleotide sequence of the GPI 8P transamidase gene (Non-patent Document 1). However, there is no report on the use of the gene as a malaria vaccine.

 [Non-Patent Document 1] Nagamune K. et al., Proc. Natl. Acad. Sci. USA, vol. 10 0, pages 10682-10687, 2003 Disclosure of the Invention

 (Problems to be solved by the invention)

 An object of the present invention is to provide a new malaria vaccine that is powerful and easy to prepare.

 (Means for solving problems)

The present inventors searched for a candidate molecule having a potent actin activity in order to develop an effective vaccine against malaria, particularly P. falciparum malaria having infectivity to humans. Since P. falciparum does not infect mammals other than primates (eg, mice), it is difficult to directly confirm the effect of the vaccine against P. falciparum by animal experiments. Therefore, the present inventors selected nine in silico cell surface proteins (GPI. Anchor proteins) of mouse malaria (P. yoelii) that are highly homologous to the gene of P. falciparum from the information of the malaria genome. After preparing the cDNA, it was incorporated into a commercially available expression vector and administered to mice as a DNA vaccine. The DNA vaccine was administered by subcutaneous injection using compressed air. After the prime booster, mice were infected with malaria parasites, and the protective effect was observed. As a result, the administration of an expression vector encoding a partial peptide of GPI 8 P transamidase strongly suppressed malaria parasite infection. It was. In the vaccine administration group, blood antibodies against Plasmodium were confirmed, and this infection control effect was induced by a specific immune response against a partial peptide of GP I 8 P transamidase expressed in vivo. It was thought to be due to this.

 Based on the above findings, the present invention has been completed.

 That is, the present invention relates to the following.

 [1] A malaria vaccine comprising any substance selected from the following (1) to (3):

(1) a polypeptide comprising the amino acid sequence of Plasmodium GP I 8 P transamidase;

 (2) a polypeptide comprising a partial amino acid sequence of Plasmodium GP I 8 P transamidase, wherein the partial amino acid sequence has a length of 6 amino acids or more and has immunogenicity; as well as

 (3) An expression vector capable of expressing the polypeptide of (1) or (2).

 [2] The vaccine according to [1], wherein the partial amino acid sequence is contained within the region of amino acids 17-240 of the amino acid sequence of Plasmodium GP I 8 P transamidase.

 [3] The vaccine according to [1], wherein the substance is the expression vector according to (3).

 [4] Any substance selected from (1) to (3) below for use in vaccination against malaria:

 (1) a polypeptide comprising the amino acid sequence of Plasmodium GP I 8 P transamidase;

 (2) a polypeptide comprising a partial amino acid sequence of the Plasmodium GP I 8 P transamidase, wherein the partial amino acid sequence has a length of 6 amino acids or more and has immunogenicity; as well as

 (3) An expression vector capable of expressing the polypeptide of (1) or (2).

 [5] The substance according to [4], wherein the partial amino acid sequence is contained within the region of amino acids 17-240 of the amino acid sequence of Plasmodium GP I 8 P transamidase.

[6] The substance according to [4], which is the expression vector according to (3). [7] A method for preventing malaria infection in a mammal, comprising administering to the mammal an effective amount of any of the following substances (1) to (3):

 (1) a polypeptide containing the amino acid sequence of the malaria parasite GP I 8 P transamidase;

 (2) a polypeptide comprising a partial amino acid sequence of Plasmodium GP I 8 P transamidase, wherein the partial amino acid sequence has a length of 6 amino acids or more and has immunogenicity; as well as

 (3) An expression vector capable of expressing the polypeptide of (1) or (2).

 [8] The method according to [7], wherein the partial amino acid sequence is contained within the region of amino acids 17-240 of the amino acid sequence of the malaria parasite GP 1.8 P transamidase.

 [9] The method of [7], wherein the substance is the expression vector of (3).

 [10] Use of any substance selected from the following (1) to (3) for producing a malaria vaccine:

 (1) a polypeptide comprising the amino acid sequence of the malaria parasite GP I 8 P transamidase;

 (2) a polypeptide comprising a partial amino acid sequence of Plasmodium GP I 8 P transamidase, wherein the partial amino acid sequence has a length of 6 amino acids or more and has immunogenicity; as well as

 (3) An expression vector capable of expressing the polypeptide of (1) or (2).

