# Patent

## Title
Malaria vaccine

## Abstract

The present invention provides a vaccine for preventing and/or treating Plasmodium falciparum infections, which comprises a polypeptide set forth in SEQ ID NO: 1 or represented by formula (1), and an adjuvant.

X1-A-B-X2-Y-X3-(Y)n-X4-(Y)n-X5  (1)

(In the formula, X1 represents the 1st to 7th amino acid residues in a polypeptide set forth in SEQ ID NO: 1; X2 represents the 73th to 177th amino acid residues; X3 represents the 178th to 258th amino acid residues; X4 represents the 259th to 289th amino acid residues; X5 represents the 290th to 334th amino acid residues; A represents an 8-mer repeat sequence contained in a 47-kd region of SERA polypeptide of Plasmodium falciparum; B represents a sequence of a serine-rich region contained in a 47-kd region of SERA polypeptide of Plasmodium falciparum; Y represents any one selected from A-A, A-B, and B; and n is an integer of 0 or 1).

## Background
TECHNICAL FIELD
The present invention relates to a high-immunogenicity polypeptide that is useful as a malaria vaccine. The present invention relates to a vaccine that is useful for preventing and treating malaria parasite infections, and a diagnostic agent for malaria parasite infections.
BACKGROUND ART
Infectious diseases annually cause huge human and social damage in developing countries and elsewhere. In particular, malaria parasite infections annually infect 500 million people and cause the death of 2 million to 3 million people; however, no effective vaccine for the prevention of this disease has been developed. Therefore, there is an urgent need to develop a malaria vaccine.
However, in spite of many vaccine clinical tests conducted over the past 30 to 40 years, none of the developed vaccines showed efficacy and all efforts for vaccine development have been frustrated. In such circumstances, as a molecule recognized by an antibody having antimalarial activity, SERA (serine repeat antigen) protein was identified in the serum of adults who have acquired protective immunity (see, for example, Non-patent Literature (NPL) 1).
Later, the development of vaccines was conducted using recombinant SERA protein as an antigen. However, because the antibody titer of the obtained anti-SERA protein antibody is lower than that of Africans who have gained protection (immunity) against malaria parasite infections, further improvement has been desired.
CITATION LIST
Non-Patent Literature


    NPL 1: Bzik D J, Li W B, Horii T, Inselburg J. Mol Biochem Parasitol. 1988 September; 30(3): 279-88


SUMMARY OF INVENTION
Technical Problem
A primary object of the present invention is to provide a high-immunogenicity polypeptide that is useful as a malaria vaccine, and to provide the use of the polypeptide as a vaccine against malaria parasite infections. Another object of the present invention is to provide an antibody to the polypeptide, and the use of the antibody or the polypeptide as a diagnostic agent for malaria parasite infections.
Solution to Problem
The present inventors previously identified that an 8-mer repeat region and/or a serine-rich region, which are present on the N-terminus of a SERA (serine repeat antigen) polypeptide, are protective epitopes of antibodies to Plasmodium falciparum. 
Now the present inventors have found a polypeptide that has excellent immunogenicity and is useful as a malaria vaccine. In addition, the present inventors have found an optimal adjuvant for enhancing the immunogenicity of a malaria vaccine. The inventors conducted further research based on these findings and accomplished the present invention.
The present invention includes the following embodiments.
(I) Polypeptide Useful as a Malaria Vaccine, and Polynucleotide Encoding the Polypeptide
(I-1) A polypeptide represented by the following formula (1):

X1-A-B-X2-Y-X3-(Y)n-X4-(Y)n-X5  (1)

(wherein
X1 represents the 1st to 7th amino acid residues in a polypeptide set forth in SEQ ID NO: 1;
X2 represents the 73th to 177th amino acid residues of the polypeptide;
X3 represents the 178th to 258th amino acid residues of the polypeptide;
X4 represents the 259th to 289th amino acid residues of the polypeptide;
X5 represents the 290th to 334th amino acid residues of the polypeptide;
A represents an 8-mer repeat sequence contained in a 47-kd region of SERA polypeptide of Plasmodium falciparum; 
B represents a sequence of a serine-rich region contained in a 47-kd region of SERA polypeptide of Plasmodium falciparum; 
Y represents any one selected from A-A, A-B, and B; and
n represents an integer of 0 or 1).
(I-2) The polypeptide according to (I-1), wherein, in formula (1), Y is A-B or B.
(I-3) The polypeptide according to (I-1), wherein, in formula (1), A is an amino acid sequence selected from SEQ ID NOS: 2 to 8, and B is an amino acid sequence set forth in SEQ ID NO: 9.
(I-4) The polypeptide according to (I-1), wherein the polypeptide represented by formula (1) is a polypeptide represented by one of the formulas (2) to (6):

X1-A1-B-X2-(A2-B)-X3-X4-X5  (2)

X1-A1-B-X2-(A2-B)-X3-(A3-B)-X4-X5  (3)

X1-A1-B-X2-(A2-B)-X3-(A3-B)-X4-(A4-B)-X5  (4)

X1-A1-B-X2-(A2-A5)-X3-(A3-A6)-X4-(A4-A7)-X5  (5)

X1-A1-B-X2-(B)-X3-(B)-X4-(B)-X5  (6)

(wherein
X1 represents the 1st to 7th amino acid residues in the polypeptide set forth in SEQ ID NO: 1;
x2 represents the 73rd to 177th amino acid residues of the polypeptide;
X3 represents the 178th to 258th amino acid residue of the polypeptide;
X4 represents the 259th to 289th amino acid residue of the polypeptide;
X5 represents the 290 to 334th amino acid residue of the polypeptide;
A1 to A7 represent amino acid sequences set forth in SEQ ID NOS: 2 to 8, respectively; and
B represents an amino acid sequence set forth in SEQ ID NO: 9).
(I-5) The polypeptide according to any one of (I-1) to (I-4), comprising an amino acid sequence set forth in one of SEQ ID NOS: 10 to 14.
(I-6) A polynucleotide encoding at least one of the polypeptides according to (I-1) to (I-5).
(II) Vaccine for Preventing and/or Treating a Plasmodium falciparum Infection
(II-1) A vaccine for preventing and/or treating a Plasmodium falciparum infection, comprising as an active ingredient at least one of the polypeptides according to (I-1) to (I-5).
(II-2) The vaccine according to (II-1), further comprising at least one adjuvant selected from the group consisting of aluminium hydroxide gel, K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), and sHZ (synthetic hemozoin adjuvant).
(II-3) The vaccine according to (II-1) or (II-2), further comprising a ligand having innate immune-stimulatory activity.
(II-4) The polypeptide according to any one of (I-1) to (I-5), which is used as a vaccine for preventing and/or treating a Plasmodium falciparum infection.
(II-5) A combination of the polypeptide according to one of (I-1) to (I-5) with at least one adjuvant selected from the group consisting of aluminium hydroxide gel, K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), and sHZ (synthetic hemozoin adjuvant), the combination being used as a vaccine for preventing and/or treating a Plasmodium falciparum infection.

(II-6) A combination of the polypeptide according to one of (I-1) to (I-5) with at least one adjuvant selected from the group consisting of aluminium hydroxide gel, K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), and sHZ (synthetic hemozoin adjuvant), and a ligand having innate immune-stimulatory activity, the combination being used as a vaccine for preventing and/or treating a Plasmodium falciparum infection.

(II-7) A vaccine for preventing and/or treating a Plasmodium falciparum infection, comprising the polypeptide set forth in SEQ ID NO: 1 and an adjuvant.

(II-8) The vaccine according to (II-7), wherein the adjuvant is at least one member selected from the group consisting of K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), and sHZ (synthetic hemozoin adjuvant), or a combination of at least one of these adjuvants with aluminium hydroxide gel.

(II-9) The vaccine according (II-7) or (II-8), further comprising a ligand having innate immune-stimulatory activity.

(II-10) A combination of a polypeptide set forth in SEQ ID NO: 1 with an adjuvant, the combination being used as a vaccine for preventing and/or treating a Plasmodium falciparum infection.

