JP2022127591A - Structural protein of parvovirus and virus-like particle vaccine - Google Patents

Abstract

To provide a new structural protein having excellent immunogenicity against parvovirus and a virus-like particle.SOLUTION: The present invention relates to a structural protein, wherein one or more and five or less RBDs are further added to an N terminus of structural protein VP1 of parvovirus having a receptor binding domain (RBD) and a phospholipase A2(PLA2) active area. There is also provided a virus-like particle (VLP) comprising the same.SELECTED DRAWING: Figure 1

Description

The present invention relates to parvoviral structural proteins and virus-like particle vaccines.

Human parvovirus B19 is known as a causative virus of erythema infectiousis (apple disease) in children, and 90% of patients are children up to 9 years old. Human parvovirus B19 also infects adults and causes various symptoms such as fever, skin rash, and arthritis. The prognosis for human parvovirus B19 infection is generally good. However, when a woman in early pregnancy is infected with human parvovirus B19, it causes miscarriage and fetal hydrops with high probability due to mother-to-child transmission.

Human parvovirus B19 belongs to the Erythrovirus genus of the Parvoviridae family, and has a single-stranded linear DNA genome consisting of about 5500 bases. The genome encodes structural proteins (VP1 and VP2), a non-structural protein (NS1) and several small proteins. Human parvovirus B19 forms viral particles with a regular icosahedral structure about 23 nm in diameter from the viral genome and two structural proteins. Since parvovirus virions do not have an envelope, they are resistant to ethanol, organic solvents and soap, and are stable to heat treatment (eg, 60° C. for 30 minutes).

To date, human parvovirus B19 vaccines and secondary infection preventive measures have not been put to practical use. Human parvovirus B19 is difficult to grow in cultured cells, and there are no viral culture systems or animal infection models, so it is difficult to produce a live attenuated virus vaccine or an inactivated virus vaccine. Vaccine candidates therefore include virus-like particles (VLPs), which have structures similar to virus particles. Since VLPs do not proliferate in the body, they are suitable for vaccines from the viewpoint of safety. On the other hand, although VLPs are expected to have high immunogenicity compared to vaccines using recombinant proteins as antigens or nucleic acid vaccines, they are not sufficiently immunogenic compared to live attenuated virus vaccines or inactivated virus vaccines. Patent Documents 1 to 3 and Non-Patent Document 1 disclose methods for producing VLPs of human parvovirus B19.

WO2011/127316 WO 90/05538 WO2014/125053

Bernstein et al, Vaccine. 2011, October 6;29(43):7357-7363

Antigens with good immunogenicity are needed for the development of vaccines against parvovirus. The present invention aims to provide new structural proteins and VLPs with good parvovirus immunogenicity.

The present invention further adds 1 to 5 receptor binding domains to the N-terminus of parvovirus structural protein VP1 having a receptor binding domain (RBD) and a phospholipase A ₂ (PLA ₂ ) active region. related to structural proteins. In addition, the structural protein according to one example of the present invention preferably has attenuated PLA ₂ activity, more preferably lacks PLA ₂ activity, as compared to wild-type VLPs by including a mutation in the PLA ₂ active region. can do.

The present invention relates to items exemplified below.
[1] A structural protein comprising a parvovirus structural protein VP1 having a receptor-binding domain and a phospholipase _A2 active region, to which one to five of the receptor-binding domains are further added to the N-terminus.
[2] The structural protein of [1], to which 1 or more and 3 or less of the receptor-binding domains are added.
[3] The structural protein of [1] or [2], wherein at least one amino acid is deleted, substituted, inserted or added in the phospholipase _A2 active region.
[4] The structural protein according to any one of [1] to [3], wherein the parvovirus is human parvovirus B19.
[5] A parvovirus structural protein having the ability to form virus-like particles and comprising the amino acid sequence according to any one of (a) to (c);
(a) an amino acid sequence set forth in any of SEQ ID NOS: 5-10;
(b) an amino acid sequence having 90% or more identity to the amino acid sequence set forth in any of SEQ ID NOS: 5-10, or (c) an amino acid sequence set forth in any of SEQ ID NOS: 5-10 Amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added.
[6] A virus-like particle comprising the structural protein according to any one of [1] to [5] and a parvovirus structural protein VP2.
[7] The virus-like particle of [6], which has a reduced phospholipase _A2 activity compared to the wild-type virus-like particle.
[8] A nucleic acid encoding the structural protein of any one of [1] to [5].
[9] A vector comprising the nucleic acid of [8].
[10] A cell introduced with the nucleic acid of [8] or the vector of [9].
[11] An immunogenic composition comprising the virus-like particle of [6] or [7], the nucleic acid of [8], or the vector of [9].
[12] A method for producing virus-like particles, comprising the steps of culturing the cells of [10] and collecting virus-like particles produced from the cells.

According to the present invention, new structural proteins and VLPs with good parvovirus immunogenicity can be provided.

FIG. 3 is a schematic diagram showing the structures of parvovirus structural proteins VP1 and VP2, and RBD-tagged VP1 and VP2. 1 is a schematic diagram showing the structures of expression plasmids of human parvovirus B19 VP1 wt and VP2 wt, and RBD-added VP1 and VP2 (also referred to as “RBD-added proteins”) prepared in Experiment 1. FIG. FIG. 10 shows CBB staining after SDS-PAGE of VLPs containing VP1 wt or RBD-attached proteins in Experiment 2. FIG. 10 is a graph showing the proportion of VP1 wt or RBD-attached protein contained in VLPs in Experiment 2. FIG. 10 is a graph showing antibody titers in sera collected from mice to which VLPs containing VP1 wt or 2RBD-VP1 were administered in Experiment 3. FIG. FIG. 4 is a graph evaluating the parvovirus-neutralizing activity of sera collected from mice administered VLPs containing VP1 wt or RBD-attached proteins in experiment 4 by reducing the infection rate. FIG. 10 is a schematic diagram showing the deletion site of the PLA ₂ active region of VP1 in Experiment 5. FIG. FIG. 10 shows CBB staining after SDS-PAGE of VLPs containing VP1 wt, 2RBD-VP1 or 2RBD-VP1 delPLA ₂ in Experiment 5. FIG. 10 is a graph showing the ratio of VP1 wt, 2RBD-VP1 or 2RBD-VP1 delPLA ₂ contained in VLPs in Experiment 5. FIG. 10 is a graph showing antibody titers in serum of mice to which VLPs containing 2RBD-VP1 or 2RBD-VP1 delPLA ₂ were administered in Experiment 6. FIG. 10 is a graph showing PLA ₂ activity of VLPs containing VP1 wt, 2RBD-VP1 or 2RBD-VP1 delPLA ₂ in Experiment 7. FIG.

