Composite polypeptide monomer, aggregate of said composite polypeptide monomer having cell penetration function, and norovirus component vaccine for subcutaneous, intradermal, percutaneous, or intramuscular administration and having said aggregate as effective component thereof

Abstract

The present invention addresses the issue of providing a norovirus component vaccine for subcutaneous, intradermal, percutaneous, or intramuscular administration which vaccine can readily immunize the target cells, an associated product of a molecular needle serving as an active ingredient of the vaccine, and a production method for the associated product. The invention provides a norovirus component vaccine containing, as an active ingredient, an associated product including a hexamer formed through bonding of two molecules of a trimer of a molecular needle represented by the following formula (1). W-L.sub.1-X.sub.n—Y (1) [wherein W represents an amino acid sequence of P domain of the capsid protein of norovirus as an immunogen; L.sub.1 represents a first linker sequence having 0 to 100 amino acids; X represents an amino acid sequence represented by SEQ ID NO: 1; Y represents an amino acid sequence of a cell introduction domain; n is an integer of 1 to 3].

Claims

1. A composite polypeptide, which comprises a polypeptide of the following formula (1):
W-L1-Xn-Y  (1) wherein W is an amino acid sequence comprising a part or the entirety of a virus structural protein as an immunogen of a vaccine against the virus; L1 is a first linker sequence having 0 to 100 amino acids; X is an amino acid sequence which comprises the amino acid sequence of SEQ ID NO: 1, or which comprises an amino acid sequence with 8 or less amino acid sequence changes as compared to SEQ ID NO: 1; Y comprises an amino acid sequence of a cell introduction domain; and n is an integer between 1 and 3, wherein the cell introduction domain of Y comprises an amino acid sequence of the following formula (2):
Y1-L2-Y2-Y3  (2) wherein Y1 is an amino acid sequence which comprises the amino acid sequence of any one of SEQ ID NOs: 2 to 5, or which comprises an amino acid sequence with 30 or less amino acid sequence changes as compared to any one of SEO ID NOs: 2 to 5; Y2 is an amino acid sequence which comprises the amino acid sequence of any one of SEQ ID NOs: 6 to 9, or which comprises an amino acid sequence with 15 or less amino acid sequence changes as compared to any one of SEQ ID NOs: 6 to 9; L2 is a second linker sequence having 0 to 30 amino acids; Y3 is an amino acid sequence for modification; and either of Y2 and Y3 may be absent, and wherein said amino acid changes are selected from the group consisting of additions, deletions, and substitutions.

2. The composite polypeptide according to claim 1, wherein L.sub.1 comprises the amino acid sequence of SEQ ID NO: 14.

3. A trimer protein comprising a composite polypeptide of claim 1 as a monomer protein, wherein the monomer proteins of said trimer protein are identical to or different from one another.

4. The trimer protein according to claim 3, which includes a parallel β-sheet structure and a helix structure of the parallel β-sheet structure, said parallel β-sheet structure formed by linking X.sub.ns and Y.sub.1s, respectively, in three molecules of the composite polypeptide, which molecules are identical to or different from one another.

5. A hexamer protein formed through association of two molecules of a trimer protein of claim 3.

6. A component vaccine for subcutaneous, intradermal, percutaneous, or intramuscular administration, wherein said vaccine comprises the hexamer protein of claim 5 as an active ingredient, and wherein W is an amino acid sequence comprising a part or the entirety of a P domain of a norovirus capsid protein.

7. A method for producing a composite polypeptide associated product, said method comprising: bringing molecules of a composite polypeptide of claim 1 into contact with one another within an aqueous liquid, to thereby form at least one of a trimer and a hexamer; and selectively isolating and recovering the trimer, the hexamer, or both.

8. The method according to claim 7, wherein the method comprises: culturing, in a liquid culture medium, a transformant into which a nucleic acid fragment encoding the composite polypeptide has been incorporated, to thereby produce molecules of the composite polypeptide through gene expression, wherein said molecules of the composite polypeptide self-associate to form at least one of a trimer and a hexamer; and selectively isolating and recovering the trimer, the hexamer, or both.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIG. 1

(2) A scheme of forming the associated products of the present invention (trimer and hexamer) from the composite polypeptide of the present invention.

(3) FIG. 2

(4) A graph showing results of size exclusion column chromatography of “PN-Saga PF,” which is an example of the associated product of the present invention.

(5) FIG. 3

(6) A gel filtration chromatographic chart in separation of the associated product of the present invention (hexamer).

(7) FIG. 4

(8) A graph showing test results of rise (in vivo) in antibody titer by subcutaneous administration of the norovirus vaccine of the present invention, indicating the value of the invention.

DETAILED DESCRIPTION OF THE INVENTION

(9) Modes for Carrying Out the Invention

(10) (1) The Composite Polypeptide of the Present Invention

(11) The composite polypeptide is represented by the following formula (1):
W-L.sub.1-X.sub.n—Y  (1)
[wherein W represents an amino acid sequence of a part or the entirety of a virus structural protein as an immunogen; L.sub.1 represents a first linker sequence having 0 to 100 amino acids; X represents an amino acid sequence represented by SEQ ID NO: 1; Y represents an amino acid sequence of a cell introduction domain; and n of X is an integer of 1 to 3].