 [11] The use of [10], wherein the partial amino acid sequence is contained within the region of amino acids 17-240 of the amino acid sequence of Plasmodium GP I 8 P transamidase.

 [12] Use of [10], wherein the substance is the expression vector of (3).

 (The invention's effect)

The present invention provides a new malaria vaccine that is powerful and easy to prepare. In particular, an expression vector that can express a partial peptide of GP I 8 P transamidase has a remarkable effect as a DNA vaccine against malaria, and is therefore useful as a cheaper and more stable malaria vaccine than conventional subunits. . Brief Description of Drawings

 [FIG. 1] The effect of an expression vector-containing vaccine on the survival rate of mice is shown (second trial). G1: GP I 8 P transamidase (PY03470), G 2: PY05000.

[Fig. 2] Shows the effect of an expression vector-containing vaccine on the survival rate of mice (3rd trial). G1: GP I 8 P transamidase (ΡΥ03470), G2: PY05000, G16: ΡΥ01490 _ο

 FIG. 3 shows the effect of an expression vector-containing vaccine on the development of protozoa in each mouse (second trial). Glm (l-7): GP I 8 P transamidase (PY03470), G 2 m (1-5): PY05000, C (l-2): control mouth.

 [Fig. 4] Shows the effect of expression vector-containing koutine on the development of protozoa in each mouse (third trial). Glm (1-2): GP I 8 P transamidase (PY03470), G2in (1-2): PY05000, G16m (1-2): PY01490, C (1-2) Nore.

 FIG. 5 shows the effect of an expression vector-containing vaccine on the survival rate of mice (4th trial). G1: GP I 8 Ρ transamidase (ΡΥ03470).

 FIG. 6 shows the effect of expression vector-containing vaccine on the development of protozoa in each mouse (4th trial). Gm (1-6): GP I 8 P transamidase (P Y03470).

 FIG. 7 shows the effect of an expression vector-containing vaccine on the survival rate of mice (5th trial). G1: GP I 8 P transamidase (PY03470).

 FIG. 8 shows the effect of an expression vector-containing vaccine on the development of protozoa in each mouse individual (5th trial). Glm (l-13): GP I 8 P transamidase (PY03470). BEST MODE FOR CARRYING OUT THE INVENTION

(I) Malaria vaccine The present invention provides a malaria actin (hereinafter sometimes referred to as the vaccine of the present invention) containing any substance selected from the following (1) to (3) force:

(1) Polypeptidyl K containing the amino acid sequence of the malaria parasite G P I 8 P transamidase;

 (2) a polypeptide comprising a partial amino acid sequence of Plasmodium GPI 8P transamidase, wherein the partial amino acid sequence has a length of 6 amino acids or more and has an immunogenicity; and

 (3) An expression vector capable of expressing the polypeptide of (1) or (2).

 In the present specification, the type of malaria is not particularly limited. Malaria is classified into P. falciparum malaria, S. falciparum malaria, A. malaria malaria, egg-shaped malaria, mouse malaria, etc. depending on the type of protozoa, and any type of malaria is included in the scope of the present invention. The malaria is preferably P. falciparum or mouse malaria.

 In the present specification, the type of malaria parasite is not particularly limited. The types of malaria parasite are P. falciparum, P. vivax, P. malariae, Oval malaria (P. ovale), Mouse malari And protozoa (P. yoelii). Each type of malaria parasite causes the corresponding type of malaria.