<Structural protein>
Parvovirus structural proteins include VP1 and VP2. VP1 has a receptor binding domain and a phospholipase _A2 active region as shown in FIG. 1 VP1 wt. The structural protein according to the present invention is a structural protein in which 1 to 5 RBDs are further added to the N-terminus of the parvovirus structural protein VP1. The present inventors selected the N-terminus of VP1 as the RBD addition site, and found that excellent immunogenicity was exhibited by adding 1 to 5 RBDs to the site. That is, the structural protein according to the present invention has two or more RBDs at the N-terminus, as exemplified by 1RBD-VP1 in FIG. In this specification, the VP1 to which the RBD is added is sometimes referred to as "RBD-added VP1".

Structural proteins according to the invention are capable of forming VLPs with good immunogenicity. Structural proteins according to the invention are also capable of generating a significant immune response alone. Immunogenicity of VLPs can be evaluated by methods well known to those skilled in the art, for example, by administering VLPs to mice and assessing whether the collected serum has the antibody of interest. IgG ELISA using a plate coated with the full-length immunogen or a partial region thereof can be used to evaluate whether or not the target antibody is present. Antibodies of interest include, for example, neutralizing antibodies, and the presence or absence of the antibodies can be evaluated by performing IgG ELISA against the neutralizing epitope region. When the parvovirus is human parvovirus B19, the neutralizing epitope region of VP1 can be anywhere known to those of skill in the art, such as the 1-90 amino acid region of SEQ ID NO:1.

The number of RBDs to be added (which may be referred to herein as "additional regions") is preferably 1 or more and 5 or less, may be 4 or less or 3 or less, or may be 2 or less. good too. VLPs containing a structural protein in which many RBDs are added to parvovirus VP1 are VLPs containing wild-type parvovirus VP1 to which RBDs are not added (herein, sometimes referred to as "wild-type VLP". ) tend to be more immunogenic. However, if too many RBDs are added, it may become difficult to form a VLP. The structural protein according to the present invention may be added with RBDs by adding 1 to 5 RBDs, preferably 1 to 3 RBDs, to the N-terminus of the parvovirus structural protein VP1. It has a strong virus-like particle-forming ability. Virus-like particle-forming ability refers to the ability to form virus-like particles. For example, RBD-attached VP1, which can be combined with the parvovirus structural protein VP2 to form VLPs, has the ability to form virus-like particles. The presence or absence of VLP formation can be confirmed by the method described in the section <VLP> below.

A linker peptide of appropriate length may connect between the added RBD and VP1, and between the added RBDs when multiple RBDs are added. The linker sequence may be, for example, 1 to 20 amino acid residues, or 10 or less amino acid residues. The linker sequence may be a glycine-rich sequence and may contain, for example, a GGGGGG sequence. In this specification, amino acid residues are sometimes represented by single-letter codes.

The human parvovirus B19 genome encodes VP1 and VP2, structural proteins that make up the virus particle. The open reading frames (ORFs) of VP1 and VP2 overlap, with VP1 encoding the RBD and PLA2 active regions upstream of the VP2 _- encoding sequence. VP1 is composed of a VP1 unique region including the RBD and PLA2 active region and a common region with VP2 in order from the N _- terminal side, as shown in FIG. The amino acid sequences of VP1 and VP2 of parvovirus B19-J35 (Accession No. AY386330) are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The RBD of the parvovirus B19 consists of 76 amino acid residues (SEQ ID NO: 3) from positions 5 to 80 of VP1. The PLA ₂ active region of the parvovirus B19 consists of 66 amino acid residues from positions 130 to 195 of VP1 (SEQ ID NO:4). As used herein, references to simply "VLP,""VP1,""VP2,""RBD," and " _PLA2 active region" refer to parvovirus VLP, VP1, VP2, RBD, and PLA2 active region, _respectively . .

The receptor for human parvovirus B19 is the P antigen on the surface of red blood cells, and human parvovirus B19 infects and proliferates P antigen-bearing cells, especially erythroid progenitor cells. Among the antibodies induced by using parvovirus as an antigen, antibodies against RBD contained in the VP1 unique region are important for protection against infection, but RBD alone does not have immunogenicity.

Structural proteins according to the invention preferably contain mutations in the PLA ₂ active region by deletion, substitution, insertion or addition of at least one amino acid in the PLA ₂ active region. Parvovirus particles do not exhibit PLA ₂ activity because they have a PLA ₂ active region inside the particle. On the other hand, VLPs, which have a structure partially different from virus particles, exhibit PLA ₂ activity because they have a PLA ₂ active region outside the particle (Ros et al, JOURNAL OF VIROLOGY, Dec. 2006, pp. 12017-12024). . Structural proteins containing mutations in the PLA ₂ active region are preferably capable of forming VLPs with attenuated, more preferably deleted, PLA ₂ activity, which facilitates reduction of inflammatory reactions, thus improving safety as vaccines. can be made

_The PLA2 active region of human parvovirus B19 has the HDXXY motif present in common secretory _PLA2s . The PLA ₂ active center of human parvovirus B19 has been reported to be amino acids 153-164 of VP1 (HDFRYSQLAKLG) (Bonsch et al, JOURNAL OF VIROLOGY, Dec. 2008, p.11784-11791). 130 (Y), 132 (G), 153 (H), 154 (D), 157 (Y), 162 (K), 168 (Y), 175 (D), 195 (D) of VP1 in the PLA ₂ active region D) th amino acid has been reported to be particularly involved in PLA ₂ activity (Deng et al, PLOS ONE, April 2013, Volume 8, Issue 4, e61440). Structural proteins according to the present invention preferably have at least one of these amino acids deleted or substituted, or any amino acid inserted or added to the PLA ₂ active region. Amino acid deletions, substitutions, insertions or additions in the PLA ₂ active region preferably do not affect VLP formation ability. It is also preferred that amino acid deletions, substitutions, insertions or additions in the PLA ₂ active region do not reduce the immunogenicity of the VLP. In the present invention, it was found that PLA ₂ activity can be attenuated or deleted while maintaining VLP-forming ability and immunogenicity by inserting a mutation into a part of the PLA ₂ active region. The site to insert the mutation, while maintaining the VLP-forming ability and immunogenicity, any site that can attenuate or delete PLA ₂ activity, including a part of the PLA ₂ active region 141st The 159th amino acid from is preferred. Preferably, at least one amino acid is deleted, substituted, inserted or added at positions 141 to 159 of VP1, and at least one amino acid at positions 141 to 159 of VP1 is deleted. is more preferable, and all of the 141st to 159th amino acids may be deleted. The partially deleted regions of the PLA ₂ active region may be connected by linker peptides of appropriate length. The linker sequence may be, for example, 1 to 20 amino acid residues, or 10 or less amino acid residues. The linker sequence may be a glycine-rich sequence and may contain, for example, a GGGGGG sequence.