(12) Examples of the immunogen W include an amino acid sequence based on a part or the entirety of the peptide structure of the virus structural protein. The wording “based on” refers to the immunogen W including not only the original peptide structure but also a modified amino acid sequence such as a detoxicated amino acid sequence obtained by modifying the original amino acid sequence.

(13) The entirety or a part of the virus structural protein is preferably a part or the entirety of a surface portion including the antigen-presenting part. An example thereof is a part or the entirety of P domain of the capsid protein, which serves as the immunogen of the norovirus vaccine of the present invention.

(14) The first linker sequence L.sub.1 is required for appropriately maintaining the distance between the immunogen W and the molecular needle part Y, to thereby suppress steric hindrance. Generally, the number of amino acid residues is preferably larger, as the molecular weight of the immunogen W increases. As described above, the number of amino acid residues is 0 to 100, preferably 10 to 40.

(15) X is an amino acid residue represented by SEQ ID NO: 1, and the amino acid sequence X.sub.n is a sequence of repeating unit Xs with the number of repetitions n (n is an integer). The mode of repetition is linear repetition. The case of X.sub.2 means “X-X” (wherein symbol “-” denotes a peptide bond). Also, in the repeated sequence X.sub.n, the amino acid sequence may be modified in the aforementioned manner. As mentioned above, the number n is an integer of 1 to 3. The number n is preferably 1 but may be 2 or 3. The repeated sequence X.sub.n (n=2 or 3) is employed for consistently maintaining the suitable distance between the molecular needle Y and the immunogen W in response to the dimension and characteristics of the immunogen W.

(16) The cell introduction domain Y is a basement of the molecular needle and is based on the tail needle of a bacteriophage (i.e., cell-penetrating part). The domain Y is a polypeptide represented by the following formula (2):
Y.sub.1-L.sub.2-Y.sub.2-Y.sub.3  (2)
[wherein Y.sub.1 represents an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5; Y.sub.2 represents an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 9; L.sub.2 represents a second linker sequence having 0 to 30 amino acids; Y.sub.3 represents an amino acid sequence for modification of interest; and Y.sub.2 or Y.sub.3 may be absent].

(17) In Y.sub.1 of formula (2), the amino acid sequence from the end of the N-terminal side to the 32 amino acid residue (32 Leu) corresponds to the amino acid sequence of a triple helix β-sheet structure of the bacteriophage T4. The N-terminal amino acid of valine (1 Val) may also be leucine (1 Leu). The remaining C-terminal amino acid sequence is an amino acid sequence of the needle protein of the bacteriophage on the C-terminal side. Examples of the amino acid sequence that can be used at C-terminus of Y.sub.1 include an amino acid sequence of gp5 of bacteriophage T4, an amino acid sequence of gpV of bacteriophage P2, an amino acid sequence of gp45 of bacteriophage Mu, and an amino acid sequence of gp138 of bacteriophage ϕ92. More specific examples of Y.sub.1 include the amino acid sequence (SEQ ID NO: 2) which has the amino acid sequence of gp5 of bacteriophage T4 on the C-terminal side, the amino acid sequence (SEQ ID NO: 3) which has the amino acid sequence of gpV of bacteriophage P2 on the C-terminal side, the amino acid sequence (SEQ ID NO: 4) which has the amino acid sequence of gp45 of bacteriophage Mu on the C-terminal side, and the amino acid sequence (SEQ ID NO: 5) which has the amino acid sequence of gp138 of bacteriophage ϕ92 on the C-terminal side. The nucleotide sequence encoding the amino acid sequence of Y.sub.1 may be selected in accordance with a generally known amino acid-nucleobase relationship.

(18) Y.sub.2 in formula (2) is an amino acid sequence of a domain “foldon” of bacteriophage T4 or an amino acid sequence of a domain “tip” of bacteriophage P2, bacteriophage Mu, or bacteriophage ϕ92. The foldon or the tip is a domain forming the tip portion of the molecular needle structure (i.e., fibritin) of bacteriophage. In formula (2), Y.sub.2 is not necessarily present. However, when it includes the amino acid sequence of the foldon or the tip, the efficiency of incorporation of the molecular needle to the cell membrane can be enhanced. Thus, the presence of Y.sub.2 is preferred. The amino acid sequence of the foldon of bacteriophage T4 is represented by SEQ ID NO: 6. The nucleotide sequence encoding the amino acid sequence may be selected in accordance with a generally known amino acid-nucleobase relationship.

(19) The amino acid sequence of the tip of bacteriophage P2 is represented by SEQ ID NO: 7. The nucleotide sequence encoding the amino acid sequence may be selected in accordance with a generally known amino acid-nucleobase relationship. The amino acid sequence of the tip of bacteriophage Mu is represented by SEQ ID NO: 8. The nucleotide sequence encoding the amino acid sequence may be selected in accordance with a generally known amino acid-nucleobase relationship. The amino acid sequence of the tip of bacteriophage ϕ92 is represented by SEQ ID NO: 9. The nucleotide sequence encoding the amino acid sequence may be selected in accordance with a generally known amino acid-nucleobase relationship.

(20) L.sub.2 serves as a second linker intervening between Y.sub.1 and Y.sub.2. The number of amino acid residues in linker L.sub.2 is 0 to 30, preferably 0 to 5. The number of amino acid residues in linker L.sub.2 of 0 means the absence of second linker L.sub.2.

