WO2021251384A1 - サイトメガロウイルスのgBとペンタマーとの融合タンパク質及び該融合タンパク質を含むワクチン - Google Patents
サイトメガロウイルスのgBとペンタマーとの融合タンパク質及び該融合タンパク質を含むワクチン Download PDFInfo
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Definitions
- the present invention relates to a fusion protein of cytomegalovirus gB and pentamer, and a vaccine containing the fusion protein for preventing or treating cytomegalovirus infection.
- Cytomegalovirus (CMV) infections include major organ disorders such as CMV pneumonia, enteritis, and retinitis that develop in immunosuppressed patients such as transplantation, AIDS, and congenital immunodeficiency, and fetuses when a pregnant woman is first infected.
- CMV infection is one of TORCH syndromes and is an important congenital infection that causes malformations or serious clinical symptoms in the foetation.
- Non-Patent Document 2 The Institute of Health has the impact that congenital CMV infections outperform Down's syndrome as a cause of congenital central nervous system disorders in developed countries, and the life of congenital infected children with residual disorders.
- QOL Quality-adjusted life years
- Pathogens that cause infectious diseases are roughly classified into Class I group pathogens that can obtain sufficient effects with conventional vaccines and Class II group pathogens that cannot obtain sufficient protective immunity with conventional vaccines or pathogen infection history.
- CMV is classified into the latter.
- the clever immune escape mechanism that they have has been pointed out as the reason why it is difficult to overcome the Class II group pathogens. Humans have developed a number of effective vaccines against Class I pathogens and have overcome the threat of infectious diseases caused by them. And the focus of future vaccine development is shifting to Class II group pathogens.
- Non-Patent Document 3 Non-Patent Document 3
- ganciclovir has also been put on the market as a small molecule drug, but its effect is limited and there is a problem of side effects. At present, there is no vaccine, and as mentioned above, there is no sufficiently effective treatment method, so its unmet needs are considered to be high.
- the Sanofi vaccine is a recombinant subunit vaccine using gB of CMV glycoprotein as an antigen, and showed an infection prevention effect of about 50% in a clinical study targeting uninfected adult women. .. Although the development was practically suspended due to the limited effect, a meaningful finding was obtained that "a certain effect can be shown (but not sufficient) only with gB antigen" (Non-Patent Document 4). ..
- Non-Patent Document 5 For the experimental proof of the effect of CMV vaccine candidates, it is necessary to consider the species specificity of CMV. Due to the species specificity of CMV, animal experiments using human cytomegalovirus (HCMV) are basically impossible. Animal experiments are carried out using mice, rats, guinea pigs, monkeys, etc., but are carried out using CMVs specific to various animal species. Regarding transplacental transmission, only guinea pigs are an animal model system that can confirm infection to the fetus without special treatment by causing viral infection to the mother, and the transplacental transmission test system for guinea pigs is widespread. It is used (Non-Patent Document 5).
- Non-Patent Document 6 recombinant guinea pig cytomegalovirus (GPCMV) gB protein + adjuvant to female guinea pigs suppressed the initial infection of female guinea pigs, and transplacental transmission to fetuses was also possible. It has been reported that it was suppressed (Non-Patent Document 6).
- GPCMV guinea pig cytomegalovirus
- Non-Patent Document 7 shows that gB suppresses transplacental transmission to fetuses in a transplacental transmission model of guinea pigs by using an adenoviral vector vaccine incorporating the gB protein of GPCMV.
- Pentamer antigen has attracted a great deal of attention in recent years as the main antigen of CMV.
- Pentamar is a cell directivity determinant of CMV and is a molecule composed of five subunits of gH, gL, UL128, UL130 and UL131 (gH / gL / UL128 / UL130 / UL131) in human CMV (non-patented). Document 15).
- Non-Patent Document 8 GPCMV lacking the pentamer gene has lost infectivity to epithelial and endothelial cells and transplacental infectivity, and by ectopically expressing the deleted gene, they are lost. Has been reported to be revived (Non-Patent Document 8).
- Non-Patent Document 9 the neutralizing ability in cell lines is clearly high, and the same is true for the neutralizing ability in nutrient membrane cells, which is considered to be important in transplacemental infection.
- Non-Patent Document 10 vegetative bud progenitor cells in the human placenta are targets of CMV, and the contribution of pentamer to the infection of the cells with CMV was hardly recognized, and the contribution of gB was clearly recognized. ..
- Non-Patent Document 11 it is stated that the contribution of pentamer to the infection and proliferation of GPMMV to placental tissue is hardly recognized by using the placental infection test system ex vivo.
- Non-Patent Document 12 the effect of the combined use of the anti-gB monoclonal antibody and the anti-pentamar monoclonal antibody is verified in vitro, and it is stated that there is an advantage of the combined use in terms of neutralizing ability and suppression of the appearance of resistant strains. The effect of the combination has not been proved for the defense ability.
- Patent Document 2 there is data that the production of some cytokines is higher than that of the single group by immunization with a divalent vaccine of gB + pentamer, but the combination group is not superior in terms of neutralizing ability, and infection. No experiments have been done. Further, Patent Document 4 discloses a modified CMV gB protein and a CMV vaccine containing the same.
- Non-Patent Document 15 clarifies the X-ray crystal structure of pentamer.
- the pentamer is a molecule having a major axis of about 18 nm having a spiral structure, gH is present at the end thereof, and a part of gH is entangled with gL to form a gH / gL domain.
- UL128 / UL130 / UL131 interact with the N-terminus of gL while taking a gently curved shape. Of UL128 / UL130 / UL131, UL131 exists at the center of the three.
- the UL 130 forms a sheet structure together with the ⁇ strand of the UL 131 on the C-terminal side thereof, and one side thereof is covered with the helix structure of the UL 131.
- Both UL128 and UL130 form a spherical structure on the N-terminal side, and they are located opposite to each other with the core structure in between.
- the C-terminal side of UL128 is a flexible linker that reaches 5 nm and interacts with gL, forming a small helix structure that fits into the groove of gL.
- pentamers have many interactions between internal molecules. However, it has been shown that the pentamer can be stabilized by binding to Fab, although it has a very flexible structure due to its small interface. Therefore, it may be possible to improve stability by introducing appropriate site-specific mutations.
- Non-Patent Document 13 clarifies the X-ray crystal structure of the extracellular domain (ect domain) of gB whose cohesiveness is improved by site-specific mutation.
- the homotrimer of gB has a spike-like structure, and its protomer is composed of five domains. Domains I and II are located adjacent to the cell membrane side, and domain III forms a coiled coil structure with a very long helix structure. Domain IV is located on the opposite side of the cell membrane, and domain V exists from domain I to domain III along the entire length of gB. The N-terminus is located near domain IV and the C-terminus is in domain V.
- Non-Patent Document 13 The three-dimensional structure of the CMV gB protein has been analyzed (Non-Patent Document 13).
- Domain II consisting of 97-108th amino acid residue and 320-414th amino acid residue, 71-87th amino acid residue, 453-525th amino acid residue and 614-643th amino acid residue It is known to have domain III consisting of amino acid residues 65-70, domain IV consisting of amino acid residues 526-613, and domain V consisting of amino acid residues 644-675. ..
- AD-1 antigen domains in gB
- AD-1 antigen domains
- AD-5 antigen domains in gB
- AD-1 is said to have the highest antigenicity. Since AD-1 is located in domain IV and the antigen is exposed in domain IV with a relatively small amount of N-type sugar chains, it is considered that the antibody is easily accessible. Further, in Patent Document 4, it is reported that the non-neutralizing antibody is concentrated in the region (head region) containing the domain IV, and the possibility of gB as a vaccine excluding the head region is reported. ..
- CMV cytomegalovirus
- gB protein enveloped glycoprotein B
- pentamer A homotrimer of gB protein composed of three gB protein constituent molecules and a heteropenter of pentamer composed of five pentamer constituent molecules are composed of at least one gB protein constituent molecule and at least one pentamer constituent molecule.
- the fusion protein according to [1] which forms a protein complex by fusing with and in a genetic engineering manner.
- the fusion protein according to [2] wherein at least two gB protein constituent molecules and at least two pentamer constituent molecules are genetically engineered to each other.
- the linker comprises an amino acid sequence in which the amino acid sequence unit set forth in SEQ ID NO: 22 is repeated 1 to 3 times.
- fusion protein according to any one of [1] to [11], wherein the gB protein is the ect domain of the CMV gB protein.
- the fusion protein according to [12] which is a domain.
- the gB protein is a gB protein variant into which a modification has been introduced that reduces aggregate formation and increases the proportion of homotrimer structures as compared to wild-type gB protein, [1]-[ 14] The fusion protein according to any one of.
- gB protein is a gB protein variant into which a modification that reduces immunogenicity in the head region is introduced as compared with the wild-type gB protein. protein.
- HCMV human cytomegalovirus
- gB protein is an ect domain of a gB protein having 80% or more sequence identity with the ect domain of the gB protein consisting of the amino acid sequence set forth in SEQ ID NO: 31. Fusion protein.
- HCMV human cytomegalovirus
- the pentamer GH which consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added.
- GL consisting of the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added in the amino acid sequence.
- UL128, which consists of the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added.
- UL130 consisting of the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added in the amino acid sequence, and one or more in the amino acid sequence set forth in SEQ ID NO: 8 or the amino acid sequence.
- UL131 consisting of an amino acid sequence in which the amino acid residue of The fusion protein according to [24] or [25], which is a pentamer protein of human cytomegalovirus (HCMV) comprising.
- HCMV human cytomegalovirus
- [28] A nucleic acid fragment encoding the fusion protein according to any one of [1] to [27].
- [30] A transformant into which the nucleic acid fragment according to [28] or the recombinant expression vector according to [29] has been introduced.
- a vaccine for preventing or treating CMV infection which comprises the fusion protein according to any one of [1] to [27].
- [32] The vaccine according to [31], wherein the infection with CMV is a congenital infection with CMV.
- the present invention in the protection against congenital infection of CMV, by using a fusion protein of gB and pentamer as an antigen, it is possible to provide a vaccine having an infection suppressing effect exceeding the effect of each single administration. As a result, the commercialization of CMV vaccine can be expected.
- FIG. 1 It is a figure which shows the result of the gel filtration chromatography of the refined product of each gB-pentamer constituent protein fusion of Example 1.
- FIG. 2 It is a figure which shows the result of the gel filtration chromatography of the refined product of each gB-pentamer constituent protein fusion of Example 1.
- FIG. 2 It is a figure which shows the result of the gel filtration chromatography of the refined product of each gB-pentamer constituent protein fusion of Example 1.
- FIG. It is a figure which shows the result of the reactivity analysis using the HCMV-gB immune serum and the pentamer immune serum of each gB-pentamar constituent protein fusion of Example 2.
- FIG. 1 It is a figure which shows the result of the gel filtration chromatography of the refined product of each gB-pentamer constituent protein fusion of Example 1.
- the fusion protein of the present invention is a fusion protein of cytomegalovirus (CMV) enveloped glycoprotein B (gB protein) and pentamer.
- CMV cytomegalovirus
- the fusion protein consists of a homotrimer of gB protein consisting of three gB protein constituent molecules and a heteropenter of pentamer consisting of five pentamer constituent molecules, and at least one gB protein constituent molecule and at least one pentamer.
- a protein complex is formed by fusing each of the constituent molecules in a genetic engineering manner.
- the constituent molecule refers to a protein constituting a homotrimer of gB protein or a heteropentamer of pentamer, and is also referred to as a subunit.
- the fusion protein is used as an antigen to prepare a vaccine useful for the prevention and treatment of CMV.
- the fusion protein is preferably a protein complex formed by genetically engineeringly fusing at least two gB protein constituent molecules and at least two pentamer constituent molecules, respectively, and more preferably three gB protein constituents. It is a protein complex formed by genetically engineeringly fusing a molecule and any three pentamer constituent molecules out of the five pentamer constituent molecules.
- Cytomegalovirus includes any CMV strain, eg, human cytomegalovirus (HCMV), guinea pig cytomegalovirus (GPCMV), mouse cytomegalovirus (MCMV), rat cytomegalovirus (RCMV) and red lizard monkey. Cytomegalovirus (RhCMV) and the like can be mentioned. It is preferably HCMV.
- the CMV gB protein in the present embodiment may be a wild-type CMV gB protein or a modified CMV gB protein.
- the wild-type CMV gB protein means a gB protein derived from any CMV strain, for example, a gB protein derived from the HCMV AD169 strain having the amino acid sequence set forth in SEQ ID NO: 1 (Registration number of GenBank: X17403.1). Examples thereof include a gB protein derived from the GPCMV 22122 strain having the amino acid sequence set forth in SEQ ID NO: 2 (Registration number of GenBank: AB592928.1).
- the modified CMV gB protein (also referred to as “gB protein variant”, “gB variant” or “modified variant”) is a wild-type CMV gB protein having at least one amino acid residue or a continuous amino acid residue.
- the gB variant has 1 or more, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, in the amino acid sequence of the wild-type CMV gB protein.
- One to five, one to three amino acid residues may have an amino acid sequence deleted, substituted or added, and substitutions, deletions (deletion) or addition may occur simultaneously.
- the addition of an amino acid includes both insertion into the original amino acid sequence and addition to the end of the original amino acid sequence.
- the amino acid residue may be simply referred to as an amino acid when it is clear that it is an amino acid residue.
- the gB variant may be a variant having a modification that does not affect the three-dimensional structure and function of the original gB protein, or may have improved properties.
- Examples of the gB variant having improved properties include a variant in which a modification is introduced to increase or decrease the formation of aggregates and increase the proportion of homotrimer structure, or an antibody-inducing ability or neutralization. Examples thereof include modifications for improving antibody-inducing ability, for example, variants into which modifications for reducing the immunogenicity of the head region of gB protein as described in Patent Document 4 have been introduced.
- Neutralizing antibody-inducing ability refers to the ability to induce a neutralizing antibody against an antigenic protein, and is a neutralizing antibody titer (neutralizing antibody titer) in immune serum obtained by inoculating a test animal with the antigenic protein. Can be evaluated.
- Negtralizing antibody means an antibody capable of eliminating the infectivity of virus particles, for example, at the concentration of the antibody (NT50) required to reduce the number of plaques of the test virus by 50%. The high level of neutralizing activity can be evaluated.