As used herein, parvoviruses may include all parvoviruses associated with mammalian species (eg, humans, dogs, chickens, cats, mice, pigs, raccoons, minks, Kilham rats, rabbits). Parvoviruses may broadly include all genera of the Parvoviridae family, eg Parvovirus, Dependovirus, Erythrovirus, Bocavirus. The parvovirus genus includes, for example, canine parvovirus, feline parvovirus. The Dependovirus genus includes adeno-associated viruses. Erythroviruses include human parvovirus B19. The Bocavirus genus includes bovine parvovirus, canine microvirus. The parvovirus is preferably of the genus Dependovirus, Erythrovirus or Bocavirus, which infects humans, more preferably human parvovirus B19. In some embodiments, the parvovirus is from the genus Parvovirus. Parvoviruses may also include isolates that have not been characterized at the time of filing.

It is known that human parvovirus B19 can be subdivided into three distinct genotypes. Nucleotide differences between these genotypes are approximately 10% and more than 20% in the promoter region. All viruses previously known as human parvovirus B19 were classified as genotype 1. Genotype 2 is found relatively infrequently. When genotype 2 is found, it is identified with much higher frequency in individuals older than approximately 40 years of age. Genotype 3 viruses are divided into two subtypes, represented by prototype strain V9 (GenBank Accession No. AX003421) and prototype strain D91.1 (GenBank Accession No. AY083234). Genotype 3 viruses have been shown to be endemic to Ghana, West Africa, and may also be present in certain areas of Brazil.

As used herein, the structural protein VP1 of parvovirus may be a structural protein corresponding to VP1 of human parvovirus B19, and the amino acid sequence described in SEQ ID NO: 1 is 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% It may have amino acid sequences with 96% or more, 97% or more, 98% or more, 99% or more or 100% identity. The parvovirus structural protein VP1 may have an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added to the amino acid sequence shown in SEQ ID NO:1. Here, several may be, for example, 2 to 20, 2 to 10, 2 to 5, or 2 to 3.

As used herein, the structural protein VP2 of parvovirus may be a structural protein corresponding to VP2 of human parvovirus B19, and the amino acid sequence set forth in SEQ ID NO: 2 is 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% It may have amino acid sequences with 96% or more, 97% or more, 98% or more, 99% or more or 100% identity. The parvovirus structural protein VP2 may have an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added to the amino acid sequence shown in SEQ ID NO:2.

As used herein, the RBD of parvovirus may be a region corresponding to the RBD of VP1 of human parvovirus B19, and the amino acid sequence described in SEQ ID NO: 3 and 80% or more, 81% or more, 82% or more, 83% % or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more , 96% or greater, 97% or greater, 98% or greater, 99% or greater or 100% identity. A parvovirus RBD may have an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added to the amino acid sequence shown in SEQ ID NO:3.

As used herein, the PLA ₂ active region of parvovirus may be a region corresponding to the PLA ₂ active region of VP1 of human parvovirus B19, and the amino acid sequence described in SEQ ID NO: 4 and 80% or more, 81% or more , 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94 % or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater or 100% identity. The parvovirus PLA ₂ active region may have an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added to the amino acid sequence shown in SEQ ID NO:4.

The structural protein according to the present invention has the ability to form virus-like particles and may be a parvovirus structural protein comprising (a) an amino acid sequence set forth in any one of SEQ ID NOS: 5-10. The amino acid sequences represented by SEQ ID NOs: 5 to 7 are amino acid sequences in which 1, 2 or 3 RBDs are added to the N-terminus of VP1 of human parvovirus B19. The amino acid sequences shown in SEQ ID NOS:8-10 are amino acid sequences obtained by deleting a part of the PLA ₂ active region from the amino acid sequences of SEQ ID NOS:5-7, respectively. A structural protein according to the invention may consist of the amino acid sequence set forth in any of SEQ ID NOS:5-10.

The structural protein according to the present invention has the ability to form virus-like particles and comprises (b) an amino acid sequence having 90% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 10. It may be a parvoviral structural protein. The structural protein according to the present invention is preferably 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99% of the amino acid sequence set forth in any of SEQ ID NOs: 5 to 10. 0.5% or greater identity.

The structural protein according to the present invention has the ability to form virus-like particles, and (c) has deletion, substitution, or deletion of one or several amino acids in the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 10. It may be a parvoviral structural protein containing inserted or added amino acid sequences.

<Nucleic acid>
A nucleic acid according to the invention encodes a structural protein according to the invention. The structural proteins described above can be produced from the nucleic acids according to the invention. Nucleic acids are preferably DNA or RNA and may be single- or double-stranded. The nucleic acid according to the present invention may contain a base sequence encoding another peptide at the 5' end of the base sequence corresponding to the structural protein according to the present invention, and a termination codon at the 3' end of the base sequence. It may contain intronic sequences.

Nucleic acids according to the present invention are not limited to the nucleic acid sequences possessed by wild-type parvoviruses, and include nucleic acids containing nucleotide sequences in which codons encoding each amino acid in the coding region are replaced with other codons encoding the same amino acids. . From the viewpoint of improving protein expression, the nucleic acid according to the present invention is a nucleic acid containing a base sequence in which codon usage is changed so as to be suitable for cell types into which the nucleic acid is introduced, preferably human cells. good too.

A nucleic acid according to this embodiment can be obtained by chemical synthesis, polymerase chain reaction (PCR), or the like.

Examples of the nucleic acid according to the present invention include a nucleic acid sequence (SEQ ID NOS: 11 to 13) in which 1 to 3 RBD nucleic acid sequences are added to a nucleic acid encoding VP1 of human parvovirus B19, and a PLA from the nucleic acid sequence of SEQ ID NO: 12. ₂ active region is partially deleted (SEQ ID NO: 14). The nucleic acid sequences shown in SEQ ID NOS: 11-14 are human codon-optimized sequences.

<Vector>
A vector according to the invention comprises a nucleic acid as described above. Vectors are nucleic acid molecules capable of amplifying and maintaining DNA and include, for example, expression vectors and cloning vectors. In one example, the nucleic acid described above can be introduced into a host cell or the like in the form of being inserted into an expression vector to express the structural protein of the present invention. The vector according to the present invention can be obtained by inserting the above-mentioned nucleic acid into an appropriate basic vector according to the usual genetic engineering techniques.