(21) Y.sub.3 is an amino acid sequence for modification and may be selectively incorporated into Y. The modification amino acid sequence is added in order to purify or protect protein (or for another reason), and examples thereof include tag proteins such as histidine tag, GST tag, and FLAG tag. An appropriate linker sequence may be incorporated into Y.sub.3. Such an additional linker sequence may also be a component of the amino acid sequence of Y.sub.3.

(22) The composite polypeptide of the present invention may be produced through a known method; specifically, a genetic engineering process or chemical synthesis. The entirety of the composite polypeptide of the present invention may be produced in a single process. Alternatively, segments of the polypeptide are individually produced, and the produced segments are linked through a chemical modification method, to produce the composite polypeptide. In one linking procedure of polypeptides by mediation of a linker (e.g., L.sub.1 or L.sub.2), a lysine residue or a cysteine residue of one polypeptide may be linked to that of another polypeptide by mediation of a linker having a succinimido group or a maleimido group.

(23) In a genetic engineering process, a nucleic acid fragment encoding the entirety of a part of the target composite polypeptide of the present invention may be expressed in host cells (e.g., Escherichia coli, yeast, insect cells, and animal cells) or in a cell-free expression system such as an E. coli extract, a rabbit reticulocyte extract, or a wheat germ extract. Any nucleic acid expression vector suited for the expression system may be used. Examples of the expression vector include pET (for expression in E. coli), pAUR (for expression in yeast), pIEx-1 (for expression in insect cells), pBApo-CMV (for expression in animal cells), and pF3A (for expression in wheat germ extract).

(24) Regarding chemical synthesis, any known method for chemically synthesizing peptides may be employed. More specifically, the entirety or a part of the composite polypeptide of the present invention may be produced through any established customary method (e.g., liquid phase peptide synthesis or solid phase peptide synthesis). Among generally known suitable solid phase peptide synthesis techniques (i.e., chemical synthesis), a Boc solid phase method or an Fmoc solid phase method may be employed. Also, as described above, a ligation technique may be employed in accordance with need. Needless to say, each amino acid may be produced through a known method, and a commercial product thereof may be used.

(25) (2) The Associated Product of the Present Invention

(26) FIG. 1 is a scheme of forming the associated products of the present invention (trimer and hexamer) from the composite polypeptide of the present invention. In FIG. 1, reference numeral 10 denotes the composite polypeptide of the present invention in the form of monomer. Reference numeral 30 denotes the trimer of the present invention, and reference numeral 60 denotes the hexamer of the present invention.

(27) The composite polypeptide 10 of the present invention is formed of a “molecular needle domain 13 corresponding to X.sub.n and Y in formula (1)” in which a “base part 131 corresponding to X.sub.n formula (1) and Y.sub.1 in formula (2)” is linked to a “foldon 132 corresponding Y.sub.2 in formula (2),” and an “immunogen 11 corresponding to W in formula (1),” wherein two components are linked together via a “linker 12 corresponding to L.sub.1 in formula (1). A linker other than linker 12, and the modification sequence corresponding to Y.sub.3 in formula (2) are not illustrated. The composite polypeptide 10 of the present invention per se substantially exhibits no function of penetrating the cell membrane of the target tissue cells.

(28) The trimer 30 is a spontaneously associated product of three molecules of the composite polypeptide 10 serving as a monomer via a spontaneous association process. In the trimer 30, 3 units of the molecular needle domain 13 are combined with association via formation of C-terminal-C-terminal bonds, to thereby provide a trimer parallel β-sheet structure, and a helix structure of the β-sheet structure per se (i.e., triple helix β-sheet structure), which corresponds to a needle structure. As a result, a molecular needle 13×3 is formed. The molecular needle 13×3 is composed of a basic part 131×3 and a foldon aggregate 132×3. In this way, a “molecular needle” which has a target tissue cell membrane penetration function is formed through trimerization and a self-assembly process. Three linkers (12.sup.1, 12.sup.2, 12.sup.3) originating from the respective monomers, and three immunogen portions (11.sup.1, 11.sup.2, 11.sup.3) linked to the respective linkers exist in the outside area of the molecular needle 13×3.

(29) The hexamer 60 is formed through linking 2 units of the trimer 30, with formation of a bond between the N-terminals of the molecular needle basic parts (13×3).sup.1 and (13×3).sup.2. The hexamer 60 also exhibits a target tissue cell membrane penetration function. Six linkers (12.sup.1, 12.sup.2, 12.sup.3, 12.sup.5, 12.sup.6, 12.sup.4 (not illustrated)) originating from the respective trimers, and six immunogen portions (11.sup.1, 11.sup.2, 11.sup.3, 11.sup.5, 11.sup.6, 11.sup.4 (not illustrated) lined to the respective linkers exist in the outside area of two molecular needles (13×3).sup.1 and (13×3).sup.2.