- the gB protein of CMV in the present embodiment may be the full length of the CMV gB protein or an antigenic fragment of the CMV gB protein.
- the total length of the gB protein includes, for example, the HCMV gB protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 above (GenBank registration number: X17403.1.). However, among the amino acid sequences shown in SEQ ID NO: 1, the 1st to 24th amino acid sequences (SEQ ID NO: 19) are signal sequences. Therefore, the total length of the CMV gB protein may be the HCMV gB protein obtained by removing the signal sequence (SEQ ID NO: 19) from the amino acid sequence set forth in SEQ ID NO: 1.
- the antigenic fragment of the gB protein is a fragment that is antigenic and can form a homotrimer. It may be, for example, an extracellular domain (ect domain) of the gB protein of CMV, or a fragment or a variant of the ect domain.
- ect domain an extracellular domain of the gB protein of CMV, or a fragment or a variant of the ect domain.
- the ect domain include a fragment of the HCMV gB protein consisting of the 25th to 706th amino acid sequences among the amino acid sequences shown in HCMV (SEQ ID NO: 1) derived from the HCMV AD169 strain, and this fragment is a wild type.
- ect domain (SEQ ID NO: 15) of the HCMV gB protein is defined as the ect domain (SEQ ID NO: 15) of the HCMV gB protein. Further, in the case of the ect domain of another CMV gB protein, it is defined at a corresponding position based on the sequence alignment with the ect domain (SEQ ID NO: 15) of HCMV derived from the above HCMV AD169 strain.
- the ect domain of the gB protein in the present embodiment may be the ect domain of the wild-type CMV gB protein or the ect domain of the modified CMV gB protein (the ect domain variant of the gB protein).
- the ect domain of the gB protein or a variant thereof includes 1 or more, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20 in the amino acid sequence shown in SEQ ID NO: 15 or the amino acid sequence. It may be the ect domain of the gB protein of HCMV consisting of an amino acid sequence in which 1 to 15, 1 to 10, 1 to 5, and 1 to 3 amino acid residues are deleted, substituted or added.
- the ect domain of the gB protein or a variant thereof is also 80% or more, for example, 85% or more, 90% or more, 93% or more, 95% or more with the ect domain of the gB protein consisting of the amino acid sequence set forth in SEQ ID NO: 15.
- it may be the ect domain of a gB protein having 98% or more sequence identity.
- sequence identity refers to the percentage of (the number of amino acid residues that match between the sequences) / (the total length of the amino acid sequence) when a plurality of sequences are aligned. For example,% Identity Matrix created by aligning the target sequence with the genetic information processing software GENETYX and excluding the gap from the calculation corresponds to the sequence identity.
- the variant of the ect domain of the gB protein may be, for example, a variant lacking domain IV.
- the 97-468 amino acid and the 631-682 amino acid of the amino acid sequence set forth in SEQ ID NO: 15 may be directly linked or may be linked via an appropriate peptide linker.
- amino acid substitutions may be further introduced into Y131A, I132A, Y133A, W216A, R432T and R434Q, for example, 97-468 amino acid of SEQ ID NO: 15.
- a variant (“gB ⁇ d4”) (SEQ ID NO: 16) in which amino acids were connected to 631-682 amino acids with 9 amino acids of GGGSGSGGG (SEQ ID NO: 20), and amino acid substitutions were introduced into Y131A, I132A, Y133A, W216A, R432T and R434Q. Can be mentioned.
- HCMV human cytomegalovirus
- the gB protein also contains 80% or more, for example 85% or more, 90% or more, 93% or more, the ect domain of the gB protein consisting of the amino acid sequence set forth in SEQ ID NO: 16. It may be the ect domain of a gB protein with 95% or more or 98% or more sequence identity.
- the gB protein variant having the amino acid sequence set forth in SEQ ID NO: 15 is based on the ect domain of the HCMV gB protein consisting of the amino acid sequence shown in SEQ ID NO: 15.
- the 132nd amino acid residue is histidine residue (His)
- the 133rd amino acid residue is arginine residue (Arg)
- the 215th amino acid residue is glutamate residue (Glu).
- the 216th amino acid residue is replaced with an alanine residue (Ala)
- the 432nd amino acid residue is replaced with a treonine residue (Thr)
- the 434th amino acid residue is replaced with a glutamine residue (Gln).
- Examples thereof include the gB protein ect domain variant (SEQ ID NO: 3).
- gB protein variant 1 or more in the amino acid sequence set forth in SEQ ID NO: 3, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 It may be the ect domain of the gB protein of human cytomegalovirus (HCMV) consisting of an amino acid sequence in which ⁇ 10, 1-5, 1-3 amino acid residues are deleted, substituted or added.
- HCMV human cytomegalovirus
- the gB protein variant also includes the ect domain of the gB protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 and 80% or more, for example, 85% or more, 90% or more, 93% or more, 95% or more or 98% or more. It may be the ect domain of the gB protein having the sequence identity of.
- Examples of the gB protein variant in which the modification for improving the antibody-inducing ability or the neutralizing antibody-inducing ability has been introduced include a gB protein variant in which a modification for reducing the immunogenicity of the head region of the gB protein has been introduced.
- a modification for reducing the immunogenicity of the head region of the gB protein has been introduced.
- S128-L138 was further deleted, R127 and G139 at the ends were connected with a glycine linker GGG, and W216-Y218 was deleted.
- gB protein variant 1 or more in the amino acid sequence set forth in SEQ ID NO: 31, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 It may be the ect domain of the gB protein of human cytomegalovirus (HCMV) consisting of an amino acid sequence in which ⁇ 10, 1-5, 1-3 amino acid residues are deleted, substituted or added.
- HCMV human cytomegalovirus
- the gB protein variant also includes the ect domain of the gB protein consisting of the amino acid sequence set forth in SEQ ID NO: 31 and 80% or more, for example, 85% or more, 90% or more, 93% or more, 95% or more or 98% or more. It may be the ect domain of the gB protein having the sequence identity of.
- the gB protein antigen of CMV may be prepared by protein purification using CMV, or can be prepared by a method of genetic engineering.
- the production method is not particularly limited, but for example, a cDNA of a wild-type gB protein is used as a template, a primer is designed, a nucleic acid is obtained by PCR, functionally linked to an expression promoter, and optionally a tag is also linked, which is appropriate. It can be obtained by introducing it into an expression vector and expressing it.
- the produced CMV gB protein antigen may be purified if necessary.
- the purification method is not particularly limited, and examples thereof include purification by an affinity chromatography column or the like.
- the modified gB protein antigen is a mutant by mutagenesis
- a primer for introducing the desired mutation is designed, the nucleic acid into which the mutation is introduced is obtained by PCR, and the nucleic acid is functionally linked to the expression promoter. It can also be obtained by ligating the tag with, introducing it into an appropriate expression vector, and expressing it.
- the modified gB protein antigen is a modified product by introducing a sugar chain (sugar chain modification)
- the usual method may be used, and the present invention is not particularly limited.
- the primer is designed so that the three consecutive amino acid sequences of the target site into which the N-type sugar chain is introduced are N-XS / T (X is an arbitrary amino acid other than proline). Then, the mutation is introduced by PCR.
- a modified CMV gB protein can be obtained by cloning and expressing the nucleic acid sequence of the desired modified gB protein and, if necessary, the nucleic acid sequence ligated with a tag such as 6 ⁇ His into an appropriate vector. Then, an N-type sugar chain is added to the asparagine at the target site of the gB variant by a usual method.
- the CMV pentamer is also referred to as a pentamer complex consisting of five different constituent molecules (subunits), a heteropentameric, or simply a pentamer. It may be a wild-type CMV pentamer or a modified CMV pentamer.
- Wild-type CMV pentamer means a pentamer derived from any CMV strain, for example, a pentamer consisting of human cytomegalovirus (HCMV) gH, gL, UL128, UL130 and UL131, guinea pig cytomegalovirus (GPCMV). Examples thereof include a pentamer composed of GP75 (gH), GP115 (gL), GP129 (UL128), GP131 (UL130) and GP133 (UL131).
- HCMV human cytomegalovirus
- GPCMV guinea pig cytomegalovirus
- Examples thereof include a pentamer composed of GP75 (gH), GP115 (gL), GP129 (UL128), GP131 (UL130) and GP133 (UL131).
- the HCMV pentamer is derived from the HCMV Merlin strain consisting of the amino acid sequences set forth in, for example, SEQ ID NO: 4 (gH), SEQ ID NO: 5 (gL), SEQ ID NO: 6 (UL128), SEQ ID NO: 7 (UL130) and SEQ ID NO: 8 (UL131).
- SEQ ID NO: 4 gH
- SEQ ID NO: 5 gL
- SEQ ID NO: 6 UL1278
- SEQ ID NO: 7 UL130
- SEQ ID NO: 8 UL131
- the GPCMV pentamer is derived from the GPCMV 22122 strain consisting of the amino acid sequences set forth in, for example, SEQ ID NO: 10 (GP75), SEQ ID NO: 11 (GP115), SEQ ID NO: 12 (GP129), SEQ ID NO: 13 (GP131) and SEQ ID NO: 14 (GP133). (Registration number of GenBank: AB592928.1) (However, since the base sequence of GP133 contains a mutation, it is modified based on the sequence information of other CMV strains). ..
- the subunit (constituent molecule) of the modified CMV pentamer may be a modified product having a modification that does not affect the three-dimensional structure and function of the original subunit, or may have improved properties. ..
- Variants of subunits with improved properties include, for example, variants in which aggregates are not formed or reduced, thereby increasing the content of pentamers, antibody induction. Examples thereof include a variant into which a modification for improving the ability or the ability to induce a neutralizing antibody has been introduced.
- the modified CMV pentamer refers to a pentamer in which at least one of the five subunits constituting the wild-type CMV pentamer is a modified protein (a variant of the subunit).
- a subunit variant is a protein in which one or more amino acid residues or contiguous amino acid residue regions are substituted, deleted (deleted) or added to the corresponding wild-type subunit, and the amino acid residue. It also includes proteins with protein modifications that are not present in the wild form, such as proteins into which sugar chains have been introduced by subunit substitution, deletion (deletion) or addition.
- the addition of an amino acid includes both insertion into the original amino acid sequence and addition to the end of the original amino acid sequence.
- the HCMV pentamer of one embodiment is GH, which consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added.
- GH consisting of the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added in the amino acid sequence.
- UL128, which consists of the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added.
- UL130 consisting of the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added in the amino acid sequence, and one or more in the amino acid sequence set forth in SEQ ID NO: 8 or the amino acid sequence.
- UL131 consisting of an amino acid sequence in which the amino acid residue of It may be a pentamer protein of HCMV containing.
- deletions, substitutions or additions of one or more amino acid residues are performed, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10. It may be a deletion, substitution or addition of 1 to 5 amino acid residues and 1 to 3 amino acid residues.
- the HCMV pentamer of one embodiment is GH having 80% or more sequence identity with gH consisting of the amino acid sequence set forth in SEQ ID NO: 4, A gL consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a gL having 80% or more sequence identity, UL128, which has 80% or more sequence identity with UL128 consisting of the amino acid sequence set forth in SEQ ID NO: 6.
- It may be a pentamer protein of HCMV containing.
- the sequence identity of 80% or more may be, for example, 85% or more, 90% or more, 93% or more, 95% or more, or 98% or more sequence identity.
- the GPMV pentamer of one embodiment is GP75, which consists of the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added.
- GP115 which consists of the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added.
- GP129 which consists of the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added.
- GP131 consisting of the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence in which one or more amino acid residues are deleted, substituted or added, and the amino acid sequence set forth in SEQ ID NO: 14 or one or more in the amino acid sequence.
- GP133 consisting of an amino acid sequence in which the amino acid residue of It may be a GPCMV pentamer protein containing.
- deletions, substitutions or additions of one or more amino acid residues are performed, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10. It may be a deletion, substitution or addition of 1 to 5 amino acid residues and 1 to 3 amino acid residues.
- the GPMV pentamer of one embodiment is GP75 having 80% or more sequence identity with GP75 consisting of the amino acid sequence set forth in SEQ ID NO: 10, GP115, which has 80% or more sequence identity with GP115 consisting of the amino acid sequence set forth in SEQ ID NO: 11.
- GP129 which has 80% or more sequence identity with GP129 consisting of the amino acid sequence set forth in SEQ ID NO: 12.
- GP131 having 80% or more sequence identity with GP131 consisting of the amino acid sequence set forth in SEQ ID NO: 13 and GP133 having 80% or more sequence identity with GP133 consisting of the amino acid sequence set forth in SEQ ID NO: 14.
- It may be a GPCMV pentamer protein containing.
- the sequence identity of 80% or more may be, for example, 85% or more, 90% or more, 93% or more, 95% or more, or 98% or more sequence identity.
- Each subunit may be full length, for example, only the ect domain. It is preferred that gH is the ect domain of the gH protein.
- the CMV pentamer antigen may be prepared by protein purification using CMV, or can be prepared by a method of genetic engineering.
- the production method is not particularly limited, but for example, the cDNAs of the five proteins constituting the wild-type CMV pentamer are used as templates, primers are designed, nucleic acids are obtained by PCR, and functionally linked to an expression promoter, in some cases. Tags can also be obtained by ligating, introducing into a suitable expression vector, expressing and folding to form a pentameric structure.
- the CMV pentamer antigen can also be expressed as a secretory protein, if desired.
- the produced CMV pentamer antigen may be purified if necessary.
- the purification method is not particularly limited, and examples thereof include purification by an affinity chromatography column or the like.
- the modified CMV pentamer antigen is a modified product by introducing a mutation or a modified product by introducing a sugar chain (sugar chain modification), it can be produced as described above.
- the fusion protein of the present invention partially has the three-dimensional structure of gB and pentamer. It is sufficient that one or more molecules of gB and pentamer constituent molecules are linked, and the combination thereof can be appropriately selected. Further, even when one or more molecules of gB and pentamer constituent molecules are linked and other molecules are expressed without fusion, they can form a complex together with the linked molecules, and thus can be said to be a gB-pentamer fusion protein. ..
