WO2017179726A1

WO2017179726A1 - Dengue vaccine antigen inducing neutralizing antibody but inhibiting induction of infection-enhancing antibody

Info

Publication number: WO2017179726A1
Application number: PCT/JP2017/015384
Authority: WO
Inventors: 英二小西; 敦史山中; ポングラマラマスータ; パナンティップピタクサジャクン
Original assignee: 国立大学法人大阪大学; マヒドンユニバーシティ
Priority date: 2016-04-15
Filing date: 2017-04-14
Publication date: 2017-10-19
Also published as: JPWO2017179726A1

Abstract

A dengue vaccine antigen, which induces a neutralizing antibody but inhibits the induction of an infection-enhancing antibody, characterized by containing an envelope protein having a mutation at the 107th or 87th amino acid in the amino acid sequence of dengue virus envelope protein. The dengue vaccine antigen according to the present invention induces a neutralizing antibody but does not inhibit the induction of an infection-enhancing antibody. Thus, the dengue vaccine antigen is expected to exhibit high preventive efficacy even at a low vaccine dose and be appropriately usable for treating or preventing dengue fever or dengue hemorrhagic fever at high safety.

Description

Dengue vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies

The present invention relates to a dengue vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies, a dengue vaccine that contains or expresses the antigen, and a method for obtaining the antigen or dengue virus that expresses the antigen. .

Dengue virus is a virus belonging to the family Flaviviridae and Flavivirus. Flaviviruses include Japanese encephalitis virus, yellow fever virus, West Nile virus and the like in addition to dengue virus. Infection with dengue virus is often subclinical, but when it develops, it exhibits a wide range of symptoms ranging from mild transient fever to death. Dengue fever is a disease in which high fever accompanied by pain such as headache, muscle pain, joint pain, orbital pain lasts for about one week. A severe type that exhibits increased vascular permeability and bleeding tendency in addition to this symptom is called dengue hemorrhagic fever.

There are four types of dengue virus, “species” in biological classification, from type 1 to type 4 (these are called serotypes), all of which cause dengue fever or dengue hemorrhagic fever. Epidemiologically, dengue fever often develops in the first infection. However, if a second infection occurs after a certain period of time from the first infection, it will be protected if it is the same serotype as the first infection, but it may become severe if it is infected with a dengue virus of a different serotype It has been known.

Dengue fever and dengue hemorrhagic fever are the most important mosquito-borne infections that the world is trying to control, but there is still no dengue vaccine approved by WHO. Currently, various strategies such as attenuated vaccines, inactivated vaccines and subunit vaccines are under development, and six candidate vaccines are in the licensed or clinical trial stage. Among the diseases caused by flavivirus, vaccines have already been marketed against yellow fever, Japanese encephalitis and tick-borne encephalitis, all of which are types of vaccines that induce neutralizing antibodies. Neutralizing antibodies are the main defense factors, and by effectively reducing the amount of virus in the blood, the virus's pathogenicity, internal movement, and transmission efficiency to vector mosquitoes are suppressed, and individuals and society are protected from disease. protect.

The development of neutralizing antibody-inducing vaccines has been promoted in the same way for dengue vaccines. However, unlike other flaviviruses, neutralizing antibodies against dengue virus show infection enhancing activity at low concentrations. The antibody-dependent infection enhancement phenomenon is the most promising hypothesis for explaining dengue severity (Non-patent Document 1). Therefore, many researchers question the safety of dengue vaccines, and as an example, when the level of neutralizing antibodies induced by the vaccine decreases with time, it becomes an infection-enhancing antibody and becomes severe due to vaccination (Non-Patent Document 2).

The most developed dengue vaccine is Sanofi Pasteur's recombinant chimeric vaccine. Phase 3 clinical trials have already been completed and licenses have been obtained in three countries. Because dengue does not have an appropriate animal model to represent human pathology, and there is no established immunological index of protection, the exact protective efficacy of a candidate vaccine can only be determined with a large number of human subjects. This phase 3 clinical trial therefore provided the world's first dengue vaccine efficacy data. Moreover, since it was a large-scale test targeting 30,000 people in 10 countries in Asia and the United States, this data is reliable data that surpassed individual differences and environmental differences. Meanwhile, other candidate vaccines are in Phase II clinical trials, which are currently the only efficacy trials in the world. Although this vaccine had good results in preclinical studies, the expectation for clinical studies was great, but the protective efficacy was about 60% as a whole (Non-patent Documents 3 and 4). It was a surprising result that despite the high induction of neutralizing antibodies in all cases, the actual protective efficacy was low. This result was observed data for 2 years after the 3rd vaccination, but recently data from 3 to 4 years after vaccination was recently released, which shows that the overall efficacy is about 45 in the 9-year-old population. % (Non-patent Document 5). In addition, some groups increased the risk of illness due to vaccination.

Although the reason for its low efficacy has been discussed, one factor is the induction of infection-enhancing antibodies. In the above flavivirus diseases in which an approved vaccine is present, the infection enhancement phenomenon is not observed in nature, and the efficacy of the vaccine exceeds 80 to 90%. On the other hand, dengue fever has an infection-increasing phenomenon, and in endemic areas all dengue antibody-positive individuals possess infection-enhancing antibodies (Non-patent Document 6), so that infection-enhancing antibodies are induced by natural infection. Is clear. Therefore, as with natural infections, infection-enhancing antibodies were also induced together with neutralizing antibodies after vaccination, and this seems to have reduced efficacy despite the high neutralizing antibody induction.