(30) Trimerization of the composite polypeptide 10 of the present invention to the trimer 30 and dimerization of the trimer 30 to the hexamer 60 proceed spontaneously in aqueous liquid, and the product exists as the trimer or the hexamer in a stable state. The stability of the trimer or hexamer is remarkably high. For example, the trimer or hexamer is stable in an aqueous liquid at 100° C., in an aqueous liquid at a pH of 2 to 11, or in an aqueous liquid containing 50 to 70 vol. % of organic solvent. In addition, the trimer or hexamer is excellent in safety. When being isolated from the aqueous liquid and dried, the trimer or hexamer is highly stable and retains an excellent cell membrane penetration function.

(31) As described above, transformation of the composite polypeptide of the present invention to the associated product of interest proceeds spontaneously. Generally, most are the hexamers (final form), but some remains as the trimers.

(32) (3) The Norovirus Vaccine of the Present Invention

(33) The associated product of the present invention (hexamer), serving as an active ingredient of the norovirus vaccine of the present invention, exhibits excellent cell penetration function and immunogenicity. Thus, when administered to target tissue cells via subcutaneous administration, intradermal administration, percutaneous administration, or intramuscular administration, the vaccine of the invention efficiently transfers a part or the entirety of P domain of the capsid protein of a norovirus (immunogen) to the target tissue cells, to thereby attain immunization. As a result, the efficacy and safety of the norovirus component vaccine via subcutaneous administration, intradermal administration, percutaneous administration, or intramuscular administration are expected to be enhanced.

(34) The norovirus vaccine of the present invention is provided as a pharmaceutical composition (liquid form) for subcutaneous administration, intradermal administration, percutaneous administration, or intramuscular administration, which contains, as an active ingredient (protective antigen), a “hexamer including, as W, the P domain of the capsid protein of norovirus,” among the associated product as described above. In the case where the associated product of the present invention (hexamer) is directly administered, the associated product is subcutaneously, intradermally, percutaneously, or intramuscularly administrated in liquid form which is prepared upon use by suspending and mixing the associated product of the present invention (hexamer) in a buffer or the like. This form is also included in the pharmaceutical composition.

(35) The norovirus vaccine of the present invention may be prepared in a form of a pharmaceutical composition by blending the associated product of the present invention (hexamer) serving as the active ingredient (protective antigen) with an optional adjuvant and an appropriate pharmaceutical carrier. The pharmaceutical carrier may be selected in accordance with the form of use. Examples of the pharmaceutical carrier which may be used in the invention include a filler, an extender, a binder, a humectant, a disintegrant, a surfactant, an excipient, and a diluent. As described above, the form of the composition is generally liquid, but may be a dry product, powder, granule, and the like which are diluted with liquid upon use.

(36) In the norovirus vaccine of the present invention, the amount of the associated product of the present invention (hexamer) is not necessarily fixed and may be appropriately chosen. Generally, it is preferably in a liquid form which contains 1 to 10 mass % of the associated product of the present invention (hexamer) upon administration. The appropriate single dose of administration (inoculation) is about 0.01 μg to about 10 mg for an adult. As needed, initial inoculation may be appropriately combined with booster inoculation. The administration (inoculation) may be carried out one or more times.

EXAMPLES

(37) The present invention will next be described in detail by way of example.

[Example 1] Preparation of the Associated Product of the Present Invention

(38) (1) Preparation of the Composite Polypeptide of the Present Invention

(39) In Example 1, four composite polypeptides falling within the scope of the present invention and having different immunogens (the following (a), (b), (c), and (d)) were prepared through a genetic engineering technique.

(40) (a) MNV-PF:

(41) a composite polypeptide falling within the scope of the present invention employing, as an immunogen, the entirety of one unit of “P domain Full (PF domain)” prepared by deleting N-terminal domain and Shell domain from “VP-1 domain” of a mouse norovirus (hereinafter may be referred to as “MNV PF”). The amino acid sequence of the immunogen is represented by SEQ ID NO: 10. The nucleotide sequence encoding the amino acid sequence may be selected in accordance with a generally known amino acid-nucleobase relationship.

(42) (b) MNV-P2:

(43) a composite polypeptide falling within the scope of the present invention employing, as an immunogen, only “P2 domain,” among capsid-inside “P1 domain” and capsid-outside “P2 domain” which constitute one unit of “PF domain (P domain Full: PF)” prepared by deleting N-terminal domain and Shell domain from “VP-1 domain” of a mouse norovirus. The amino acid sequence of the immunogen is represented by SEQ ID NO: 11. The nucleotide sequence encoding the amino acid sequence may be selected in accordance with a generally known amino acid-nucleobase relationship.

(44) (c) Saga PF:

(45) a composite polypeptide falling within the scope of the present invention employing, as an immunogen, the entirety of one unit of “P domain” (P domain Full: PF)—one viral capsid antigen of human norovirus. The amino acid sequence of the immunogen is represented by SEQ ID NO: 12. The nucleotide sequence encoding the amino acid sequence may be selected in accordance with a generally known amino acid-nucleobase relationship.

(46) (d) Saga P2:

(47) a composite polypeptide falling within the scope of the present invention employing, as an immunogen, only capsid-outside “P2 domain,” among capsid-inside “P1 domain” and capsid-outside “P2 domain which constitute one unit of “P domain” (P domain Full: PF)—one viral capsid antigen of human norovirus. The amino acid sequence of the immunogen is represented by SEQ ID NO: 13. The nucleotide sequence encoding the amino acid sequence may be selected in accordance with a generally known amino acid-nucleobase relationship.