A vector according to the present invention may further comprise a nucleic acid encoding VP2. The RBD-added VP1 gene and VP2 gene may be contained in different vectors or may be contained on the same vector. When the RBD-added VP1 gene and the VP2 gene are contained on the same vector, both the RBD-added VP1 and VP2 can be expressed in the host cell by introduction of one vector.

Vectors can be, for example, bacterial plasmid-derived vectors, yeast plasmid-derived vectors, viral vectors, cosmid vectors, phagemid vectors, artificial chromosome vectors, and the like. Vectors include pBR322, pUC plasmid vectors, pET-based plasmid vectors, and the like. Specifically, when Escherichia coli is used as a host cell, pUC19, pUC18, pUC119, pBluescriptII, pET28a(+), pET32 and the like can be mentioned. Examples of mammalian cells used as host cells include pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo, EB virus vectors, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors. be able to.

The expression vector may have a promoter sequence for expressing the integrated gene. The nucleic acid to be incorporated into the vector is inserted downstream of the promoter so that the promoter is operable. Any promoter may be used as long as it is suitable for the host used for gene expression. For example, when the host is an animal cell, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex viral thymidine kinase) promoter and the like are used. Among them, CMV promoter, SRα promoter and the like are preferable. When the host is E. coli, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter and the like are preferred. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferred. When the host is yeast, Gal1/10 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferred. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferred. When the host is a plant cell, CaMV35S promoter, CaMV19S promoter, NOS promoter and the like are preferred.

As the expression vector, in addition to the above, those containing an enhancer, a splicing signal, a terminator, a poly A addition signal, a drug resistance gene, a selection marker such as an auxotrophic complementary gene, a replication origin, etc. may be used depending on the purpose. can be done.

For example, a vector having an autonomous replication origin (ori) is maintained intracellularly as an episome when introduced into a host cell. A vector integrated with the ori of SV40 can greatly increase the copy number of the vector in the cell, for example, when introduced into a COS cell transformed with an ori-deleted SV40 genome.

<Cell>
A cell according to this embodiment is a cell into which the nucleic acid described above has been introduced. A nucleic acid may be introduced into a cell in the form contained in a vector. Cells into which nucleic acids have been introduced can express the structural proteins of the invention. The nucleic acid may be maintained extrachromosomally within the cell or may be integrated into the chromosome of the host cell. The nucleic acid according to the present invention or the vector according to the present invention is preferably maintained in a state in which the gene can be expressed under the control of a promoter that functions in the host cell.

Methods for introducing nucleic acids into cells include chemical methods such as calcium phosphate method, DEAE-dextran method and cationic liposome method; biological methods such as adenovirus vector, vaccinia virus vector, retrovirus vector and HVJ liposome; Physical techniques such as poration, microinjection, and gene gun are exemplified. An appropriate introduction method can be selected depending on the cells to be introduced.

Cells into which the nucleic acid is introduced can be eukaryotic or prokaryotic cells, including bacteria, fungi, plant cells, animal cells, and insect cells. The cells may be yeast, E. coli, mammalian, avian or fish cells, such as humans, cows, horses, sheep, monkeys, pigs, mice, rats, hamsters, guinea pigs, rabbits, dogs, etc. included. Cells well known to those skilled in the art can be used as cells into which nucleic acids are introduced, and HEK 293T cells, CHO cells and the like are used, for example.

<VLP>
A VLP according to the invention comprises a structural protein according to the invention and a parvovirus structural protein VP2. A parvoviral VLP according to the invention is a non-replicating, non-infectious viral shell that does not contain a viral genome. In general, when a protein or peptide is added to VP1, which is a structural protein of viruses, it may become difficult to form VLPs. can be formed automatically when expressed in A VLP according to the invention may be formed from a combination of any parvoviral RBD-tagged VP1 and any parvoviral VP2. The presence of VLPs can be confirmed by methods known to those skilled in the art, such as a method of directly observing particles with a transmission electron microscope (TEM) and a method of confirming the presence of a characteristic density band by density gradient centrifugation. Due to the wide variation in particle size and shape of VLPs, it is possible to confirm whether or not a subject is a VLP by multiple means. VLPs according to the invention are immunogenic.

VLPs according to the present invention preferably have reduced PLA ₂ activity, more preferably no PLA ₂ activity, compared to wild-type VLPs. VLPs with attenuated PLA ₂ activity can have improved safety when administered to a subject. Attenuation of PLA ₂ activity is, for example, half or less, preferably 1/3 or less, 1/5 or less, or 1/10 or less of the PLA ₂ activity of a VLP containing native (wild-type) VP1. . PLA ₂ activity can be measured, for example, by the kit used in the Examples below. A VLP with attenuated or deleted PLA ₂ activity has a mutation in at least a part of the PLA ₂ active region of VP1 in the structural protein described above, preferably at least one amino acid at positions 141 to 159 of VP1. is deleted, substituted, inserted or added, more preferably at least one of the 141st to 159th amino acids of VP1 is deleted, for example, all of the 141st to 159th amino acids are deleted. It can be obtained by using it as a component of VLP. The partially deleted regions of the PLA ₂ active region may be connected by linker peptides of appropriate length. The linker sequence may be, for example, 1 to 20 amino acid residues, or 10 or less amino acid residues. The linker sequence may be a glycine-rich sequence and may contain, for example, a GGGGGG sequence.

The VLP may contain RBD-attached VP1 in an amount equal to that of VP2, but may contain an amount less than VP2. The quantitative ratio of RBD-attached VP1 and VP2 in the VLP is, for example, 5:95 to 40:60, and may be 30:70, 20:80 or 10:90. The quantitative ratio of RBD-added VP1 and VP2 in VLP is determined, for example, by the amount of RBD-added VP1 expression vector and VP2 expression vector introduced into cells, the timing of introduction of these vectors, the type of promoter contained in the vector, and other transcription factors. It can be prepared by a preparation factor or the like. The protein ratio can be measured by absorbance, fluorescence, polyacrylamide gel electrophoresis, or the like.

<Method for producing VLP>
Methods for producing VLPs are known in the art. One aspect comprises the steps of culturing cells into which a nucleic acid encoding a structural protein of the present invention or a vector containing the nucleic acid has been introduced, and recovering VLPs produced from the cells.

A cell is a cell capable of expressing a structural protein according to the invention. The cell has been introduced with a nucleic acid or vector containing a nucleic acid encoding parvoviral VP2. By culturing cells containing the RBD-added VP1 gene and the VP2 gene in the same cell, the RBD-added VP1 and VP2 are expressed in the same cell to form a VLP. The RBD-added VP1 gene and VP2 gene may be contained in the same vector or may be contained in different vectors. Preferably, the RBD-added VP1 gene and the VP2 gene are subject to different promoters or different transcriptional regulation. The RBD-added VP1 gene and the VP2 gene may be introduced into the cell at the same timing or at different timings.