- the gB-pentamer fusion protein may be linked in any combination.
- the subunit of pentamer that is genetically engineered with the gB protein constituent molecule is not particularly limited, and one gB protein constituent molecule and any one of the subunits of pentamer are genetically engineered.
- the combination of the subunits of the pentamer to be genetically engineered with each of the two or three gB protein constituent molecules is not particularly limited, and for example, the three gB protein constituent molecules of the homotrimer are not particularly limited.
- the constituent subunits of the pentamer fused with each may be gL, UL128 and UL130, and the constituent subunits of the pentamer fused with the gB protein may be UL128, UL130 and UL131.
- the genetic engineering fusion is a fusion in which the gB protein and the pentamer are bound in this order from the N-terminal side (that is, the C-terminal of the gB protein constituent molecule and the N-terminal of the pentamer constituent molecule). It may be a fusion in which and are linked in this order, or a fusion in which pentamer and gB protein are bound in this order from the N-terminal side.
- a fusion protein in which pentamer and gB protein are bound in this order from the N-terminal side that is, a fusion protein in which pentamer is bound to the N-terminal side of gB protein tends to be difficult to form aggregates, and is therefore preferable. That is, at least one of the genetically engineered fusions is preferably a fusion in which the pentamer constituent molecule is bound to the N-terminal side of the gB protein constituent molecule, and at least two of the genetically engineered fusions are fused.
- the gB protein and the pentamer may be directly bound, but it is preferably bound via an appropriate linker and / or tag. It is considered that the fusion protein has a linker of an appropriate length between the gB protein and the pentamer so that each of them can form an appropriate structure without causing steric hindrance to each other. Further, the fusion protein can be purified by affinity purification by having a tag between the gB protein and the pentamer. Further, the fusion protein is not limited to between the gB protein and the pentamer, and may have a linker and / or a tag on the N-terminal side or the C-terminal side of the fusion protein.
- the linker is not particularly limited and can be appropriately designed by those skilled in the art.
- a 5-amino acid linker (SEQ ID NO: 22) having Gly-Gly-Gly-Gly-Ser as a unit can be mentioned, and for example, it consists of an amino acid sequence in which the amino acid sequence unit shown in SEQ ID NO: 22 is repeated 1 to 3 times.
- the linker is mentioned.
- the tag is not particularly limited and can be appropriately selected by those skilled in the art.
- a His tag in which a plurality of histidine residues are linked for example, a His tag having the amino acid sequence set forth in SEQ ID NO: 30
- a His tag having the amino acid sequence set forth in SEQ ID NO: 30 can be mentioned.
- the fusion protein of gB and pentamer of CMV can be produced by genetically engineeringly fusing gB and pentamer as described in Examples.
- nucleic acid fragment, recombinant expression vector, transformant The nucleic acid fragment of the present invention is a nucleic acid fragment encoding a fusion protein of gB of CMV of the present invention and pentamer, and the recombinant expression vector of the present invention is a recombinant expression vector containing the nucleic acid fragment of the present invention.
- the transformant of the present invention is a transformant into which the nucleic acid fragment of the present invention or the recombinant expression vector of the present invention has been introduced.
- the nucleic acid fragment encoding the fusion protein is not particularly limited as long as it can be finally expressed as a fusion protein to which gB and pentamer are bound when the nucleic acid fragment is transcribed and expressed in a host.
- it can be obtained by functionally linking a nucleic acid fragment encoding a gB protein and a nucleic acid fragment encoding each subunit of the pentamer. It is preferable that these nucleic acid fragments do not contain codons such as stop codons that stop transcription in the middle.
- Nucleic acid fragments encoding wild-type gB proteins are, for example, various CMVs using probe DNA that can be designed based on a gene encoding wild-type gB having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. It can be obtained by Southern hybridization to the DNA of the above, or by PCR using the DNA of various CMVs as a template using a primer set that can be designed based on the gene.
- the nucleic acid fragment encoding the variant of the gB protein can be obtained, for example, by subjecting it to error prone PCR or the like using a DNA consisting of the base sequence represented by SEQ ID NO: 1 as a template. Alternatively, it can also be obtained by introducing a site-specific mutation.
- nucleic acid fragment encoding the gB protein a nucleic acid fragment encoding an amino acid sequence designed based on the amino acid sequences of SEQ ID NOs: 1-3, 15, 16, 31 and the like can be obtained by artificial synthesis.
- Nucleic acid fragments encoding wild-type or modified pentamer subunits eg, SEQ ID NOs: 4-8, or SEQ ID NOs: 10-14, can be obtained in the same manner.
- the obtained nucleic acid fragment may be cleaved as it is or with an appropriate restriction enzyme or the like, incorporated into a vector by a conventional method, the obtained recombinant expression vector is introduced into a host cell, and then a usual base sequence analysis method is used.
- the base sequence of the nucleic acid fragment can be determined by analysis using a base sequence analyzer such as.
- the vector that can be used as the recombinant expression vector is not particularly limited, and examples thereof include a pCAGGS vector using a CAG promoter and a pCMV vector using a CMV promoter.
- the transformant can be obtained by introducing the above nucleic acid fragment or recombinant expression vector into a host cell.
- the introduction may be done by a conventional method.
- the host cell is not particularly limited, and examples thereof include CHO cells, HEK293 cells, SP2 / 0 cells, BHK cells, NS0 cells and the like.
- the fusion protein of the present invention is expressed by culturing the transformant in an appropriate medium and inducing its expression as needed, and by recovering it and purifying it as necessary, the fusion protein of the present invention is expressed. Can be obtained.
- the vaccine of the present invention comprises the fusion protein of the present invention.
- the vaccine of one embodiment is a vaccine for preventing or treating a congenital infection of CMV containing a fusion protein of cytomegalovirus (CMV) enveloped glycoprotein B (gB protein) and pentamer as an antigen. That is, the vaccine of the present embodiment is a subunit vaccine composed of a fusion protein having the functions of two types of antigen proteins.
- CMV cytomegalovirus
- gB protein cytomegalovirus
- the content of the protein antigen in the vaccine of the present embodiment may be 0.1 to 1000 ⁇ g for each of the CMV gB protein antigen and the CMV pentamer antigen, and is preferably 1 to 100 ⁇ g.
- the dosage form of the vaccine of the present embodiment may be, for example, liquid, powder (lyophilized powder, dry powder), capsule, tablet, or frozen state.
- the CMV vaccine of the present embodiment may contain a pharmaceutically acceptable carrier.
- a carrier usually used for vaccine production can be used without limitation, and specific examples thereof include saline solution, buffered saline solution, dextrose, water, glycerol, isotonic aqueous buffer solution and combinations thereof. Be done.
- the vaccine may further appropriately contain an emulsifier, a preservative (eg, thimerosal), an tonicity agent, a pH adjuster, an inactivating agent (eg, formalin) and the like.
- an adjuvant in order to further enhance the immunogenicity of the vaccine of this embodiment.
- the adjuvant include an aluminum adjuvant or an oil-in-water emulsion adjuvant containing squalene (AS03, MF59, etc.), CpG and 3-O-deacylated-4'-monophosphoryl lipid A (MPL), and other Toll-like receptors.
- body ligands saponin-based adjuvants
- polymer-based adjuvants such as poly ⁇ -glutamic acid
- polysaccharides such as chitosan and inulin.
- the vaccine of the present embodiment can be obtained by mixing an antigen, which is a fusion protein of CMV gB and pentamer, with a carrier, an adjuvant, or the like, if necessary.
- the adjuvant may be one that is mixed at the time of use.
- the administration route of the vaccine of this embodiment is, for example, transdermal administration, sublingual administration, instillation administration, intradermal administration, intramuscular administration, oral administration, enteral administration, nasal administration, intravenous administration, subcutaneous administration, and peritoneal administration. Oral administration or inhalation administration from the mouth to the lungs may be performed.
- the vaccine administration method of the present embodiment may be, for example, a syringe, a percutaneous patch, a microneedle, a transplantable sustained release device, a syringe with a microneedle, a needleless device, or a method of spraying. good.
- transplacental infection of CMV can be prevented or treated.
- Prevention of transplacental infection is to suppress the vertical transmission of CMV to the foetation by administering a vaccine to the mother, or to suppress the onset of various symptoms caused by congenital infection. These can be evaluated by a nucleic acid amplification method using fetal amniotic fluid or a newborn's body fluid, a newborn's head ultrasonic examination, a head CT examination, a head MRI examination, an auditory screening, or the like.
- Treatment of transplacental infection is to suppress the onset and progression of various symptoms caused by congenital infection by administering a vaccine to a child with congenital infection.
- the vaccine of the present embodiment is preferably administered to a female or a female child of childbearing age. From the viewpoint of herd immunity, it is possible to target men, boys, and the elderly. The number of administrations is 1 to 3 times, but it is desirable to inoculate multiple times at intervals of 2 months to several years. It is also possible to measure the antibody titer in the blood and target a person who is negative for the antibody or has a low antibody titer.
- Example 1 A gene encoding the ect domain (SEQ ID NO: 15) of HCMV gB derived from the AD169 strain was artificially synthesized and cloned into pCAGGS1-dhfr-neo (Patent Document 3). It was designed so that a 9-amino acid His-tag (SEQ ID NO: 30) was added to the C-terminal. Based on this, amino acid substitutions of I132H, Y133R, T215E, W216A, R432T and R434Q (SEQ ID NO: 3) were designated as gB1-682-fm3Mv9 (hereinafter referred to as "gBv9”) (Patent Document 4). . When expressing gBv9, the signal sequence of HCMV-gB (MESRIWCLVVCVNLCIVCLGAAVS) (SEQ ID NO: 19) was inserted at the N-terminus.
- gBv9 the signal sequence of HCMV-gB
- Expi293 expression system (Life Technologies) was used.
- the expression plasmid was transfected into the cells and the culture supernatant was collected in 4-6 days. From the culture supernatant containing gBv9, purification was performed using Ni NTA agarose (QIAGEN), and dialysis was performed on PBS + 0.5M arginine to obtain a refined product of the ectodomain of HCMV gB protein.
- gB ⁇ d4 SEQ ID NO: 16
- PCR polymerase chain reaction
- the signal sequence of HCMV-gB (MESRIWCLVVCVNLCIVCLGAAVS) (SEQ ID NO: 19) was inserted at the N-terminal, and the 9-amino acid His-tag (SEQ ID NO: 30) was inserted at the C-terminal. Expression and purification were carried out in the same manner as for gBv9.
- the gene encoding UL128 (SEQ ID NO: 6), the gene encoding UL130 (SEQ ID NO: 7), and the gene encoding UL131 (SEQ ID NO: 8) were artificially synthesized and cloned into pCAGGS1-dhfr-neo.
- a DNA fragment encoding a fusion protein in which gBv9 was added via a 5-amino acid linker (GGGGS) (SEQ ID NO: 22) was prepared by PCR and cloned into pCAGGS1-dhfr-neo.
- GGGGS 5-amino acid linker
- the one in which the signal peptide substituted UL128 and gBv9 were linked was N-UL128-gB
- the one in which the signal peptide substituted UL130 and gBv9 were linked was N-UL130-gB
- the one in which the signal peptide substituted UL131 and gBv9 were linked was N-. It was set to UL131-gB.
- the signal sequence of HCMV-gB (MESRIWCLVVCVNLCIVCLGAAVS) (SEQ ID NO: 19) was inserted into the N-terminal of gBv9, and the 5-amino acid linker (GGGGS) (GGGGS) (SEQ ID NO: 30) was removed from the C-terminal side of gBv9 except for His-tag (SEQ ID NO: 30).
- C-UL128-gB is the signal-removed UL128 and gBv9 linked
- C-UL130-gB is the signal-removed UL130 and gBv9 linked
- C-UL131-gB is the signal-removed UL131 and gBv9 linked.
- a DNA fragment encoding a fusion protein in which signal-removing UL128, signal-removing UL130, or signal-removing UL131 was inserted at the same position except for 373aa-381aa of gB ⁇ d4 (SEQ ID NO: 16) was prepared by PCR and pCAGGS1-. It was cloned into dhfr-neo. In each case, the signal sequence of HCMV-gB (MESRIWCLVVCVNLCIVCLGAAVS) (SEQ ID NO: 19) was inserted at the N-terminal.
- the one with the signal-removing UL128 inserted was designated as ⁇ d4-UL128-gB
- the one with the signal-removing UL130 inserted was designated as ⁇ d4-UL130-gB
- the one with the signal-removing UL131 inserted was designated as ⁇ d4-UL131-gB.
- ⁇ d4-UL128-gB / ⁇ d4-UL130-gB / ⁇ d4-UL131-gB When three types of -UL130-gB and ⁇ d4-UL131-gB were co-expressed ( ⁇ d4-UL128-gB / ⁇ d4-UL130-gB / ⁇ d4-UL131-gB), a total of 12 types of gB and pentamer-constituting proteins were added. Obtained a refined product of the fusion protein of.
- a fusion protein of gB or a part thereof and a pentamer constituent protein or a part thereof, a monomer or a multimer obtained by expressing these fusion proteins in a cell and the same.
- Genes encoding these fusion proteins or DNA fragments thereof may be collectively referred to as "gB-pentamer constituent protein fusions”.
- ⁇ Gel filtration chromatography of refined protein products The properties of the obtained refined product were evaluated by gel filtration chromatography. As the column, Superdex 200 Increase 5/150 GL (GE Healthcare) was used, and each refined product having a concentration of 100 ⁇ g / mL was applied. The flow rate was 0.4 mL / min, the mobile phase was D-PBS (Wako), and the absorbance at a wavelength of 280 nm was measured.
- Example 2 Antigen preparation>
- UL128, UL130, or UL131 was fused to the N-terminal side of gB as follows.
- Each gB-pentamer constituent protein fusion was designed.
- UL128 (C162S) -gB was prepared by adding a C162S amino acid substitution to UL128 (SEQ ID NO: 6) and further adding gBv9 to the C-terminal side via a 5-amino acid linker (GGGGS) (SEQ ID NO: 22).
- UL128 (C162S) - ⁇ d4 was prepared by adding a C162S amino acid substitution to UL128 (SEQ ID NO: 6) and further adding gB ⁇ d4 to the C-terminal side via a 15-amino acid linker (GGGGGSGGGGSGGGGS) (SEQ ID NO: 23).