(48) <Production of Plasmid for Forming “PN-Saga PF” Via Expression>

(49) “PN-Saga PF” is a composite polypeptide represented by the aforementioned formula (1):
W-L.sub.1-X.sub.n—Y  (1)
wherein Y represents a cell introduction domain represented by formula (2):
Y.sub.1-L.sub.2-Y.sub.2-Y.sub.3  (2).

(50) In the above formulas, the immunogen W is “Saga PF” represented by amino acid sequence SEQ ID NO: 13; the first linker L.sub.1 is amino acid sequence SEQ ID NO: 15; the repeating unit of the repeating sequence X.sub.n is amino acid sequence SEQ ID NO: 1; the number n is 1; the amino acid sequence of the molecular needle base Y.sub.1 is amino acid sequence SEQ ID NO: 2; the second linker L.sub.2 is “SVE”; the amino acid sequence of the foldon Y.sub.2 is amino acid sequence SEQ ID NO: 6; and the amino acid sequence of the modification sequence Y.sub.3 is amino acid sequence SEQ ID NO: 16 (VEHHHHHH).

(51) As mentioned below, expression plasmid “plasmid pKN1-1” for forming a composite polypeptide in which the immunogen W in the above formulas (1) and (2) is substituted by GFP (green fluorescent protein: SEQ ID NO: 17) was produced. The composite polypeptide expression plasmid of interest was produced by use of plasmid pKN1-1.

(52) Specifically, a plasmid for forming “PN-Saga PF” was produced through the following procedure.

(53) Firstly, the gene fragment corresponding to 461th to 484th amino acid residues of the wac protein of T4 phage was amplified through PCR from the T4 phage genome, followed by cloning to pUC18, to thereby yield a gene encoding the foldon. Subsequently, the plasmid was cut by restriction enzymes EcoRI and SalI, and the product was inserted into a plasmid pET29b (Novagen) treated by EcoRI and XhoI, to thereby yield plasmid pMTf1-3. Also, the gene fragment corresponding to 474th to 575th amino acid residues of gp5 of the T4 phage was amplified through PCR from the T4 phage genome, followed by cloning to pUC18, to thereby yield a gene encoding gp5. Subsequently, the resultant plasmid was cut by restriction enzymes EcoRI and SalI, and the product was inserted into the aforementioned plasmid pMTf1-3 treated by EcoRI and XhoI, to thereby yield plasmid pKA176. Separately, the GFP expression vector provided by Takahasi of Gunma University was cut by restriction enzymes NdeI and EcoRI, to thereby yield a gene encoding the GFP. The product was inserted into the aforementioned plasmid pKA176 treated by restriction enzymes NdeI and EcoRI, to thereby yield plasmid pKN1-1 (plasmid for GFP-gp5f expression).

(54) On the basis of pKN1-1 serving as the plasmid for GFP-gp5f expression, “plasmid pKN1-SagaPF (or simply referred to as “pKN1-SagaPF”) serving as a “PN-Saga PF expression plasmid” was produced by replacing the GFP gene moiety of “plasmid pKN1-1” by Saga PF gene as if cassette exchange was done.

(55) More specifically, a linear plasmid fragment (i.e., receptor plasmid fragment) was produced by removing the GFP gene from plasmid pKN1-1. Specifically, an anti-sense primer (SEQ ID NO: 18) for inverted PCR was synthesized immediately upstream of the GFP gene. Furthermore, a sense primer complimentary to the nucleotide sequence of “MDELYKE” (SEQ ID NO: 14) moiety of the linker sequence L.sub.1 (SEQ ID NO: 15) added to the C-terminal of the GFP protein (SEQ ID NO: 17), was synthesized. Then, by use of the aforementioned antisense primer and the sense primer, inverted PCR was performed with the plasmid pKN1-1 as a template, whereby a linear plasmid fragment free of GFP was yielded as a PCR amplification product (first PCR amplification product).

(56) Subsequently, the Saga PF moiety was amplified, and the product (Saga PF) was incorporated into the above-amplified linear plasmid fragment by use of an In-Fusion cloning kit, as if a cassette was to be inserted. This was carried out through the following PCR process.

(57) The specific procedure is as follows. Firstly, 35-nucleotide sense primer (SEQ ID NO: 20) was synthesized so that the sequence thereof was a 15-nucleotide sense chain nucleotide sequence on the linear vector 5′ side and a 20-nucleotide nucleotide sequence on the Saga PF gene sense chain 5′ side. In addition, a 35-nucleotide anti-sense primer (SEQ ID NO: 21) was synthesized so that sequence thereof was “a nucleotide sequence of the Saga PF gene 3′-terminal part (corresponding to 7 amino acid residues on the C-terminal amino acid sequence SEQ ID NO: 10 from which the aforementioned linker sequence (SEQ ID NO: 14) had been deleted) and a nucleotide sequence corresponding to MDELY (SEQ ID NO: 30) of the linker sequence MDELYKE (SEQ ID NO: 14).” Subsequently, the Saga PF fragment was amplified by use of the anti-sense primer and the sense primer and pHuNoV-Saga 1F (human norovirus GII.4 Saga-1 full-length clone) as a template, to thereby yield a PCR amplification product (second PCR amplification product). The sequence on the sense primer side (SEQ ID NO: 22) of the PCR product overlapped with the sequence on the anti-sense primer side of the linear vector PCR product, and the sequence on the anti-sense primer side (SEQ ID NO: 23) of the PCR product overlapped with the sequence on the sense primer side of the linear vector PCR product. The primer which is served as a cassette to be inserted is designed so that the sequence of the primer overlaps with the liner vector sequence at both terminals, and, while the overlapping sequences are employed to maintain the orientation, the primer is joined to the vector by means of the In-Fusion cloning kit.