Cell culture can be carried out according to known methods depending on the type of host. For example, when culturing Escherichia coli or Bacillus, a liquid medium is preferable as the medium used for culturing. In addition, the medium preferably contains carbon sources, nitrogen sources, inorganic substances, and the like necessary for cell growth. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose; examples of nitrogen sources include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. inorganic or organic substances such as; inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like, respectively. Yeast extract, vitamins, growth promoting factors and the like may be added to the medium. The pH of the medium is preferably from about 5 to about 8. Cultivation of E. coli is usually carried out at about 15 to about 43°C. If necessary, aeration and stirring may be performed. Cultivation of Bacillus is usually carried out at about 30 to about 40°C. If necessary, aeration or stirring may be performed.
Examples of media for culturing yeast include Burkholder's minimal medium, SD medium containing 0.5% casamino acids, and the like. The pH of the medium is preferably from about 5 to about 8. Cultivation is usually carried out at about 20°C to about 35°C. Aeration or agitation may be performed as necessary.
As a medium for culturing insect cells, for example, Grace's Insect Medium to which an additive such as inactivated 10% fetal bovine serum is appropriately added is used. The pH of the medium is preferably from about 6.2 to about 6.4. Cultivation is usually carried out at about 27°C. Aeration or agitation may be performed as necessary.
As the medium for culturing animal cells, for example, minimum essential medium (MEM) containing about 5 to about 20% fetal bovine serum, Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 medium, 199 medium, etc. are used. . The pH of the medium is preferably about 6 to about 8. Cultivation is usually carried out at about 30°C to about 40°C. Aeration or agitation may be performed as necessary.
As a medium for culturing plant cells, MS medium, LS medium, B5 medium and the like are used. The pH of the medium is preferably from about 5 to about 8. Cultivation is usually carried out at about 20°C to about 30°C. Aeration or agitation may be performed as necessary.

In the step of recovering VLPs, the VLPs may be recovered from the host cell culture supernatant, host cell lysate, host cell homogenate, or a combination thereof. The recovered VLPs are selected from the group consisting of density gradient centrifugation (e.g., sucrose cushions, sucrose gradients, PEG precipitation, pelleting, etc.), chromatographic methods (ion exchange chromatography, gel filtration chromatography, etc.), and combinations thereof. It can be purified using any purification method.

<Immunogenic composition>
The present invention provides immunogenic compositions comprising the VLPs described above. An immunogenic composition may comprise a single type of VLP, or two or more different VLPs, and one or more additional polypeptides, proteins, other viral VLPs, etc., depending on the purpose. A structural protein according to the invention or a VLP comprising it can be administered as an antigen, eg in a prophylactic or therapeutic immunogenic composition, either individually or in combination. An immunogenic composition may be administered in two or more boosts (e.g., a "prime" administration followed by one or more "boosts") to achieve the desired effect. ). The same composition may be administered in one or more priming steps and one or more boosting steps. Different compositions may be used for priming and boosting.

The invention may also provide an immunogenic composition comprising a nucleic acid encoding a structural protein of the invention and a nucleic acid encoding parvovirus VP2. In immunogenic compositions containing nucleic acids encoding RBD-tagged VP1 and VP2, the nucleic acid can be any nucleic acid described herein, such as linear DNA or RNA, plasmid DNA, mRNA, self-replicating RNA, and the like. It's okay. Depending on the purpose, the nucleic acid may contain one or more modified bases to improve stability and/or resistance to nuclease degradation. Depending on the purpose, the nucleic acid may be combined with one or more components, such as cationic microparticles or nanoparticles, cationic lipids, etc., to facilitate uptake of the nucleic acid by cells and/or reduce nuclease degradation. may also include

Immunogenic compositions may generally comprise one or more pharmaceutically acceptable excipients or vehicles such as water, saline, glycerol, ethanol and the like, alone or in combination. Immunogenic compositions may typically include one or more pharmaceutically acceptable carriers in addition to the above components. Such carriers include any carrier that does not itself induce the production of harmful antibodies in an individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes, etc.). Such carriers are well known to those skilled in the art. Additionally, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be included. A discussion of pharmaceutically acceptable ingredients can be found in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, available at ISBN: 0683306472.

Pharmaceutically acceptable salts can also be used in the immunogenic compositions of the invention. Examples of salts include inorganic salts and salts of organic acids. Examples of inorganic salts include hydrochlorides, hydrobromides, phosphates, and sulfates. Examples of salts of organic acids include acetates and propionic acids. salts, malonates, benzoates, and the like.

Depending on the purpose, the antigen is adsorbed to, entrapped in, or otherwise liposomal and particulate carriers such as poly(D,L-lactide co-glycolide) (PLG) microparticles or nanoparticles. can be associated with it. Antigens can be conjugated to carrier proteins to enhance immunogenicity. Ramsay et al. (2001) Lancet 357(9251):195-196; Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Buttery & Moxon (2000) J R Coll Physicians Lond 34:163-. 168; Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii; Goldblatt (1998) J. Am. Med. Microbiol. 47:563-567; EP 0477508; U.S. Pat. No. 5,306,492; WO98/42721; Conjugate Vaccines (Cruse et al. ) Bioconjugate Techniques ISBN: 0123423368 or 012342335X.

Immunogenic compositions of the invention can be administered in conjunction with other immunomodulatory agents. For example, an immunogenic composition of the invention may include an adjuvant. Examples of adjuvants include aluminum salts such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate, or combinations thereof, Freund's adjuvant, retinoic acid, oil-in-water emulsion, inorganic salts, saponins, other VLPs, microbial derivatives (for example, intestinal bacteria). non-toxic derivatives of lipopolysaccharide (LPS), lipid A derivatives, immunostimulatory oligonucleotides, ADP-ribosylating toxins and their detoxified derivatives), cytokines (including interleukins), bioadhesives and mucoadhesives ( esterified hyaluronic acid microspheres, including poly(acrylic acid)), microparticles (e.g., particles of about 100 nm to about 150 μm in diameter formed from poly(α-hydroxy acid)), liposomes, polyoxyethylene ethers and known adjuvants such as polyoxyethylene ester formulations, polyphosphazenes, muramyl peptides, imidazoquinolone compounds and the like. These adjuvants may be included alone or in combination. The immunogenic composition of the invention can also be premixed with an adjuvant prior to administration. One aspect of the invention is the use of the above structural proteins or VLPs comprising the same in the manufacture of immunogenic compositions.

