WO2018088509A1

WO2018088509A1 - Malaria vaccine

Info

Publication number: WO2018088509A1
Application number: PCT/JP2017/040535
Authority: WO
Inventors: 晃久福島; 敬文坪井; 英造高島; ひかる長岡
Original assignee: 大日本住友製薬株式会社; 国立大学法人愛媛大学
Priority date: 2016-11-11
Filing date: 2017-11-10
Publication date: 2018-05-17
Also published as: JP2020028219A

Abstract

The present invention provides a polypeptide and a malaria vaccine comprising said polypeptide, the polypeptide comprising any of the following amino acid sequences: (a) an amino acid sequence represented by SEQ ID NO. 4; (b) an amino acid sequence represented by SEQ ID NO. 4, in which 1-10, preferably 1-5, and more preferably 1-3 amino acids have been substituted, deleted, added or inserted; and (c) an amino acid sequence having at least 95%, preferably at least 97%, and more preferably at least 99% sequence identity with SEQ ID NO. 4.

Description

Malaria vaccine

This application claims priority with respect to Japanese Patent Application No. 2016-220515, which is hereby incorporated by reference in its entirety.
The present invention relates to an antigen for a vaccine for preventing infection with malaria parasites or onset of malaria infection.

Malaria infection is an infectious disease caused by Plasmodium protozoa such as Plasmodium falciparum 寄生 as a parasitic pathogen, and is widespread in tropical and subtropical regions. Malaria infections are caused by malaria parasites that invade the human body through an anopheles through the sporozoite, liver, and erythrocyte stages. In each period, malaria parasites originate in the body. Protein is produced. If a vaccine that induces the production of an antibody using the protein as an antigen can be obtained, it is possible to attack the malaria parasite and suppress its infection and growth in the body after the infection. Currently, research and development of malaria vaccines are being carried out all over the world, but the current situation is that they have not yet been put into practical use. GPI-anchored micronemal antigen las (PlasmoDB gene code: PF3D7_0828800plasm (http://plasmodb.org/)), also called GAMA, is one of the proteins that are predicted to be expressed in the merozoite phase of Plasmodium falciparum The use as a malaria vaccine antigen has been studied (Non-patent Documents 1 and 2 and Patent Document 1).

International Publication No. 2012/011381

The problem to be solved by the present invention is to provide a polypeptide useful as a malaria vaccine antigen.

As a result of intensive studies, the present inventors have found that an antibody obtained using a fragment of the protein GAMA derived from the malaria parasite as an antigen exhibits a growth inhibitory activity of the malaria parasite, and has completed the present invention. That is, the present invention relates to:
[1] A polypeptide comprising any of the following amino acid sequences:
(A) the amino acid sequence represented by SEQ ID NO: 4,
(B) an amino acid sequence in which 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3 amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by SEQ ID NO: 4, and (C) an amino acid sequence having a sequence identity of 95% or more, more preferably 97% or more, more preferably 99% or more with the amino acid sequence represented by SEQ ID NO: 4.
[2] A polypeptide comprising the polypeptide according to 1 above linked to a carrier.
[3] The polypeptide according to 2 above, wherein the carrier is a virus particle, a lipid particle, or a carrier protein.
[4] The polypeptide according to any one of the above 1 to 3, for use as a malaria vaccine antigen.
[5] A malaria vaccine comprising the polypeptide according to any one of 1 to 4 above.
[6] Further, it contains at least one malaria vaccine antigen selected from CSP, TRAP, MSP1, AMA-1, SERA5, Ripr, EBA175, RH5, Pfs25 and Pfs230, or is used in combination with the at least one malaria vaccine antigen. 6. The malaria vaccine according to 5 above, wherein
[7] The malaria according to the above 5 or 6, further comprising a vaccine antigen against at least one infection selected from polio, diphtheria, pertussis and tetanus, or administered in combination with the at least one vaccine antigen vaccine.
[8] The malaria vaccine according to any one of 5 to 7 above, for preventing malaria parasite infection, preventing onset after infection with malaria parasite, or treating malaria infection.
[9] A polynucleotide comprising a nucleic acid sequence encoding the polypeptide according to 1 above.
[10] A polynucleotide comprising the polynucleotide according to 9 above linked to a promoter and / or regulatory element that enables expression in a host cell.
[11] An expression vector comprising the polynucleotide according to 9 or 10 above.
[12] A malaria vaccine comprising the polynucleotide according to 9 or 10 or the expression vector according to 11 above.
[13] A recombinant host cell transformed with the vector according to 11 above.
[14] The host cell according to 13 above, which is a bacterium, yeast, insect cell, or mammalian cell.
[15] An antibody that specifically recognizes the polypeptide according to 1 above.
[16] A pharmaceutical composition comprising the polypeptide according to any one of 1 to 4 above, the polynucleotide according to 9 or 10 above, the expression vector according to 11 above, or the antibody according to 15 above as an active ingredient. .

According to the present invention, it is possible to provide a polypeptide useful as a malaria vaccine antigen, a malaria vaccine containing the polypeptide, and the like.

It is a graph which shows the malaria parasite growth inhibition rate of the polyclonal IgG antibody obtained by immunizing a rabbit with GAMA or a GAMA fragment.

1. Definitions In this specification, the abbreviations of amino acids, (poly) peptides, (poly) nucleotides, etc. are defined by IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984). )], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office) and conventional symbols in the field.