(II-11) A combination of a polypeptide set forth in SEQ ID NO: 1, an adjuvant, a ligand having innate immune-stimulatory activity, the combination being used as a vaccine for preventing and/or treating a Plasmodium falciparum infection.

(II-12) The combination according to (II-10) or (II-11) wherein the adjuvant is at least one member selected from the group consisting of K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), and sHZ (synthetic hemozoin adjuvant), or a combination of at least one of these adjuvants with aluminium hydroxide gel.

(III) Diagnostic Agent for a Plasmodium falciparum Infection

(III-1) An antibody to the polypeptide according to one of (I-1) to (I-5).

(III-2) The antibody according to (III-1) having affinity for polypeptides set forth in SEQ ID NO: 23 to 37.

(III-3) The antibody according to (III-1) having affinity for polypeptides set forth in SEQ ID NO: 23 to 25.

(III-4) A diagnostic agent for a Plasmodium falciparum infection, comprising, as an active ingredient, the polypeptide according to one of (I-1) to (I-5), or the antibody according to one of (III-1) to (III-3).

Advantageous Effects of Invention
(i) The polypeptide represented by formula (1) (hereinafter sometimes simply referred to as “polypeptide (1)”) has high immunogenicity and can maintain high anti-SE36 antibody titers over a long period of time when used as a vaccine. Accordingly, immunity against malaria parasites can be acquired, and the acquired immunity can be maintained for a long period of time.

(ii) According to the polypeptide (1), sufficient immunity can be acquired with a dose that is 1/10th to 1/100th the amount of an antigen polypeptide required to be used to produce an antibody to malaria parasites in vivo. Due to such a small dose, the polypeptide (1) is useful from an economic viewpoint as well.

(iii) The use of the polypeptide (1) as a vaccine can induce an antibody to a protective epitope. Therefore, the polypeptide (1) of the present invention can be used as a malaria vaccine having high immunogenicity (particularly a Plasmodium falciparum malaria vaccine).

(iv) According to the present invention, a combination of the polypeptide set forth in SEQ ID NO: 1 (hereinafter sometimes referred to as “polypeptide (2)” or “original SE36 polypeptide”) with an optimal adjuvant can be provided. Although the polypeptide (2) itself also has higher antigenicity than a natural SERA polypeptide, a combination of the polypeptide (2) with specific adjuvant or adjuvants can exhibit more remarkable excellent antigenicity. Accordingly, the combined use of the polypeptide (2) with specific adjuvant(s) (e.g., a combination of the polypeptide (2) with a human TLR9 ligand adjuvant, or with aluminium hydroxide gel and a human TLR9 ligand adjuvant, particularly K3 (K-type CpG adjuvant), can induce even higher anti-SE36 antibody titers.




BRIEF DESCRIPTION OF DRAWINGS
FIG. 1-1 shows the amino acid sequences of polypeptide (2) (original SE36 polypeptide) and polypeptides (1) (SE36-1 to SE36-3).
FIG. 1-2 shows the amino acid sequences of polypeptides (1) (SE36-4 to SE36-5) and a negative SE36 polypeptide.
FIG. 1-3 shows the base sequence of a nucleotide encoding the amino acid sequence of the polypeptide (2) (original SE36 polypeptide).
FIG. 1-4 shows the base sequence of a nucleotide encoding the amino acid sequence of the polypeptide (1) (SE36-1).
FIG. 1-5 shows the base sequence of a nucleotide encoding the amino acid sequence of the polypeptide (1) (SE36-2).
FIG. 1-6 shows the base sequence of a nucleotide encoding the amino acid sequence of the polypeptide (1) (SE36-3).
FIG. 1-7 shows the base sequence of a nucleotide encoding the amino acid sequence of the polypeptide (1) (SE36-4).
FIG. 1-8 shows the base sequence of a nucleotide encoding the amino acid sequence of the negative SE36 polypeptide.
FIG. 1-9 shows the amino acid sequence of an epitope mapping polypeptide.
FIG. 2 shows the location and positional relationship between the 8-mer repeat region and the serine-rich region in the polypeptide (2) (original SE36 polypeptide), polypeptides (1) (SE36-1 to 36-5 polypeptides), and negative SE36 polypeptide.
FIG. 3 shows the results of measurement of antibody titers of the whole IgG against SE36 after inoculation of the polypeptide (2) (original SE36 polypeptide), polypeptides (1) (SE36-1 to SE36-5 polypeptides), and negative SE36 polypeptide.
FIG. 4 shows the results of epitope mapping of the polypeptide (2) (original SE36 polypeptide), and polypeptides (1) (SE36-1 to SE36-5 polypeptides) (Test Example 2). In FIG. 4, 1 to 15 on the abscissa show the polypeptides used for mapping 15 epitopes set forth in SEQ ID NO: 23 to 37.
FIG. 5 shows the results of antibody induction by combined use of the polypeptide (2) (original SE36 polypeptide) and adjuvant(s) (aluminium hydroxide gel (Alum) alone, K3 alone, or aluminium hydroxide gel (Alum) and K3).
FIG. 6 is a graph showing changes over time in antibody titers of the whole IgG measured after combined use of various adjuvants (aluminium hydroxide gel (Alum); aluminium hydroxide gel (Alum) and K3; aluminium hydroxide gel (Alum) and D35; and aluminium hydroxide gel (Alum) and sHZ) with the polypeptide (2) (original SE36 polypeptide) versus the use of the polypeptide alone (2) (original SE36 polypeptide).
FIG. 7 is a graph showing the results of epitope mapping in Test Example 3. In FIG. 7, the polypeptides of SEQ ID NO: 23 to 37 used for mapping 15 epitopes are shown as 1 to 15 on the abscissa. Used as adjuvants for the polypeptide (2) were aluminium hydroxide gel (Alum) only (top-left figure), aluminium hydroxide gel and K3 (Alum+K3) (K-type CpG adjuvant) (top-right figure), aluminium hydroxide gel and D35 (D-type CpG adjuvant) (Alum+D35) (bottom-left figure), or aluminium hydroxide gel and sHZ (synthetic hemozoin adjuvant) (Alum+sHZ) (bottom-right figure).
FIG. 8 shows the 14-day measurement results of the number of malaria parasite-infected red blood cells in the blood (parasitemia (%)) measured after immunization of squirrel monkeys with the polypeptide (2) and aluminium hydroxide gel (Alum) (n=2) (left figure); polypeptide (2) (SE36), aluminium hydroxide gel (Alum), and K3 (n=3) (center figure); and aluminium hydroxide gel (Alum) and K3 (n=2) (right figure), after subsequent inoculation with a malaria parasite. In FIG. 8, “dt” indicates ethical death of squirrel monkeys.