The strain used as a vaccine antigen is usually a strain isolated from nature. The technology has been passed down since the days of Jenner and Pasteur. However, in the case of a dengue vaccine, since the natural strain itself induces an infection-enhancing antibody, it is not appropriate to use it as a vaccine antigen as it is. Two successful cases have been reported so far in which the neutralization antibody induction ability was left behind by artificial alteration of the envelope antigen epitope, leading to a decrease in infection-enhancing antibody induction (Non-patent Documents 7 and 8). Therefore, it is possible to control the induction of infection-enhancing antibodies by epitope modification. However, both are strategies for estimating mutant amino acids with the help of bioinformatics in methods of structural biology and protein computational science, and proof that the estimated amino acids are actually living proteins There is no. If it is not a harmonized envelope (E) protein in the virus particle structure, problems in vaccine production such as immunogenicity and low yield arise. Specifically, for example, when producing an inactivated vaccine, it is necessary to proliferate a large amount of infectious virus particles in cells.

An object of the present invention is to provide a dengue vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies, and dengue vaccines that contain or express the antigens. Another object of the present invention is to provide a method for obtaining a dengue vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies, or a dengue virus that expresses the antigens.

The present invention includes the following inventions in order to solve the above problems.
[1] A dengue vaccine antigen that induces neutralizing antibody but suppresses induction of infection-enhancing antibody, and includes an envelope protein having a mutation in the 107th or 87th amino acid of the amino acid sequence of the dengue virus envelope protein Dengue vaccine antigen characterized by
[2] The dengue vaccine antigen according to [1], wherein leucine at position 107 is substituted with one selected from phenylalanine, tryptophan, methionine, proline, alanine, valine and isoleucine.
[3] The dengue vaccine antigen according to [2], wherein leucine at position 107 is substituted with phenylalanine.
[4] The above-mentioned [1] to [3], wherein the envelope protein having a mutation at the 107th amino acid comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4 The dengue vaccine antigen according to any one of the above.
[5] The aspartic acid at position 87 is substituted with one selected from glutamic acid, tyrosine, cysteine, arginine, histidine, lysine, serine, threonine, glutamine, asparagine and glycine. Dengue vaccine antigen according to 1].
[6] The dengue vaccine antigen according to [5], wherein aspartic acid at position 87 is substituted with asparagine.
[7] The above-mentioned [1], [5] or [5], wherein the envelope protein having a mutation at the 87th amino acid sequence comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 Dengue vaccine antigen according to [6].
[8] A dengue vaccine comprising or expressing the dengue vaccine antigen according to any one of [1] to [7].
[9] An envelope protein having a mutation in the 107th or 87th amino acid of the amino acid sequence of a dengue virus envelope protein, a nucleic acid encoding the envelope protein, a vector containing the nucleic acid encoding the envelope protein, or the envelope protein The dengue vaccine according to [8] above, which contains a dengue virus that expresses.
[10] The dengue vaccine according to [8] or [9] above, comprising a vaccine component against pathogens other than dengue virus.
[11] A method for obtaining a dengue vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies, or a dengue virus that expresses the antigens, comprising the following steps (1) to (5) Features method:
(1) A step of obtaining a monoclonal antibody having an infection enhancing activity but not a neutralizing activity, using a dengue virus envelope protein as an antigen,
(2) obtaining a monoclonal antibody having neutralizing activity by changing the class or subclass of the monoclonal antibody obtained in (1),
(3) obtaining a mutant dengue virus that is not neutralized by the monoclonal antibody by culturing dengue virus-infected cells in the presence of the monoclonal antibody having neutralizing activity obtained in (2);
(4) a step of confirming a mutation occurring in the envelope protein of the mutant dengue virus obtained in (3), and (5) that the envelope protein of the mutant dengue virus obtained in (3) suppresses the induction of the infection-enhancing antibody. The process of confirming.
[12] Dengue vaccine antigen that induces neutralizing antibody but suppresses induction of infection-enhancing antibody, and includes dengue virus envelope protein having mutations at

amino acids

107 and 87 of the amino acid sequence and amino acid 2 A dengue vaccine antigen comprising a dengue virus precursor membrane protein having no mutation.
[13] A dengue vaccine comprising or expressing the dengue vaccine antigen according to [12].

The present invention also includes the following inventions.
[14] The dengue vaccine antigen according to any one of [1] to [7] and [12] or the dengue vaccine according to any one of [8] to [10] and [13] A pharmaceutical composition for preventing or treating dengue fever or dengue hemorrhagic fever.
[15] The administration of the dengue vaccine antigen according to any one of [1] to [7] and [12] or the dengue vaccine according to any one of [8] to [10] and [13]. A method for preventing or treating dengue fever or dengue hemorrhagic fever.
[16] The dengue vaccine antigen according to any one of the above [1] to [7], [12] or the above [8] to [10], for preventing and / or treating dengue fever or dengue hemorrhagic fever 13] Use of the dengue vaccine according to any one of the above.
[17] The dengue vaccine antigen according to any one of the above [1] to [7], [12] or the above [8] to [10] for use in the prevention and / or treatment of dengue fever or dengue hemorrhagic fever [13] The dengue vaccine according to any one of the above.
[18] The dengue vaccine antigen according to any one of [1] to [7] and [12] or the above [8] to [8] for producing a medicament for preventing and / or treating dengue fever or dengue hemorrhagic fever Use of the dengue vaccine in any one of 10] and [13].

The present invention can provide a dengue vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies, and dengue vaccines that contain or express the antigens. In addition, it is possible to provide a method for obtaining a dengue vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies or dengue viruses that express the antigens. Since the dengue vaccine of the present invention can suppress the induction of infection-enhancing antibodies, there is another concern in that it can exert protective efficacy by enhancing the effect of neutralizing antibody activity that is inhibited in the presence of infection-enhancing antibodies. It is very useful in that it can be prevented from becoming serious.