GPI-anchored micronemal antigen (also called GAMA) is an amino acid sequence represented by SEQ ID NO: 2, PlasmoDB gene code: PF3D7_0828800 (http://plasmodb.org/) or NCBI Reference Sequence: XP_001349238 (https: // www. ncbi.nlm.nih.gov/) is a protein that is predicted to be expressed in the merozoite phase of Plasmodium falciparum. As used herein, GAMA includes a protein comprising the amino acid sequence of SEQ ID NO: 2 and a protein comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2.
Here, as the substantially identical amino acid sequence,
(A) an amino acid sequence in which 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3 amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by SEQ ID NO: 2; (B) an amino acid sequence having a sequence identity of 95% or more, more preferably 97% or more, more preferably 99% or more with the amino acid sequence represented by SEQ ID NO: 2,
Is mentioned.

In the present specification, the GAMA gene is not particularly limited as long as it is a polynucleotide encoding the GAMA, and may include both DNA and RNA. Specific examples of GAMA genes include SEQ ID NO: 1, PlasmoDB gene code: PF3D7_0828800 (http://plasmodb.org/)) and NCBI Reference Sequence: XM_001349202 (https://www.ncbi.nlm.nih.gov/ The polynucleotide shown by the base sequence described in).

As used herein, “sequence identity” refers to sequence identity between two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the protein to be compared may have an addition or a deletion (for example, a gap or the like) in the optimal alignment of the two sequences. Such sequence identity is calculated, for example, by creating an alignment using Vector NTI using the Clustal W algorithm (Nucleic Acid Res.,: 22 (22): 4673-4680 (1994)). be able to. The sequence identity is measured using sequence analysis software, specifically Vector NTI, GENETYX-MAC or an analysis tool provided by a public database. The public database is generally available at, for example, a homepage address http://www.ddbj.nig.ac.jp.

2. Polypeptide TECHNICAL FIELD This invention relates to the fragment which consists of a part of amino acid sequence of the above-mentioned GAMA useful as a malaria vaccine antigen. Specifically, the 602nd to 715th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2, that is, the amino acid sequence represented by SEQ ID NO: 4, or a polymorphism consisting of an amino acid sequence substantially identical to the amino acid sequence. It is a peptide (hereinafter sometimes referred to as the polypeptide of the present invention).
Here, as an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 4,
(A) an amino acid sequence in which 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3 amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by SEQ ID NO: 4, and (B) an amino acid sequence having a sequence identity of 95% or more, more preferably 97% or more, more preferably 99% or more with the amino acid sequence represented by SEQ ID NO: 4,
Is mentioned.

The number of amino acid mutations and mutation sites are preferably selected so that the resulting polypeptide retains immunological activity equivalent to that of a polypeptide comprising the original amino acid sequence. Indicators that determine how many amino acid residues need to be substituted, deleted, added or inserted without loss of immunological activity are computer programs well known to those skilled in the art, such as DNA Star. It can be found using software. For example, the number of mutations is typically within 5% of all amino acids, preferably within 3% of all amino acids, and more preferably within 1% of all amino acids. Further, the amino acid to be substituted is preferably selected so that the polypeptide obtained after the substitution retains immunological activity equivalent to that of the polypeptide consisting of the amino acid sequence before substitution. The amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution in terms of amino acid polarity, charge, solubility, hydrophobicity, hydrophilicity, amphiphilicity, etc. from the viewpoint of maintaining the structure of the protein. . For example, Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are amino acids classified as non-polar amino acids, and Gly, Ser, Thr, Cys, Tyr, Asn and Gln are mutually uncharged amino acids. Asp and Glu are amino acids that are classified as acidic amino acids, and Lys, Arg, and His are amino acids that are classified as basic amino acids. Therefore, amino acids belonging to the same group can be appropriately selected using these as indices.

That is, in a preferred embodiment, the polypeptide of the present invention comprises
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4,
(B) consisting of an amino acid sequence in which 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3 amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by SEQ ID NO: 4 A polypeptide having immunological activity equivalent to that of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4, and (c) 95% or more of the amino acid sequence represented by SEQ ID NO: 4, more preferably 97% or more, More preferably, a polypeptide comprising an amino acid sequence having a sequence identity of 99% or more and having an immunological activity equivalent to that of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4,
A polypeptide selected from

In the present specification, “immunological activity” in “an immunological activity equivalent to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: X” (where X is an integer) means immunity against malaria parasites. Refers to response inducing activity. A polypeptide having “an immunological activity equivalent to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: X” means 70%, 80% of the effect of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: X, It means a polypeptide that shows 90% or more than 95% effect. The immunological activity of the polypeptide can be confirmed by methods well known to those skilled in the art. For example, the antiprotozoal activity of an antibody obtained by immunizing the polypeptide according to the method described in the Examples This can be confirmed by examining. Alternatively, it can be confirmed by administering the polypeptide to an animal model of malaria infection.

The polypeptide of the present invention can be appropriately linked to a carrier well known to those skilled in the art by chemical covalent bonding (conjugate) via a cross-linked structure or hybridized as a chimeric peptide. Conjugates and hybrids are also within the scope of the present invention.

Examples of carriers used herein include virus-like particles, lipid particles such as liposomes, keyhole limpet hemocyanin, or carrier proteins such as bovine serum albumin, CRM197 or Pseudomonas aeruginosa extracellular toxin A.