DESCRIPTION OF EMBODIMENTS
1. SERA Polypeptide Variant
The polypeptides (1) and (2) of the present invention, which are useful as vaccines for malaria parasite infections, are described below in detail. Herein, the polypeptides (1) and (2) may be collectively referred to as “the polypeptide of the present invention”.
(1-1) SERA Polypeptide
SERA (serine-repeat antigen) is a protein antigen with a molecular weight of 115 kd consisting of 989 amino acids in total and expressed by a Pf gene at the intra-erythrocyte stage. The structure of SERA consists of 3 domains, i.e., 47 kd-50 kd-18 kd, in order of the N-terminal to the C-terminal direction. The SERA working as a precursor for these domains is expressed by 4 exons comprising a total of 5868 bases, processed and cleaved at the intra-erythrocyte stage during merozoite release to yield the above-described domains (Molecular and Biochemical Parasitology, 86, pp. 249-254, 1997; and Experimental Parasitology, 85, pp. 121-134, 1997). In addition, the data on the full length of the SERA gene (DNA) and the amino acid sequence encoded by this gene are open to the public and available from GenBank (Accession Number: J04000; www.ncbi.nlm.nih.gov). The N-terminal region of SERA (hereinafter referred to as “47 kd region”) consists of 382 amino acids in total. The homology search between the Pf strains relative to the sequence indicates that SERA is varied since in some regions there is amino acid deletion or addition, or amino acid variation (non-synonymous substitution) at about 20 positions (Molecular and Biochemical Parasitology, supra; and Experimental Parasitology, supra).
(1-2) Polypeptide (2)
Polypeptide (2) is the original SE36 polypeptide set forth in SEQ ID NO: 1.
The polypeptide (2) is derived from an SE47′ antigen (Vaccine, 14, pp. 1069-1076, 1996; hereinafter simply referred to as “SE47′”), which is based on a 47-kd region of SERA of the above-described Honduras-1 strain of Plasmodium falciparum (Pf) (hereafter simply referred to as “Hond-1”). The polypeptide (2) consists of a total of 334 amino acids, in which the sequence starts from the N-terminal methionine (the 1st amino acid) of 382 amino acids constituting 47 kd of SERA of Hond-1, and in the ordinal number towards the C-terminal, the 16th amino acid codon (aspartic acid) is substituted with an initiation codon (methionine), and a translation stop codon is inserted after the 382nd amino acid (glutamic acid), and further the 33 polymerized serine residues (193rd to 225th serines) occupying the serine repeat region are deleted. The amino acid sequence of the polypeptide (2) is specifically shown as SEQ ID NO: 1 in FIG. 1-1. The polypeptide (2) set forth in SEQ ID NO: 1 has higher immunogenicity than the natural SERA polypeptide.
(1-3) Polypeptide (1)
Polypeptide (1) is an improved SE36 polypeptide obtained by further improving the polypeptide set forth in SEQ ID NO: 1 (2) to have higher immunogenicity.
The polypeptide (1) is represented by the following formula (1):

X1-A-B-X2-Y-X3-(Y)n-X4-(Y)n-X5  (1)

wherein

X1 represents the 1st to 7th amino acid residues in the polypeptide set forth in SEQ ID NO: 1;

X2 represents the 73th to 177th amino acid residues of the polypeptide;

X3 represents the 178th to 258th amino acid residues of the polypeptide;

X4 represents the 259th to 289th amino acid residues of the polypeptide;

X5 represents the 290th to 334th amino acid residues of the polypeptide;

Y represents any one selected from A-A, A-B, and B; and

n represents an integer of 0 or 1.

In formula (1), A represents an 8-mer repeat sequence contained in a 47-kd region of SERA polypeptide of Plasmodium falciparum. In the present invention, any sequence that corresponds to the 8-mer repeat sequence can be used as sequence A, even if there is amino acid variation (non-synonymous substitution) among Pf strains. Examples of amino acid sequences that can be preferably used as sequence A include the following specific sequences A1 to A7 (shown with the N-terminus at the left).







A1:


(SEQ ID NO: 2)


TGESQTGNTGGGQAGNTVGDQAGSTGGSPQGSTGASQPGS


 


A2:


(SEQ ID NO: 3)


TGESQTGNTGGGQAGNTGGDQAGSTGGSPQGSTGASPQGSTGASPQGSTG


 


ASQPGS


 


A3:


(SEQ ID NO: 4)


TGESQTGNIGGGQAGNTVGDQAGNIVGDQAGSTGGSPQGSTGASQPGS


 


A4:


(SEQ ID NO: 5)


TGESQTGNIGGGQAGNTGGGQAGNTVGDQAGSTGGSPQGSTGASQPGS


 


A5:


(SEQ ID NO: 6)


TGESQTGNTGGGQVGNTGGGQAGSTGGSPQGSTGASQPGSSEPSNPVS


 


A6:


(SEQ ID NO: 7)


TGESQTGNTGGGQAGNTVGGQAGNTGGGQAGNTGGDPQGSTGGSQPGS


 


A7:


(SEQ ID NO: 8)


TGESQTGNAGGGQAGNTVGDQAGSTGGSPQGSTGASPQGSTGASPQGSTG


 


ASQPGS.






In formula (1), A linked to X1 is preferably A1 (SEQ ID NO: 2).
In formula (1), B represents the amino acid sequence of a serine-rich region in the 47-kd region of the SERA polypeptide of Plasmodium falciparum. In the present invention, any sequence that corresponds to the amino acid sequence of the serine-rich region can be used as sequence B, even if there is amino acid variation (non-synonymous substitution) among Pf strains. Examples of amino acid sequences that can be preferably used as sequence B include the following specific sequence (shown with the N-terminus at the left)









B:
SEPSNPVSSGHSVSTVSVSQTSTSS.
(SEQ ID NO: 9)






In formula (1), A and B can be suitably selected from the above specific sequences (SEQ ID NO: 2 to 8, and SEQ ID NO: 9) and used in combination. The amino acid sequence A may be any one selected from the above amino acid sequences shown as A1 to A7. The amino acid sequence of one polypeptide may contain, as the amino acid sequence A, two or more of the same amino acid sequence selected from A1 to A7, or any combination of different amino acid sequences selected from A1 to A7.
Y may be suitably selected from A-A, A-B, and B. Preferably, Y is either A-B or B.
Examples of preferable polypeptides (1) in the present invention include polypeptides (1) set forth in SEQ ID NO: 10 to 14, i.e., SE36-1 to SE36-5. FIG. 2 shows a schematic diagram thereof. As in formula (1), SE36-1 to SE36-5 can be represented by the following formulas:

Formula: X1-A-B-X2-Y-X3-(Y)n-X4-(Y)n-X5  (1)

X1-A1-B-X2-(A2-B)-X3-(Y)0-X4-(Y)0-X5  SE36-1:

X1-A1-B-X2-(A2-B)-X3-(A3-B)-X4-(Y)0-X5  SE36-2:

X1-A1-B-X2-(A2-B)-X3-(A3-B)-X4-(A4-B)-X5  SE36-3:

X1-A1-B-X2-(A2-A5)-X3-(A3-A6)-X4-(A4-A7)-X5  SE36-4:

X1-A1-B-X2-(B)-X3-(B)-X4-(B)-X5  SE36-5:

(in the above formulas, (Y)0 means that there is no amino acid residue corresponding to Y; that is, (Y)0 indicates a single bond).