(58) Finally, the first PCR amplification product and the second PCR amplification product were linked through fusion of respective overlapping sequences by means of the In-Fusion cloning kit, whereby pKN1-Saga PF was produced.

(59) The thus-produced “pKN1-Saga PF” (i.e., a “PN-Saga PF” expression plasmid) was incorporated into E. coli, to thereby mass-produce “PN-Saga PF” through expression. The product was purified and employed in the following experiments.

(60) <Ultracentrifugal Analysis of “PN-Saga PF”>

(61) The thus-prepared “PN-Saga PF” was subjected to ultracentrifugal analysis. In the specific procedure, 1.2-μM “PN-Saga PF” was subjected to dialysis with 0.1M sodium phosphate buffer (pH: 7.0), and the product was ultracentrifuged by means of Optimal XL-I analytical ultracentrifuge (Beckman) at 500,000 rpm and 20° C.

(62) FIG. 2 shows the results of analysis (size exclusion column chromatography) of “PN-Saga PF.” As shown in FIG. 2, most of the composite polypeptides “PN-Saga PF” were found to be spontaneously self-associated, and exist in the form of a “PN-Saga PF” trimer and a hexamer formed from 2 molecules of the trimer.

[Example 2] Test of Introduction of “PN-Saga P2” into Cells

(63) The procedure of producing “PN-Saga PF” expression plasmid was repeated, except that the immunogen W was “PN-Saga P2” represented by amino acid sequence SEQ ID NO: 13, to thereby produce a “PN-Saga P2” expression plasmid (pKN1-Saga P2). The formation, purification and ultracentrifugal analysis of “PN-Saga P2” were performed in the same manner as employed in the case of “PN-Saga PF.” For replacement of the GFP gene, a sense primer SEQ ID NO: 24 and an anti-sense primer SEQ ID NO: 25 were used for PCR. The thus-obtained PCR product was incorporated into the expression vector PCR product by means of the In-Fusion cloning kit in the same manner as mentioned above.

(64) As a result, most of the composite polypeptides “PN-Saga P2” were also found to be spontaneously self-associated, and exist in the form of a “PN-Saga P2” trimer and a hexamer formed from 2 molecules of the trimer.

(65) Then, the PN-Saga P2 was added to a supernatant of an HeLa cell culture, so as to investigate the possibility for PN-Saga P2 including the Saga P2 domain to be introduced into target cells. P2 was detected through immunofluorescence using an anit-Saga-1 VLP antibody.

(66) As a result, Saga P2 protein introduced into the HeLa cells was detected as a green fluorescent signal at many spots, whereby it was confirmed that PN-Saga P2 functioned as had been expected (not illustrated, but a photograph can be submitted upon request).

[Example 3] Studies on “PN-MNV”

(67) The procedure of producing “PN-Saga PF” expression plasmid was repeated, except that the immunogen W was “MNV PF” represented by amino acid sequence SEQ ID NO: 10 or “MNV P2” represented by amino acid sequence SEQ ID NO: 11, to thereby produce a “PN-MNV PF” expression plasmid (pKN1-MNVPF) and a “PN-MNV P2” expression plasmid (pKN1-MNVP2). The procedures of formation, purification, and ultracentrifugal analysis of “PN-MNV PF” and “PN-MNV P2” were performed in the same manner as employed in the case of “PN-Saga PF.” For replacement of the GFP gene by the gene of P domain Full, sense primer SEQ ID NO: 26 and anti-sense primer SEQ ID NO: 27 were used in PCR, and for replacement of the GFP gene by the P2 gene of P domain, sense primer SEQ ID NO: 28 and anti-sense primer SEQ ID NO: 29 were used in PCR.

(68) As a result, similar to the case of “PN-Saga PF,” most of each of “PN-MNV PF” and “PN-MNV P2” was found to be spontaneously self-associated, and exist in the form of a trimer thereof and a hexamer formed from 2 molecules of the trimer.

(69) Subsequently, introduction of the above “PN-MNV P2” associated product (the associated product of the present invention, also referred to as “PMNVP2 associated product”) into HeLa cells as a target was carried out. The results were observed under a confocal microscope.

(70) Firstly, the PMNVP2 associated product was dyed with a fluorescent dye (ATTO520). Specifically, a 7.5 μM suspension of the PMNVP2 associated product in 0.1 M sodium phosphate buffer (pH: 8.3) and a 300 μM ATTO solution in DMSO were mixed at a volume ratio of 2:1 and at ambient temperature so that the total volume was 450 μL. The dyeing reaction was performed at ambient temperature. After completion of dyeing reaction, the product was washed thrice with 0.1 M sodium phosphate buffer (pH: 7.0) by means of Centricon.