Examples of the crosslinking agent for forming a crosslinked structure (linker), that is, a chemical covalent bond for linking the polypeptide of the present invention and the carrier, include a homobifunctional crosslinking agent and a heterobifunctional crosslinking agent. For example, homobifunctional cross-linking agents include N, N'-disuccinimidyl bissuberate, 1,4-bismaleimidobutane (1,4-bis (maleimido) butane), Heterobifunctional cross-linking agents include 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-succinimidyl sodium 4- (N-maleimidomethyl) 4-cyclohexane- 1-carboxylate Sodium Salt) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

The hybrid of the polypeptide of the present invention and the carrier can be produced in the same manner as the polypeptide of the present invention based on the amino acid sequence of the polypeptide of the present invention and the carrier or the nucleic acid sequence encoding the same.

3. Polynucleotides An embodiment of the present invention includes a polynucleotide comprising a nucleic acid sequence encoding the polypeptide of the present invention (hereinafter referred to as the polynucleotide of the present invention). Specifically, the 602nd to 715th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2, that is, the amino acid sequence represented by SEQ ID NO: 4, or a polymorphism consisting of an amino acid sequence substantially identical to the amino acid sequence. The polynucleotide is not particularly limited as long as it is a polynucleotide encoding a peptide, and may be either DNA or RNA, and includes both a single-stranded nucleic acid sequence and a double-stranded nucleic acid sequence.
Here, as a polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising substantially the same amino acid sequence,
(A) an amino acid sequence in which 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3 amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by SEQ ID NO: 4, and (B) an amino acid sequence having a sequence identity of 95% or more, more preferably 97% or more, more preferably 99% or more with the amino acid sequence represented by SEQ ID NO: 4,
And a polynucleotide comprising a nucleic acid sequence encoding

An example of the polynucleotide of the present invention is a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 3. A further example of the polynucleotide of the present invention includes a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 6. In the present specification, the nucleic acid sequence shown in the sequence listing such as SEQ ID NO: 3 is a DNA sequence for convenience, but in the case of an RNA sequence, thymine (T) is interpreted as uracil (U).

The polynucleotide of the present invention may be linked to a promoter and / or regulatory element that enables expression of the polypeptide of the present invention in a host cell, comprises the polynucleotide of the present invention as a protein coding region, and Polynucleotides linked to / or regulatory elements are also included within the scope of the present invention.

When the polynucleotide of the present invention is double-stranded, a recombinant expression vector for expressing the polypeptide of the present invention can be prepared by inserting the polynucleotide of the present invention into an expression vector. That is, the category of the present invention includes an expression vector containing the polynucleotide of the present invention.

The expression vector can be appropriately selected according to the host to be used, purpose, etc., and examples thereof include plasmids, phage vectors, virus vectors and the like. For example, when the host is E. coli, examples of the vector include plasmid vectors such as pUC118, pUC119, pBR322, and pCR3, and phage vectors such as λZAPII and λgt11. When the host is yeast, examples of the vector include pYES2, pYEUra3, and the like. When the host is an insect cell, examples include bacmid using pFastBac1 and pAcSGHisNT-A. When the host is an animal cell, plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.1, pRC / RSV, pRc / CMV, and viral vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors Is mentioned. When the host is a plant, vectors such as tobacco mosaic virus vectors and Agrobacterium vectors can be mentioned.

The vector may appropriately have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a marker gene for selection, and a terminator. In addition, a sequence expressed as a fusion protein with Gp67, thioredoxin, His tag, GST (glutathione S-transferase) or the like may be added so that isolation and purification can be facilitated. In this case, a GST fusion protein vector (such as pGEX4T) having an appropriate promoter (such as lac, tac, trc, trp, CMV, SV40 early promoter) that functions in the host cell, or a vector having a tag sequence such as Myc, His, etc. (Eg, pcDNA3.1 / Myc-His), and a vector (pET32a) that expresses a fusion protein with thioredoxin and His tag can be used.

By transforming a host with the expression vector prepared above, a transformed cell or a transformed plant containing the expression vector can be prepared. Examples of the host used here include Escherichia coli, yeast, insect cells, mammalian cells, plant cells, and individual plants. Examples of E. coli include DH10Bac strain, E. coli. HB101 strain, C600 strain, JM109 strain, DH5α strain, AD494 (DE3) strain and the like of E. coli K-12 strain. Examples of yeast include Saccharomyces cerevisiae. Examples of animal cells include L929 cells, BALB / c3T3 cells, C127 cells, CHO cells, COS cells, Vero cells, Hela cells, and 293-EBNA cells. Insect cells include sf9. Examples of the plant include Nicotiana ianabenthamiana.

As a method for introducing the expression vector into the host, a normal method suitable for the host may be used. Specifically, calcium phosphate method, DEAE-dextran method, electroporation method, method using lipid for gene transfer (Cellfectin II, Lipofectamine, Lipofectin; Gibco-BRL), Agrobacterium method, microinjection method, particle gun method Etc. After the introduction, the transformant introduced with the expression vector can be selected by culturing in a normal medium containing a selection marker.

The polypeptide of the present invention can be produced by culturing or growing the transformant obtained as described above under suitable conditions. The obtained polypeptide can be further isolated and purified by general biochemical purification means. Examples of the purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography and the like. Further, when the polypeptide of the present invention is expressed as a fusion polypeptide with the aforementioned thioredoxin, His tag, GST, etc., it can be isolated and purified by a purification method utilizing the properties of these fusion polypeptide and tag. it can.