Among SE36-1 to SE36-5, SE36-3 to SE36-5 are preferable, SE36-3 and SE36-5 are more preferable, and SE36-3 is particularly preferable.
(1-4) Synthesis of the Polypeptide of the Present Invention
The polypeptide of the present invention can be synthesized by known methods, and the synthesis method is not particularly limited. For example, the polypeptide (2) can be synthesized by the following method. After a DNA fragment (hereinafter sometimes referred to as “SE36 gene (DNA)”) encoding the polypeptide (2) is synthesized and cloned, an expression vector for the synthesized gene clone is constructed. The vector is transfected into a host (for example, Escherichia coli), and the obtained transformant is cultured.
The expression efficiency of SE36 gene (DNA) having native Pf codons in Escherichia coli is low. Therefore, when E. coli is used as a host, it is desirable to convert all naturally occurring Pf codons encoding the amino acid sequence of the polypeptide of the present invention into Escherichia coli codons to achieve efficient production of the polypeptide as in the present invention.
Although the synthesis of the polypeptide (2) is explained below, the polypeptide (1) can also be synthesized in a similar manner.
Synthesis and Cloning of SE36 Gene (DNA)
A theoretical base sequence of an SE36 gene (DNA) of Pf (SE36 gene having Pf codons) and an amino acid sequence coded thereby are available from well-known gene database publication organizations, such as DDBJ, GenBank, and EMBL on the Internet. The amino acid sequence of the polypeptide (2) is encoded by the nucleotide sequence set forth in SEQ ID NO: 16 (FIGS. 1 to 3). Accordingly, SE36 gene (DNA) encoding the amino acid sequence (SEQ ID NO: 1) of the polypeptide (2) is synthesized based on SEQ ID NO: 16. For example, DNA encoding the amino acid sequence of the polypeptide (1) is synthesized based on the nucleotide sequences set forth in SEQ ID NO: 17 to 22 that encode the amino acid sequences (SEQ ID NO: 10 to 14) of SE36-1 to SE36-5.
Theoretical conversion of Pf codons into Escherichia coli codons may be conducted referring to, for example, the codon usage database of GenBank and publications of the present inventors (the above-mentioned Vaccine; and Molecular Biochemistry of Parasitology, 63, 265-273, 1994). In the conversion into Escherichia coli codons, care should be taken to not damage the antigenicity due to non-synonymous substitution of amino acids except for substitution of the N-terminal amino acid with an initiation codon Met, because the native amino acid sequence of naturally occurring Pf is considered to be important.
In the synthesis of DNA, a commercially available DNA synthesizer, such as a DNA/RNA synthesizer (Applied Biosystems Model 392, a product of PE Co., USA) or an ASU-102U DNA synthesizer (a product of Biosset Ltd., USA) can be used. By using such a synthesizer, sense (+) and antisense (−) DNA fragments, in which about 100 to about 200 nucleotides are polymerized, are separately synthesized, and each DNA fragment thus synthesized is purified, for example, by polyacrylamide electrophoresis. Subsequently, a complementary strand (pair) of the purified single-stranded DNA fragment is annealed to give a synthetic double-stranded DNA fragment.
In cloning of the synthetic double-stranded DNA fragment, a known or commercially available cloning vector of which the host is Escherichia coli (various vectors disclosed in “Cloning Vectors: A Laboratory Manual”, 1-1 to 1-D-1-8, P. H. Pouwels, et al., Elsevier 1988), for example, a combination of a plasmid pBluescript II SK and E. coli XL1-BLue (Stratagene, USA) may be used. In such cloning, for example, a restriction enzyme-digested fragment of the above-mentioned DNA fragment is inserted into the restriction enzyme sites of a vector digested with the same enzyme, and the thus constructed vector is transferred to a host to give a transformant clone. Subsequently, each clone containing the double-stranded DNA fragment is amplified by culturing the transformant, and each base sequence can be determined by means of the chain terminator method (dideoxy method) or the Maxam-Gilbert method. In this procedure, a commercially available DNA sequencer, e.g., ABI PRISM 3700 (a product of PE Co., USA), may be used. Based on the results, approximately 5 to 10 clones of double-stranded DNA fragments covering the full length of SE36 gene DNA are selected.
The cloning of the full length of an SE36 gene (DNA) may be achieved by sequentially ligating the aforementioned double-stranded DNA fragments together. For example, when 8 clones (8 pairs) are obtained in the above operation, these double-stranded DNA fragments can be ligated one by one to give a full-length SE36 gene DNA. Then, the full-length DNA is cloned in the same manner as mentioned above. In carrying out the ligation, it is desirable to introduce a cohesive site for ligation by restriction enzymes at both ends of each fragment of the double-stranded DNAs. In such introduction, the codon base sequence(s) has to be adjusted so that the amino acid sequence of the native Pf is not changed. In the polypeptide (1), the cohesive site for ligation by restriction enzymes may remain in the ligation site of the full-length DNA, or may be translated as is and be present in an amino acid sequence.
Construction of Expression System of SE36 Gene
In producing an expression vector for an SE36 gene (DNA), a known or commercially available expression vector of which the host is Escherichia coli (variously disclosed in “Cloning Vectors: A Laboratory Manual”; supra), such as, a combination of plasmid pET-3a and Escherichia coli BL21 (DL3) pLysS or Escherichia coli BL21 (DL3) pLysE (a product of Stratagene, USA) may be used. In producing such a vector, for example, a restriction enzyme-digested fragment containing SE36 gene (DNA) is cut out from the above cloning vector and then inserted into the restriction enzyme site of a vector cleaved with the same enzyme. Alternatively, the expression vector for an SE36 gene may also be prepared from pET-SE47′ (Vaccine, supra). In this case, for example, the whole or partial region that encodes the serine repeat region is cleaved by a restriction enzyme from pET-SE47′ or an SE47′ synthetic gene contained therein, and the expression vector for an SE36 gene is prepared in the same manner as above. Subsequently, the resulting expression vector is transferred to a host to give a transformant. From such transformants, the most appropriate one as an expression system for mass production of the polypeptide SE36 can be screened in view of industrial applicability.
Mass production of the polypeptide of the present invention is achieved by culturing the aforementioned transformant of Escherichia coli. In view of enhancement of productivity of the polypeptide of the invention, in such a culture, it is possible to improve or reform a manipulation such as the use of an inducer, e.g., IPTG (isopropyl-1-thio-β-D-galactopyranoside), avoidance of catabolite repression, culture medium composition, culture temperature and period, removal of proteases in host cells, and the like. These may be achieved by alteration of an expression vector, change of a promoter or host organism, etc.
Purification of Polypeptide
When the polypeptide of the present invention is secreted outside the cells, the culture solution after removal of the cells from the aforementioned transformant culture is used as a starting material for extraction of the SE36 polypeptide. When SE36 is accumulated in the cells, the cells may be recovered from the culture, for example, by centrifugation, filtration, etc., and extracted to give the above SE36 polypeptide. At the first step of extraction, the cells may be destroyed by digestion with an enzyme, destruction with osmotic pressure, sudden pressure and decompression, sonication, use of various homogenizers, etc. The destroyed cells are then fractionated by physical means such as low-speed centrifugation, ultra-centrifugation, filtration, a molecular sieve, membrane concentration, etc.; or by chemical means such as a precipitating agent, a solubilizing agent, an adsorbent and desorption agent, a dispersing agent, etc.; or by physicochemical means such as electrophoresis, column chromatography, a support, dialysis, salting-out, etc. These techniques may be used in combination. In applying these techniques, physicochemical conditions, such as temperature, pressure, pH, and ion strength, can be suitably set.
Polypeptide of the Present Invention and Confirmation of Its Antigenicity