FIG. 1 is a diagram showing the results of confirming the infection enhancing activity and neutralizing activity of a monoclonal antibody against dengue type 1 virus Mochizuki strain, and the left shows neutralizing activity at a high concentration and infection enhancing activity at a low concentration. As a result of a representative example of a monoclonal antibody (D1-I-15C12), the result of a representative example of a monoclonal antibody (D1-IV-7F4) showing only neutralizing activity in the center, the right shows a monoclonal antibody showing only infection enhancing activity This is a result of a representative example (D1-V-3H12). FIG. 2 is a diagram showing the results of confirming the neutralizing activity against dengue type 1 virus Mochizuki strains of antibodies (3H12-IgG2a and 3H12-IgG2b) substituted with a subclass of 3H12 antibody that showed only infection-enhancing activity. FIG. 3 shows enhanced infection of 3H12 antibody against escape mutant virus obtained by subculturing Vero cells infected with Dengue type 1 virus Mochizuki strain in the presence of an antibody (3H12-IgG2b) showing neutralizing activity. It is a figure which shows the result which confirmed activity. FIG. 4 shows enhanced infection of the 3H12 antibody against single infectious particles (SRIP) having mutations at position 87 (E87) and position 107 (E107) of the envelope protein found in escape mutant viruses. It is a figure which shows the result which confirmed activity. FIG. 5 shows immunization of mice with plasmid DNA containing DNA encoding the envelope protein of Dengue type 1 virus Mochizuki strain having a mutation in E87 and / or E107. It is a figure which shows the result of having confirmed the neutralization activity with respect to a dengue type 1 virus Mochizuki strain, and the infection enhancement activity, A is the result of the neutralization activity, B is the result of the infection enhancement activity. FIG. 6 shows immunization of mice with plasmid DNA containing DNA encoding the envelope protein of Dengue type 1 virus Mochizuki strain having a mutation in E87 and / or E107. It is a figure which shows the result of having confirmed the neutralization activity with respect to dengue type 2 virus (New Guinea strain C), dengue type 3 virus (H87 strain) or dengue type 4 virus (H241 strain), and the infection enhancing activity, A is neutralization As a result of activity, B is the result of infection-enhancing activity. FIG. 7 shows a case in which a mutation was introduced into E87 or E107 of the envelope protein (E antigen) of dengue type 2 virus (New Guinea C strain), dengue type 3 virus (H87 strain), and dengue type 4 virus (H241 strain). It is a figure which shows the result of having confirmed the infection enhancement activity of 3H12 antibody with respect to infectious particle | grains (SRIP). FIG. 8 shows an envelope protein (E antigen) in which mutations are introduced into each of E87 or E107 of the envelope proteins of Dengue type 2 virus (New Guinea C strain), Dengue type 3 virus (H87 strain), and Dengue type 4 virus (H241 strain). ) Is used to immunize mice, and the antibody in the obtained serum confirms the infection enhancing activity against the corresponding serotype virus. FIG. 9 shows immunization of mice with plasmid DNA expressing proteins in which E107 or E87 amino acids in the E region of dengue type 1 virus Mochizuki strain were introduced with single point mutations into various amino acids. It is a figure which shows the result of the infection enhancement activity with respect to all the serotype viruses of the antibody of. FIG. 10 is a diagram showing the results of confirming the infection-enhancing activity of the 3H12 antibody against SRIP in which a mutation was introduced into E87 or E107 of the dengue virus envelope protein of different strains. FIG. 11 shows dengue type 1 virus (genotype I, BDV-1), dengue type 2 virus (genotype American, BDV-2), dengue type 3 virus (genotype I, BDV-3), and dengue type 4 virus. All serotype viruses of the antibodies in the sera obtained by immunizing mice using plasmid DNA expressing envelope proteins in which mutations were introduced into E87 or E107 of each envelope protein of (genotype III, BDV-4) It is a figure which shows the result of the neutralization activity with respect to. FIG. 12 shows dengue type 1 virus (genotype I, BDV-1), dengue type 2 virus (genotype American, BDV-2), dengue type 3 virus (genotype I, BDV-3), and dengue type 4 virus. All serotype viruses of the antibodies in the sera obtained by immunizing mice using plasmid DNA expressing envelope proteins in which mutations were introduced into E87 or E107 of each envelope protein of (genotype III, BDV-4) It is a figure which shows the result of the infection enhancement activity with respect to. FIG. 13 shows dengue type 1 virus (genotype I, BDV-1), dengue type 2 virus (genotype American, BDV-2), dengue type 3 virus (genotype I, BDV-3), and dengue type 4 virus. Serum antibodies obtained by immunizing mice using a dengue tetravalent vaccine mixed with plasmid DNA expressing envelope proteins having mutations in E87 or E107 of each envelope protein of (genotype III, BDV-4) It is a figure which shows the result of the neutralization activity with respect to all the serotype viruses. FIG. 14 shows dengue type 1 virus (genotype I, BDV-1), dengue type 2 virus (genotype American, BDV-2), dengue type 3 virus (genotype I, BDV-3), and dengue type 4 virus. Serum antibodies obtained by immunizing mice using a dengue tetravalent vaccine mixed with plasmid DNA expressing envelope proteins having mutations in E87 or E107 of each envelope protein of (genotype III, BDV-4) It is a figure which shows the result of the infection enhancement activity with respect to all the serotype viruses of all. FIG. 15 shows a method for expressing a mouse using plasmid DNA expressing a protein in which a mutation is introduced into the second amino acid of the precursor membrane (prM) protein of dengue type 1 virus (Mochizuki strain) and / or envelope proteins E87 and E107. It is a figure which shows the result of the neutralization activity with respect to all the serotype viruses of the antibody in the sera obtained after immunization. FIG. 16 shows that a mouse is treated with a plasmid DNA expressing a protein in which a mutation is introduced into both the amino acid at position 2 of the precursor membrane (prM) protein of dengue type 1 virus (Mochizuki strain) and / or envelope proteins E87 and E107. It is a figure which shows the result of the infection enhancement activity with respect to all the serotype viruses of the antibody in the obtained sera after immunization. FIG. 17 shows that the 3H12 antibody against single infectious particles (SRIP) having mutations in the amino acid position 2 of the precursor membrane (prM) protein of dengue type 1 virus (Mochizuki strain) and / or envelope proteins E87 and E107. It is a figure which shows the result of having confirmed the infection enhancement activity.