Plasmodium GPI 8 P transamidase is a known gene, and its nucleotide sequence and amino acid sequence are also known. The amino acid sequence of the GPI 8 P transamidase of Plasmodium falciparum can include the amino acid sequence represented by SEQ ID NO: 2, and is encoded by the nucleotide sequence represented by SEQ ID NO: 1, for example. In addition, the amino acid sequence of GPI 8 P transamidase of Plasmodium falciparum is an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or attached in the amino acid sequence represented by SEQ ID NO: 2. Thus, naturally occurring amino acid sequences are also included. It is known that the nucleotide arrangement IJ composing the malaria parasite gene is mutated by natural mutation. Polypeptide having an amino acid sequence different from SEQ ID NO: 2 caused by such natural mutation is also a GPI 8 P transamidase of P. falciparum. include. Examples of the amino acid sequence include: (1) one or more (preferably 1 to 30 pieces, more preferably 1 to 10 pieces, more preferably 1 to several pieces) in the amino acid sequence represented by SEQ ID NO: 2. (2-5) amino acid sequences from which amino acids have been deleted, (2) one or more amino acid sequences represented by SEQ ID NO: 2 (preferably 1-30, more preferably 1-10, More preferably, an amino acid sequence to which 1 to several (2 to 5) amino acids are added. (3) One or more (preferably 1 to 30 amino acids, more preferably, to the amino acid sequence shown in SEQ ID NO: 2. An amino acid sequence in which 1 to 10 amino acids, more preferably 1 to several (2 to 5) amino acids are inserted; (4) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 (preferably 1 to 30 amino acids, more preferably 1 to 10 amino acids, and still more preferably 1 to several (2 to 5) amino acids Is an amino acid sequence in which is substituted with another amino acid, or (5) an amino acid sequence in which the mutations in (1) to (4) above are combined (in this case, the sum of the mutated amino acids is preferably 1 to 30 More preferably, it is 1 to 10, more preferably 1 to several (2 to 5), and a naturally occurring amino acid sequence is included.

Further, the amino acid sequence of GPI 8 P transamidase of mouse malaria parasite includes the amino acid sequence represented by SEQ ID NO: 4, and is encoded by the nucleotide sequence represented by SEQ ID NO: 3, for example. Further, the amino acid sequence of GPI 8 P transamidase of mouse malaria parasite is an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4. And naturally occurring amino acid sequences are also included. Nucleotide sequences that constitute the malaria parasite gene are known to be mutated by spontaneous mutation. A polypeptide having an amino acid sequence different from SEQ ID NO: 4 caused by such a spontaneous mutation is also included in the GPI 8 P transamidase of the mouse malaria parasite. Examples of the amino acid sequence include: (1) one or a plurality of amino acid sequences represented by SEQ ID NO: 4 (preferably 1 to 30 pieces, more preferably 1 to 10 pieces, more preferably 1 to numbers (2 (2) amino acid sequence from which amino acid is deleted, (2) one or more (preferably about 1-30, more preferably 1-10) in the amino acid sequence shown in SEQ ID NO: 4, More preferably, an amino acid sequence to which 1 to several (2 to 5) amino acids are added, (3) an amino acid represented by SEQ ID NO: 4 An amino acid sequence in which one or more (preferably 1 to 30, more preferably 1 to 10, more preferably 1 to several (2 to 5)) amino acids are inserted in the acid sequence, (4) One or more amino acids in the amino acid sequence represented by SEQ ID NO: 4 (preferably 1 to 30, more preferably 1 to 10, more preferably 1 to several (2 to 5) amino acids) Or (5) an amino acid sequence in which the mutations in (1) to () above are combined (in this case, the total of the mutated amino acids is preferably 1 to 30 More preferably 1 to 10, more preferably 1 to several (2 to 5), and a naturally occurring amino acid sequence is included.

 Nucleotide sequences and amino acid sequences of GPI 8 P transamidase orthologs of other types of malaria parasites also use the nucleotide sequence and amino acid sequence information disclosed in the sequence listing in this specification, and publicly known sequence databases. Thus, appropriate primers and probes can be designed and determined easily using conventional genetic engineering techniques such as RT-PCR and plaque hybridization.

 Since the gene sequence of the malaria parasite GPI 8 P transamidase differs depending on the type of malaria parasite, it is preferable to change the origin of the GPI 8 P transamidase used in the vaccine according to the type of malaria to be targeted. . That is, when producing a vaccine against P. falciparum, P. falciparum G P I 8 P transamidase is preferably used. When producing a vaccine against Plasmodium falciparum malaria parasite, the G.sub.P. In the case of producing a vaccine against Plasmodium falciparum malaria parasite, the G.sub.P. When producing a vaccine against egg-shaped malaria, the egg-shaped malaria parasite G P I 8 P transamidase is preferably used. When producing a vaccine against mouse malaria, the mouse malaria parasite G P I 8 P transamidase is preferably used.