Detection and size confirmation of the obtained polypeptide of the present invention may be achieved, for example, by determination of sedimentation coefficient, molecular sieve, SDS-polyacrylamide electrophoresis, etc. Antigenicity of the polypeptide of the invention may be confirmed by means of an antigen-antibody reaction using a polyclonal or monoclonal antibody to SERA 47 kd, such as, Western blot analysis, ELISA, agglutination reaction, fluorescent antibody technique, radioimmunoassay, and the like. In addition, immunogenicity of the polypeptide of the present invention and the ability of an anti-SE36 antibody to inhibit Pf parasite growth can be confirmed, for example, by means of an antigen-antibody reaction using the serum of a patient suffering from Pf malaria or an experimental small animal immunized with said polypeptide, such as a rat or mouse, or by growth inhibition (i.e., a neutralization reaction) of Pf merozoites within erythrocytes, or determination of the blood Pf number in an anti-SE36 antibody carrier.
2. Malaria Vaccine
The present invention provides a malaria vaccine comprising the polypeptide of the present invention. Vivax malaria, malariae malaria, ovale malaria, and falciparum malaria are known as malaria parasite infections. The vaccine of the present invention is suitable for use as a falciparum malaria vaccine because it has remarkably high specificity as a falciparum malaria antigen. However, the use of the vaccine is not limited thereto, and the vaccine can be used for other malaria parasite infections.
An example method for preparing a vaccine using the polypeptide (2) is described below. However, a vaccine can also be prepared by using the polypeptide (1) in a similar manner.
(2-1) Preparation of a Vaccine
As an antigen, the polypeptide (2) purified above is dissolved in a solvent, such as isotonic PBS (phosphate buffer saline), to give a vaccine stock solution.
The above antigen for vaccine may be immobilized with a conventional inactivating agent to stabilize the steric structure. Examples of inactivating agents that can be used include formalin, phenol, glutaric dialdehyde, β-propiolactone, and the like, which may be added before or after preparation of the vaccine stock solution. When formalin is used, the amount to be added is about 0.005 to 0.1% (v/v), the inactivation temperature is about 4 to 38° C., and the inactivation period is about 5 to 180 days. If the antigenicity is damaged by inactivation, ingenuity is required to moderate the inactivation conditions. Such moderation may be achieved, for example, by reduction of the amount of inactivating agent used, addition of a neutral or basic amino acid, lowering of the inactivation temperature, etc. Free formaldehyde remaining unchanged after the inactivation step may be, if required, neutralized with addition of an equivalent of sodium hydrogen sulfite or removed by dialysis.
In order to induce mucous or local immunity by oral or nasal inoculation of a vaccine, the polypeptide (2) (i.e., antigen) may be processed or modified. For this purpose, a drug delivery system (DDS) technique using, for example, liposome, emulsion, microcapsules, micro-spheres, polylactic acid, polyglycolic acid, etc., may be applied. The preparation thus obtained is used as a vaccine stock solution in the subsequent step.
The vaccine stock solution is diluted, for example, with the above-mentioned PBS to adjust the amount of the antigen in the vaccine so that antibody production is induced and immunity is established. In this process, it is possible to add a stabilizer for increasing the heat resistance of the vaccine and to add an adjuvant as an auxiliary for enhancing antigenicity. As a stabilizer, for example, sugars or amino acids may be used. Mineral oil, vegetable oil, alum, aluminum compounds (e.g., aluminium hydroxide gel), bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, etc., may be used as an adjuvant. Examples of adjuvants that can be preferably used include, as described below, aluminium hydroxide gel and human TLR9 ligand adjuvants. Specific examples of human TLR9 ligand adjuvants include K3 (K-type CpG adjuvant), D35 (D-type CpG ODN adjuvant), and sHZ (synthetic hemozoin adjuvant).
Subsequently, the resulting vaccine is dispensed into vials in an appropriate amount, such as in vials of about 1 to 20 ml, and the vials are tightly closed or sealed for use as vaccine preparations. Such vaccine preparations may be used in a liquid state, or formed into dry preparations by lyophilization after dispensing and used.
(2-2) Assay of Vaccines
Assay of vaccines, which is related to production process control and quality control, is conducted in accordance with the Japanese Rules for “Minimum Requirements for Biological Products” based on the Pharmaceutical Affairs Law (Law No. 145 enacted in 1960), Article 42, Section 1; WHO recommendation on “Requirements for Biological Substances” (WHO Technical Report Series (TRS), No. 889, pp. 105 to 111, 1999), etc. A malaria vaccine has not yet been put to practical use, and there is no standard for pharmaceutical preparations. The assay, therefore, may be conducted in accordance with a standard for an analogous vaccine, such as the variety of rules on safety and efficacy as described in WHO recommendation on “Requirements for Hepatitis B Vaccines Made by Recombinant DNA Techniques” (the aforementioned TRS, No. 786, 1898, and No. 889, 1999), and “Requirements for Japanese Encephalitis Vaccine (Inactivated) for Human Use” (the aforementioned TRS, No. 771, 1988), etc. For example, the assay for sterilization, denial of abnormal toxicity, protein content, purity, hydrogen ion concentration, confirmation of antigens, antigenic polypeptides, and the like may be conducted in accordance with the rules for a variety of required or recommended tests. A product lot that has passed all of the above tests may be put to practical use as a qualified malaria vaccine preparation.
(2-3) How to Use the Vaccine
The vaccine can be inoculated according to methods common in the art, and the method of vaccine inoculation is not particularly limited. For example, the vaccine can be subcutaneously inoculated at a dose of about 0.25 to 0.5 ml. Such inoculation may be preferably performed 1 to 3 times at intervals of about 2 to 4 weeks.
3. Adjuvants and Vaccine Comprising a Combination of the Adjuvants
(3-1) In preparation of the vaccine, the polypeptide (1) may be used together with a combination of known adjuvants. Such adjuvants are not particularly limited, and any adjuvant that can enhance the immunizing effect of the present invention can be used. Examples of usable adjuvants include aluminium hydroxide gel and human TLR9 ligand adjuvants such as K3 (K-type CpG adjuvant), D35 (D-type CpG ODN adjuvant), and sHZ (synthetic hemozoin adjuvant). In addition to these adjuvants, ligands having innate immune-stimulatory activity can also be used as adjuvants.
In the present invention, adjuvants may be used singly or in combination of two or more selected from such adjuvants. Aluminium hydroxide gel has the property of forming an insoluble antigen-adjuvant complex. Due to local accumulation of this antigen-adjuvant complex, combined use of aluminium hydroxide gel with other adjuvant(s) as mentioned above is preferable.
(3-2) In preparation of the vaccine, the polypeptide (2) may also be used together with known adjuvants as exemplified above. Examples of adjuvants that can be preferably used include K3 (K-type CpG adjuvant), D35 (D-type CpG ODN adjuvant), and sHZ (synthetic hemozoin adjuvant), which are human TLR9 ligand adjuvants. However, when the polypeptide (2) is used with a specific combination of adjuvants, particularly excellent effects can be achieved.
Examples of combinations of adjuvants with which the polypeptide (2) can provide particularly excellent immunogenicity include, in addition to combinations of the above-mentioned humans TLR9 ligand adjuvants, (a) a combination of aluminium hydroxide gel and K3 (K-type CpG adjuvant), (b) a combination of aluminium hydroxide gel and D35 (D-type CpG adjuvant), and (c) a combination of aluminium hydroxide gel and sHZ (synthetic hemozoin adjuvant). Among these, (a) a combination of aluminium hydroxide gel and K3 (K-type CpG adjuvant) can maintain high anti-SE36 antibody titers for a long period of time. Accordingly, use of the polypeptide (2) with a combination of specific adjuvants as mentioned above is effective as a malaria vaccine.
4. Diagnostic Agent