- GB ⁇ d4 was added to the C-terminal side of the UL128 (SEQ ID NO: 6), UL130 (SEQ ID NO: 7) or UL131 (SEQ ID NO: 8) gene via a 15-amino acid linker (GGGGSGGGGGSGGGGS) (SEQ ID NO: 23), of which N
- the one with UL128 on the terminal side was designated as UL128- ⁇ d4
- the one with UL130 on the N-terminal side was designated as UL130- ⁇ d4
- the one with UL131 on the N-terminal side was designated as UL131- ⁇ d4.
- gBVC37 SEQ ID NO: 31
- Patent Document 4 the signal sequence of HCMV-gB (MESRIWCLVVCVNLCIVCLGAAVS) (SEQ ID NO: 19) was inserted at the N-terminus.
- UL128 (C162S) -VC37 was prepared by adding a C162S amino acid substitution to UL128 (SEQ ID NO: 6) and adding gBVC37 to the C-terminal side via a 5-amino acid linker (GGGGS) (SEQ ID NO: 22).
- GBVC37 was added to the C-terminal side of the UL128 (SEQ ID NO: 6), UL130 (SEQ ID NO: 7) or UL131 (SEQ ID NO: 8) gene via a 5-amino acid linker (GGGGS) (SEQ ID NO: 22), of which N
- the one with UL128 on the terminal side was designated as UL128-VC37
- the one with UL130 on the N-terminal side was designated as UL130-VC37
- the one with UL131 on the N-terminal side was designated as UL131-VC37.
- a DNA fragment encoding each gB-penter constituent protein fusion was prepared by PCR and cloned into pCAGGS1-dhfr-neo to obtain a gB-penter constituent protein fusion expression plasmid.
- gH a gene encoding gH (1-715aa, SEQ ID NO: 4) and a gene encoding gL (1-278aa, SEQ ID NO: 5) were artificially synthesized. It was cloned into pCAGGS1-dhfr-neo. Here, it was designed so that a 9-amino acid His-tag (SEQ ID NO: 30) was added to the C-terminal of gH (hereinafter, when it is referred to as gH, it refers to the sequence to which this His-tag is added).
- gH / gL / UL128 / UL130 / UL131 correspond to the ect domain of the HCMV pentamer and are hereinafter referred to as "pentamer".
- Expression was carried out in the same manner as for the HCMV gB protein, and a culture supernatant of each gB-pentamer constituent protein fusion was obtained. Purification of the pentamer was carried out in the same manner as for the HCMV gB protein.
- Guinea pigs (Hartley) were immunized using gBv9 and pentamer as antigens. Each antigen was prepared with physiological saline (Otsuka Pharmaceutical Co., Ltd.) so as to be 5 ⁇ g / animal, and 10 v / v% Alum (Invivogen vac-alu250) and 50 ⁇ g / animal CpG ODN1826 (Eurofin) were used as an adjuvant. ..
- the prepared antigen solution was intramuscularly inoculated (100 ⁇ L / one leg, both legs) into guinea pigs (3 females / group) 3 times at 2-week intervals, and 2 weeks after the final immunization, the whole was collected by cardiac blood sampling under isoflurane inhalation anesthesia. Blood was collected. The obtained blood was serum-separated in a separation tube containing a coagulation promoter. After serum separation, sera for 3 animals were pooled to obtain gBv9 immune serum and pentamer immune serum. Using this, the reactivity of the gB-pentamar constituent protein fusion to the gBv9 immune serum and the pentamer immune serum was evaluated.
- the 1% BSA / PBS solution was discarded, the obtained culture supernatant was diluted 10-fold, 100 ⁇ L was added to the well of the plate, and the mixture was incubated at room temperature. After 1 hour, the cells were washed with PBST, 100 ⁇ L of various immune sera were added to the well of the plate, and the mixture was incubated at room temperature. After 1 hour, the cells were washed with PBST, 100 ⁇ L of the detection antibody HRP-Rabbit anti Guinea Pig IgG (Invitrogen 614620) was added to the well of the plate, and the mixture was incubated at room temperature.
- HRP-Rabbit anti Guinea Pig IgG Invitrogen 614620
- the cells were washed with PBST and developed by adding 100 ⁇ L of TMB (SIGMA T-4444) to the well of the plate. After 30 minutes, the reaction was stopped with 1N sulfuric acid, and the color development value (OD 450 nm / 650 nm) was measured with a microplate reader (molecular device).
- ⁇ Gel filtration chromatography of refined protein products The properties of the obtained refined product were evaluated by gel filtration chromatography. As the column, Superdex 200 Increase 5/150 GL (GE Healthcare) was used, and 50 ⁇ L of each refined product having a concentration of 100 ⁇ g / mL was applied. The flow rate was 0.4 mL / min, and the migration Buffer was D-PBS (Wako), and the absorbance at a wavelength of 280 nm was measured.
- FIG. 4 The results of the reactivity analysis using HCMV-gB immune serum (gBv9 immune serum) and pentamer immune serum are shown in FIG. According to FIG. 4, UL128 (C162S) -gB / N-UL130-gB / N-UL131-gB, UL128 (C162S) -VC37 / UL130-VC37 / UL131-VC37, UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4.
- the gB-pentamer-constituting protein fusion of any combination of / UL131- ⁇ d4 and N-UL128-gB / N-UL130-gB / N-UL131-gB is reactive to both HCMV-gB immune serum and pentamer immune serum.
- a point mutation of C162S was introduced into UL128, an improvement in reactivity with pentamer immune serum was observed.
- the gB-pentamar-constituting protein fusion having the gB moiety of gBVC37 or gB ⁇ d4 had a greater reaction with the pentamer-immune serum than the gB-pentamar-constituting protein fusion having the gB portion of gBv9.
- the HCMV gB protein and the pentamer have reactivity with the HCMV-gB immune serum and the pentamer immune serum even in the wild type sequence, and further, appropriate mutations are made to the HCMV gB protein or the pentamer. It was shown that by introduction, the pentamer moiety can be expressed as a gB-pentamer-constituting protein fusion in a form that maintains the original structure.
- Example 3-1 Antigen preparation>
- the structure as gB and pentamer was maintained by co-expressing three kinds of gB-pentamer constituent protein fusion expression plasmids in which a mutation was introduced into the region corresponding to the HCMV gB protein or pentamer. It was shown that the gB-pentamer constituent protein fusion could be expressed. Therefore, for the purpose of co-expressing three or more kinds of HCMV gB protein or pentamer constituent protein or gB-pentamar constituent protein fusion in combination, the following gB-pentamar constituent protein fusion expression plasmids are further designed. did.
- gH Cloning of gH (SEQ ID NO: 4) into pCAGGS1-dhfr-neo was designated as gH (His-). Further, a DNA fragment encoding a gL (SEQ ID NO: 5) to which a C144S amino acid substitution was added was cloned into pCAGGS1-dhfr-neo, and the DNA fragment was designated as gL (C144S).
- gBVC37 or gB ⁇ d4 was added to the C-terminal side of gL (SEQ ID NO: 5) via a 15 amino acid linker (GGGGSGGGGGSGGGGS) (SEQ ID NO: 23).
- GGGGSGGGGGSGGGGS 15 amino acid linker
- ⁇ d4 on the C-terminal side is designated as gL- ⁇ d4.
- gBVC37 was ligated to the C-terminal side of UL128 (SEQ ID NO: 6) or UL130 (SEQ ID NO: 7) or UL131 (SEQ ID NO: 8) via a 15-amino acid linker (GGGGSGGGGGSGGGGS) (SEQ ID NO: 23).
- GGGGSGGGGGSGGGGS 15-amino acid linker
- the one with UL128 on the N-terminal side is UL128-VC37-L15aa
- the one with UL130 on the N-terminal side is UL130-VC37-L15aa
- the one with UL131 on the N-terminal side is UL131-VC37-L15aa.
- the body-encoding DNA fragment was cloned into pCAGGS1-dhfr-neo.
- Co-expression was performed by combining these various gB-pentamer constituent protein fusion expression plasmids.
- the combined expression plasmids were a total of 31 types shown in Table 1. Expression was carried out in the same manner as for the HCMV gB protein, and a culture supernatant of each gB-pentamer constituent protein fusion was obtained. Further, purification was carried out in the same manner as for the HCMV gB protein to obtain a refined product of each gB-pentamer constituent protein fusion.
- UL128 (C162S) -VC37 / UL130 / UL131 has lower reactivity with pentamer serum than UL128 (C162S) -VC37 / UL130-VC37 / UL131-VC37, and UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131.
- UL128 (C162S) - ⁇ d4 / UL130 / UL131 has lower reactivity with pentamer serum, gH (His-) / gL (C144S) / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131- ⁇ d4. It was observed that gH (His-) / gL (C144S) / UL128 (C162S) - ⁇ d4 / UL130 / UL131 had lower reactivity with pentamer serum. From this, it was considered that 3 of the pentamer-constituting molecules should be expressed as a gB-pentamer-constituting protein fusion.
- ⁇ Gel filtration chromatography of refined protein products The properties of the obtained refined product were evaluated by gel filtration chromatography. As the column, Superdex 200 Increase 5/150 GL (GE Healthcare) was used, and 50 ⁇ L of each refined product having a concentration of 100 ⁇ g / mL was applied. The flow rate was 0.4 mL / min, and the migration Buffer used D-PBS (Wako) to detect the absorbance at a wavelength of 280 nm.
- FIGS. 10 to 13 The evaluation results are shown in FIGS. 10 to 13.
- a gB-pentamer-constituting protein fusion in which one of the pentamer-constituting molecules is fused with VC37 or ⁇ d4 as gB, the other four pentamer-constituting proteins, and VC37 or unfused with the pentamer-constituting protein. It was confirmed that the co-expressed ⁇ d4 had a high content other than aggregates (Fig. 10), but gB was obtained by fusing one of the pentamer-constituting molecules with the pentamer-constituting molecule and VC37 or ⁇ d4 as gB.
- gB-pentamer constituent protein fusion was immunized by the same method as in Example 2 to obtain an immune serum. This serum was used to evaluate the binding antibody titer and neutralizing antibody titer of HCMV against gB protein, pentamer.
- gBv9 or pentamer was first diluted with PBS (Wako) to 1 ⁇ g / mL, 100 ⁇ L was placed in MaxiSorp plate (Nunc), and the mixture was allowed to stand overnight at 4 ° C. to solidify the antigen. After solid phase, the plate was washed with PBS, 300 ⁇ L / well of 1% BSA / PBS solution was added, and the mixture was allowed to stand for 1 hour or more for blocking.
- TMB (sigma: T4444) was added, and after allowing to stand at room temperature for 30 minutes, the reaction was stopped by adding 1N sulfuric acid, and the color development value (OD. 450 nm / 650 nm) was measured.
- a fibroblast neutralization test was performed using MRC-5 cells, and an epithelial cell neutralization test was performed using ARPE-19 cells.
- the HCMV AD-169 strain was subcultured with MRC-5 cells as a test virus, and in the epithelial cell neutralization test, the HCMV AD-169 strain was subcultured with ARPE-19 cells. I used a substitute.
- the fibroblast neutralization test was performed using MRC-5 according to the following procedure.
- an MRC-5 suspension was prepared, seeded in CellCarrier-96 (PerkinElmer: 6055700) at 2 ⁇ 10 4 cells / well, and cultured overnight in a CO 2 incubator at 5% CO 2 concentration and 37 ° C.
- the immune serum was diluted with virus dilution medium (MEM medium containing 10 mM HEPES, 0.21% BSA, Pencillin-Streptomycin) so that the HCMV AD-169 strain had a final concentration of 200 TCID 50 / mL.
- the virus-serum mixture was prepared by adding the mixture and allowing it to stand at 37 ° C.
- a virus-serum mixture was prepared in the same manner.
- the cultured MRC-5 cells were washed with PBS, a virus-serum mixture or virus solution was added at 30 ⁇ L / well, and then centrifuged at 400 g at room temperature for 30 minutes. Then, the virus-serum mixture or virus solution is removed, washed with MEM medium containing Penicillin-Streptomycin, and the medium is added at 100 ⁇ L / well to a 5% CO 2 concentration in a CO 2 incubator at 37 ° C. It was cultured in the evening.
- the cells were washed with the same medium, 100 ⁇ L / well of 50% acetone / PBS solution was added, and the cells were allowed to stand at room temperature for 20 minutes. Then, 50% acetone / PBS solution was removed, washed with 0.5% BSA / PBS solution, 100 ⁇ L / well of 0.1% Triton X-100 / PBS solution was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then, the cells were washed again with 0.5% BSA / PBS solution, and 100 ⁇ L / well of CMV pp72 / 86 antibody (Santa Cruz: sc-69748) diluted 100-fold with 0.5% BSA / PBS solution was added, 37.
- the measurement was performed with an Image Express Micro (molecular device), the total number of cells was counted by Hoechst staining, the number of HCMV-infected cells was counted by Alexa Fluor staining, and the infection rate was calculated by (HCMV-infected cell number) / (total cell number). did. Furthermore, the infection inhibition rate (%) was calculated by setting 1- (infection rate of test well) / (infection rate of positive control). This result is shown in FIG. 15 (a). From this result, it was shown that the administration of the gB-pentamer constituent protein fusion can induce a neutralizing antibody against the invasion of HCMV into fibroblasts.
- Example 3-2-1 Antigen preparation> Since it was shown by the examination of Example 3-1 that the structure as gB and pentamer was maintained even when 5 kinds of various gB-pentamer constituent protein fusion expression plasmids were combined and co-expressed, further gL C144 and UL128 were shown. In order to investigate the effect of the point mutation of C162, the following gB-pentamer constituent protein fusion expression plasmids were designed.
- a DNA fragment encoding a fusion protein in which a C144S amino acid substitution was added to gL (SEQ ID NO: 5) and a linker of 15 amino acids (GGGGSGGGGGSGGGGS) (SEQ ID NO: 23) and ⁇ d4 were added to the C-terminal side was prepared by PCR, and the DNA fragment was prepared.
- the cloned product to pCAGGS1-dhfr-neo was designated as gL (C144S) - ⁇ d4.