<Administration of Immunogenic Composition>
The method of administering the immunogenic composition of the present invention is not particularly limited, and may be parenteral administration such as rectal, intranasal, intradermal, subcutaneous, intramuscular, or intravenous administration, or oral administration. good. The dosage form of the immunogenic composition of the present invention is not particularly limited, and injections, emulsions, suppositories, patches, eye drops, nasal drops, capsules, tablets, granules, powders, syrups and the like can be used. mentioned. The number of administrations and the administration interval can be determined as appropriate.

The immunogenic compositions of the invention are administered to mammals such as mice, baboons, chimpanzees, or humans. Compositions of the invention can also be administered in a manner compatible with the particular composition used, and in an amount effective to induce an immune response.

The specific dosage of the protein in the immunogenic composition is not particularly limited, but may vary, including the species, age, and general condition of the mammal to which the immunogenic composition is administered, as well as the mode of administration of the composition. can depend on factors. An effective amount of an immunogenic composition of the invention can be readily determined using only routine experimentation. In vitro and in vivo models can be used to identify appropriate doses. Generally, 0.5 mg, 0.75 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 5 mg, 10 mg, 20 mg or 50 mg of a parvoviral polypeptide or VLP is administered to a large mammal, e.g., a baboon. , chimpanzees, or humans. Depending on the purpose, co-stimulatory molecules or adjuvants can also be administered before, after, or at the same time as administration of the composition.

The parvovirus-specific immune response generated by administering the composition of the present invention can be enhanced by varying the dose, route of administration, frequency of administration, or the like.

<Test to determine efficacy of immune response>
One way of assessing the efficacy of the immune response involves monitoring the immune response to the antigens in the composition of the invention after administration of the composition.

One way of examining the efficacy of the immune response is to monitor the immune response to the antigens in the composition of the invention after administration of the composition (such as monitoring IgG1 and IgG2a production levels). and/or monitoring mucosal immune responses, such as monitoring IgA production levels.

Currently, there are no approved animal models for evaluating protective immunity induced by parvovirus (eg, human parvovirus B19) immunogenic compositions. However, the ability of an immunogenic composition to induce an immune response can be assessed in suitable animals.

<Use of Immunogenic Composition as Pharmaceutical>
The invention also provides a composition of the invention for use as a medicament. Preferably, the medicament is capable of generating an immune response in a mammal (ie is an immunogenic composition), more preferably a vaccine. The present invention also provides a method of inducing a parvovirus immune response by administering a vaccine comprising the VLPs described above to a mammal. The invention also provides use of a composition of the invention in the manufacture of a medicament for raising an immune response in a mammal. Preferably the medicament is a vaccine. Vaccines are preferably used to prevent and/or treat erythema infectiosum in children, hydrops fetalis or miscarriage in pregnant women.

The invention provides methods for inducing or enhancing an immune response using the compositions described herein. The immune response is preferably protective and may include humoral and/or cellular immunity (including systemic and mucosal immunity). An immune response includes a booster response.

The invention also provides a method for raising an immune response in a mammal comprising administering an effective amount of the composition of the invention. The immune response is preferably protective and preferably involves humoral and/or cellular immunity. Preferably, the immune response comprises one or both of a TH1 immune response and a TH2 immune response.

Preferably, the mammal is human. The human may be a child, adolescent or adult, preferably a preschool child or adolescent, more preferably an infant or pre-pregnant female.

<Kit>
A kit according to the invention comprises a parvoviral VLP according to the invention. A kit may comprise an immunogenic composition comprising a VLP, nucleic acid or vector according to the invention. The immunogenic composition may be in liquid form or may be lyophilized. A kit may include one or more containers. Suitable containers for compositions include, for example, bottles, vials, syringes, and test tubes. The container may be made from a variety of materials including glass or plastic. The container may have a sterile access port. The container can be, for example, an intravenous solution bag or a vial having a needle-pierceable stopper.

The kit may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline (PBS), Ringer's solution, or dextrose solution. It also contains other materials useful to the end user, including other pharmaceutically acceptable formulation solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices. You can The kit may further comprise a third component comprising an adjuvant.

The kit may also include a package insert containing instructions for methods of inducing immunity or for treating an infection. A kit may include a delivery device, eg, a syringe with or without an injection needle, prefilled with an immunogenic composition of the invention.

The invention may also provide diagnostic kits using the structural proteins or VLPs produced by the above methods as antigens to determine seropositivity for parvovirus. Diagnostic kits contain, in addition to the structural proteins or VLPs described herein, one or more ancillary reagents for determining whether an individual is seropositive for antibodies that bind parvovirus. you can Suitable ancillary reagents include, for example, buffers, secondary or detection antibodies (eg, labeled anti-human antibodies, labeled anti-canine antibodies), detection agents, and the like. The kit may further contain instructions for performing the diagnostic assay.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these.

Experiment 1: Cloning of RBD-added VP1 expression plasmid Human codon-optimized nucleic acids (hVP1: SEQ ID NO: 15) and (hVP2: SEQ ID NO: 16) for VP1 and VP2 of parvovirus B19-J35 (Accession No. AY386330) ) was inserted into the pAAV-IRES-Puro eukaryotic expression vector, resulting in pAAV-IRES-Puro-hVP1 and pAAV-IRES-Puro-hVP2 in FIG. BamH1 and EcoR1 were used as restriction enzyme sites. One, two or three RBDs (SEQ ID NO: 17) were added to the N-terminus of the hVP1 gene to form pAAV-IRES-Puro-1RBD-hVP1, pAAV-IRES-Puro-2RBD-hVP1 and pAAV-IRES in FIG. - Puro-3RBD-hVP1 was generated. The nucleic acid sequences of 1RBD-hVP1, 2RBD-hVP1 and 3RBD-hVP1 are shown in SEQ ID NOS: 11-13. As described above, structural proteins in which 1 to 3 RBDs are further added to the N-terminus of parvovirus VP1, specifically 1RBD-VP1: SEQ ID NO: 5, 2RBD-VP1: SEQ ID NO: 6, 3RBD shown in FIG. - VP1: an expression plasmid containing a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:7 was obtained.

As a comparative example, one or two RBDs were added to the N-terminus of the hVP2 gene to prepare pAAV-IRES-Puro-1RBD-hVP2 and pAAV-IRES-Puro-2RBD-hVP2 shown in FIG. The nucleic acid sequences of 1RBD-hVP2 and 2RBD-hVP2 are shown in SEQ ID NO: 18 and SEQ ID NO: 19, respectively. As a comparative example, one RBD was added to the C-terminus of the hVP1 gene to prepare pAAV-IRES-Puro-hVP1-1RBD shown in FIG. The nucleic acid sequence of hVP1-1RBD is shown in SEQ ID NO:20. RBDs, RBD and hVP1, or RBD and hVP2 were connected by a nucleic acid sequence containing the linker sequence (amino acid sequence: GGGGGG, SEQ ID NO:22) set forth in SEQ ID NO:21. The gene sequences of all plasmids were determined by the Sanger sequencing method. As a result, expression plasmids containing the nucleic acid sequences encoding the amino acid sequences shown in FIG. 1, 1RBD-VP2: SEQ ID NO: 23, 2RBD-VP2: SEQ ID NO: 24 and VP1-1RBD: SEQ ID NO: 25, were obtained.