The antibody to the polypeptide (1) or (2) can be used for the diagnosis of malaria parasite infections. More specifically, the present invention can provide a diagnostic agent for malaria parasite infections, comprising an antibody to the polypeptide (1) or (2) as an active ingredient. The antibody to the polypeptide (1) or (2) can be obtained, for example, by intraperitoneally, subcutaneously, or intramuscularly inoculating the polypeptide (1) or (2) to an animal, such as a rabbit, guinea pig, or mouse, to generate an antibody and isolate the antibody from the serum of the animal. Such an antibody can be used for detection of an antigen. If the antigen is detected in a sample, for example, serum, of a patient suspected of a malaria parasite infection, the patient can be diagnosed as being infected with a malaria parasite.
An anti-SE36 antibody can be detected by a precipitation reaction, agglutination reaction, neutralization reaction, fluorescent antibody technique, enzyme immunoassay, radioimmunoassay, or the like, using the polypeptide (1) or (2) as an antigen and using the serum, etc., of a patient suspected of a malaria parasite infection as a sample. Accordingly, if the antigen is detected in the sample, the patient is diagnosed as being infected with a malaria parasite.
The antigen and antibody used in diagnosis according to the present invention may be diluted with a solvent, such as the above-mentioned PBS, so that the content of the antigen and antibody in the diagnostic agent becomes the amount necessary for the antigen/antibody reaction.
5. Method for Preventing and/or Treating Malaria Parasite Infections
The vaccine of the present invention has high antibody titers as mentioned above. Therefore, malaria parasite infections can be effectively prevented by pre-administering the vaccine to a subject, such as person in need of prevention against malaria parasite infection.
Because the vaccine of the present invention is particularly effective against Plasmodium falciparum infections, Plasmodium falciparum infections can be effectively treated by administering the vaccine to a patient infected with Plasmodium falciparum. Thus, the present invention further provides a method for preventing and/or treating malaria using a malaria vaccine. The malaria vaccine preparation method, dosage, etc., are as described above.
As described above, administration of the polypeptide (1) or (2) of the present invention together with adjuvants as mentioned above can further enhance malaria preventive and/or therapeutic effects.
EXAMPLES
The present invention is described below in more detail with reference to Test Examples. However, the scope of the invention is not limited to these Examples.
Test Example 1
The present inventors previously concluded from the results of epitope mapping directed to SE36 (SEQ ID NO: 1) that either one of or both of the 8-mer repeat and serine-rich regions that are present in the N-terminal region are important as protective epitopes. (The document that discloses this will be described here.)
Based on the above finding, five kinds of polypeptide (1) (SEQ ID NO: 10 to 14), each containing a plurality of these regions, were designed in this Test Example. At the same time, an original SE36 polypeptide (polypeptide (2): SEQ ID NO: 1), and SE36 without the N-terminal region (negative SE36: SEQ ID NO: 15) as a negative control were designed. FIGS. 1-1 to 1-2 show the amino acid sequences of these polypeptides. FIGS. 1-3 to 1-9 (SEQ ID NO: 16 to 22) show the base sequences of nucleotides encoding the amino acid sequences. FIG. 2 shows a schematic diagram of the structures of five kinds of polypeptide (1), polypeptide (2), and negative SE36 polypeptide.
The method for preparing the polypeptides used in Test Example 1 is described below. Although a method of preparing the polypeptide (2) is described in detail, other polypeptides can also be produced in a similar manner.
Construction of Polypeptide Expression System
The expression system of the polypeptide (2) (SEQ ID NO: 1) was constructed in the following manner.
The DNA base sequence of the full-length SE36 gene that had been theoretically converted from Pf codons to Escherichia coli codons was divided into 8 fragments. For each divided fragment, a sense (+) strand and an antisense (−) strand were synthesized to obtain 16 single-stranded DNA fragments in total (8 pairs), which were annealed to give 8 pairs of double-stranded DNA. These sequences were ligated to each other to give a full-length of SE36 gene, from which an expression vector was constructed.
In this operation, the basic procedure for cloning and ligation of the synthetic DNA fragments was conducted in accordance with the method of Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).
The above single-stranded DNA fragments were individually synthesized using an Applied Biosystems Model 392 DNA/RNA synthesizer (a product of PE Co., USA). These synthesized fragments were purified by electrophoresis on 10% (w/v) polyacrylamide (containing 50 mM Tris-borate salt, pH 8.3, 1 mM EDTA, and 8M urea). Then, 20 pmoles of the + and − complementary strands of each purified DNA fragment were mixed, and then heated in a buffer solution (20 μl of 20 mM Tris-HCl, pH 7.0, 50 mM NaCl, and 2 mM MgCl2) at 85° C. for 5 minutes.
Further, the complementary regions of both the strands above were annealed by lowering the temperature to 55° C. at a rate of 5° C./5 minutes and then to 25° C. at a rate of 5° C./10 minutes using a Zymoreactor II (a product of ATTO Co., Japan). After annealing, an equal amount of a buffer solution (20 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 5 mM dithiothreitol (DTT), 1 mM each of 4 species of nucleoside-5′-triphosphate (NTP), and 3 units of T4 DNA polymerase) was added, and the mixture was kept at 4° C. for 5 minutes, 25° C. for 5 minutes, and then at 37° C. for 120 minutes.
For construction of SE36 gene, the resulting double-stranded DNA fragments were individually digested with restriction enzymes KpnI and BamHI, and then cloned and multiplied with pBluescript II SK+ and Escherichia coli XL1-Blue. The base sequences of the above DNA fragments in each of the clones were determined by the dideoxy method, and 8 clones covering the full length of SE36 gene were screened. The synthesized double-stranded DNA fragments of these 8 clones (8 pairs) were ligated to give a full length of SE36 double-stranded DNA.
In this operation, using a base sequence designed in such a manner that the amino acid sequence of the native Pf was not altered, the restriction enzyme sites for ligation were introduced to both ends of each pair of DNA. Subsequently, the full length of an SE36 gene was cloned with pBluescript II SK+, and then transfected to Escherichia coli XL1-Blue for proliferation. The base sequence was determined by the dideoxy method. The results are shown in SEQ ID NO: 10 in the Sequence Listing.
Subsequently, the fragments of the above clone digested with restriction enzymes NdeI and BamHI were inserted and ligated into the NdeI-BamHI cleavage sites of a plasmid pET-3a to construct a SE36 expression vector pET-SE36. This expression vector was transfected to Escherichia coli BL21 (DE3) pLysS to give a transformant, Escherichia coli BL21 (DE) pLysS/pET-SE37, which was designated as Escherichia coli BL/SE36.
Expression and Purification of the Polypeptide
The Escherichia coli BL/SE36 obtained above was incubated on an LB medium (Bacto-trypton 1% (w/v), Bacto-yeast extract 0.5% (w/v), and NaCl 1% (w/v)) containing 50 μg/m of ampicillin at 37° C. for 18 hours to give seeds. The seeds (50 ml) were inoculated on fresh LB medium (5 L) and incubated at 37° C. When the number of cells reached 1×108/ml, IPTG (isopropyl-β-D-thiogalactopyranoside) was added at a final concentration of 50 μg/ml, and further incubated at 37° C. for 3 hours. After the incubation, the mixture was centrifuged (5,000 rpm, 10 minutes) to collect the cells. 3.2 g of cell paste was obtained. The paste was suspended into 9.6 ml of an ice-cold lysis buffer solution (50 mM Tris-HCl, pH 8.0, and 1 mM EDTA). Then, the procedures (1) to (6) were conducted at 4° C. in the order as described.
(1) Sonication
The above cell paste was disrupted by treatment with ultrasonic waves (19.5 kHz, 50 W) 6 times for 20 seconds. The supernatant after centrifugation (15,000 rpm, 30 minutes) was collected and placed in a beaker of 20-ml volume.
(2) Salting-Out with Ammonium Sulfate (I)
To the supernatant in the beaker was added 2.37 g of (NH4)2SO4 crystals with stirring to achieve a saturation of 35%(W/W). The mixture was further stirred for 30 minutes for salting-out. Subsequently, the mixture was centrifuged (12,000 rpm, 10 minutes), and the supernatant was discarded. The precipitate was suspended in 9 ml of an ice-cold ammonium sulfate solution (a lysis buffer solution as described above containing 1.1 M (NH4)2SO4) at an ammonium sulfate saturation of 30% (w/w). The resulting suspension was centrifuged (12,000 rpm, 10 minutes) and the supernatant was discarded. The precipitate was suspended again into 8.8 ml of a lysis buffer solution (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM 2-mercaptoethanol, 9 M urea, and 1% (w/v) TWEEN 80 and recovered. Half the volume (4.4 ml) of the recovered suspension was heated at 60° C. for 10 minutes, then again ice-cooled and filtered through a 0.45-μm filter (a product of Millipore, USA).