The partial amino acid sequence of the malaria parasite GPI 8 P transamidase contained in the polypeptide (2) is required to have immunogenicity. “Amino acid sequence has immunogenicity” means that a polypeptide comprising the amino acid sequence is administered to a mammal. This means that a specific immune response (production of specific antibodies, proliferation of specific T cells, etc.) to the polypeptide can be induced in the mammal.

 The length of the partial amino acid sequence of the malaria parasite GPI 8 P transamidase contained in the polypeptide (2) above is usually 6 amino acids or more, preferably 10 amino acids or more, and the vaccinated mammal As long as a specific immune response against the malaria parasite GPI 8 P transamidase (or the malaria parasite itself) can be induced in the body, the length is not limited. In order to increase the antigen specificity of the induced immune response, the partial amino acid sequence is preferably longer in length, and the partial amino acid sequence has a length of, for example, 50 amino acids or more, preferably 100 amino acids or more. More preferably, it is at least 200 amino acids. The partial amino acid sequence contained in the polypeptide of (2) above may be derived from any part of the malaria parasite GPI 8 P transamidase, but preferably the partial sequence is malaria parasite GPI 8 P transamidase. The amino acid sequence of the amidase is contained within the region of amino acids 17-2440. By using a partial amino acid sequence in the region, a stronger protective effect against infection can be obtained.

In addition to the amino acid sequence of the malaria parasite GPI 8 P transamidase or a partial sequence thereof, the polypeptide of (1) or (2) is 1 or 2 or more (for example, 1 to 500, preferably 1 About 1 to 100, more preferably about 1 to 15). Such amino acid additions are permissible as long as the polypeptide induces a specific immune response against the malaria parasite GPI 8 P transamidase. The amino acid sequence to be added is not particularly limited, and examples thereof include a tag for facilitating detection and purification of the polypeptide. Tags include Flag tag, histidine tag, C-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (eg, GFP, YFP, RFP, CFP, BFP, etc.), Immunoglobulin Fc Tags etc. can be illustrated. The amino acid sequence is added at the N-terminal and Z- or C-terminal of the amino acid sequence of the malaria parasite GPI 8 P transamidase or its partial sequence. In the expression vector of (3) above, the polynucleotide (DNA or RA, preferably DNA) encoding the polypeptide of (1) or (2) above, and promoter activity in the mammalian cells to be administered. It is operably linked downstream of the promoter that can be exerted. That is, the expression vector (3) can express the polypeptide (1) or (2) as a transcription product under the control of a promoter. By administering the expression vector of (3) to a mammal, the polypeptide of (1) or (2) is produced in the body of the mammal, and the polypeptide of (1) or (2) is produced in the mammal. A specific immune response to the peptide is induced.

 The promoter to be used is not particularly limited as long as it can function in mammalian cells to be administered. As the promoter, a p o 1 I-type promoter, a poll I-type promoter, a poll I I I-type promoter, or the like can be used. Specifically, SV40-derived early promoters, viral promoters such as cytomegalovirus LTR, mammalian constituent protein gene promoters such as actin gene promoter, and the like are used.

 The expression vector (3) preferably contains a transcription termination signal, that is, a terminator region downstream of the polynucleotide encoding the polypeptide (1) or (2). Furthermore, a selection marker gene for selection of transformed cells (a gene conferring resistance to drugs such as tetracycline, ampicillin, and kanamycin, a gene that complements an auxotrophic mutation, etc.) can be further contained.

 The type of vector used for the expression vector in the present invention is not particularly limited, and examples of vectors suitable for administration to mammals such as humans include viral vectors and plasmid vectors. Examples of virus vectors include retroviruses, adenoviruses, adeno-associated viruses and the like. In view of ease of production and handling and economic efficiency, a plasmid vector is preferably used.