Experiment 2: Preparation of Parvovirus VLPs Equal masses were simultaneously introduced into the cells and expressed. For gene introduction, 9 μl of Polyethylenemine (Polyscuebces, Inc., Cat#24765-2, 1 mg/ml, pH 8.0) was used per 1 μg of plasmid. After transfection, cells were cultured at 37° C. under 5% CO ₂ for 48 hours.

The cultured Lenti-X 293T cells were collected together with the medium by pipetting, and the cells and supernatant were separated by centrifugation (1,750 g, 4° C., 3 minutes). A cell pellet was obtained by removing the supernatant. Penicillin/streptomycin-added DMEM (SIGMA, Cat # D6429-500ML) or PBS was added to the cells, and the cells were suspended well and centrifuged (1,750 g, 4° C., 3 minutes) to separate the cells and the supernatant. Remove to obtain a cell pellet. Penicillin-streptomycin-added DMEM (or PBS) containing 0.1% Triton X-100 (Alfa Aesar, Cat# A16046) was added to the cell pellet at 500 μl per 10 cm dish, and mixed well using a vortex. was mixed with shaking for 10 minutes at room temperature. Cell debris and supernatant were separated by centrifugation (32,300 g, 4° C., 60 minutes) to obtain a supernatant (protein extract) containing parvovirus VLPs.

Parvovirus VLPs were purified according to the following procedure. A continuous gradient solution from 10% sucrose to 50% sucrose was prepared in 35.6 ml polypropylene tubes (himac). The protein extract was gently added on top of the continuous gradient solution of sucrose. Ultracentrifugation (hitachi P32ST rotor, 100,000 g, 4° C.) was performed for 4 hours, and the protein extract was placed in a continuous density gradient solution of sucrose. A continuous gradient solution of sucrose was fractionated from the bottom into 1.5 ml fractions.

To confirm fractions containing VLPs, SDS-PAGE and Western blotting were performed. After electrophoresis, proteins in the gel were stained with Coomassie Brilliant Blue (CBB). In addition, proteins in the gel after electrophoresis were transferred to a polyvinylidene fluoride (PVDF) membrane, and VLPs were detected using an anti-parvovirus VP2 antibody (Abcam, ab64295) that recognizes VP1 and VP2.

Fractions containing VLPs were placed in Slide-A-Lyzer Dialysis Cassette G2 20000 MWCO (Thermo Fisher Scientific) and added to approximately 70 volumes of PBS solution (140 mM NaCl, 2.7 mM KCl, 10 mM PO ₄ ³⁻ , pH 7). .4), and the VLP-containing fraction solution was replaced with PBS at 4°C. The VLP solution after dialysis was collected and subjected to ultrafiltration using a syringe filter with a pore size of 0.45 μm (Merck Millipore, Cat# SLHVR33RS).

The VLP-containing solution was bound at a flow rate of 1 ml/min to a column (HiTrap Q HP column, GE Healthcare, Cat#29-0513-25) packed with 1 ml of prewashed and equilibrated Q Sepharose beads. Bound proteins were eluted using a NaCl gradient. Chromatography was performed as follows.
Buffer A: PBS (140 mM NaCl, 2.7 mM KCl, 10 mM PO ₄ ^3- , pH 7.4)
Buffer B: PBS containing 1 M NaCl (140 mM NaCl, 2.7 mM KCl, 10 mM PO ₄ ³⁻ , pH 7.4)
(1) The inside of the column was washed at a flow rate of 1 ml/min using Buffer A in an amount three times the column volume.
(2) 20 times the column volume of the buffer was passed through the column at a flow rate of 1 ml/min with a gradient of 0-100% buffer B to elute the protein.
(3) The inside of the column was washed at a flow rate of 1 min/ml using Buffer B in an amount 5 times the volume of the column.
(4) Fractionation was performed in 1 ml during elution and fractions were saved.

After SDS-PAGE, CBB staining was performed in order to confirm that no protein showing band intensity equal to or exceeding that of the VP1 and VP2 protein bands was detected (FIG. 3). FIG. 4 shows the result of calculating the proportion of RBD-added protein in each VLP (total of RBD-added protein and VP2) from band intensity. From the above results, it was confirmed that the intended VLP was produced. Formation of VLPs was also confirmed by an electron microscope.

Experiment 3: Evaluation of Immunogenicity of VLPs BALB/c mice were used to evaluate the immunogenicity of VP1 wt VLPs (n=5) and 2RBD-VP1 VLPs (n=6). Immunization was performed by intramuscular injection of 10 μg aluminum adjuvant and 5 μg VLP. The first immunization was performed at 7 weeks of age, and the second immunization was performed 14 days later at 9 weeks of age. Fourteen days after the second immunization, the third immunization was performed at 11 weeks of age. Fourteen days after the third immunization, the mice were bled at 13 weeks of age to obtain serum samples.

For each treatment group, IgG ELISA was performed using plates coated with the 1-90 amino acid region of VP1 (SEQ ID NO: 1), a neutralizing epitope region. Mouse immune serum as a primary antibody, Anti-Mouse IgG (whole molecule)-Peroxidase antibody produced in goat affinity isolated antibody (Sigma-Aldrich, catalog number: A4416-1ML) as a secondary antibody, BioFX (registered trademark) Detection was by TMB One Component HRP Microwell Substrate (Surmodics, catalog number: TMBW-1000-01). The results are shown in FIG. Mice administered with VLPs containing VP1 in which RBD was added to the N-terminus of VP1 (2RBD-VP1 VLPs) showed higher antibody titers than mice administered with VLPs containing VP1 wt (VP1 wt VLPs). In addition, VLPs containing VP1 with RBD added to the N-terminus of VP1 showed little variation among individual mice. It was found that adding an RBD to the N-terminus of VP1 increased the immunogenicity of VLPs.

Experiment 4: Evaluation of Neutralizing Activity of Antibodies Induced by VLPs Various VLPs obtained in Experiment 2 were administered to BALB/c mice (n=3-6) for immunization. Immunization was performed by the same procedure as Experiment 3, and serum samples were obtained. In order to evaluate the neutralizing activity of neutralizing antibodies contained in mouse serum, the following neutralization test was performed.