(3) Column Purification (I)
The filtrate was chromatographed on a column of SEPHACRYL S-300 (26/60) equilibrated with a GF buffer solution (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM 2-mercaptoethanol, and 8M urea) (3.5 ml/fraction; flow rate=0.3 ml/minute; 4° C.). Each of the fractions 22-43 was subjected to SDS-polyacrylamide electrophoreses. Based on their migration patterns, the fractions 32-37 containing a large amount of SE36 protein were pooled. The remaining resuspended solution (4.4 ml) was also treated in the same manner as above, then combined with the pooled fractions above, and used in the subsequent operation (4).
(4) Column Purification (II)
The resulting pooled fractions were kept at room temperature, and (NH4)2SO4 was added thereto with stirring at an amount of 0.093 g per ml of the pooled fractions to achieve a final ammonium sulfate concentration of 0.7 M. On the other hand, an aqueous column with 13 ml of OCTYL SEPHAROSE (a product of Pharmacia Biotech) was equilibrated with a 10-fold volume of an HIC buffer solution (a GF buffer solution as mentioned above containing 0.7 M (NH4)2SO4). The ammonium sulfate-adjusted pooled fractions were poured onto the column at a rate of 0.5 ml/minute. Subsequently, the HIC buffer solution was added to the column until the absorbance decreased, and then for confirmation, the column was eluted with a GF buffer solution that does not contain (NH4)2SO4 to elute the adsorbed components. The fractions not adsorbed on the column were placed in a dialysis bag and dialyzed against 1 L of 20-mM Tris-HCl buffer solution (pH 8.0)(containing 1 mM EDTA) at 4° C. for 10 hours. During the dialysis, this outside solution was changed twice.
(5) Salting-Out with Ammonium Sulfate (II)
The dialysis bag after completion of the dialysis was further dialyzed against 0.3 L of a 50% (w/w) saturated (NH4)2SO4 solution (containing 20 mM Tris-HCl (pH 8.0) and 1 mM EDTA) at 4° C. for 10 hours to obtain proteins as a precipitate. The precipitate was collected by centrifugation (12,000 rpm, 10 minutes) and suspended into 2 ml of GF buffer solution.
(6) Column Purification (III)
The suspension obtained above was heated at 60° C. for 10 minutes and then cooled back to 4° C. This was filtered through a 0.45-μm filter. The filtrate was chromatographed on a column of the above-mentioned S-300 (26/60) equilibrated with a GF buffer solution 2 (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 20 mM 2-mercaptoethanol, and 8 M urea) at a flow rate of 0.3 ml/minute. In the same manner as in the above item (3), each fraction was applied to SDS-polyacrylamide electrophoresis to screen fractions of the SE36 protein, which were collected to pool 12 ml fraction. This fraction was added to a dilution buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 2 M urea) with stirring so as to give a solution of the SE36 protein at a concentration of 25 μg/ml. The diluted solution was dialyzed against 2 L of PBS (9 mM NaHPO4, 3 mM NaH2PO4, and 137 mM NaCl (pH 7.4)) at 4° C. for 10 hours. During this operation, the outside solution was changed twice. Subsequently, the inside solution after dialysis was concentrated with CENTPREP 30, and filtered through a Durapore 0.22 μm-filter (a product of Millipore, USA) for sterilization to give 10 ml sterile specimen containing 1 mg/ml of the SE36 protein. This was stored as a stock solution for an SE36 vaccine at 4° C.
Determination of the Amino Acid Sequence
The amino acid sequence of the polypeptide (2) thus obtained was determined by Edman degradation using an Applied Biosystems 473A protein sequencer (a product of PE Co., USA). The polypeptide (2) was confirmed to have the amino acid sequence set forth in SEQ ID NO: 1.
Test Example 2
Using the polypeptides (1) (SE36-1 to SE36-5 polypeptides), polypeptide (2), and negative SE36 polypeptide as antigens, a test was performed in the following manner.
Mice were immunized with each of the purified polypeptides (antigens), and induction of antibody titers was monitored. More specifically, thirty-five C57BL/6_tlr4KO mice were divided into 7 groups of 5 mice each, and each group was inoculated with a mixture of one antigen with aluminium hydroxide gel and a K3 adjuvant by subcutaneous injection. FIG. 3 shows the results.
The immunizing amount was defined as 13 μg of aluminium hydroxide gel and 50 μg of K3 per μg of the antigen. As indicated by an arrow in FIG. 3, a second immunization was performed two weeks after the first immunization. Every week, the blood was sampled to obtain the serum, and anti-SE36-IgG antibody titers were determined by an ELISA assay. The ELISA assay was performed in the following manner.
Preparation of ELISA Plate
SE36 was diluted to 1 μg/ml with a coating buffer (pH 9.6, containing 3.4 g of Na2CO3 and 5.7 g of NaHCO3 in 1 L of distilled water). The diluted SE36 was dispensed into each well of a 96-well MaxiSorp NUNC-Immuno plate (442404: Nunc) using 100 μl per well. The plate was washed twice with a wash buffer (PBS(−) containing 0.05% TWEEN 20), and then blocked with a blocking buffer (5% skim milk and PBS(−) containing 0.05% Tween 20) and incubated at 4° C. for 2 hours. The blocked ELISA plate was washed with washing buffer 4 to 5 times and stored at −20° C. until use.
Reaction with Test Sera
(a) Standard Serum
Plates with 12 (row)×8 (column) wells were used. A blocking buffer was dispensed into each well of the first row in an amount of 150 μl, and into each well of the subsequent rows in an amount of 100 μl. 1.5 μl of a standard serum was placed into the wells of the first row, and the serum and the blocking buffer were mixed thoroughly (1/100 dilution). 50 μl in each well of the first row was transferred to the wells of the adjacent row and mixed thoroughly (1/3 dilution). This operation was repeated until the wells of the last row (whereby the serum can be serially diluted to 1/100, 1/300, 1/900, 1/2700, etc.).
(b) Test Sera
Each sample (serum) was diluted with the blocking buffer (to 1/100, 1/500, 1/1000, etc.: the sample was diluted in such a manner that the O.D. values fell within the standard curve). The diluted sample was dispensed into each well in an amount of 100 μl per well. The sample was incubated at room temperature for 2 to 3 hours, or at 4° C. overnight, and then washed with the wash buffer 4 to 5 times.
Reaction with a Secondary Antibody
The secondary antibody (Anti-Mouse IgG (Goat Anti-Mouse IgG (H+L))) (1031-05: SouthernBiotech) was diluted with a blocking buffer to a dilution factor. The resulting mixture was dispensed in an amount of 100 μl per well and incubated at room temperature for 2 to 3 hours, or at 4° C. overnight, followed by washing with the wash buffer 4 to 5 times. The dilution factor of the second antibody can be set as desired.
Detection
TMB (3,3′,5,5′-tetramethylbenzidine: Sigma: T8665-100ML), which is a chromogenic substrate, was dispensed in an amount of 50 μl per well. When the sample had turned an appropriately dark blue, 1 mol/l sulfuric acid (Nacalai Tesque: 95626-06) was dispensed in an amount of 50 μl per well. Detection was performed at 450 nm using a plate reader, and background was subtracted at 540 nm.
As shown in FIG. 3, all the polypeptides (1) (SE36-1 to SE36-5) provided higher antibody titers than the polypeptide (2). In particular, SE36-3 to SE36-5 induced high antibody titers. These results indicate that the polypeptide (1) of the present invention, i.e., an improved SE36 polypeptide, can induce an anti-SE36 antibody several ten to several hundred times higher than the amount induced by the polypeptide (2).
Epitope Mapping
Epitope mapping was performed using the serum obtained in the 4th week. For the epitope mapping, the wells of plates were coated with fifteen peptides set forth in SEQ ID NOS: 23 to 37 at a concentration of 0.03 mM (see FIGS. 1 to 9), and the ELISA assay was performed. FIG. 4 shows the results. The results of epitope mapping indicate that the polypeptides (1) (SE36-1 to SE36-5) highly promote induction of an antibody to the N-terminal region that is important as a protective epitope.
More specifically, induction of the antibody to the N-terminal region of a SE36 polypeptide by using the polypeptide (2) (original SE36 polypeptide) as an antigen is as shown in the upper-left graph of FIG. 4. The results of FIG. 4 show that the polypeptides (1), i.e., SE36-1 to SE36-5 (particularly SE36-3 to SE36-5) can remarkably enhance the induction of the antibody to the N-terminal region of a SE36 polypeptide (epitope polypeptides 1 to 3 (polypeptides set forth in SEQ ID NOS: 23 to 25)).