[Dengue vaccine antigen]
The present invention provides dengue vaccine antigens that induce neutralizing antibodies but suppress induction of infection-enhancing antibodies. In the present specification, “neutralizing antibody” means an antibody having an activity of inhibiting dengue virus infection of cells, and “infection enhancing antibody” means an antibody having an activity of enhancing dengue virus infection of cells. . Here, “inducing neutralizing antibody but suppressing induction of infection-enhancing antibody” means that the dengue vaccine antigen of the present invention has neutralizing activity against dengue virus and has suppressed infection-enhancing activity. Hereinafter, it may be simply referred to as “vaccine antigen” or “dengue virus antigen”.

The vaccine antigen of the present invention only needs to contain an envelope protein in which the amino acid at

position

107 or 87 of the amino acid sequence of the dengue virus envelope protein is mutated (hereinafter, for convenience, the vaccine antigen is referred to as the aspect A). Sometimes referred to as vaccine antigen). That is, the vaccine antigen of aspect A of the present invention includes an envelope protein in which the amino acid at position 107 of the dengue virus envelope protein is mutated and an amino acid at position 87 in the amino acid sequence of the dengue virus envelope protein. The thing containing an envelope protein is mentioned. The amino acid sequence of the basic wild-type dengue virus envelope protein (dengue virus envelope protein having no mutation at the 107th and 87th amino acids) may be any amino acid sequence as long as it is the amino acid sequence of the dengue virus envelope protein. Moreover, the vaccine antigen of the present invention may contain an envelope protein derived from dengue virus of any serotype of type 1, type 2, type 3, and type 4. That is, in the present invention, when the term “dengue virus” is simply used, the serotype is not particularly limited and includes all serotypes. As an example, the amino acid sequence of the envelope protein of the dengue type 1 virus Mochizuki strain is shown in SEQ ID NO: 1.

Dengue virus is a virus belonging to the genus Flavivirus (flavivirus), and has a + strand RNA of about 11 kb in length as a genome. There is one open reading frame on this genome, and three structural proteins (C, prM, E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) are in this order. It is coded. The third E protein from the upstream (5 ′ side) is an envelope protein. The base sequence of the genome of the dengue type 1 virus Mochizuki strain is shown in SEQ ID NO: 2, and the amino acid sequence of the full-length protein encoded by the genome is shown in SEQ ID NO: 3, respectively. In the amino acid sequence of the full-length protein of Mochizuki strain (SEQ ID NO: 3), the envelope protein corresponds to positions 281 to 775, and the base sequence encoding the envelope protein is from position 935 to the base sequence represented by SEQ ID NO: 2. It corresponds to the 2419th place. Therefore, the 107th position of the amino acid sequence represented by SEQ ID NO: 1 corresponds to the 387th position of the amino acid sequence represented by SEQ ID NO: 3. The boundary between the prM protein and the envelope protein (E protein), and the boundary between the envelope protein (E protein) and the NS1 protein are both cleaved by a signalase from the host cell.
Table 1 below shows typical dengue virus strains of each serotype and their sequence information (accession number). Table 2 also shows the strains shown in Table 1 including information on the country of detection, year of isolation, and genotype.

As long as the vaccine antigen of aspect A of the present invention induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies, the mutation at position 107 of the envelope protein may be any mutation, but position 107 It is preferable that the amino acid is a mutation in which another amino acid is substituted. The combination of the amino acid before substitution and the amino acid after substitution is not limited as long as it is a vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies. Preferably, the leucine at position 107 of the envelope protein is a vaccine antigen substituted with another amino acid, more preferably a vaccine where the leucine at position 107 of the envelope protein is replaced with another hydrophobic amino acid It is an antigen. Other hydrophobic amino acids include phenylalanine, tryptophan, methionine, proline, alanine, valine and isoleucine. Therefore, in the vaccine antigen of aspect A of the present invention, the leucine at position 107 is more preferably substituted with one selected from phenylalanine, tryptophan, methionine, proline, alanine, valine and isoleucine. More preferably, it is a vaccine antigen having a mutation in which leucine at position 107 of the envelope protein is substituted with phenylalanine. Further, it may be a vaccine antigen in which the leucine at position 107 of the envelope protein is substituted with a hydrophilic amino acid, such as glutamic acid, tyrosine, cysteine, arginine, histidine, lysine, serine, threonine, glutamine, asparagine, etc. Illustrated are substituted vaccine antigens.

As long as the vaccine antigen of aspect A of the present invention induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies, the mutation at position 87 of the envelope protein may be any mutation, but position 87 It is preferable that the amino acid is a mutation in which another amino acid is substituted. The combination of the amino acid before substitution and the amino acid after substitution is not limited as long as it is a vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies. Preferably, the aspartic acid at position 87 of the envelope protein is a vaccine antigen substituted with an amino acid containing another polar amino acid, and the aspartic acid at position 87 of the envelope protein is glutamic acid, tyrosine, cysteine, arginine, More preferably, it is substituted with one selected from histidine, lysine, serine, threonine, glutamine, asparagine and glycine. In addition, it is more preferable that the aspartic acid at position 87 of the envelope protein is substituted with a hydrophobic amino acid such as phenylalanine, tryptophan, methionine, proline, alanine, valine, isoleucine and the like. More preferably, the vaccine antigen has a mutation in which aspartic acid at position 87 of the envelope protein is substituted with asparagine.

The vaccine antigen of aspect A of the present invention includes an envelope protein having an amino acid mutation at a position other than position 107 or position 87 as long as it induces neutralizing antibodies but can suppress induction of infection-enhancing antibodies. It may be. Positions other than the 107th and 87th positions are not particularly limited, and the type of amino acid to be substituted is not particularly limited. Further, the number of amino acid mutations at positions other than the 107th position or the 87th position is not limited, and may be 1, 2, 3, 4, or 5. The upper limit of the number of mutations is not particularly limited, but is preferably 50 or less including the amino acid mutation at

position

107 or 87, more preferably 40 or less, further preferably 30 or less, and further preferably 20 or less. More preferably, 10 or less.