The vaccine of the present invention can be provided as a pharmaceutical composition containing any carrier, for example, a pharmaceutically acceptable carrier, in addition to the polypeptide of (1) or (2) or the expression vector of (3). Examples of pharmaceutically acceptable carriers include excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, Perfumes such as acids and menthol, preservatives such as sodium benzoate and sodium hydrogen sulfite, stabilizers such as citrate and sodium citrate, suspending agents such as methylcellulose and polybutylpyrrole, surfactants, etc. Dispersants, diluents such as water and physiological saline, base waxes and the like can be mentioned, but the invention is not limited to them.

 In order to facilitate introduction of the expression vector into cells, the vaccine of the present invention may further contain a reagent for nucleic acid introduction. When a viral vector is used as the expression vector, retronectin, fibronectin, polyprene, or the like can be used as a gene introduction reagent. If a plasmid vector is used as the expression vector, lipofectin, lipofectamine, DOGS (transfectam), D0PE, D0TAP, DDAB, DHDEAB, HDEAB, polyprene, or poly (ethylenemine) (PEI) Cationic lipids such as can be used.

 In order to enhance the effect of acupuncture, the vaccine of the present invention can further contain an adjuvant. Examples of the adjuvant include mineral oil oil and aluminum gel. .

The vaccine of the present invention can be administered to mammals orally or parenterally. Polypeptides and expression vectors are preferably administered parenterally because they can be degraded in the stomach. Examples of preparations suitable for oral administration include solutions, capsules, sashing agents, tablets, suspensions, and emulsions. Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, topical injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants In addition, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Also, aqueous and non-aqueous sterile suspensions can be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The product should be sealed in a unit dose or multiple doses like ampoules or vials. be able to. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use. The content of active ingredient in the pharmaceutical composition is typically about 0 total pharmaceutical composition. 1 1 0 0 wt ° / _0, preferably from about 1 to 9 9 wt%, more preferably about 1 0-9 About 0% by weight.

 The dose of the vaccine of the present invention varies depending on the subject to be administered, the administration method, the dosage form, etc., but when the active ingredient is the polypeptide (1) or (2) above, it is usually The peptide is administered in the range of 1 g to l 00 0 per dose, preferably in the range of 20 g to lOO ^ g, usually 2 to 3 times over a period of 4 weeks to 18 months. When the active ingredient is the expression vector of (3) above, usually the expression vector per adult is in the range of l / g to 100 g, preferably in the range of 20 μg to 100 g Usually, it is administered 2 to 3 times over 4 weeks to 18 months.

 By administering the vaccine of the present invention to a mammal, a specific immune response (specific antibody production, specific T cell proliferation, etc.) to the malaria parasite GPI 8 P transamidase is induced, and the mammal is infected with malaria. To gain resistance to.

 The mammal to which the vaccine of the present invention is administered is usually a mammal that can be infected with the target malaria parasite. For example, Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum and Oval malaria parasites infect primates (humans, monkeys, monkeys, marmoset, aged orangutans, chimpanzees, etc.). For the purpose of preventing infection with malaria parasites, the vaccine of the present invention is administered to primates. In addition, mouse malaria parasites infect rodents (such as mice, rats, hamsters, guinea pigs, etc.). Be administered. (I I) Combination vaccine

In addition, a suitable malaria vaccine can be provided by using an appropriate amount of the above (1) or (2) polypeptide and the above (3) expression vector. Can be provided. That is, the present invention provides a malaria vaccine comprising a combination of the polypeptide (1) or (2) above and the expression vector (3) above.

 When the polypeptide and the expression vector are used in combination, the administration time of the polypeptide and the expression vector is not limited, and the polypeptide and the expression vector are administered to the administration target. They can be administered at the same time or at different times. The dosage of the polypeptide and the expression vector is not particularly limited as long as it can prevent the onset of malaria when used in the combination vaccine of the present invention, and the administration subject, administration route, disease, combination It can be suitably selected by matching.