- various gB-pentamer constituent protein fusion expression plasmids were combined and co-expressed.
- the combined expression plasmids are gH (His-) / gL- ⁇ d4 / UL128- ⁇ d4 / UL130- ⁇ d4 / UL131, gH (His-) / gL (C144S) - ⁇ d4 / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / There were a total of three types: UL131, gH (His-) / gL- ⁇ d4 / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131. Further, purification was carried out in the same manner as for the HCMV gB protein to obtain a refined product of each gB-pentamer constituent protein fusion.
- gB ⁇ d4 or pentamer or gH (His-) / gL- ⁇ d4 / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131 or saline is immunized with guinea pig (Hartley), and gB ⁇ d4 immune serum and Pentamar immune serum and gH (His-) / gL- ⁇ d4 / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131 immune serum and saline immune serum were obtained.
- the flow rate was 0.4 mL / min, and the migration Buffer used D-PBS (Wako) to detect the absorbance at a wavelength of 280 nm.
- the results are shown in FIG. From this result and FIG. 12 (d), it was shown that the point mutation introduction of C144 of gL and C162 of UL128 did not change the separation pattern by gel filtration chromatography.
- Each recombinant protein was diluted with PBS (SIGMA) to 0.2 or 0.3 mg / mL and particle size analysis was performed with Zetasizer Nano ZS (Malvern).
- a quartz cell was used for the measurement, and the measurement condition was a temperature of 25.0 ° C. This is shown in FIG.
- the peaks (a), (b), and (c) in FIG. 21 show the results of three times, and the numbers shown in the table are the average values of the three times. From this result, it was shown that the point mutation introduction of C144 of gL and C162 of UL128 did not affect the particle size distribution.
- Example 3-2-2 ⁇ Antigen preparation> Co-expression was performed by combining various gB-pentamer constituent protein fusion expression plasmids.
- the combined expression plasmids are gH (His-) / gL / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131- ⁇ d4, gH (His-) / gL / UL128- ⁇ d4 / UL130- ⁇ d4 / UL131- ⁇ d4.
- all purification was carried out in the same manner as for gB protein of HCMV to obtain a refined protein product.
- a quartz cell was used for the measurement, and the measurement condition was a temperature of 25.0 ° C. This is shown in FIG.
- the peak in FIG. 29 shows the results of 3 times, and the numbers shown in the table are the average values of 3 times. From this result, it was shown that the point mutation introduction of C162 of UL128 did not affect the particle size distribution.
- Example 3-2-3 Co-expression was performed by combining various gB-pentamer constituent protein fusion expression plasmids.
- the combined expression plasmids are gBv9, pentamer, gH (His-) / gL- ⁇ d4 / UL128- ⁇ d4 / UL130- ⁇ d4 / UL131 and gH (His-) / gL / UL128- ⁇ d4 / UL130- ⁇ d4 / UL131- ⁇ d4.
- all purification was carried out in the same manner as for gB protein of HCMV to obtain a refined protein product.
- guinea pig Hartley
- gBv9 immune serum pentamer immune serum
- pentamer immune serum gH (His-) / gL- ⁇ d4 / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131 immune serum
- gH (His-) / gL / UL128- ⁇ d4 / UL130- ⁇ d4 / UL131- ⁇ d4 immune serum and salon immune serum were obtained.
- Example 3-1 The evaluation result of the neutralizing antibody titer is shown in FIG.
- Example 4 ⁇ Evaluation of stability of gB-pentamer fusion protein> Co-expression was performed by combining various gB-pentamer constituent protein fusion expression plasmids.
- the combined expression plasmids are gBv9, pentamer, gH (His-) / gL- ⁇ d4 / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131, gH (His-) / gL / UL128 (C162S) - ⁇ d4 / UL130- With a total of 6 types: ⁇ d4 / UL131- ⁇ d4, gH (His-) / gL / UL128- ⁇ d4 / UL130- ⁇ d4 / UL131- ⁇ d4, gH (His-) / gL- ⁇ d4 / UL128- ⁇ d4 / UL130- ⁇ d
- the stability of the obtained recombinant gB, recombinant pentamer, an equal amount mixture of recombinant gB and recombinant pentamer, and the recombinant gB-pentamar constituent protein fusion was evaluated by dynamic light scattering. Accelerated tests were performed by diluting each recombinant protein with PBS (SIGMA) to 0.2 mg / mL and allowing it to stand at 37 ° C. for 0 days, 1 day, 3 days, and 7 days.
- PBS PBS
- a protease inhibitor (Wako, 165-26021) was added to the protein that was allowed to stand for 1 day or more, and the protein after each test was subjected to particle size analysis by Zetasizer Nano ZS (Malvern).
- a quartz cell was used for the measurement, and the measurement condition was a temperature of 25.0 ° C.
- particles having a particle size ( ⁇ 100 nm) considered to be the target product are "single particles", and particles larger than the target product and having a particle size of less than 1000 nm are “aggregated (small)”. Those having a diameter exceeding 1000 nm were referred to as "aggregation (large)”.
- Figures 22, 23 and 32 show the distribution of each particle. From this result, it was observed that the content of aggregate (large) in the pentamer increased with time, but gH (His-) / gL- ⁇ d4 / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131, gH (His-) / gL / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131- ⁇ d4, gH (His-) / gL / UL128- ⁇ d4 / UL130- ⁇ d4 / UL131- ⁇ d4, and gH (His-) / Most of gL- ⁇ d4 / UL128- ⁇ d4 / UL130- ⁇ d4 / UL131 remained as a monomer.
- Example 5 Antigen preparation> As the stimulating antigen, gBv9, pentamer, gH (His-) / gL- ⁇ d4 / UL128 (C162S) - ⁇ d4 / UL130- ⁇ d4 / UL131, gH (His-) / gL- ⁇ d4 / UL128 in the same procedure as in Example 4. - ⁇ d4 / UL130- ⁇ d4 / UL131, gH (His-) / gL / UL128- ⁇ d4 / UL130- ⁇ d4 / UL131- ⁇ d4 were expressed and purified.
- Cell-mediated immunity evaluation using human PBMC was performed by ELISpot assay using Human IFN- ⁇ ELISpot PLUS (MABTECH 3420-4HST-2). For human PBMC, 23 donors with IFN ⁇ induction to HCMV antigen stimulation were selected from CTL.
- CTL Anti-Aggregate Wash (20 ⁇ ) (CTL) is warmed and thawed in a water bath set at 37 ° C for 10 min, diluted with RPMI-1640 to prepare CTL Anti-Aggregate Wash (1 ⁇ ), and 37 ° C until use. It was allowed to stand for 20 minutes or more in the CO 2 incubator of.
- CTL-Test Medium 1 vol% of L-Glutamine (100 ⁇ ) was added and the mixture was allowed to stand in a CO 2 incubator at 37 ° C. for 20 min or more.
- the vial containing PBMC was warmed in a water bath at 37 ° C.
- CTL Anti-Aggregate Wash (1 ⁇ ) was gently added to prepare a cell solution. Centrifuge the cell solution (330 g, 10 min, RT) to remove the supernatant, add 10 mL of CTL Assay-Aggregate Wash (1 ⁇ ), centrifuge again (330 g, 10 min, RT) to remove the supernatant, and then remove the supernatant at 37 ° C. It was diluted with CTL-Test Medium that had been allowed to stand in a CO 2 incubator and used for ELISPOT assay.
- the required number of strips of Human IFN- ⁇ ELISpot PLUS was washed 4 times with 200 ⁇ L / well PBS, 200 ⁇ L / well of CTL-Test Medium was added, and the mixture was allowed to stand at room temperature for 30 min or more.
- the CTL-Test Medium was removed from the plate, 100 ⁇ L / well of the cell suspension was added, and then 100 ⁇ L / well of the antigen solution diluted to 2 ⁇ g / mL was added and suspended.
- the plate was shielded from light and statically cultured for 12 to 48 hours in a 5% CO 2 incubator at 37 ° C. After culturing, the cells were removed together with the medium and washed 5 times with 200 ⁇ L / well PBS.
- the detection antibody solution (7-6-1-biotin) attached to Human IFN- ⁇ ELISpot PLUS was diluted with PBS-0.5% FBS to 1 ⁇ g / mL, 100 ⁇ L / well was added, and the mixture was allowed to stand at room temperature for 2 hours. After the reaction, the mixture was washed 5 times with 200 ⁇ L / well of PBS, and the labeled antibody solution (Streptavidin-HRP) attached to Human IFN- ⁇ ELISpot PLUS was diluted 1000-fold with PBS-0.5% FBS and 100 ⁇ L / well was added. It was allowed to stand at room temperature for 1 hr.
- the average value of the number of spots for 2 wells was used as the measurement result.
- the average value of the number of spots for 4 wells was used as the measurement result only for the negative control.
- the average value of the number of spots is greater than 0 and less than 5, it is "0", when it is 5 or more and less than 50, it is “1”, when it is 50 or more and less than 100, it is “2”, and when it is 100 or more, it is "3". Scores were given and the scores of all donors were totaled for each antigen. A graph of this is shown in FIG.
- Example 6 Preparation of GPCMV-gB> Guinea pig cytomegalovirus (GPCMV), which is infectious to guinea pigs, was used for evaluation using a guinea pig transplacental infection model system. Since the recombinant GPCMV gB protein may contain aggregates, a modified GPCMV gB protein having improved properties and containing no aggregates was prepared.
- GPCMV Guinea pig cytomegalovirus
- a signal sequence was added to the gB ect domain (1-656aa) derived from GPCMV 22122 strain, and an amino acid mutation for improving properties was further introduced into gB (SEQ ID NO: 17, 1-692aa; GPCMV-gB ect domain variant). ) was artificially synthesized and cloned into pCAGGS1-dhfr-neo. It was designed so that a 9-amino acid His-tag (SEQ ID NO: 30) was added to the C-terminal of gB.
- Expi293 expression system (Life Technologies) was used. The expression plasmid was transfected into the cells and the culture supernatant was collected in 4-6 days.
- GPCMV pentamer International Application PCT / JP2019 / 047966
- GPCMV-gB-pentamer fusion protein ⁇ Preparation and property analysis of GPCMV-gB-pentamer fusion protein>
- 111-475aa and 641-692aa of the GPCMV-gB ect domain variant (SEQ ID NO: 17) were connected with 9 amino acids of GGGSGSGGG (SEQ ID NO: 20), and a His-tag sequence (SEQ ID NO: 20) was further attached to the C-terminal.
- GPCMV- ⁇ d4 SEQ ID NO: 32 was added with 30), and a DNA fragment encoding GPCMV- ⁇ d4 was prepared by PCR, and the DNA fragment was cloned into pCAGGS1-dhfr-neo.
- a DNA fragment encoding a fusion protein in which GPCMV- ⁇ d4 is linked to the C-terminal side of GP115, GP129, GP131 or GP133 via a 15-amino acid linker (GGGGSGGGGGSGGGGS) (SEQ ID NO: 23) based on the above gene is provided.
- the DNA fragments prepared by PCR and cloned into pCAGGS1-dhfr-neo were designated as GP115- ⁇ d4, GP129- ⁇ d4, GP131- ⁇ d4, or GP133- ⁇ d4, respectively.
- various gB-pentamer constituent protein fusion expression plasmids were combined and co-expressed.
- the combined expression plasmids were GP75 (His-) / GP115- ⁇ d4 / GP129- ⁇ d4 / GP131- ⁇ d4 / GP133, GP75 (His-) / GP115 / GP129- ⁇ d4 / GP131- ⁇ d4 / GP133- ⁇ d4. Expression and purification were carried out in the same manner as for GPCMV-gB.
- GP133- ⁇ d4 a DNA fragment encoding a fusion protein in which GPCMV- ⁇ d4 was ligated via a 15-amino acid linker (GGGGSGGGGGSGGGGS) (SEQ ID NO: 23) on the C-terminal side of GP133 was prepared by PCR.
- the DNA fragment cloned into pCAGGS1-dhfr-neo was designated as GP133- ⁇ d4.
- GP129 (C167S) - ⁇ d4 was prepared by adding C167S modification, which is considered to dissociate the disulfide bond sites of GP115 and GP129, based on GP129- ⁇ d4.
- ⁇ Placenta infection protection test> For Heartley guinea pigs (female, 5 weeks old), GPCMV-gB, GPCMV pentamer, GP75 (His-) / GP115- ⁇ d4 / GP129- ⁇ d4 / GP131- ⁇ d4 / GP133 and GP129 (C167S) - ⁇ d4 / GP131- ⁇ d4 / GP133- ⁇ d4 (GPCMV- ⁇ d4-ULs) was immunized.
- Each antigen was prepared with physiological saline (Otsuka Pharmaceutical Co., Ltd.) so as to be 0.0016 ⁇ g / animal, and 10 v / v% Alum (Invivogen vac-alu250) and 50 ⁇ g / animal CpG ODN1826 were used as an adjuvant.
- the prepared antigen solution was intramuscularly inoculated into guinea pigs twice at 2-week intervals (100 ⁇ L / one leg, both hind limbs), and mated with Hartley guinea pigs ( ⁇ , 9 weeks old or older) 2 weeks after the second immunization. , Made a pregnant guinea pig.
- the PBS-administered group was also mated in the same manner.
- wild-type GPCMV was subcutaneously administered at 1 ⁇ 10 6 pfu / animal at 4 weeks of gestation, and 3 weeks later, placenta was collected after euthanasia with pentobarbital sodium. The collected placenta was crushed by gentleMACS (Miltenyi Biotec), and DNA was extracted by MagNA Pure96 (Roche).
- the GPCMV gp83 gene and the cell-derived ⁇ actin gene were quantified using the probe and primer set (international application PCT / JP2019 / 047966) shown in Table 2 using a 7500 Fast real-time PCR system (Thermo).
- Gp83 gene of 105 cells per namely the viral copy number shown in FIG. 26. At this time, determination limit was 105 cells per 10 copies.
- the results of FIG. 26 show that GP75 (His-) / GP115- ⁇ d4 / GP129- ⁇ d4 / GP131- ⁇ d4 / GP133 have higher infection protection than GPCMV-gB, GPCMV pentamer and GPCMV- ⁇ d4-ULs. rice field.