As an aspect of the vaccine antigen of aspect A of the present invention, it is preferable that the vaccine antigen contains an envelope protein in which the amino acid at

position

107 or 87 of the amino acid sequence of the envelope protein of the Dengue 1 virus Mochizuki strain is mutated. Examples of such a vaccine antigen include a vaccine antigen comprising an envelope protein consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5. The amino acid sequence represented by SEQ ID NO: 4 is an amino acid sequence obtained by substituting leucine at position 107 with phenylalanine in the amino acid sequence of the envelope protein of the dengue type 1 virus Mochizuki strain represented by SEQ ID NO: 1, and is represented by SEQ ID NO: 5. The amino acid sequence is an amino acid sequence in which aspartic acid at position 87 is substituted with asparagine in the amino acid sequence of the envelope protein of the dengue type 1 virus Mochizuki strain represented by SEQ ID NO: 1. It has been confirmed that an envelope protein comprising these amino acid sequences is an envelope protein that induces neutralizing antibodies but can suppress induction of infection-enhancing antibodies.

Examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 4 include an amino acid sequence in which leucine at position 107 is substituted with phenylalanine in the amino acid sequence of dengue type 1 virus other than Mochizuki. Further, for example, in the amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence in which 1 to several amino acids are deleted, substituted or added at positions other than the 87th and 107th positions can be mentioned. Examples of the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 include an amino acid sequence in which aspartic acid at position 87 is substituted with asparagine in the amino acid sequence of dengue type 1 virus other than Mochizuki. In addition, for example, in the amino acid sequence represented by SEQ ID NO: 5, an amino acid sequence in which 1 to several amino acids are deleted, substituted or added at positions other than the 87th and 107th positions can be mentioned. “One to several amino acids are deleted, substituted or added” means that the number can be deleted, substituted or added by a known mutant polypeptide production method such as site-directed mutagenesis (preferably, 20 amino acids or less, more preferably 10 amino acids or less, further preferably 7 amino acids or less, further preferably 5 amino acids or less, more preferably 3 amino acids or less, still more preferably 2 amino acids or less, and even more preferably 1 amino acid). , Means substituted or added. Such a mutant protein is not limited to a protein having a mutation artificially introduced by a known mutant polypeptide production method, and may be a protein obtained by isolating and purifying a naturally occurring mutant protein. . A substantially identical amino acid sequence is at least 60% identical, preferably at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 , 96%, 97%, 98%, 99% or 99.5% identical amino acid sequence (provided that amino acid sequence substantially identical to SEQ ID NO: 4 is phenylalanine at position 107 and substantially identical to SEQ ID NO: 5 The identical amino acid sequence is at position 87 asparagine). The sequence identity can be determined according to a known method.

An envelope protein consisting of an amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 4 or 5 is treated with a neutralizing antibody in the same manner as an envelope protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5. It must be an envelope protein that induces but can suppress the induction of infection-enhancing antibodies. It is described in, for example, Examples that an envelope protein consisting of an amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 4 or 5 induces neutralizing antibodies and suppresses induction of infection-enhancing antibodies. This can be confirmed by performing the experiment.

The present invention also includes, as another embodiment of the dengue vaccine antigen, a precursor membrane (prM) protein adjacent to the upstream of the envelope protein in addition to the dengue virus envelope protein, which induces neutralizing antibodies but induces infection-enhancing antibodies. Provide something to suppress. Hereinafter, for convenience, the vaccine antigen may be referred to as the vaccine antigen of aspect B. The amino acid sequences of the basic wild-type dengue virus precursor membrane (prM) protein and the envelope protein may be any amino acid sequence as long as they are dengue virus-derived amino acid sequences, type 1, type 2, type 3, and type 4. Any of these serotypes may be derived from dengue virus, and the combination of serotypes is not particularly limited. Preferably, the prM protein and the envelope protein are the same serotype.

The vaccine antigen of embodiment B is characterized in that both the 107th and 87th amino acids of the envelope protein are mutated and the second amino acid of the prM protein is not mutated. Here, the mutation at position 107 and the mutation at position 87 may be any mutation, and the mutation in the vaccine antigen of aspect A can be referred to. As the vaccine antigen of aspect B, specifically, for example, leucine at position 107 is substituted with one selected from phenylalanine, tryptophan, methionine, proline, alanine, valine and isoleucine, and asparagine at position 87 Dengue vaccine antigen in which the acid is substituted with one selected from glutamic acid, tyrosine, cysteine, arginine, histidine, lysine, serine, threonine, glutamine, asparagine and glycine, and the amino acid at position 2 of the prM protein is not mutated A dengue vaccine antigen in which leucine at position 107 is substituted with phenylalanine, aspartic acid at position 87 is replaced with asparagine, and the amino acid at position 2 of the prM protein is not mutated is more preferable.

In addition to the 107th and 87th amino acid mutations, the envelope protein in the vaccine antigen of aspect B may have amino acid mutations at other positions. The position is not particularly limited, and the type of amino acid to be substituted is not particularly limited. Further, the number of amino acid mutations is not particularly limited as long as the number is the same as that in the aspect A.

The prM protein in the vaccine antigen of aspect B may have an amino acid mutation at other positions except that the amino acid at the second position is not mutated. The position is not particularly limited, and the type of amino acid to be substituted is not particularly limited. Further, the number of amino acid mutations is not particularly limited, and may be 1, 2, 3, 4 or 5, for example, and the upper limit is preferably 50 or less, more preferably 40 or less, and 30 The number is more preferably, 20 or less is more preferable, and 10 or less is more preferable.