(71) The HeLa cells were preliminarily cultured in 5.0×10.sup.4 cells/100 μL (DMEM) at 37° C. under 5% CO.sub.2. Then, the preculture medium was changed to a new medium (total volume: 90 μL) prepared by mixing DMEM (phenol red (-)) and 0.1M sodium phosphate buffer (pH: 8.3) at a volume ratio of 2:1. The ATTO520-added PMNVP2 associated product was added to the new culture system so that the total PMNVP2 concentration was 5 μM. For introducing the PMNVP2 associated product into the HeLa cells, culturing was performed for 30 minutes under the same culture conditions as mentioned above.

(72) Incorporation of the PNMVP2 associated product into HeLa cells was observed under the following conditions using a confocal microscope (laser confocal microscope A1 (Nikon)).

(73) (1) Wavelength of Laser Light:

(74) Excitation: 405 nm laser/observation (a 450 nm/50 emission filter) and

(75) Excitation: 488 nm laser/observation (a 525 nm/50 emission filter)

(76) (2) Laser Power

(77) Laser power: 405 nm: 1.4, 488 nm: 0.4

(78) As a result, the fluorescent dye-added PNMVP2 associated product was observed as a large number of small bright spots around cell nuclei as dark portions. Thus, the PNMVP2 associated product was found to be introduced into HeLa cells for a very short incubation time of 30 minutes (not illustrated, but a photograph can be submitted upon request).

[Example 4] Mouse Immunization Test

(79) (1) Materials

(80) “PN-MNV PF” associated product (PNVPF associated product)

(81) E. coli BL21 (DE3), which had been transformed with the “PN-MNV PF” expression plasmid (pKN1-MNVPF) and used in Example 3, was cultured at 37° C. for 6 hours in an LB medium (200 mL, kanamycin concentration: 30 μg/mL). Subsequently, an aliquot (30 mL) of the culture liquid was added to a 3 L LB medium (kanamycin concentration: 30 μg/mL), and the mixture was cultured at 37° C. When the turbidity (OD 600) reached 0.8 or higher, an IPTG solution was added so as to adjust the final concentration of 1 mM. The resultant mixture was incubated overnight at 20° C. The incubated product was centrifuged at 4° C. and 8,000 rpm, to thereby collect the bacterial cells. The collected bacterial cells were instantly frozen with liquid nitrogen and stored at −80° C.

(82) One tablet of complete, EDTA-free (Roche) was dissolved in Buffer A (0.1M Tris-HCl (pH: 8), 0.5M NaCl, 1 mM DTT, and 5 mM imidazole), and the above bacterial cells (22.4 g) were suspended in the solution. The bacterial cells were broken by ultrasonication. The set of the above operations was performed on ice. Then, the product was centrifuged at 17,500 rpm and 4° C. for 50 minutes, and the supernatant was filtered by means of a 0.8 μm filter. The thus-purified supernatant was further purified through Ni affinity column [HisTrap TMHP Colum (GE Healthcare)] at 4° C. Elution was performed with Buffer A and Buffer B (0.1M Tris-HCl (pH: 8), 0.5M NaCl, 1 mM DTT, and 500 mM imidazole) under linear gradient conditions (5 to 500 mM imidazole). Samples were monitored at 280 nm, and fractions of interest were recovered.

(83) The combined sample obtained through Ni affinity purification was concentrated to a volume of about 10 mL. The product was filtered by means of a 0.2 μm filter. The filtrate was subjected to gel filtration purification by use of HiLoad 26/60 Superdex 200 column (GE Healthcare). The elution was performed by use of Buffer C (20 mM Tris-HCl, 0.5M NaCl, and 1 mM TCEP). The chart of FIG. 3 shows the results.

(84) The molecular weight of each of the fractions shown in FIG. 3 (each fraction is assigned a fraction number along the horizontal axis) was confirmed through Native SDS PAGE (not illustrated). Among fractions in which a band corresponding to a molecular weight of about 300,000 Da (similar to that of the hexamer of interest) was detected, the 36th fraction, which exhibited the sharpest band, was selected. The fraction was employed as a fraction of the PNVPF associated product (hexamer). The trimer thereof may be readily isolated by subjecting the 38th to 40th fractions exhibiting a molecular weight of about ½ of the above molecular weight to a customary procedure such as purification through ion chromatography.

(85) The P domain (MNVPF) of the capsid protein of the mouse norovirus was prepared on the basis of a known gene sequence through a customary method as a recombinant protein produced from E. coli as a host. More specifically, a sense primer having, on the N-terminal side of the PF domain of the mouse norovirus, an overlapping portion (15 nucleotides) with the upstream side of the cloning site of the expression vector pET6×HN, and an anti-sense primer having, on the C-terminal side, an overlapping portion (15 nucleotides) with the downstream side of the cloning site of the expression vector pET6×HN, were synthesized according to the protocol of In-Fusion cloning kit. Then, PCR was performed by use of the primers, and the thus-obtained PCR product was cloned to pET6×HN by means of the In-Fusion cloning kit, to thereby yield pET6×HN-MNVPF. The pET6×HN-MNVPF was introduced into E. coli BL21, and MNVPF was expressed through a customary method. The resultant E. coli cells were broken, and the entire proteins contained therein were solubilized by use of guanidine. As a result, all the proteins were converted to a linear form. By use of 6 HN tags added to MNVPF contained in the solubilized protein, the product was bound to a column of TALON resin according to the manual thereof. Thereafter, MNVPF was dialyzed with PBS(-), and the solvent was completely changed to PBS(-). After adjustment of the concentration, mice were immunized with the thus-purified MNVPF solution in the following manner.