In addition, the polynucleotide of the present invention can be administered to animals by incorporating it into a gene therapy vector or the like. Gene therapy vectors incorporating the polynucleotides of the present invention are also within the category of expression vectors of the present invention.

Examples of gene therapy vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai viruses, vaccinia viruses, pox viruses, polioviruses, Synbis viruses and the like. Of these, retrovirus, adenovirus, adeno-associated virus, and vaccinia virus are particularly preferred. In addition, plasmids that are non-viral vectors can also be used.

A cell in which the polynucleotide of the present invention is expressed can produce the polypeptide of the present invention in vivo. That is, the polynucleotide of the present invention or the expression vector of the present invention can itself be used as a malaria vaccine.

4). Production of Polypeptide The polypeptide of the present invention is produced by a gene recombination method in which a polynucleotide encoding the polypeptide is expressed in a suitable host cell, a method using a cell-free synthesis system, a method of chemical synthesis. Etc., and can be produced by methods well known to those skilled in the art. This will be described below.

The polypeptide of the present invention is based on the gene sequence information of SEQ ID NO: 1 or SEQ ID NO: 3, DNA cloning, expression vector construction, transfection into the host, transformant culture, and recovery of the polypeptide from the culture It can obtain by operation of. Examples of the expression vector and host include those described above. These operations are performed according to methods known to those skilled in the art and methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM.Glover, IRL PRESS (1985)), etc. It can be carried out. The category of the present invention also includes transformed cells or transformed plants that express the polypeptide of the present invention.

In addition, the polypeptide of the present invention can be produced using a cell-free protein synthesis system. As the cell-free protein synthesis system, methods well known to those skilled in the art, such as the method described in International Publication No. 05/030954 using wheat germ extract, can be used as appropriate. In the method described in WO05 / 030954, components containing ribosomes and the like are extracted from wheat germ, and transferred to this extract or translation template, nucleic acid serving as substrate, amino acid, energy source, various ions, buffer solution , And other effective factors are added in vitro. Among these, one using RNA as a template (hereinafter sometimes referred to as “cell-free translation system”) and one using DNA and further adding an enzyme required for transcription such as RNA polymerase (this is performed) (Hereinafter sometimes referred to as “cell-free transcription / translation system”). In the production of the polypeptide of the present invention, the RNA transcribed from the DNA containing the polynucleotide of the present invention is used as a translation template, or the DNA containing the polynucleotide of the present invention is used as a transcription template for preparing a translation template in a test tube. Can be used. In addition to the polynucleotide of the present invention, the translation template may contain an RNA polymerase recognition sequence (for example, SP6, T3 or T7 promoter) and a sequence (for example, an Ω sequence or an E01 sequence) that enhances the translation activity in the synthesis system. As the wheat germ extract-containing liquid used here, a liquid marketed as WEPRO (registered trademark; manufactured by Cell Free Science) can be used. Alternatively, a wheat germ extract can be prepared according to the method described in, for example, Johnston, F. B. et al., Nature, 179, 160-161 (1957). In addition, as a method for extracting the wheat germ extract-containing liquid from the isolated germ, for example, Erickson, A. H. et al., (1996) Meth. In Enzymol., 96, 38-50, etc. The method can be used. In addition, the method described in International Publication No. 03/064671 is exemplified. Specifically, as shown in the Examples of the present specification, DNA containing a polynucleotide consisting of the nucleic acid sequence represented by SEQ ID NO: 6 or RNA transcribed therefrom can be used. Nucleotides are also mentioned as an aspect of the present invention.

Alternatively, the polypeptide of the present invention can be produced according to a method used in ordinary peptide chemistry. Specifically, literature (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press1976Inc., New York, 1976; Peptide synthesis, Maruzen (stock) ), 1975; Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1985; Chemical Development, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991). For example, the polypeptide of the present invention is produced by a method of producing by a solid phase synthesizer using the Fmoc method or Boc method, or a method of producing by sequentially condensing Boc-amino acids or Z-amino acids by a liquid phase synthesis method. (Fmoc represents a 9-fluorenylmethoxycarbonyl group, Boc represents a t-butoxycarbonyl group, and Z represents a benzyloxycarbonyl group). The obtained polypeptide can be purified according to a method used in ordinary peptide chemistry. For example, it can be purified by various chromatography (for example, silica gel column chromatography, ion exchange column chromatography, gel filtration, or reverse phase chromatography) or recrystallization. For example, the recrystallization solvent includes alcohol solvents such as methanol, ethanol or 2-propanol, ether solvents such as diethyl ether, ester solvents such as ethyl acetate, aromatic hydrocarbon solvents such as benzene or toluene, acetone A ketone solvent such as hexane, a hydrocarbon solvent such as hexane, an aprotic solvent such as dimethylformamide or acetonitrile, water, or a mixed solvent thereof can be used. As other purification methods, the methods described in Experimental Chemistry Course (The Chemical Society of Japan, Maruzen) vol. 1 can be used.

5). Antibody One embodiment of the present invention includes an antibody that specifically recognizes the polypeptide of the present invention (hereinafter referred to as the antibody of the present invention). As used herein, the term “antibody” includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, and antibodies that have antigen-binding properties such as those produced by Fab fragments, Fab expression libraries, and the like. Some are included.