Test Example 3
As shown below, adjuvants (aluminium hydroxide gel, K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), and sHZ (synthetic hemozoin adjuvant) were used in combinations, and their immunogenicity-enhancing effects on the polypeptide (2) were evaluated.
(1) Enhancement of Antibody Induction Capacity by Combined Use of Adjuvants
Immunization was performed using the polypeptide (2) alone as the antigen or using the polypeptide (2) with aluminium hydroxide gel, the polypeptide (2) with K3, or the polypeptide (2) with aluminium hydroxide gel and K3. The test method was performed in the same manner as in Test Example 2. Four weeks after the first immunization, anti-SE36-IgG antibody titers were measured. Table 5 shows the results. The results of FIG. 5 show that the combined use of aluminium hydroxide gel and K3 as adjuvants (indicated as “Alum+K3” in FIG. 5) can remarkably induce an anti-SE36 antibody as compared to using the antigen alone (indicated as “none” in FIG. 5), using only aluminium hydroxide gel as the adjuvant (indicated as “Alum” in FIG. 5), and using only K3 as the adjuvant (indicated as “K3” in FIG. 5).
(2) Enhancement of Antibody Induction Capacity by Combined Use of Adjuvants
The present inventors further evaluated the enhancement of immunogenicity of the polypeptide (2) by adjuvants other than the combination of aluminium hydroxide gel and K3. In this test, antibody titers in test animals were evaluated over a long period of time. The specific test method is as shown below.
Twelve cynomologus monkeys were divided into 4 groups of 3 monkeys. The monkeys were immunized by subcutaneous injection using the following as adjuvants for the polypeptide (2): aluminium hydroxide gel only; aluminium hydroxide gel and K3 (K-type CpG adjuvant); aluminium hydroxide gel and D35 (D-type CpG adjuvant); and aluminium hydroxide gel and sHZ (synthetic hemozoin adjuvant).
For the immunization, vaccines prepared by adding 500 μg of either K3 or D35, or 1.5 mM sHZ to the polypeptide (2) (10 μg) and aluminium hydroxide gel (125 μg) were used. Immunization was performed at the time points indicated by arrows in FIG. 6 (day 0, day 22, and day 101). On days 0, 7, 14, 22, 28, 36, 42, 56, 73, 86, 101, 112, 140, 175, 205, 238, 268, and 365, blood was drawn, and the serum was collected. The anti-SE36-IgG antibody titers in the serum were measured by an ELISA assay. The ELISA assay was performed in the same manner as in Test Example 2. In Test Example 3, anti-monkey IgG (whole molecule)-peroxidase, antibody produced in a rabbit (A2054: Sigma), was used as a second antibody. FIG. 6 shows the results.
The results of this test show that compared to the use of aluminium hydroxide gel alone, combined use of aluminium hydroxide gel with K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), or sHZ (synthetic hemozoin adjuvant) can enhance the immunogenicity of the polypeptide (2), and that in particular, combined use of aluminium hydroxide gel with K3 can provide high antibody titers and also maintain this effect over a long period of time.
Epitope Mapping
Epitope mapping was performed using the sera with the highest antibody titers (indicated with an asterisk (*) in FIG. 6). For the epitope mapping, the wells of the plates were coated with fifteen peptides shown in FIGS. 1 to 9 (SEQ ID NOS: 23 to 37) (concentration: 0.03 mM) and subjected to the ELISA assay. A comparison was made using one animal having the highest antibody titer in each group. The results show that addition of K3 (K-type CpG adjuvant) can promote antibody titers to the N-terminal region (FIG. 7: circled portions).
The results of this Test Example suggest that addition of a TLR9 ligand adjuvant (K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), or sHZ (synthetic hemozoin adjuvant)) can enhance the immunogenicity of the polypeptide (2), and the preparation containing a TLR9 ligand adjuvant can be used as a more effective malaria vaccine.
In the above Test Example, the vaccinated animals were all healthy, and no unusual weight loss; abnormality in behavior, excrement, or appearance; or death was observed. More specifically, safety of the vaccine used in this Test Example was confirmed.
Test Example 4
Squirrel monkeys were immunized with a combination of the polypeptide (2) with adjuvants (aluminium hydroxide gel and K3 (K-type CpG adjuvant)), and the protective effects against malaria parasite were evaluated.
Seven squirrel monkeys were randomly divided into three groups. Each of the three groups of squirrel monkeys was immunized by subcutaneous injection with polypeptide (2) and aluminium hydroxide gel (n=2); polypeptide (2), aluminium hydroxide gel and K3 (n=3) (K-type CpG adjuvant); or aluminium hydroxide gel and K3 (n=2) (K-type CpG adjuvant) twice at three-week intervals. The immunization was performed in such a manner that the total dosage of the components, i.e., (polypeptide (2) (10 μg), aluminium hydroxide gel (125 μg), and K3 (500 μg)), was 0.5 ml. After the immunization, a live malaria parasite was inoculated in a concentration of 5×108 via the femoral vein. The number of malaria parasite-infected red blood cells in each squirrel monkey was determined every day for 14 days.
FIG. 8 shows the results. In one of the monkeys (n=2) in the group administered with polypeptide (2) and aluminium hydroxide gel, and both monkeys (n=2) in the group administered with aluminium hydroxide gel and K3, the number of malaria parasite-infected red blood cells reached as high as about 40 to 50%, and these monkeys died ethically by day 9, as shown in FIG. 8. In contrast, in all three monkeys in the group administered with polypeptide (2), aluminium hydroxide gel, and K3 (n=3), the number of malaria parasite-infected red blood cells was lower than 30%, and none of the monkeys in this group died.
These results show that compared to the use of aluminium hydroxide gel alone, combined use of K3 (K-type CpG adjuvant) with aluminium hydroxide gel can more effectively enhance the immunogenicity of the polypeptide (2) and inhibit the growth of the malaria parasite, thus preventing the death of infected subjects.
SEQUENCE LISTING FREE TEXT
SEQ ID NO: 1 is the amino acid sequence of polypeptide (2).
SEQ ID NO: 2 is the amino acid sequence of A1.
SEQ ID NO: 3 is the amino acid sequence of A2.
SEQ ID NO: 4 is the amino acid sequence of A3.
SEQ ID NO: 5 is the amino acid sequence of A4.
SEQ ID NO: 6 is the amino acid sequence of A5.
SEQ ID NO: 7 is the amino acid sequence of A6.
SEQ ID NO: 8 is the amino acid sequence of A7.
SEQ ID NO: 9 is the amino acid sequence of B.
SEQ ID NO: 10 is the amino acid sequence of an SE36-1 polypeptide.
SEQ ID NO: 11 is the amino acid sequence of an SE36-2 polypeptide.
SEQ ID NO: 12 is the amino acid sequence of an SE36-3 polypeptide.
SEQ ID NO: 13 is the amino acid sequence of an SE36-4 polypeptide.
SEQ ID NO: 14 is the amino acid sequence of an SE36-5 polypeptide.
SEQ ID NO: 15 is the amino acid sequence of a negative SE36-6 polypeptide.
SEQ ID NO: 16 is the base sequence of a nucleotide encoding the amino acid sequence of polypeptide (2).
SEQ ID NO: 17 is the base sequence of a nucleotide encoding the amino acid sequence of an SE36-1 polypeptide.
SEQ ID NO: 18 is the base sequence of a nucleotide encoding the amino acid sequence of an SE36-2 polypeptide.
SEQ ID NO: 19 is the base sequence of a nucleotide encoding the amino acid sequence of an SE36-3 polypeptide.
SEQ ID NO: 20 is the base sequence of a nucleotide encoding the amino acid sequence of an SE36-4 polypeptide.
SEQ ID NO: 21 is the base sequence of a nucleotide encoding the amino acid sequence of an SE36-5 polypeptide.
SEQ ID NO: 22 is the base sequence of a nucleotide encoding the amino acid sequence of a negative SE36 polypeptide.
SEQ ID NO: 23 to 37 are the amino acid sequences of epitope mapping polypeptides 1 to 15.

## Claims

1. An immunogenic composition against Plasmodium falciparum comprising an isolated polypeptide of SEQ ID NO: 1 and an adjuvant, wherein the adjuvant is a combination of aluminium hydroxide gel and the K-type CpG adjuvant, K3.

2. The immunogenic composition of claim 1, wherein the polypeptide is purified.

3. A method of immunizing a subject against Plasmodium falciparum comprising administering an immunogenic amount of the immunogenic composition of claim 1.

4. A method of immunizing a subject against Plasmodium falciparum comprising administering an immunogenic amount of the immunogenic composition of claim 2.