(86) Besides, a 1-mL syringe with a 26G injection needle (product of TERMO), and mice (Japan SLC, C57BL/6J, 6-week, female) were provided.

(87) (2) Method

(88) The test systems employing the following two protective antigens were established.

(89) (a) administration of MNVPF (3.12 μg/200 μL PBS)

(90) (b) administration of hexamer (13.2 μg/200 μL PBS)

(91) Each protective antigen diluted solution (total amount: 200 μL) was subcutaneously injected to each mouse at 5 sites (40 μL/site) of the back thereof by means of a syringe with a 26G injection needle. Three weeks after the injection, blood was collected through the tail of the mouse (primarily immune serum). Then, the equal volume of the same antigen diluted solution was subcutaneously injected again to each mouse at the back thereof. Two weeks after the injection, blood was collected through the tail of the mouse (secondarily immune serum).

(92) <Preparation of Serum Samples from Collected Blood>

(93) Each of the thus-collected blood samples was centrifuged by means of a desktop centrifuge at 15,000 rpm for 1 minute. The supernatant was transferred to an Eppendorf microtube and centrifuged again at 15,000 rpm for 1 minute. The supernatant was transferred to another Eppendorf microtube and employed as a sample for ELISA. ELISA was carried out under the following conditions.

(94) <Conditions of ELISA>

(95) (a) Material

(96) Virus-like hollow particles (VLP) of mouse norovirus

(97) Mouse serum

(98) Anti-norovirus monoclonal antibody (G3B9λm IgG 2a, positive control)

(99) ELISA plate (F96 MAXI SORP NUNC-IMMUNO PLATE, Thermo SCIENTIFIC)

(100) 96-well dilution plate (96-well Round Bottom Non-Treated, CORNING)

(101) Tween 20

(102) BSA

(103) PBS

(104) PBST (0.1% Tween 20-added PBS)

(105) PBSB (1% BSA-added PBS)

(106) PBSTB (1% BSA, 0.1% Tween 20-added PBS)

(107) Goat anti-mouse IgG HRP label (Southern Biotech)

(108) OPD substrate (Sigma, 10 mg tablet of o-phenylenediamine dihydrochloride)

(109) H.sub.2O.sub.2

(110) OPD buffer (36 g Na.sub.2HPO.sub.4.12H.sub.2O+9.6 g citrate (citric acid)+1 L D.sub.2W, pH: 5.0)

(111) 2N H.sub.2SO.sub.4

(112) Plate reader (BIORAD iMark)

(113) (b) Method

(114) A mouse norovirus VLP was mixed with PBS to thereby prepare a 1-μg/mL liquid thereof, and the virus liquid was added to an ELISA plate at 50 μL/well. The ELISA plate was allowed to stand overnight at 4° C. and then washed 4 times with PBST. PBST remaining on the plate was completely wiped off, and new PBSB was added to the plate at 80 μL/well. The plate was allowed to stand for 2 hours at room temperature. Separately, a mouse serum was 20-fold diluted with PBSB by use of a dilution plate. The thus-diluted serum was stepwise (3-fold) diluted, to thereby provide 8 dilution series in total. After the above standing for 2 hours, PBSB was removed from the ELISA plate, and the diluted serum was added to the ELISA plate at 50 μL/well. The plate was allowed to stand at room temperature for 4 hours, and the diluted serum was removed from the ELISA plate. The plate was washed 4 times with PBST, and PBST remaining on the plate was completely wiped off. Next, an HRP-labeled anti-mouse IgG antibody (5,000-fold diluted with PBSTB) was added to the plate at 50 μL/well, and the plate was allowed to stand for 2 hours at room temperature. Thereafter, the HRP-labeled antibody was removed from the ELISA plate, and the plate was washed 5 times with PBST, followed by completely wiping off remaining PBST. Separately, an OPD substrate (10 mg) was dissolved in an OPD buffer (20 mL), and H.sub.2O.sub.2 (10 μL) was added thereto, followed by inversion mixing. The OPD substrate solution was added to the ELISA plate at 50 μL/well, and the plate was allowed to stand at room temperature for color development. Thereafter, color development was stopped by adding 2N H.sub.2SO.sub.4 to the plate at 50 μL/well. OD490 was measured by means of a plate reader, and the measurements were compared with a positive control to perform concentration calculation.

(115) (3) Results

(116) FIG. 4 is a graph showing the results of ELISA after the above initial inoculation. In FIG. 4, the vertical axis represents the IgG antibody titer (U/ml). As shown in FIG. 4, the antibody titer was found to significantly rise in one sample among the 4 samples in the case where the PMNVPF associated product (hexamer) had been inoculated. A tendency of rising in antibody titer was also observed in 2 of the remaining 3 samples. In the case of MNVPF (control), no substantial rise was observed in antibody titer.

(117) That is, the associated product of the present invention (hexamer) having MNVPF as an immunogen was found to be very excellent as a protective antigen.