- Example 7 ⁇ Transplacental infection protection test>
- GPCMV-gB GPCMV pentamer
- GP75 (His-) / GP115- ⁇ d4 / GP129- ⁇ d4 / GP131- ⁇ d4 / GP133 GPCMV pentamer
- GP75 (His-) / GP115 / GP129- ⁇ d4 / GP131- ⁇ d4 / GP133- ⁇ d4 was expressed and prepared.
- GPCMV-gB and GPCMV pentamer 0.1 ⁇ g was mixed to make 0.2 ⁇ g / animal.
- the PBS-administered group was also mated in the same manner.
- wild-type GPCMV was subcutaneously administered at 1 ⁇ 10 7 pfu / animal at 4 weeks of gestation to attack. Three weeks after the attack, the mother and fetus were dissected after being euthanized with pentobarbital sodium, and the salivary glands and placenta were collected from the mother and the pancreas was collected from the fetus.
- the maternal salivary glands and placenta were crushed with gentleMACS (Miltenyi Biotec) together with an appropriate amount of PBS.
- the fetal pancreas was disrupted with FastPrep24 (MP Biomedicals) with an appropriate amount of PBS.
- DNA extraction was performed on each homogenated solution with MagNA Pure96 (Roche). From the resulting DNA, in the same manner as in Example 6, it was subjected to viral copy number quantification of 105 cells per. At this time, determination limit was 105 cells per copy.
- GP75 (His-) / GP115- ⁇ d4 / GP129- ⁇ d4 / GP131- ⁇ d4 / GP133, GP75 (His-) / GP115 / GP129- ⁇ d4 / GP131- ⁇ d4 / GP133- ⁇ d4 have an effect on both the mother and the fetus. From the above, it is strongly suggested that the vaccine antigen of the present invention is useful not only for the prevention of congenital infection but also for the prevention of cytomegalovirus infection in healthy subjects, transplant patients, AIDS patients and the like. Was done.
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Abstract
Description
[1] サイトメガロウイルス(CMV)のエンベロープ糖タンパク質B(gBタンパク質)とペンタマーとの融合タンパク質。
[2] 3つのgBタンパク質構成分子からなるgBタンパク質のホモ三量体と、5つのペンタマー構成分子からなるペンタマーのヘテロ五量体とが、少なくとも1つのgBタンパク質構成分子と少なくとも1つのペンタマー構成分子とがそれぞれ遺伝子工学的に融合することによって、タンパク質複合体を形成している、[1]に記載の融合タンパク質。
[3] 少なくとも2つのgBタンパク質構成分子と、少なくとも2つのペンタマー構成分子とがそれぞれ遺伝子工学的に融合している、[2]に記載の融合タンパク質。
[4] 3つのgBタンパク質構成分子と、5つのペンタマー構成分子のうちのいずれか3つのペンタマー構成分子とがそれぞれ遺伝子工学的に融合している、[2]又は[3]に記載の融合タンパク質。
[5] 3つのgBタンパク質構成分子と、ペンタマー構成分子であるgL、UL128及びUL130とがそれぞれ遺伝子工学的に融合している、[2]~[4]のいずれかに記載の融合タンパク質。
[6] 3つのgBタンパク質構成分子と、ペンタマー構成分子であるUL128、UL130及びUL131とがそれぞれ遺伝子工学的に融合している、[2]~[4]のいずれかに記載の融合タンパク質。
[7] 遺伝子工学的な融合のうち少なくとも1つの融合は、gBタンパク質構成分子のN末端側にペンタマー構成分子が結合している融合である、[2]~[6]のいずれかに記載の融合タンパク質。
[8] 遺伝子工学的な融合のうち少なくとも2つの融合は、gBタンパク質構成分子のN末端側にペンタマー構成分子が結合している融合である、[3]~[6]のいずれかに記載の融合タンパク質。
[9] 3つの遺伝子工学的な融合がすべて、gBタンパク質構成分子のN末端側にペンタマー構成分子が結合している融合である、[4]~[6]のいずれかに記載の融合タンパク質。
[10] gBタンパク質構成分子とペンタマー構成分子との間にリンカー及び/又はタグを有する、[2]~[9]のいずれかに記載の融合タンパク質。
[11] リンカーが配列番号22記載のアミノ酸配列単位を1~3回繰り返したアミノ酸配列からなるリンカーである、[10]に記載の融合タンパク質。
[12] gBタンパク質がCMV gBタンパク質のエクトドメインである、[1]~[11]のいずれかに記載の融合タンパク質。
[13] gBタンパク質が配列番号15に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるヒトサイトメガロウイルス(HCMV)のgBタンパク質のエクトドメインである、[12]に記載の融合タンパク質。
[14] gBタンパク質が、配列番号15に記載のアミノ酸配列からなるgBタンパク質のエクトドメインと80%以上の配列同一性を有するgBタンパク質のエクトドメインである、[12]又は[13]に記載の融合タンパク質。
[15] gBタンパク質がドメインIVを欠失したgBタンパク質改変体である、[1]~[14]のいずれかに記載の融合タンパク質。
[16] gBタンパク質が配列番号16に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるヒトサイトメガロウイルス(HCMV)のgBタンパク質のエクトドメインである、[15]に記載の融合タンパク質。
[17] gBタンパク質が、配列番号16に記載のアミノ酸配列からなるgBタンパク質のエクトドメインと80%以上の配列同一性を有するgBタンパク質のエクトドメインである、[15]又は[16]に記載の融合タンパク質。
[18] gBタンパク質が、野生型gBタンパク質に比べて、凝集体の形成を低減させ、ホモ三量体構造の割合を増加させる改変が導入されたgBタンパク質改変体である、[1]~[14]のいずれかに記載の融合タンパク質。
[19] gBタンパク質が配列番号3に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるヒトサイトメガロウイルス(HCMV)のgBタンパク質のエクトドメインである、[18]に記載の融合タンパク質。
[20] gBタンパク質が、配列番号3に記載のアミノ酸配列からなるgBタンパク質のエクトドメインと80%以上の配列同一性を有するgBタンパク質のエクトドメインである、[18]又は[19]に記載の融合タンパク質。
[21] gBタンパク質が、野生型gBタンパク質に比べて、ヘッド領域の免疫原性を低減させる改変が導入されたgBタンパク質改変体である、[1]~[14]のいずれかに記載の融合タンパク質。
[22] gBタンパク質が配列番号31に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるヒトサイトメガロウイルス(HCMV)のgBタンパク質のエクトドメインである、[21]に記載の融合タンパク質。
[23] gBタンパク質が、配列番号31に記載のアミノ酸配列からなるgBタンパク質のエクトドメインと80%以上の配列同一性を有するgBタンパク質のエクトドメインである、[21]又は[22]に記載の融合タンパク質。
[24] ペンタマーが、ヒトサイトメガロウイルス(HCMV)のgH、gL、UL128、UL130及びUL131からなる、[1]~[23]のいずれかに記載の融合タンパク質。
[25] gHがgHタンパク質のエクトドメインである、[24]に記載の融合タンパク質。
[26] ペンタマーが、
配列番号4に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるgH、
配列番号5に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるgL、
配列番号6に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるUL128、
配列番号7に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるUL130、及び
配列番号8に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるUL131
を含むヒトサイトメガロウイルス(HCMV)のペンタマータンパク質である、[24]又は[25]に記載の融合タンパク質。
[27] ペンタマーが、
配列番号4に記載のアミノ酸配列からなるgHと80%以上の配列同一性を有するgH、
配列番号5に記載のアミノ酸配列からなるgLと80%以上の配列同一性を有するgL、
配列番号6に記載のアミノ酸配列からなるUL128と80%以上の配列同一性を有するUL128、
配列番号7に記載のアミノ酸配列からなるUL130と80%以上の配列同一性を有するUL130、及び
配列番号8に記載のアミノ酸配列からなるUL131と80%以上の配列同一性を有するUL131
を含むHCMVのペンタマータンパク質である、[24]~[26]のいずれかに記載の融合タンパク質。
[28] [1]~[27]のいずれかに記載の融合タンパク質をコードする核酸断片。
[29] [28]に記載の核酸断片を含む組換え発現ベクター。
[30] [28]に記載の核酸断片又は[29]に記載の組換え発現ベクターが導入された形質転換体。
[31] [1]~[27]のいずれかに記載の融合タンパク質を含む、CMVの感染を予防又は治療するためのワクチン。
[32] CMVの感染がCMVの先天性感染である、[31]に記載のワクチン。
本発明の融合タンパク質は、サイトメガロウイルス(CMV)のエンベロープ糖タンパク質B(gBタンパク質)とペンタマーとの融合タンパク質である。当該融合タンパク質は、3つのgBタンパク質構成分子からなるgBタンパク質のホモ三量体と、5つのペンタマー構成分子からなるペンタマーのヘテロ五量体とが、少なくとも1つのgBタンパク質構成分子と少なくとも1つのペンタマー構成分子とがそれぞれ遺伝子工学的に融合することによってタンパク質複合体を形成したものである。ここで、構成分子とは、gBタンパク質のホモ三量体又はペンタマーのヘテロ五量体を構成するタンパク質をいい、サブユニットともいう。当該融合タンパク質は抗原としてCMVの予防及び治療に有用なワクチンの作製に用いられる。
配列番号4に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるgH、
配列番号5に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるgL、
配列番号6に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるUL128、
配列番号7に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるUL130、及び
配列番号8に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるUL131
を含むHCMVのペンタマータンパク質であってよい。ここで、1以上のアミノ酸残基の欠失、置換若しくは付加は、例えば、1~50個、1~40個、1~30個、1~20個、1~15個、1~10個、1~5個、1~3個のアミノ酸残基の欠失、置換若しくは付加であってよい。
配列番号4に記載のアミノ酸配列からなるgHと80%以上の配列同一性を有するgH、
配列番号5に記載のアミノ酸配列からなるgLと80%以上の配列同一性を有するgL、
配列番号6に記載のアミノ酸配列からなるUL128と80%以上の配列同一性を有するUL128、
配列番号7に記載のアミノ酸配列からなるUL130と80%以上の配列同一性を有するUL130、及び
配列番号8に記載のアミノ酸配列からなるUL131と80%以上の配列同一性を有するUL131
を含むHCMVのペンタマータンパク質であってよい。ここで、80%以上の配列同一性は、例えば85%以上、90%以上、93%以上、95%以上又は98%以上の配列同一性であってよい。
配列番号10に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるGP75、
配列番号11に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるGP115、
配列番号12に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるGP129、
配列番号13に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるGP131、及び
配列番号14に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるGP133
を含むGPCMVのペンタマータンパク質であってよい。ここで、1以上のアミノ酸残基の欠失、置換若しくは付加は、例えば、1~50個、1~40個、1~30個、1~20個、1~15個、1~10個、1~5個、1~3個のアミノ酸残基の欠失、置換若しくは付加であってよい。
配列番号10に記載のアミノ酸配列からなるGP75と80%以上の配列同一性を有するGP75、
配列番号11に記載のアミノ酸配列からなるGP115と80%以上の配列同一性を有するGP115、
配列番号12に記載のアミノ酸配列からなるGP129と80%以上の配列同一性を有するGP129、
配列番号13に記載のアミノ酸配列からなるGP131と80%以上の配列同一性を有するGP131、及び
配列番号14に記載のアミノ酸配列からなるGP133と80%以上の配列同一性を有するGP133
を含むGPCMVのペンタマータンパク質であってよい。ここで、80%以上の配列同一性は、例えば85%以上、90%以上、93%以上、95%以上又は98%以上の配列同一性であってよい。
本発明の核酸断片は、本発明のCMVのgBとペンタマーとの融合タンパク質をコードする核酸断片であり、本発明の組換え発現ベクターは、本発明の核酸断片を含む組換え発現ベクターであり、本発明の形質転換体は、本発明の核酸断片又は本発明の組換え発現ベクターが導入された形質転換体である。
本発明のワクチンは、本発明の融合タンパク質を含む。一実施形態のワクチンは、サイトメガロウイルス(CMV)のエンベロープ糖タンパク質B(gBタンパク質)とペンタマーとの融合タンパク質を抗原として含むCMVの先天性感染を予防又は治療するためのワクチンである。すなわち、本実施形態のワクチンは2種類の抗原タンパク質の機能を併せ持つ融合タンパク質からなるサブユニットワクチンである。
<抗原調製>
AD169株に由来するHCMV gBのエクトドメイン(配列番号15)をコードする遺伝子を人工合成し、pCAGGS1-dhfr-neo(特許文献3)にクローニングした。C末端には9アミノ酸のHis-tag(配列番号30)が付加されるように設計した。これをベースに、I132H、Y133R、T215E、W216A、R432T及びR434Qのアミノ酸置換を行ったもの(配列番号3)をgB1-682-fm3Mv9(以下、「gBv9」と呼ぶ)とした(特許文献4)。gBv9を発現する際は、N末端にHCMV-gBのシグナル配列(MESRIWCLVVCVNLCIVCLGAAVS)(配列番号19)を挿入した。