Methods for producing antibodies are already known to those skilled in the art, and the antibodies of the present invention can also be produced according to known methods (Current protocols Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons . Section 11.12-11.13; Antibodies: A Laboratory Manual, Second Edition, Edward A. Greenfield, Cold Spring Harber Laboratory Press, New York 2013).

Specifically, a polyclonal antibody can be produced by using the polypeptide of the present invention produced according to a method well known to those skilled in the art, such as a genetic recombination method, a cell-free synthesis system method, a chemical synthesis method, and the like. It is possible to immunize a non-human animal and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, a monoclonal antibody can be obtained from hybridoma cells prepared by immunizing a non-human animal such as a mouse with the polypeptide of the present invention and fusing the obtained spleen cells and myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11; Antibodies: A Laboratory Manual, Second Edition, Edward A. Greenfield, Cold Spring Harber Laboratory Press)

Preparation of an antibody against the polypeptide of the present invention can also be performed by enhancing the immunological reaction using various adjuvants depending on the host. Such adjuvants include Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol, BCG (calmet) -Human adjuvants such as (Guerin gonococci) and Corynebacterium parvum.

As described above, an antibody that specifically recognizes the polypeptide of the present invention, and further an antibody that neutralizes the activity of GAMA can be easily prepared by appropriately immunizing an animal using the polypeptide of the present invention by a conventional method. it can. Applications of the antibody include suppression of the onset of serious diseases caused by malaria parasite infection, affinity chromatography, immunological diagnosis, and the like. The immunological diagnosis can be appropriately selected from immunoblotting, radioimmunoassay (RIA), enzyme immunoassay (ELISA), fluorescence or luminescence assay.

6). The pharmaceutical composition of the present invention, the polynucleotide of the present invention, the expression vector of the present invention, or the antibody specifically recognizing the polypeptide of the present invention prevents malaria parasite infection, prevents onset after malaria parasite infection, Or it is useful for the treatment of malaria infection. That is, a pharmaceutical composition comprising the polypeptide of the present invention, the polynucleotide of the present invention, the expression vector of the present invention, or the anti-polypeptide antibody of the present invention is also an aspect of the present invention. In certain embodiments, the pharmaceutical composition of the invention is a malaria vaccine.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or a suitable adjuvant so that the acquired immunity is effectively established. Alternatively, the polypeptide of the present invention, the polynucleotide of the present invention, the expression vector of the present invention, or the composition comprising the anti-polypeptide antibody of the present invention and a carrier is mixed with a composition containing an adjuvant, or administered or used in combination. Can also be administered. That is, the pharmaceutical composition of the present invention may be provided as a kit in combination with an adjuvant composition.

As adjuvants, those described in the literature (Nature Medicine, 19, 1597-1608, 2013) and the like can be applied. Specifically, viruses, fungus-derived components or derivatives thereof, cytokines, plant-derived components or their Derivatives, marine organism-derived components or derivatives thereof, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin and pluronic polyol, polyanions and the like can be mentioned.

In the above, “cell-derived component or derivative thereof” specifically includes, for example, (i) dead bacteria, (ii) bacterial cell wall skeleton (abbreviated as Cell Wall Skeleton, CWS), (iii) It is classified into specific components derived from bacterial cells or derivatives thereof.
Here, (i) bacteria killed bacteria include, for example, streptococcal powder (eg, Pisibanil; Chugai Pharmaceutical Co., Ltd.), killed bacteria cocktail (eg, Broncasma Berna; Sanwa Chemical Laboratory), or death of M. tuberculosis Examples include bacteria.
(Ii) As CWS derived from bacteria, CWS derived from the genus Mikebacteria (for example, CWS of BCG strain which is Mycobacterium genus Mycobacterium tuberculosis), CWS derived from the genus Nocardia (for example, CWS of Nocardia Nobra), or Examples include CWS derived from Corynebacterium.
(Iii) Specific components derived from bacterial cells or derivatives thereof include, for example, polysaccharides derived from Mycobacterium tuberculosis that are polysaccharides derived from bacterial cells (for example, Answer; Zeria Shinyaku Kogyo Co., Ltd.) and polysaccharides derived from basidiomycetes (for example, Lentinan; Ajinomoto, Krestin; Sankyo Corporation, basidiomycete Kawaratake), muramyl dipeptide (MDP) related compound, lipopolysaccharide (LPS), lipid A related compound (MPL), glycolipid trehalose dimycolate (TDM), bacteria DNA derived from the source (for example, CpG oligonucleotide), nucleic acid derived from a virus, or derivatives thereof (for example, poly I: C).

These bacterial cell-derived components and derivatives thereof are obtained if they are already commercially available, or known literatures (for example, CancerCanRes., 33, 2187-2195 (1973), J. Natl. Cancer Inst. , 48, 831-835 (1972, J. Bacteriol., 94, 1736-1745 (1967), Gann, 69, 619-626 (1978), J. Bacteriol., 92, 869-879 (1966), J. Natl. Cancer Inst., 52, 95-101 (1974)) and the like.

In the above, “cytokine” includes, for example, IFN-α, IL-12, GM-CSF, IL-2, IFN-γ, IL-18, or IL-15. These cytokines may be natural products or gene recombinant products. These cytokines can be obtained and used if they are already on the market. In the case of a gene recombinant product, for example, based on each nucleotide sequence registered in a database such as GenBank, EMBL, or DDBJ, a desired gene is cloned by an ordinary method and ligated to an appropriate expression vector. Expression and production can be achieved by transforming host cells with the recombinant expression vector.