 The administration mode of the polypeptide and the expression vector is not particularly limited as long as the polypeptide and the effort vector are combined at the time of administration. Examples of such administration modes include (1) administration of a single preparation obtained by simultaneously formulating the polypeptide and the expression vector, and (2) administration of the polypeptide and the expression vector. Simultaneous administration of two types of preparations obtained by separately formulating the drug from the same administration route, (3) Same type of two preparations obtained by separately formulating the above polypeptide and the above expression vector (4) Simultaneous administration by different administration routes of two preparations obtained by separately formulating the polypeptide and the expression vector, (5) The polypeptide Administration of two types of preparations obtained by separately preparing tide and the above expression vector at different administration routes (for example, administration in the order of the polypeptide → the expression vector, or vice versa) Administration in the order of That. Hereinafter, these administration forms are collectively abbreviated as the combined vaccine of the present invention.

 The combination vaccine of the present invention can be formulated according to conventional means by mixing with a pharmaceutically acceptable carrier in the same manner as the vaccine of the present invention of (I) above.

When the polypeptide and the expression vector are formulated simultaneously and used as a single pharmaceutical composition, the content of the polypeptide in the combination vaccine of the present invention varies depending on the form of the formulation. generally about 0.1 to 9 9 total pharmaceutical composition. 9% by weight, preferably from about 1 to 9 9 wt _^0/0, more preferably about 1 0-9 about 0 wt%. In addition, the content of the expression vector in the combination vaccine of the present invention varies depending on the form of the preparation and the like, but is usually about 0.1 to 99.9% by weight of the whole pharmaceutical composition, preferably About 1 to 99% by weight, more preferably about 10 to 90% by weight.

 The mixing ratio of the polypeptide to the expression vector in the combination vaccine of the present invention can be appropriately selected depending on the administration subject, administration route, disease and the like.

 The dose of the combination actin of the present invention varies depending on the type of the polypeptide and the expression vector, administration route, symptom, patient age, etc., and can be appropriately selected. ) Of the vaccine of the present invention.

 The same content may be used when the above-mentioned polypeptide and the above-mentioned expression vector are formulated separately.

 When the polypeptide and the expression vector are separately formulated and administered together, the pharmaceutical composition containing the polypeptide and the pharmaceutical composition containing the expression vector are administered at the same time. However, the pharmaceutical composition containing the polypeptide may be administered after the pharmaceutical composition containing the expression vector is administered first, or the pharmaceutical composition containing the polypeptide. May be administered first, followed by administration of the pharmaceutical composition containing the expression vector. Preferably, the first vaccination is performed by administration of a pharmaceutical thread containing the expression vector, and the second and subsequent vaccinations are performed by administration of a pharmaceutical composition containing the polypeptide. When administered with a time difference, the time difference varies depending on the active ingredient, dosage form, and method of administration. For example, when a pharmaceutical composition containing the above expression vector is administered first, the expression vector is included. After administering the pharmaceutical composition to be administered, a method of administering the pharmaceutical composition containing the above polypeptide usually within 2 weeks to 12 months, preferably within 4 weeks to 6 months can be mentioned. When the pharmaceutical composition containing the polypeptide is administered first, it is usually within 2 weeks to 12 months, preferably within 4 weeks to 6 months after administering the pharmaceutical composition containing the polypeptide. Examples thereof include a method for administering a pharmaceutical yarn composition containing the above expression vector.

By administering the combination vaccine of the present invention to a mammal, a specific immune response to the malaria parasite GPI 8 P transamidase (specific antibody production, proliferation of specific T cells) Etc.) are strongly induced and the mammal acquires resistance to malaria infection. The embodiment of the mammal to which the combination vaccine of the present invention is administered is the same as the above-described vaccine of the present invention (I).

 Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited to the examples shown below. Example

 (Example 1)

Nine mouse surface proteins (GPI anchor proteins) of mouse malaria (P. yoelii), which have high homology with the gene of P. falciparum malaria, were selected in silico from the information on the malaria genome (Table 1) and these cDNAs were prepared. Later, it was incorporated into a commercially available pVAXDEST200 vector and administered under the skin of 5-week-old female C57BL / 6 mice using a needleless pneumatic syringe shimajet. The dose was 4 per mouse (4 g / ΙΟΟ μ 1). After a total of 3 similar administrations at 2 week intervals, 10 days later, 1 million Plasmodium yoelii 17XL was administered by intraperitoneal infection, and the subsequent protozoalemia and the number of survivors were observed.