UL130(配列番号7)のシグナル配列(1-25アミノ酸)を除き、同じ位置にMRLPAQLLGLLMLWVPGSSG(配列番号21)のアミノ酸配列を挿入した「シグナルペプチド置換UL130」、又は、
UL131(配列番号8)のシグナル配列(1-18アミノ酸)を除き同じ位置にMRLPAQLLGLLMLWVPGSSG(配列番号21)のアミノ酸配列を挿入した「シグナルペプチド置換UL131」
のC末端側に5アミノ酸のリンカー(GGGGS)(配列番号22)を介してgBv9を付加した融合タンパク質をコードするDNA断片をPCRによって作製し、pCAGGS1-dhfr-neoにクローニングした。このうち、シグナルペプチド置換UL128とgBv9を連結したものをN-UL128-gB、シグナルペプチド置換UL130とgBv9を連結したものをN-UL130-gB、シグナルペプチド置換UL131とgBv9を連結したものをN-UL131-gBとした。
UL128(配列番号6)のシグナル配列(1-27アミノ酸)を除いたシグナル除去UL128、又は、
UL130(配列番号7)のシグナル配列(1-25アミノ酸)を除いたシグナル除去UL130、又は、
UL131(配列番号8)シグナル配列(1-18アミノ酸)を除いたシグナル除去UL131
を連結し、さらにそのC末端側に9アミノ酸のHis-tag(配列番号30)を付加した融合タンパク質をコードするDNA断片をPCRによって作製し、pCAGGS1-dhfr-neoにクローニングした。このうち、シグナル除去UL128とgBv9を連結したものをC-UL128-gB、シグナル除去UL130とgBv9を連結したものをC-UL130-gB、シグナル除去UL131とgBv9を連結したものをC-UL131-gBとした。
取得した精製品の性状はゲル濾過クロマトグラフィーによって評価した。カラムはSuperdex200 Increase 5/150 GL(GE Healthcare)を使用し、濃度100μg/mLの各精製品をアプライした。流速は0.4mL/minで移動相はD-PBS(Wako)を使用し、波長280nmの吸光度を測定した。
ゲル濾過クロマトグラフィーの結果を図1~3に示す。全ての発現物において、凝集体が確認された。しかし、N-UL128-gB、N-UL130-gB、N-UL131-gB及びN-UL128-gB/N-UL130-gB/N-UL131-gBを発現した場合に比べ、C-UL128-gB、C-UL130-gB、C-UL131-gB及びC-UL128-gB/C-UL130-gB/及びC-UL131-gBを発現した場合、並びに、Δd4-UL128-gB、Δd4-UL130-gB、Δd4-UL131-gB及びΔd4-UL128-gB/Δd4-UL130-gB/Δd4-UL131-gBを発現した場合は、凝集体含量が増加していることが確認された。このことからUL128、UL130又はUL131をHCMVgBタンパク質と融合する場合、HCMV gBタンパク質のN末端側に融合した方が凝集体を形成しにくく、凝集体以外の含量が増加すると示唆された。
<抗原調製>
実施例1の検討によりUL128、UL130、又はUL131をgBのN末端側に融合すると凝集体含量の低下が認められたため、UL128、UL130、又はUL131をgBのN末端側に融合させた次のような各gB-ペンタマー構成タンパク質融合体をデザインした。
UL128(配列番号6)にC162Sアミノ酸置換を加え、更にC末端側に5アミノ酸のリンカー(GGGGS)(配列番号22)を介してgBv9を付加したものをUL128(C162S)-gBとした。
UL128(配列番号6)にC162Sアミノ酸置換を加え、更にC末端側に15アミノ酸のリンカー(GGGGSGGGGSGGGGS)(配列番号23)を介してgBΔd4を付加したものをUL128(C162S)-Δd4とした。
UL128(配列番号6)、UL130(配列番号7)又はUL131(配列番号8)遺伝子のC末端側に15アミノ酸のリンカー(GGGGSGGGGSGGGGS)(配列番号23)を介してgBΔd4を付加し、このうち、N末端側がUL128のものをUL128-Δd4、N末端側がUL130のものをUL130-Δd4、N末端側がUL131のものをUL131-Δd4とした。
gBv9のS128aa-L138aaを欠損させ、末端のR127とG139をグリシンリンカーGGGでつなぎ、W216aa-Y218aaを欠損させ、N型糖鎖を4か所導入(D77N、I79T/E544N、P546T/L588N、P589G/K609N、R610T、M611T)したものをgBVC37(配列番号31)とした(特許文献4)。gBVC37を発現する際はN末端に HCMV-gBのシグナル配列(MESRIWCLVVCVNLCIVCLGAAVS)(配列番号19)を挿入した。
UL128(配列番号6)にC162Sアミノ酸置換を加え、C末端側に5アミノ酸のリンカー(GGGGS)(配列番号22)を介してgBVC37を付加したものをUL128(C162S)-VC37とした。
UL128(配列番号6)、UL130(配列番号7)又はUL131(配列番号8)遺伝子のC末端側に5アミノ酸のリンカー(GGGGS)(配列番号22)を介してgBVC37を付加し、このうち、N末端側がUL128のものをUL128-VC37、N末端側がUL130のものをUL130-VC37、N末端側がUL131のものをUL131-VC37とした。
N-UL128-gB/N-UL130-gB/N-UL131-gB、
UL128(C162S)-gB/N-UL130-gB/N-UL131-gB、
UL128(C162S)-VC37/UL130-VC37/UL131-VC37、
UL128(C162S)-Δd4/UL130-Δd4/UL131-Δd4、
UL128-Δd4/UL130-Δd4/UL131-Δd4、
gH/gL/UL128/UL130/UL131(ペンタマー)、
gBv9
の計7種類であった。gH/gL/UL128/UL130/UL131はHCMVペンタマーのエクトドメインに相当し、以下「ペンタマー」と呼ぶ。発現はHCMV gBタンパク質と同様の方法で行い、各gB-ペンタマー構成タンパク質融合体の培養上清を得た。また、ペンタマーの精製はHCMV gBタンパク質と同様の方法で行った。
gBv9、ペンタマーを抗原として、モルモット(Hartley)に免疫を行った。各抗原は5μg/匹になるように、生理食塩水(大塚製薬)で調製し、アジュバントとして10v/v%のAlum(Invivogen vac-alu250)及び50μg/匹のCpG ODN1826(ユーロフィン)を使用した。調製した抗原液を2週間間隔で3回、モルモット(雌 3匹/群)に筋肉内接種(100μL/片足、両足投与)し、最終免疫の2週間後にイソフルラン吸入麻酔下での心臓採血により全採血した。得られた血液は凝固促進剤入り分離管で血清分離した。血清分離後、3匹分の血清をプールし、gBv9免疫血清とペンタマー免疫血清を得た。これを用いて、gB-ペンタマー構成タンパク質融合体の、gBv9免疫血清及びペンタマー免疫血清に対する反応性評価を行った。
取得したgB-ペンタマー構成タンパク質融合体の培養上清の、各種免疫血清に対する反応性(結合活性)をELISAによって評価した。Rabbit anti 6 His Ab(BETHYL A190-114)をPBS(SIGMA)で1μg/mLに希釈し、MaxiSorp plate(Nunc)に100μL入れ、4℃でオーバーナイトインキュベートすることによって固相化した。固相化後、plateをPBSで洗浄し、1%BSA/PBS液を300μL/well添加し、1時間以上静置してブロッキングを行った。ブロッキング後に1%BSA/PBS液を捨て、取得した培養上清を10倍希釈し、plateのwellに100μL加え、室温でインキュベーションした。1時間後、PBSTで洗浄し、各種免疫血清をplateのwellに100μL加え、室温でインキュベーションした。1時間後、PBSTで洗浄し、検出抗体HRP-Rabbit anti Guinea Pig IgG(Invitrogen 614620)をplateのwellに100μL加え、室温でインキュベーションした。1時間後、PBSTで洗浄し、TMB(SIGMA T-4444)をplateのwellに100μL加えることによって発色させた。30分後、1N 硫酸で反応を停止させ、マイクロプレートリーダー(モレキュラーデバイス)で発色値(O.D.450nm/650nm)を測定した。
取得した精製品の性状はゲル濾過クロマトグラフィーによって評価した。カラムはSuperdex200 Increase 5/150 GL(GE Healthcare)を使用し、濃度100μg/mLの各精製品を50μLアプライした。流速は0.4mL/minで泳動BufferはD-PBS(Wako)を使用し、波長280nmの吸光度を測定した。
HCMV-gB免疫血清(gBv9免疫血清)及びペンタマー免疫血清を用いた反応性解析の結果を図4に示す。図4によれば、UL128(C162S)-gB/N-UL130-gB/N-UL131-gB、UL128(C162S)-VC37/UL130-VC37/UL131-VC37、UL128(C162S)-Δd4/UL130-Δd4/UL131-Δd4、N-UL128-gB/N-UL130-gB/N-UL131-gBのいずれの組み合わせのgB-ペンタマー構成タンパク質融合体もHCMV-gB免疫血清及びペンタマー免疫血清両方に対する反応性を有していた。また、UL128に対してC162Sの点変異を導入した場合、ペンタマー免疫血清に対する反応性の向上が認められた。さらに、gB-ペンタマー構成タンパク質融合体のgB部分がgBv9のものに比べ、gB部分をgBVC37若しくはgBΔd4としたgB-ペンタマー構成タンパク質融合体の方がペンタマー免疫血清との反応が大きかった。
<抗原調製>
実施例2の検討により、HCMV gBタンパク質若しくはペンタマーに相当する領域に変異を導入したgB-ペンタマー構成タンパク質融合体発現プラスミドを3種組み合わせて共発現することで、gB及びペンタマーとしての構造を保ったgB-ペンタマー構成タンパク質融合体を発現できていることが示された。よってHCMV gBタンパク質又はペンタマーの構成タンパク質又はgB-ペンタマー構成タンパク質融合体のうち3種以上のタンパク質を組み合わせて共発現させることを目的として、さらに次の各gB-ペンタマー構成タンパク質融合体発現プラスミドをデザインした。
取得したgB-ペンタマー構成タンパク質融合体の培養上清若しくは精製品の、各種免疫血清に対する反応性(結合活性)を実施例2と同様の方法により評価した。ただし、培養上清は100倍に希釈したものを使用している点、また培養上清の代わりに精製品を用いて試験を行う場合は1μg/mLで固相抗体と反応させている点が実施例2と異なる。
取得した精製品の性状はゲル濾過クロマトグラフィーによって評価した。カラムはSuperdex200 Increase 5/150 GL(GE Healthcare)を使用し、濃度100μg/mLの各精製品を50μLアプライした。流速は0.4mL/minで泳動BufferはD-PBS(Wako)を使用し、波長280nmの吸光度を検出した。
取得したgB-ペンタマー構成タンパク質融合体を実施例2と同様の方法により免疫し、免疫血清を取得した。この血清を用いて、HCMVのgBタンパク質、ペンタマーに対する結合抗体価及び中和抗体価を評価した。
<抗原調製>
実施例3-1の検討により各種gB-ペンタマー構成タンパク質融合体発現プラスミドを5種組み合わせて共発現してもgB、ペンタマーとしての構造を保っていることが示されたため、さらにgLのC144及びUL128のC162の点変異の影響を精査すべく、次の各gB-ペンタマー構成タンパク質融合体発現プラスミドをデザインした。gL(配列番号5)にC144Sアミノ酸置換を加えC末端側に15アミノ酸のリンカー(GGGGSGGGGSGGGGS)(配列番号23)、Δd4を付加した融合タンパク質をコードするDNA断片をPCRによって作製し、該DNA断片をpCAGGS1-dhfr-neoにクローニングしたものをgL(C144S)-Δd4とした。発現時には各種gB-ペンタマー構成タンパク質融合体発現プラスミドを組み合わせて共発現を行った。組み合わせた発現プラスミドは、gH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131、gH(His-)/gL(C144S)-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131、gH(His-)/gL-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131の計3種類であった。また、精製はHCMV gBタンパク質と同様の方法で行い、各gB-ペンタマー構成タンパク質融合体の精製品を得た。
実施例2と同様の方法により、gBΔd4又はペンタマー又はgH(His-)/gL-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131又はsalineをモルモット(Hartley)に免疫し、gBΔd4免疫血清及びペンタマー免疫血清及びgH(His-)/gL-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131免疫血清及びsaline免疫血清を得た。
取得したgB-ペンタマー構成タンパク質融合体について、gBΔd4、ペンタマー(PC)、gH(His-)/gL-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131(Δd4/PC)及びsaline免疫血清に対する反応性(結合活性)を実施例2と同様の方法によって評価した。これを図16~19に示す。この結果から、gLのC144及びUL128のC162の点変異は血清との反応性に影響しないことが示唆された。
取得したgH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131、及び、gH(His-)/gL(C144S)-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131の精製品の性状をゲル濾過クロマトグラフィーによって評価した。カラムはSuperdex200 Increase 5/150 GL(GE Healthcare)を使用し、濃度100μg/mLの各精製品を50μLアプライした。流速は0.4mL/minで泳動BufferはD-PBS(Wako)を使用し、波長280nmの吸光度を検出した。結果を図20に示す。この結果及び図12(d)から、gLのC144及びUL128のC162の点変異導入は、ゲルろ過クロマトグラフィーによる分離パターンに変化を及ぼさないことが示された。
取得したgH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131、gH(His-)/gL(C144S)-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131、gH(His-)/gL-Δd4/ UL128(C162S)-Δd4/UL130-Δd4/UL131融合タンパク質の粒子径分布を動的光散乱によって評価した。各組換えタンパクをPBS(SIGMA)で0.2又は0.3mg/mLに希釈し、Zetasizer Nano ZS(Malvern)により粒子径分析を実施した。測定には石英セルを用い、測定条件は温度25.0℃であった。これを図21に示す。なお、図21の(a)、(b)、(c)のピークは3回の結果を示し、表に示す数字は3回の平均値である。この結果から、gLのC144及びUL128のC162の点変異導入は、粒子径分布に影響を及ぼさないことが示された。
<抗原調製>
各種gB-ペンタマー構成タンパク質融合体発現プラスミドを組み合わせて共発現を行った。組み合わせた発現プラスミドは、gH(His-)/gL/UL128(C162S)-Δd4/UL130-Δd4/UL131-Δd4、gH(His-)/gL/UL128-Δd4/UL130-Δd4/UL131-Δd4の計2種類であった。また、精製は全てHCMVのgBタンパク質と同様の方法で行い、タンパク質精製品を得た。
取得した精製品の性状はゲル濾過クロマトグラフィーによって評価した。カラムはSuperdex200 Increase 5/150 GL(GE Healthcare)を使用し、濃度100μg/mLの各精製品を50μLアプライした。流速は0.4mL/minで泳動BufferはD-PBS(Wako)を使用し、波長280nmの吸光度を検出した。結果を図28に示す。この結果から、UL128のC162の点変異導入は、ゲルろ過クロマトグラフィーによるピークの溶出位置には変化を及ぼさないことが示された。