In the above, “plant-derived component or derivative thereof” includes saponin-derived components such as Quil A (Accurate Chemical & Scientific Corp), QS-21 (Aquila Biopharmaceuticals グリ inc.), Glycyrrhizin (SIGMA-ALDRICH, etc.), and the like. It is done.

In the above, examples of the “marine organism-derived component or derivative thereof” include α-galactosylceramide, which is a glycolipid derived from sponge.

As the dosage form of the pharmaceutical composition of the present invention, specifically, an oil emulsion (emulsion preparation) liposome preparation, a particulate preparation bound to beads having a diameter of several μm, a preparation bound to lipid, Examples include dosage forms such as sphere preparations and microcapsule preparations.

Examples of the oil emulsion (emulsion preparation) include water-in-oil (w / o) emulsion preparation, oil-in-water (o / w) emulsion preparation, and water-in-oil-in-water (w / o / w) emulsion. Examples include preparations. Here, the water-in-oil (w / o) emulsion preparation takes a form in which an active ingredient is dispersed in a dispersed phase of water. The oil-in-water (o / w) emulsion preparation takes a form in which an active ingredient is dispersed in a water dispersion medium. The water-in-oil-in-water (w / o / w) emulsion preparation takes a form in which an active ingredient is dispersed in the innermost water dispersion phase. Such an emulsion preparation can be prepared with reference to, for example, JP-A-8-985 and JP-A-9-122476.

In the above description, the liposome preparation is a fine particle in which an active ingredient is encapsulated in a lipid or bilayer liposome in an aqueous phase or in a membrane. Major lipids for making liposomes include phosphatidylcholine, sphingomyelin, and the like, and dicetyl phosphate, phosphatidic acid, phosphatidylserine, etc. are added thereto to charge the liposomes and stabilize them. Examples of the liposome preparation method include ultrasonic method, ethanol injection method, ether injection method, reverse phase evaporation method, French press extraction method and the like.

In the above description, the microsphere preparation is a fine particle composed of a homogeneous polymer matrix in which an active ingredient is dispersed in the matrix. Examples of the matrix material include biodegradable polymers such as albumin, gelatin, chitin, chitosan, starch, polylactic acid, and polyalkylcyanoacrylate. As a method for preparing a microsphere preparation, a known method (Eur. J. Pharm. Biopharm. 50: 129-146, 2000, Dev. Biol. Stand. 92: 63-78, 1998, Pharm. Biotechnol. 、 10: 1 -43, 1997) etc., and not particularly limited.

In the above, the microcapsule preparation is a fine particle having an active ingredient as a core substance and covered with a coating substance. Examples of the coating material used for the coating substance include film-forming polymers such as carboxymethyl cellulose, cellulose acetate phthalate, ethyl cellulose, gelatin, gelatin / gum arabic, nitrocellulose, polyvinyl alcohol, and hydroxypropyl cellulose. Examples of the method for preparing the microcapsule preparation include a coacervation method and an interfacial polymerization method.

The pharmaceutical composition of the present invention is useful for preventing infection with malaria parasites, preventing onset after infection with malaria parasites, or treating malaria infections. Here, “preventing malaria parasite infection” means preventing malaria parasite from infecting a host. “Preventing development after infection with plasmodium” refers to prevention of various symptoms exhibited by the host due to infection with plasmodium, including symptoms such as high fever, headache, nausea, impaired consciousness, and renal failure. . “Treatment of malaria infection” means to cure or ameliorate symptoms caused by malaria parasite infection.

The administration form, administration route and the like of the pharmaceutical composition to the subject can be appropriately determined according to the purpose and the subject. For example, it can be administered intravenously, arterial, subcutaneously, intramuscularly, transdermally, nasally, orally in an appropriate dosage form such as an injection, transdermal agent, inhalation / nasal agent, oral agent and the like. In general, intramuscular administration of an injection is widely used as a vaccine, and such administration form and administration route can be applied to the pharmaceutical composition of the present invention.

Methods for introducing the polynucleotide or expression vector of the present invention into cells include viral vector methods and other methods (Nikkei Science, April 1994, 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23. -48 (1994), experimental medicine special edition, 12 (15), (1994), and cited references thereof, etc.) can be applied.

Examples of the method using a viral vector include DNA viruses or RNA viruses such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai viruses, vaccinia viruses, poxviruses, polioviruses, and synbisviruses. Examples thereof include a method for introducing and incorporating a polynucleotide. Of these, methods using retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and the like are particularly preferred. Examples of other methods include a method of directly administering an expression plasmid (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method, a calcium phosphate method, an electroporation method, and the like, and the DNA vaccine method and the liposome method are particularly preferable. .

In order for the polynucleotide or expression vector of the present invention to act as a vaccine, the polynucleotide or the like is directly introduced into the body, and certain types of cells are collected from humans and introduced outside the body to introduce the polynucleotide or the like. There is a ex vivo method to return cells to the body (Nikkei Science, April 1994 issue, 20-45 pages, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Extra Number, 12 (15), ( 1994), and references cited therein). Among them, the in vivo method is more preferable.