Serum was mixed in an equal volume with a virus titer of 8×10 ⁷ IU and allowed to react for 1 hour at room temperature. 8×10 ⁴ cord blood-derived erythrocyte progenitor cells positive for CD36 expression were mixed with the reaction solution and allowed to stand at 4° C. for 2 hours. After that, the cells were cultured at 37° C. in 5% CO ₂ , and 70 hours after the start of culture (72 hours after infection), the cells were collected together with the culture medium and centrifuged (300 g, 5 minutes) to obtain cell pellets. . Cells were suspended in 4% paraformaldehyde, reacted at room temperature for 15 minutes, washed with PBS, and centrifuged (300 g, 5 minutes) to obtain cell pellets. Cells were suspended in PBS containing 0.1% Triton-X and Human Fc Block (Becton Dickinson, Cat#564220), reacted at room temperature for 30 minutes, then PBS was added to mix the cells well, and centrifuged ( 300 g, 5 min) to obtain a cell pellet. The cells were suspended in a 10% BSA/PBS solution containing a rabbit polyclonal antibody (homemade) against VP2 and 0.1% Triton-X, and allowed to react at room temperature for 1 hour. PBS was added, the cells were mixed well, and a cell pellet was obtained by centrifugation (300 g, 5 minutes). The cell pellet was suspended in a 10% BSA/PBS solution containing Alexa Fluor 488-modified anti-rabbit IgG antibody (Invitrogen, #A-11008) and 0.1% Triton-X, and allowed to react for 1 hour at room temperature in the dark. . PBS was added to well suspend the cells, and a cell pellet was obtained by centrifugation (300 g, 5 minutes). The cells were suspended in a PBS solution and the number of VP2-positive cells was measured using CytoFLEX (Beckman Coulter). The ratio of VP2-positive cells to the total number of cells was calculated, and the value was used as the infection rate to evaluate the neutralizing activity.

The results are shown in FIG. Antibodies induced by VLPs containing 1RBD-VP2, 2RBD-VP1 and 3RBD-VP1 with RBD attached to the N-terminal side of VP1 greatly reduced the virus infection rate. Antibodies induced by VLPs containing 1RBD-VP1 or 2RBD-VP2, in which RBD was added to the N-terminus of VP2, could hardly reduce the virus infection rate. Antibodies induced by VLPs containing VP1-RBD with an RBD added to the C-terminus of VP1 slightly reduced viral infection rate. It was found that by adding RBD to the N-terminal side of VP1, VLPs containing this protein can induce antibodies with high neutralizing activity.

Experiment 5: Preparation of VLP containing PLA ₂ active region-deleted VP1 A structure in which part of the PLA ₂ active region (VP1: 141st to 159th in SEQ ID NO: 1, underlined in FIG. 7) is deleted from 2RBD-VP1 An expression plasmid for the protein (2RBD-VP1 delPLA ₂ ) was prepared based on 2RBD-VP1 prepared in Experiment 1. A glycine linker (GGGGGG) was introduced at the deletion site. The amino acid sequence of 2RBD-VP1 delPLA ₂ is shown in SEQ ID NO:9, and the nucleic acid sequence is shown in SEQ ID NO:14.

2RBD-VP1 delPLA ₂ and VP2 were co-expressed in cells in the same manner as in Experiment 2, and the obtained VLPs were purified. FIG. 8 shows the results of CBB staining of purified VLPs in the same manner as in Experiment 2. Also, the ratio of VP1 in VLPs (total of VP1 and VP2) is shown in FIG. From this, it was confirmed that the desired VLP was produced.

Experiment 6: Evaluation of immunogenicity of PLA ₂ active region deleted VLPs The immunogenicity of VLPs containing 2RBD-VP1 delPLA ₂ obtained in Experiment 5 was evaluated. BALB/c mice (n=5) were immunized in the same procedure as in Experiment 3, and the obtained serum samples were coated with the 1-90 amino acid region of the VP1 protein, which is the neutralizing epitope region. IgG ELISA was performed. The results are shown in FIG. Partial deletion of the PLA ₂ active region implicated in proinflammatory effects was found not to affect immunogenicity.

Experiment 7: Evaluation of PLA ₂ activity of PLA ₂ active region-deleted VLP The VLP containing 2RBD-VP1 delPLA ₂ obtained in Experiment 5 was evaluated for PLA ₂ activity. A sPLA ₂ Assay Kit (Cayman Chemical Company, #765001) was used for the evaluation of PLA ₂ activity, and the test was carried out according to the following procedure. 10 μl of sPLA ₂ DTNB (#765010), 10 μl of sample and 5 μl of sPLA ₂ Assay Buffer (#765010) were mixed and 200 μl of sPLA ₂ Diheptanoyl Thio-PC (#765015) was added. After stirring the plate, absorbance (414 nm) was measured using a plate reader, and PLA ₂ activity was calculated based on the absorbance value. The results are shown in FIG. It was confirmed that partial deletion of the PLA ₂ region significantly attenuated the PLA ₂ activity of VLPs.

Claims (12)

A structural protein comprising a parvoviral structural protein VP1 having a receptor-binding domain and a phospholipase _A2 active region, to which at least 1 but not more than 5 of said receptor-binding domains are further added to the N-terminus. 2. The structural protein of claim 1, wherein no less than 1 and no more than 3 said receptor binding domains have been added. 3. The structural protein of claim 1 or 2, wherein at least one amino acid has been deleted, substituted, inserted or added in said phospholipase _A2 active region. The structural protein of any one of claims 1-3, wherein said parvovirus is human parvovirus B19. A parvovirus structural protein having the ability to form virus-like particles and comprising the amino acid sequence according to any one of (a) to (c);
(a) an amino acid sequence set forth in any of SEQ ID NOS: 5-10;
(b) an amino acid sequence having 90% or more identity to the amino acid sequence set forth in any of SEQ ID NOS: 5-10, or (c) an amino acid sequence set forth in any of SEQ ID NOS: 5-10 Amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added. A virus-like particle comprising the structural protein according to any one of claims 1 to 5 and the structural protein VP2 of parvovirus. 7. The virus-like particle of claim 6, having attenuated phospholipase _A2 activity relative to wild-type virus-like particles. A nucleic acid encoding a structural protein according to any one of claims 1-5. A vector comprising the nucleic acid of claim 8 . A cell into which the nucleic acid according to claim 8 or the vector according to claim 9 has been introduced. An immunogenic composition comprising a virus-like particle according to claim 6 or 7, a nucleic acid according to claim 8, or a vector according to claim 9. A method for producing virus-like particles, comprising the steps of culturing the cells according to claim 10 and recovering virus-like particles produced from the cells.

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