In one aspect of the vaccine antigen of aspect B, the amino acids at

positions

107 and 87 of the amino acid sequence of the prM protein and envelope protein having no mutation at the second position of the amino acid sequence of the prM protein of the dengue type 1 virus Mochizuki strain Examples include those containing a mutated envelope protein. In the amino acid sequence of the full-length protein of Mochizuki strain (SEQ ID NO: 3), the prM protein corresponds to positions 115 to 280, and the envelope protein corresponds to positions 281 to 775. The base sequence encoding the prM protein is located at positions 437 to 934 of the base sequence shown in SEQ ID NO: 2, and the base sequence encoding the envelope protein is located at positions 935 to 934 of the base sequence shown in SEQ ID NO: 2. It corresponds to 2419th place. Examples of such a vaccine antigen include a vaccine antigen containing a protein having the same or substantially the same amino acid sequence as that shown in SEQ ID NO: 6.

As the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 6, for example, in the amino acid sequence of dengue type 1 virus other than Mochizuki, there is no mutation at the second position of the amino acid sequence of prM protein, and Examples include amino acid sequences in which leucine at position 107 of the amino acid sequence of the envelope protein is substituted with phenylalanine and aspartic acid at position 87 with asparagine. In addition, for example, in the amino acid sequence represented by SEQ ID NO: 6, 1 to several amino acids are deleted, substituted or added at positions other than position 2 of the prM protein,

positions

107 and 87 of the envelope protein. The amino acid sequence made up is mentioned. “One to several amino acids are deleted, substituted or added” means that the number can be deleted, substituted or added by a known mutant polypeptide production method such as site-directed mutagenesis (preferably, 20 amino acids or less, more preferably 10 amino acids or less, further preferably 7 amino acids or less, further preferably 5 amino acids or less, more preferably 3 amino acids or less, still more preferably 2 amino acids or less, and even more preferably 1 amino acid). , Means substituted or added. Such a mutant protein is not limited to a protein having a mutation artificially introduced by a known mutant polypeptide production method, and may be a protein obtained by isolating and purifying a naturally occurring mutant protein. . A substantially identical amino acid sequence is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 6, preferably at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, Amino acid sequence that is 97%, 98%, 99%, or 99.5% identical (provided that the 2nd position of the prM protein is not mutated, the 107th position of the envelope protein is phenylalanine, and the 87th position is asparagine) Can be mentioned. The sequence identity can be determined according to a known method. In addition, a protein comprising an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 6 induces a neutralizing antibody but induces an infection-enhancing antibody, similar to the protein comprising the amino acid sequence represented by SEQ ID NO: 6. It is required to have an action of suppressing the above. Such an effect can be confirmed, for example, by performing an experiment described in the examples.

[Dengue vaccine]
The present invention provides a dengue vaccine containing or expressing the dengue vaccine antigen of the present invention. Here, the dengue vaccine antigen may be the dengue vaccine antigen of aspect A or the dengue vaccine antigen of aspect B. The dengue vaccine of the present invention induces neutralizing antibodies against dengue virus, but since the induction of infection-enhancing antibodies is suppressed, it can effectively prevent the onset of dengue fever and suppress the seriousness that is concerned. Very useful in that it can. Dengue vaccines that have been developed so far are used as vaccine antigens without basically modifying the envelope protein of strains isolated from the natural world, and thus induce infection-enhancing antibodies together with neutralizing antibodies in the same manner as natural infections. This is considered to be a cause of low vaccine efficacy, and is a reason why there are concerns about the seriousness resulting from vaccination. Therefore, this invention contributes to raising the protective efficacy and safety | security of a dengue vaccine markedly. In addition, since neutralizing antibody activity antagonizes infection enhancing activity, neutralizing antibody activity increases due to suppression of infection enhancing activity. From this, even if it is a low vaccine dose, a high protective efficacy can be expected, and it is highly useful in that the cost can be reduced.

In one embodiment of the dengue vaccine of the present invention, as a vaccine component, for example, an envelope protein having a mutation at the 107th or 87th amino acid in the amino acid sequence of the dengue virus envelope protein (hereinafter referred to as “mutant envelope protein A”). ), A nucleic acid encoding the mutant envelope protein A, a vector containing a nucleic acid encoding the mutant envelope protein A, a dengue virus expressing the mutant envelope protein A, and the like.

As the mutant envelope protein A, the full length of the envelope protein or a fragment thereof may be used. When using a fragment of the mutant envelope protein A, the amino acid sequence of the envelope protein contains a non-mutant amino acid at position 107 and a mutant amino acid at position 87, or a mutant amino acid at position 107 and a non-mutated amino acid at position 87. It is preferable to use a fragment having a length that contains the three-dimensional structure of the envelope protein. For example, a fragment having 300 or more amino acid residues, preferably 350 or more, more preferably 400 or more, and still more preferably 450 or more can be suitably used. Mutant envelope protein A may contain an amino acid sequence other than the envelope protein. Examples of amino acid sequences other than the envelope protein include marker sequences, tag sequences, amino acid sequences of other proteins encoded in the dengue virus genome, and the like.

Mutant envelope protein A is constructed by constructing a recombinant expression vector into which a gene encoding mutant envelope protein A is inserted by known genetic engineering techniques so that the gene can be expressed, and introducing the vector into a suitable host cell for expression as a recombinant protein. Can be produced by purification. In addition, the mutant envelope protein A of the present invention comprises a gene encoding the mutant envelope protein A of the present invention and a known In vitro transcription / translation system (for example, a cell-free protein synthesis system derived from rabbit reticulocytes, wheat germ or Escherichia coli, etc. ) Can be used. As for the base sequence of the gene encoding the mutant envelope protein A, the base sequence of the dengue virus genome is obtained from a known database (NCBI, etc.), and the amino acid at position 107 of the envelope protein is mutated based on the obtained base sequence. Alternatively, the amino acid at position 87 of the envelope protein can be designed to have a mutation.