When administered by the in vivo method, for example, it can be in the form of a preparation such as a solution, but is generally an injection containing the polynucleotide or expression vector of the present invention, which is an active ingredient, etc. A pharmaceutically acceptable carrier (carrier) may be added. Further, it may be a liposome or membrane fusion liposome (Sendai virus (HVJ) -liposome or the like) containing the polynucleotide or expression vector of the present invention. In this case, a liposome such as a suspension, a freezing agent, or a centrifugal concentrated freezing agent. It can be in the form of a formulation. The pharmaceutical composition of the present invention may be in the form of a cell culture solution infected with a virus into which an expression vector containing the polynucleotide of the present invention has been introduced.

The dose and schedule of active ingredients in various preparations can be appropriately adjusted depending on the purpose of administration, the age, weight, etc. of the subject. For example, the dosage of the polypeptide of the present invention is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 100 mg, more preferably 0.01 mg to 10 mg.

The administration schedule of the pharmaceutical composition of the present invention can be appropriately adjusted according to the purpose of administration, the age, weight, etc. of the subject. For example, the number of administrations may be once, or it may be administered several times (for example, 2 to 5 times) at intervals of several days or weeks. In addition, as the first-stage administration, one or a plurality of times (for example, 2 to 5 times) are administered, and after a period required for maintaining or enhancing the immune response (for example, after 1 to 10 years), One or a plurality of times (for example, 2 to 5 times) may be administered as the second phase administration.

In the pharmaceutical composition of the present invention, one or more other malaria vaccine antigens known to those skilled in the art can be appropriately selected and combined with the polypeptide of the present invention. The pharmaceutical composition of the present invention may contain other malaria vaccine antigens in addition to the polypeptide of the present invention, and may be administered in combination with other malaria vaccine antigens. When used in combination, the pharmaceutical composition of the present invention and the composition containing other malaria vaccine antigens may be mixed or administered separately. That is, the pharmaceutical composition of the present invention may be provided in the form of a kit comprising a combination of a composition containing the polypeptide of the present invention and a composition containing another malaria vaccine antigen. Other malaria vaccine antigens include CSP (circumsporozoite protein), TRAP (thrombospondin-related protein protein), MSP1 (merozoite surface protein-1), AMA-1 (apical protein membrane antigen 1), SERA5 (serine protein repeat antigen 5), Ripr (Rh5 interacting protein), EBA175 (erythrocyte binding antigen 175), RH5 (reticulocyte-binding protein homologue 5), Pfs25 (Plasmodium falciparum surface protein 25) and Pfs230 (Plasmodium falciparum protein) Can be mentioned. Examples of the analog include a fragment peptide (partial peptide) of an antigen protein, an antigen protein or a fragment peptide thereof in which one or more, preferably one or several amino acids are substituted, deleted, added or inserted, Examples thereof include a polypeptide in which one or a plurality, preferably one or several, of an antigen protein or a fragment peptide thereof is fused.

In the pharmaceutical composition of the present invention, one or more other infectious disease vaccine antigens known to those skilled in the art can be appropriately selected and combined with the polypeptide of the present invention. The pharmaceutical composition of the present invention may contain other infectious disease vaccine antigens in addition to the polypeptide of the present invention, and may be administered in combination with other infectious disease vaccine antigens. When used in combination, the pharmaceutical composition of the present invention and the composition containing other infectious disease vaccine antigens may be mixed or administered separately. That is, the pharmaceutical composition of the present invention may be provided in the form of a kit comprising a composition comprising the polypeptide of the present invention and a composition comprising another infectious disease vaccine antigen. Examples of the infectious diseases include polio, diphtheria, pertussis, tetanus and the like. These infectious disease vaccine antigens can be appropriately selected from those known to those skilled in the art, and examples include pertussis protective antigen, diphtheria toxoid, tetanus toxoid, and inactivated poliovirus.

The present invention also includes a method for preventing infection with malaria parasites, a method for preventing onset after infection with malaria parasites, or a method for treating malaria infections, comprising administering an effective amount of the pharmaceutical composition of the present invention to a human. . As used herein, “effective amount” means an amount sufficient to exert a desired preventive or therapeutic effect.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1
A pEU-E01-MCS vector in which a His-tagged sequence was added to the C-terminus of GAMA synthetic DNA codon-modified in wheat, and a His-tag was added to the C-terminus of 8 types of fragments subcloned from GAMA codon-modified DNA. Transcription was carried out using the pEU-E01-GST-TEV-N2 vector incorporating the sequence as a template. All peptides were synthesized using a sequence in which a sequence encoding Met at the N-terminus and 6 × His at the C-terminus was added as a template.

The wheat codon sequence encoding the 25th to 715th amino acids of the amino acid sequence shown in SEQ ID NO: 2 is represented by SEQ ID NO: 5, and the wheat codon sequence encoding the 602nd to 715th amino acids (SEQ ID NO: 4) is represented by SEQ ID NO: 6 respectively. The total amount of mRNA obtained was subjected to protein synthesis using a wheat germ cell-free protein synthesis kit WEPRO (registered trademark) 7240H (Cell Free Science). The resulting protein synthesis reaction solution was affinity purified using nickel sepharose 6 Fast Flow (GE Healthcare). Antigen solution containing 0.14 mg of polypeptide was mixed with the same volume of Freund's complete adjuvant and sensitized subcutaneously in rabbits. Further, 4 weeks after the initial sensitization, an antigen solution containing 0.1 mg of the polypeptide was mixed with the same volume of Freund's complete adjuvant and boosted subcutaneously. Blood was collected 6 weeks after the first sensitization, and serum was prepared. IgG was purified from serum using HiTrap protein G G Sepharose column (GE Healthcare), normal human erythrocytes were added, and IgG that nonspecifically bound to erythrocytes was removed was used as a polyclonal antibody. Polyclonal antibodies, human erythrocytes, and plasmodium-infected erythrocytes were mixed and cultured for 25 hours, and the protozoan nucleus was stained with Cyber Green, and then the number of erythrocyte-infected erythrocytes was quantified using FACS. The malaria parasite growth inhibition rate was calculated by calculating the number of malaria parasite-infected erythrocytes in each antibody-treated group when the number of malaria parasite-infected erythrocytes in the antibody-untreated group was taken as 100, and dividing this from 100.