table 1

In Table 1, the gene ID indicates the ID number in The Plasmodium Genome Resource (http: .// 爾 · p 丄 asmodb.org/plasmo/home.jsp). The amino acid sequence represented by SEQ ID NO: 6 corresponds to the partial amino acid sequence of the GPI 8 P transamidase of the malaria parasite (17th to 240th amino acids). As a result, in the control group, all mice died of malarialemia in about 5 days, but in the group (G1) to which the expression vector encoding the mouse malaria GP I 8 P transamidase was administered, three independent experiments were performed. In the tested studies, 50%, 57% and 100% mice were alive, respectively (Table 2, Figures 1 and 2). In the group (G2) to which the expression vector encoding PY05000 was administered, the survival rate of mice was slightly increased, but even when the expression vector for other genes was administered, there was almost no effect on the survival rate. It was very good.

 In the GP I 8 P transamidase group (G1), the onset of protozoa was strongly suppressed (Figures 3 and 4). In the PY05000 group (G2), the onset of protozoa was somewhat suppressed, but administration of other gene expression vectors hardly suppressed the onset of protozoa.

 In two additional experiments, the same effect was confirmed (Figs. 5-8).

 In the GP I 8 P transamidase group (G 1), blood antibodies against malaria parasites (especially malaria parasites in the blood stage) were confirmed. This was thought to be due to the induction of a specific immune response against 8P transamidase.

 Based on the above, it was confirmed that an expression vector encoding a malaria parasite GP I 8 P transamidase or a partial peptide thereof was extremely effective as a malaria vaccine.

 Table 2

Industrial applicability The present invention provides a new malaria vaccine that is powerful and easy to prepare. In particular, an expression vector capable of expressing a partial peptide of GP I 8 P transamidase has a remarkable effect as a DNA vaccine against malaria, and is therefore useful as a cheaper and more stable malaria vaccine than conventional subunits. is there. This application is based on Japanese Patent Application No. 2007-239488 filed in Japan (filing date: September 14, 2007), the contents of which are incorporated in full herein.

Claims

The scope of the claims 1. Malaria vaccine containing any substance selected from the following (1) to (3): (1) a polypeptide comprising the amino acid sequence of Plasmodium GP I 8 P transamidase;  (2) a polypeptide comprising a partial amino acid sequence of Plasmodium GP I 8 P transamidase, wherein the partial amino acid sequence has a length of 6 amino acids or more and has immunogenicity; as well as  (3) An expression vector capable of expressing the polypeptide of (1) or (2). 2. The vaccine according to claim 1, wherein the partial amino acid sequence is contained within the region of amino acids 17-240 of the amino acid sequence of the malaria parasite GP I 8 P transamidase. 3. The vaccine according to claim 1, wherein the substance is the expression vector of (3). 4. Any of the following substances selected from (1) to (3) for use in vaccination against malaria infection:  (1) a polypeptide containing the amino acid sequence of the malaria parasite GP I 8 P transamidase;  (2) a polypeptide comprising a partial amino acid sequence of Plasmodium GP I 8 P transamidase, the partial amino acid sequence having a length of 6 amino acids or more and having an immunogenicity; and Perfect  (3) An expression vector capable of expressing the polypeptide of (1) or (2). 5. The substance according to claim 4, wherein the partial amino acid sequence is contained within the region of amino acids 17-240 of the amino acid sequence of Plasmodium GP I 8 P transamidase. 6. The substance according to claim 4, which is the expression vector of (3). 7. A method for preventing malaria infection in a mammal, comprising administering to the mammal an effective amount of any substance selected from the following (1) to (3) force:  (1) a polypeptide comprising the amino acid sequence of Plasmodium GP I 8 P transamidase;  (2) a polypeptide comprising a partial amino acid sequence of Plasmodium GP I 8 P transamidase, wherein the partial amino acid sequence has a length of 6 amino acids or more and has an immunogenicity; and  (3) An expression vector capable of expressing the polypeptide of (1) or (2). 8. The method according to claim 7, wherein the partial amino acid sequence is contained within the region of amino acids 17 to 240 of the amino acid sequence of the malaria parasite GP I 8 P transamidase. 9. The method according to claim 7, wherein the substance is the expression vector of (3),