The nucleic acid encoding the mutant envelope protein A is obtained by using known genetic engineering techniques such as PCR and site-directed mutagenesis based on the base sequence of the gene encoding the mutant envelope protein A designed as described above. It can be acquired by using. The nucleic acid can be present in the form of RNA (eg, genomic RNA, mRNA) or in the form of DNA (eg, genomic DNA, cDNA). The nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be any of double-stranded DNA, double-stranded RNA, or a hybrid of DNA and RNA. In the case of a single strand, it may be either a coding strand (sense strand) or a non-coding strand (antisense strand). The polynucleotide constituting the nucleic acid of the present invention may be fused to a polynucleotide encoding a tag label (tag sequence or marker sequence) on the 5 'side or 3' side. The dengue vaccine of the present invention can be implemented as a nucleic acid vaccine using as a vaccine component a plasmid into which a nucleic acid encoding a mutant envelope protein A has been inserted. In addition, the dengue vaccine of the present invention can be implemented as a vector vaccine (recombinant bacterial vaccine, recombinant virus vaccine) containing a bacterial vector or a viral vector into which a nucleic acid encoding the mutant envelope protein A is inserted as a vaccine component.

As another aspect of the dengue vaccine of the present invention, as a vaccine component, for example, the amino acid sequence at position 2 in the amino acid sequence of the dengue virus prM protein has no mutation in the amino acid sequence at position 107 and position 87 of the envelope protein. Mutant protein (hereinafter referred to as “mutant protein B”) including an envelope protein having a mutation in an amino acid, a nucleic acid encoding the mutant protein B, a vector including a nucleic acid encoding the mutant protein B, a dengue virus expressing the mutant protein B, etc. Can be suitably contained.

As the mutant protein B, the full length of the prM protein and the envelope protein may be used, or a fragment thereof may be used. When the fragment is used, the amino acid at position 2 in the amino acid sequence of the prM protein is not mutated, and the amino acid at position 107 and the amino acid at position 87 are both mutated amino acids in the amino acid sequence of the envelope protein. It is preferable to use a fragment having a length that maintains the three-dimensional structure. For example, a fragment having 300 or more amino acid residues, preferably 350 or more, more preferably 400 or more, and still more preferably 450 or more can be suitably used. Mutant protein B may also contain other amino acid sequences other than prM protein and envelope protein. Examples of other amino acid sequences include marker sequences, tag sequences, amino acid sequences of other proteins encoded by the dengue virus genome, and the like.

Mutant protein B can be produced in the same manner as mutant envelope protein A, and can be produced, for example, by a known genetic engineering technique. As for the base sequence of the gene encoding the mutant protein B, the base sequence of the dengue virus genome is obtained from a known database (NCBI, etc.). Based on the base sequence obtained, the 107th and 87th amino acids of the envelope protein are obtained. Can be designed to have mutations.

Nucleic acid encoding mutant protein B should use known genetic engineering techniques such as PCR and site-directed mutagenesis based on the base sequence of the gene encoding mutant protein B designed as described above. It can be obtained by. The nucleic acid can be present in the form of RNA (eg, genomic RNA, mRNA) or in the form of DNA (eg, genomic DNA, cDNA). The nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be any of double-stranded DNA, double-stranded RNA, or a hybrid of DNA and RNA. In the case of a single strand, it may be either a coding strand (sense strand) or a non-coding strand (antisense strand). The polynucleotide constituting the nucleic acid of the present invention may be fused to a polynucleotide encoding a tag label (tag sequence or marker sequence) on the 5 'side or 3' side. The dengue vaccine of the present invention can be implemented as a nucleic acid vaccine comprising a plasmid into which a nucleic acid encoding a mutant protein B is inserted as a vaccine component. In addition, the dengue vaccine of the present invention can be implemented as a vector vaccine (recombinant bacterial vaccine, recombinant virus vaccine) having a bacterial vector or viral vector into which a nucleic acid encoding the mutant protein B is inserted as a vaccine component.

The dengue vaccine of the present invention can be implemented as a dengue vaccine comprising a dengue virus that expresses mutant envelope protein A or mutant protein B as a vaccine component. Dengue virus expressing mutant envelope protein A or mutant protein B can be obtained by using the “method for obtaining dengue vaccine antigen that induces neutralizing antibody but suppresses induction of infection-enhancing antibody or dengue virus that expresses the antigen” described later. Can be acquired.

The dengue vaccine of the present invention may contain one or more adjuvants. When the dengue vaccine of the present invention contains an adjuvant, it can be appropriately selected from known adjuvants. Specifically, for example, aluminum adjuvant (for example, aluminum salt such as aluminum hydroxide, aluminum phosphate, aluminum sulfate or a combination thereof), Freund's adjuvant (complete or incomplete), TLR ligand (for example, CpG, Poly (I : C), Pam3CSK4, etc.), BAY, DC-chol, pcpp, monophosphoryl lipid A, QS-21, cholera toxin, formylmethionyl peptide and the like. Preferably, it is an aluminum adjuvant, a TLR ligand, or a combination thereof. When the vaccine of the present invention contains an adjuvant, the compounding amount of the adjuvant is not particularly limited, and may be appropriately selected depending on the type of the adjuvant. For example, when aluminum adjuvant (aluminum hydroxide) and CpG are used in combination, about 1 to 100 times the amount of aluminum adjuvant and about 1 to 50 times the amount of CpG may be blended in the mass ratio with the dengue vaccine of the present invention. preferable.

The dengue vaccine of the present invention may further contain a vaccine component against pathogens other than dengue virus. Pathogen vaccine components other than dengue virus are not particularly limited, and examples thereof include vaccine components that have already been used as mixed vaccines. Specifically, for example, diphtheria toxoid, pertussis toxoid, pertussis antigen, tetanus toxoid, inactivated poliovirus, attenuated measles virus, attenuated rubella virus, attenuated mumps virus, Haemophilus influenzae type b polysaccharide antigen, hepatitis B virus HBs Examples include antigens, inactivated hepatitis A virus antigens, and the like.

The dengue vaccine of the present invention can be administered orally or parenterally. Examples of parenteral administration include intraperitoneal administration, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, intranasal administration, transdermal administration, transmucosal administration, sublingual administration, and inhalation administration. Preferred is parenteral administration, and more preferred is intradermal administration, subcutaneous administration or intramuscular administration.