取得したgH(His-)/gL/UL128(C162S)-Δd4/UL130-Δd4/UL131-Δd4、gH(His-)/gL/UL128-Δd4/UL130-Δd4/UL131-Δd4融合タンパク質の粒子径分布を動的光散乱によって評価した。各組換えタンパクをPBS(SIGMA)で0.2又は0.3mg/mLに希釈し、Zetasizer Nano ZS(Malvern)により粒子径分析を実施した。測定には石英セルを用い、測定条件は温度25.0℃であった。これを図29に示す。なお、図29のピークは3回の結果を示し、表に示す数字は3回の平均値である。この結果から、UL128のC162の点変異導入は、粒子径分布に影響を及ぼさないことが示された。
<抗原調製>
各種gB-ペンタマー構成タンパク質融合体発現プラスミドを組み合わせて共発現を行った。組み合わせた発現プラスミドは、gBv9、ペンタマー、gH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131及びgH(His-)/gL/UL128-Δd4/UL130-Δd4/UL131-Δd4の計4種類であった。また、精製は全てHCMVのgBタンパク質と同様の方法で行い、タンパク質精製品を得た。
実施例2と同様の方法により、gBv9、ペンタマー、gH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131、gH(His-)/gL/UL128-Δd4/UL130-Δd4/UL131-Δd4、又は、salineをモルモット(Hartley)に免疫し、gBv9免疫血清、ペンタマー免疫血清、gH(His-)/gL-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131免疫血清、gH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131免疫血清、gH(His-)/gL/UL128-Δd4/UL130-Δd4/UL131-Δd4免疫血清及びsaline免疫血清を得た。
取得したgB-ペンタマー構成タンパク質融合体について、gBv9免疫血清、ペンタマー免疫血清、gH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131免疫血清、gH(His-)/gL/UL128-Δd4/UL130-Δd4/UL131-Δd4免疫血清及びsaline免疫血清に対する反応性(結合活性)を実施例2と同様の方法によって評価した。結合抗体価の評価結果を図30に示す。この結果から、gB-ペンタマー構成タンパク質融合体を投与することにより、gB抗体及びペンタマー抗体を誘導しうると考えられた。
取得したgBv9免疫血清、ペンタマー免疫血清、gH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131免疫血清、gH(His-)/gL/UL128-Δd4/UL130-Δd4/UL131-Δd4免疫血清及びsaline免疫血清を用いて、実施例3-1と同様の方法によって、中和抗体価を評価した。ただし、MRC-5細胞を用いた線維芽細胞中和試験、ARPE-19細胞を用いた上皮細胞中和試験のいずれもウイルスとしてARPE-19細胞にて継代馴化したHCMV AD-169株を使用した点が実施例3-1と異なる。中和抗体価の評価結果を図31に示す。
<gB-ペンタマー融合タンパク質の安定性評価>
各種gB-ペンタマー構成タンパク質融合体発現プラスミドを組み合わせて共発現を行った。組み合わせた発現プラスミドは、gBv9、ペンタマー、gH(His-)/gL-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131、gH(His-)/gL/UL128(C162S)-Δd4/UL130-Δd4/UL131-Δd4、gH(His-)/gL/UL128-Δd4/UL130-Δd4/UL131-Δd4、gH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131の計6種類であった。また、精製は全てHCMVのgBタンパク質と同様の方法で行い、タンパク質精製品を得た。
<抗原調製>
刺激抗原として、実施例4と同様の手順でgBv9、ペンタマー、gH(His-)/gL-Δd4/UL128(C162S)-Δd4/UL130-Δd4/UL131、gH(His-)/gL-Δd4/UL128-Δd4/UL130-Δd4/UL131、gH(His-)/gL/UL128-Δd4/UL130-Δd4/UL131-Δd4を発現精製した。
ヒトPBMCを用いた細胞性免疫評価は、Human IFN-γ ELISpotPLUS(MABTECH 3420-4HST-2)を用いたELISpot assayにより行った。ヒトPBMCはCTL社製のうち、HCMV抗原刺激に対するIFNγ誘導が認められるドナー23名のものを選択した。
<GPCMV-gBの調製>
モルモット経胎盤感染モデル系を用いて評価するため、モルモットに感染性を示すモルモットサイトメガロウイルス(GPCMV)を用いた。組換えGPCMV gBタンパク質は凝集体を含む場合があるため、凝集体を含まない、性状改善した改変GPCMV gBタンパク質を作製した。
次に、GPCMV 22122株に由来するペンタマーのエクトドメインを調製した。GPCMVペンタマーのエクトドメインの可溶性発現に関しては報告例がなかったため、HCMVペンタマーのエクトドメインの可溶性発現の報告例(非特許文献14)を参考に、以下に述べるようにデザインし、発現プラスミドを構築した。
次に、GPCMV-gBエクトドメイン改変体(配列番号17)の111-475aaと641-692aaとをGGGSGSGGG(配列番号20)の9アミノ酸でつなぎ、さらに、このC末端にHis-tag配列(配列番号30)を付加したものをGPCMV-Δd4(配列番号32)とし、GPCMV-Δd4をコードするDNA断片をPCRによって作製し、該DNA断片をpCAGGS1-dhfr-neoにクローニングした。また、GP75のエクトドメインをコードする遺伝子のC末端にHis-tagが付加されないものをデザインし、該タンパク質をコードするDNA断片をPCRによって作製し、該DNA断片をpCAGGS1-dhfr-neoにクローニングしたものをGP75(His-)とした。
次に、上記遺伝子をもとに、GP133のC末端側に15アミノ酸のリンカー(GGGGSGGGGSGGGGS)(配列番号23)を介してGPCMV-Δd4を連結した融合タンパク質をコードするDNA断片をPCRによって作製し、該DNA断片をpCAGGS1-dhfr-neoにクローニングしたものを、GP133-Δd4とした。また、GP129-Δd4をベースにGP115とGP129のジスルフィド結合部位を解離させると考えられるC167S改変を加えたものをGP129(C167S)-Δd4とした。発現時には各種gB-ペンタマー構成タンパク質融合体発現プラスミドを組み合わせて共発現を行った。組み合わせた発現プラスミドは、GP129(C167S)-Δd4/GP131-Δd4/GP133-Δd4であった。発現及び精製はGPCMV-gBと同様の方法で行い、GP129(C167S)-Δd4/GP131-Δd4/GP133-Δd4(GPCMV-Δd4-ULs)精製品を得た。
Hartleyモルモット(雌、5週齢)に対し、GPCMV-gB、GPCMVペンタマー、GP75(His-)/GP115-Δd4/GP129-Δd4/GP131-Δd4/GP133及びGP129(C167S)-Δd4/GP131-Δd4/GP133-Δd4(GPCMV-Δd4-ULs)を免疫した。各抗原は0.0016μg/匹になるように生理食塩水(大塚製薬)で調製し、アジュバントとして10v/v%のAlum(Invivogen vac-alu250)及び50μg/匹のCpG ODN1826を使用した。調製した抗原液を2週間間隔で2回、モルモットに筋肉内接種(100μL/片足、後肢両足投与)し、2回免疫の2週後からHartleyモルモット(♂、9週齢以上)と交配を行い、妊娠モルモットを作製した。この時、PBS投与群も同様にして交配を行った。免疫群、PBS群(saline群)ともに、妊娠4週齢時点で野生型GPCMVを1×106pfu/匹皮下投与し、3週後にペントバルビタールナトリウムにて安楽殺後、胎盤を採取した。採取した胎盤はgentleMACS(ミルテニーバイオテク)により破砕し、MagNA Pure96(Roche)にてDNA抽出を行った。得られたDNAは7500FastリアルタイムPCRシステム(Thermo)を用い、表2に示すプローブ、プライマーセット(国際出願PCT/JP2019/047966号)にてGPCMV gp83遺伝子、細胞由来のβactin遺伝子の定量を行った。
<経胎盤感染防御試験>
実施例6と同様の方法により、GPCMV-gB、GPCMVペンタマー、GP75(His-)/GP115-Δd4/GP129-Δd4/GP131-Δd4/GP133、GP75(His-)/GP115/GP129-Δd4/GP131-Δd4/GP133-Δd4を発現、調製した。
Claims (32)
- サイトメガロウイルス(CMV)のエンベロープ糖タンパク質B(gBタンパク質)とペンタマーとの融合タンパク質。
- 3つのgBタンパク質構成分子からなるgBタンパク質のホモ三量体と、5つのペンタマー構成分子からなるペンタマーのヘテロ五量体とが、少なくとも1つのgBタンパク質構成分子と少なくとも1つのペンタマー構成分子とがそれぞれ遺伝子工学的に融合することによって、タンパク質複合体を形成している、請求項1に記載の融合タンパク質。
- 少なくとも2つのgBタンパク質構成分子と、少なくとも2つのペンタマー構成分子とがそれぞれ遺伝子工学的に融合している、請求項2に記載の融合タンパク質。
- 3つのgBタンパク質構成分子と、5つのペンタマー構成分子のうちのいずれか3つのペンタマー構成分子とがそれぞれ遺伝子工学的に融合している、請求項2又は3に記載の融合タンパク質。
- 3つのgBタンパク質構成分子と、ペンタマー構成分子であるgL、UL128及びUL130とがそれぞれ遺伝子工学的に融合している、請求項2~4のいずれか一項に記載の融合タンパク質。
- 3つのgBタンパク質構成分子と、ペンタマー構成分子であるUL128、UL130及びUL131とがそれぞれ遺伝子工学的に融合している、請求項2~4のいずれか一項に記載の融合タンパク質。
- 遺伝子工学的な融合のうち少なくとも1つの融合が、gBタンパク質構成分子のN末端側にペンタマー構成分子が結合している融合である、請求項2~6のいずれか一項に記載の融合タンパク質。
- 遺伝子工学的な融合のうち少なくとも2つの融合が、gBタンパク質構成分子のN末端側にペンタマー構成分子が結合している融合である、請求項3~6のいずれか一項に記載の融合タンパク質。
- 3つの遺伝子工学的な融合がすべて、gBタンパク質構成分子のN末端側にペンタマー構成分子が結合している融合である、請求項4~6のいずれか一項に記載の融合タンパク質。
- gBタンパク質構成分子とペンタマー構成分子との間にリンカー及び/又はタグを有する、請求項2~9のいずれか一項に記載の融合タンパク質。
- リンカーが配列番号22記載のアミノ酸配列単位を1~3回繰り返したアミノ酸配列からなるリンカーである、請求項10に記載の融合タンパク質。
- gBタンパク質がCMV gBタンパク質のエクトドメインである、請求項1~11のいずれか一項に記載の融合タンパク質。
- gBタンパク質が配列番号15に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるヒトサイトメガロウイルス(HCMV)のgBタンパク質のエクトドメインである、請求項12に記載の融合タンパク質。
- gBタンパク質が、配列番号15に記載のアミノ酸配列からなるgBタンパク質のエクトドメインと80%以上の配列同一性を有するgBタンパク質のエクトドメインである、請求項12又は13に記載の融合タンパク質。
- gBタンパク質がドメインIVを欠失したgBタンパク質改変体である、請求項1~14のいずれか一項に記載の融合タンパク質。
- gBタンパク質が配列番号16に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるヒトサイトメガロウイルス(HCMV)のgBタンパク質のエクトドメインである、請求項15に記載の融合タンパク質。
- gBタンパク質が、配列番号16に記載のアミノ酸配列からなるgBタンパク質のエクトドメインと80%以上の配列同一性を有するgBタンパク質のエクトドメインである、請求項15又は16に記載の融合タンパク質。
- gBタンパク質が、野生型gBタンパク質に比べて、凝集体の形成を低減させ、ホモ三量体構造の割合を増加させる改変が導入されたgBタンパク質改変体である、請求項1~14のいずれか一項に記載の融合タンパク質。
- gBタンパク質が配列番号3に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるヒトサイトメガロウイルス(HCMV)のgBタンパク質のエクトドメインである、請求項18に記載の融合タンパク質。
- gBタンパク質が、配列番号3に記載のアミノ酸配列からなるgBタンパク質のエクトドメインと80%以上の配列同一性を有するgBタンパク質のエクトドメインである、請求項18又は19に記載の融合タンパク質。
- gBタンパク質が、野生型gBタンパク質に比べて、ヘッド領域の免疫原性を低減させる改変が導入されたgBタンパク質改変体である、請求項1~14のいずれか一項に記載の融合タンパク質。
- gBタンパク質が配列番号31に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるヒトサイトメガロウイルス(HCMV)のgBタンパク質のエクトドメインである、請求項21に記載の融合タンパク質。
- gBタンパク質が、配列番号31に記載のアミノ酸配列からなるgBタンパク質のエクトドメインと80%以上の配列同一性を有するgBタンパク質のエクトドメインである、請求項21又は22に記載の融合タンパク質。
- ペンタマーが、ヒトサイトメガロウイルス(HCMV)のgH、gL、UL128、UL130及びUL131からなる、請求項1~23のいずれか一項に記載の融合タンパク質。
- gHがgHタンパク質のエクトドメインである、請求項24に記載の融合タンパク質。
- ペンタマーが、
配列番号4に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるgH、
配列番号5に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるgL、
配列番号6に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるUL128、
配列番号7に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるUL130、及び
配列番号8に記載のアミノ酸配列又は該アミノ酸配列において1以上のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなるUL131
を含むヒトサイトメガロウイルス(HCMV)のペンタマータンパク質である、請求項24又は25に記載の融合タンパク質。 - ペンタマーが、
配列番号4に記載のアミノ酸配列からなるgHと80%以上の配列同一性を有するgH、
配列番号5に記載のアミノ酸配列からなるgLと80%以上の配列同一性を有するgL、
配列番号6に記載のアミノ酸配列からなるUL128と80%以上の配列同一性を有するUL128、
配列番号7に記載のアミノ酸配列からなるUL130と80%以上の配列同一性を有するUL130、及び
配列番号8に記載のアミノ酸配列からなるUL131と80%以上の配列同一性を有するUL131
を含むHCMVのペンタマータンパク質である、請求項24~26のいずれか一項に記載の融合タンパク質。 - 請求項1~27のいずれか一項に記載の融合タンパク質をコードする核酸断片。
- 請求項28に記載の核酸断片を含む組換え発現ベクター。
- 請求項28に記載の核酸断片又は請求項29に記載の組換え発現ベクターが導入された形質転換体。
- 請求項1~27のいずれか一項に記載の融合タンパク質を含む、CMVの感染を予防又は治療するためのワクチン。
- CMVの感染がCMVの先天性感染である、請求項31に記載のワクチン。
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