The full length GAMA and 8 types of fragment (antigen peptides) tested are as follows.
GAMA full (full length GAMA): peptide consisting of the 25th to 715th amino acid sequences of SEQ ID NO: 2
GAMA 1-1: Peptide consisting of the 25th to 129th amino acid sequence of SEQ ID NO: 2
GAMA 1-2: Peptide consisting of the amino acid sequence from the 130th to the 241st amino acid sequence of SEQ ID NO: 2
GAMA 1-3: Peptide consisting of the amino acid sequence from position 242 to position 372 of SEQ ID NO: 2
GAMA 1-4: A peptide consisting of the amino acid sequence of the 465th to 601st amino acids of SEQ ID NO: 2.
GAMA 1-5: Peptide consisting of the amino acid sequence of the 602nd to 715th amino acids of SEQ ID NO: 2 (SEQ ID NO: 4)
GAMA 2-1: peptide consisting of the 76th to 184th amino acid sequence of SEQ ID NO: 2
GAMA 2-2: A peptide consisting of the amino acid sequence from the 185th to the 306th amino acid sequence of SEQ ID NO: 2.
GAMA 2-3: Peptide consisting of the amino acid sequence from 533 to 658 of SEQ ID NO: 2

FIG. 1 shows the results of measuring the malaria parasite growth inhibitory activity of a rabbit polyclonal antibody obtained by immunization with full-length GAMA and 8 types of fragment (antigen peptide). From these results, it was found that the GAMA1-5 peptide exhibits plasmodium growth inhibitory activity, and exhibits higher plasmodium growth inhibitory activity than full-length GAMA.

The amino acid sequence of GAMA1-5 is shown below.

GAMA1-5
EENELVNNFSTKYVLIYEKMKLQELKEMEESKLKMKYSKTNLSALQVTNPQNNKDKNDASNKNNNPNNSSTPLIAVVTDLSGEKTEDIINNNVDIATLSVGVQNTFQGPNAKAG (SEQ ID NO: 4)

The polypeptide, polynucleotide, expression vector, and antibody of the present invention are useful for preventing infection with malaria infection or onset of malaria infection.

SEQ ID NO: 3: Nucleic acid sequence encoding the amino acids 602 to 715 of SEQ ID NO: 2 SEQ ID NO: 4: The amino acids 602 to 715 of SEQ ID NO: 2 SEQ ID NO: 5: SEQ ID NO: 2 Wheat codon sequence encoding the 25th to 715th amino acid sequence SEQ ID NO: 6: Wheat codon sequence encoding the 602nd to 715th amino acid sequence of SEQ ID NO: 2

Claims

A polypeptide consisting of any of the following amino acid sequences:
(A) the amino acid sequence represented by SEQ ID NO: 4,
(B) an amino acid sequence in which 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3 amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by SEQ ID NO: 4, and (C) an amino acid sequence having a sequence identity of 95% or more, more preferably 97% or more, more preferably 99% or more with the amino acid sequence represented by SEQ ID NO: 4.
A polypeptide comprising the polypeptide according to claim 1 linked to a carrier.
The polypeptide according to claim 2, wherein the carrier is a virus particle, a lipid particle, or a carrier protein.
The polypeptide according to any one of claims 1 to 3, for use as a malaria vaccine antigen.
A malaria vaccine comprising the polypeptide according to any one of claims 1 to 4.
Furthermore, it contains at least one malaria vaccine antigen selected from CSP, TRAP, MSP1, AMA-1, SERA5, Ripr, EBA175, RH5, Pfs25 and Pfs230, or is administered in combination with the at least one malaria vaccine antigen. The malaria vaccine according to claim 5.
The malaria vaccine according to claim 5 or 6, further comprising a vaccine antigen against at least one infection selected from polio, diphtheria, pertussis and tetanus, or administered in combination with the at least one vaccine antigen.
The malaria vaccine according to any one of claims 5 to 7 for preventing infection with malaria parasites, preventing onset after infection with malaria parasites, or treating malaria infections.
A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of claim 1.
A polynucleotide comprising the polynucleotide according to claim 9 linked to a promoter and / or a regulatory element that enables expression in a host cell.
An expression vector comprising the polynucleotide according to claim 9 or 10.
A malaria vaccine comprising the polynucleotide according to claim 9 or 10, or the expression vector according to claim 11.
A recombinant host cell transformed with the vector according to claim 11.
The host cell according to claim 13, which is a bacterium, yeast, insect cell, or mammalian cell.
An antibody that specifically recognizes the polypeptide according to claim 1.
A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 4, the polynucleotide according to claim 9 or 10, the expression vector according to claim 11, or the antibody according to claim 15 as an active ingredient. object.