The dengue vaccine of the present invention is formulated by appropriately combining a nucleic acid encoding mutant envelope protein A or mutant protein B or mutant envelope protein A or mutant protein B, a pharmaceutically acceptable carrier, and an additive as appropriate. Product (medicine). Specifically, preparations for oral administration such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions; parenterals such as injections, infusions, suppositories, ointments, patches It can be set as the formulation for administration. What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ | adopted in the pharmaceutical field | area. Carriers or additives that can be blended are not particularly limited. For example, various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorption Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.

Examples of additives used in solid preparations for oral administration include excipients such as lactose, mannitol, glucose, microcrystalline cellulose, and corn starch; binders such as hydroxypropylcellulose, polyvinylpyrrolidone, and magnesium aluminate metasilicate Dispersing agents such as corn starch; disintegrating agents such as calcium calcium glycolate; lubricants such as magnesium stearate; solubilizing agents such as glutamic acid and aspartic acid; stabilizers; hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, etc. Water-soluble polymers such as cellulose, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, etc .; sucrose, powdered sugar, sucrose, fructose, glucose, lactose, reduced maltose water candy, powder returned Sweeteners such as maltose syrup, glucose fructose liquid sugar, honey, sorbitol, maltitol, mannitol, xylitol, erythritol, aspartame, saccharin, sodium saccharin; coating agents such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate Can be mentioned.
Liquid preparations for oral administration are prepared by dissolving, suspending or emulsifying in commonly used diluents. Examples of the diluent include purified water, ethanol, a mixed solution thereof and the like. Furthermore, this liquid agent may contain a wetting agent, a suspending agent, an emulsifier, a sweetening agent, a flavoring agent, a fragrance, a preservative, a buffering agent and the like.

Examples of additives used for injections for parenteral administration include isotonic agents such as sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, glucose, propylene glycol; phosphate buffer Buffers such as acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, epsilon aminocaproate buffer; methyl paraoxybenzoate, ethyl paraoxybenzoate, paraoxybenzoic acid Preservatives such as propyl, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax; hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol Thickeners such as sodium hydrogen sulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc .; pH adjustment of hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc. Agents and the like. For injections, suitable solubilizers, for example, alcohols such as ethanol; polyalcohols such as propylene glycol and polyethylene glycol; nonionic surfactants such as polysorbate 80, polyoxyethylene hydrogenated castor oil 50, lysolecithin, and pluronic polyol You may mix | blend an agent etc. further. Liquid preparations such as injections can be stored after removing moisture by freezing or lyophilization. The freeze-dried preparation is used by adding distilled water for injection at the time of use and re-dissolving it.

The dengue vaccine of the present invention can be administered to any animal (human, non-human) having an immune system, but the natural host of dengue virus is known to be human and non-human primates (monkeys). Therefore, it is preferable to administer humans and non-human primates (monkeys). Among these, the dengue vaccine of the present invention is preferably intended for human children (including newborns) and adults.

The number of administrations and the administration interval of the dengue vaccine of the present invention are not particularly limited. For example, a single dose may be administered, or multiple doses may be administered at intervals of about 2 days to about 8 weeks. When the dengue vaccine of the present invention is a protein vaccine or a nucleic acid vaccine, the dose of the vaccine varies depending on the administration subject, administration method, etc., but the single dose is preferably about 0.01 μg to about 100 mg, about 0 More preferably, it is 1 μg to about 10 mg, and more preferably about 1 μg to about 1 mg. When the dengue vaccine of the present invention is a virus vaccine, the dose of the vaccine varies depending on the administration subject, administration method, etc., but it is preferable that the single dose is about 1 × 10 ² PFU to about 1 × 10 ⁹ PFU. About 1 × 10 ³ PFU to about 1 × 10 ⁸ PFU, more preferably about 1 × 10 ⁴ PFU to about 1 × 10 ⁷ PFU.

The present invention includes a method for preventing or treating dengue fever or dengue hemorrhagic fever, which comprises administering an effective amount of the dengue vaccine of the present invention to an animal.

[Method of obtaining dengue vaccine antigen that induces neutralizing antibody but suppresses induction of infection-enhancing antibody or dengue virus that expresses the antigen]
The present invention provides a method for obtaining a dengue vaccine antigen that induces a neutralizing antibody but suppresses induction of an infection-enhancing antibody or a dengue virus that expresses the antigen. A method for obtaining a dengue vaccine antigen that induces neutralizing antibodies of the present invention but suppresses induction of infection-enhancing antibodies or dengue viruses that express the antigens (hereinafter referred to as “methods of the present invention”) includes the following steps (1 ) To (5) may be used.
Step (1): Obtaining a monoclonal antibody having infection-enhancing activity but not neutralizing activity using dengue virus envelope protein as an antigen Step (2): Class or subclass of monoclonal antibody obtained in step (1) Step (3) of obtaining a monoclonal antibody having neutralizing activity by changing the above, culturing dengue virus-infected cells in the presence of the monoclonal antibody having neutralizing activity obtained in step (2), Step (4) for obtaining a mutant dengue virus that is not neutralized by the monoclonal antibody: Step (5) for confirming a mutation occurring in the envelope protein of the mutant dengue virus obtained in step (3) (obtained in step (3)) Of a modified dengue virus suppresses the induction of infection-enhancing antibodies Step of confirming

In step (1), a monoclonal antibody having an infection-enhancing activity but no neutralizing activity is obtained using the dengue virus envelope protein as an antigen. In this step, an envelope protein having no mutation is used as an antigen. Monoclonal antibodies are obtained by immunizing a mammal (eg, a mouse) according to a normal immunization method using a dengue virus envelope protein as an antigen, and then immunizing the resulting immune cells (eg, spleen cells) with a conventional cell fusion method (eg, polyethylene glycol (PEG) method). ) With a known parent cell (eg, mouse myeloma cell line SP2, NA1, etc.), selecting a monoclonal antibody-producing hybridoma by a conventional screening method, and purifying it from the hybridoma culture supernatant by a known method. Can be acquired. It can be confirmed, for example, by the method described in Example 1 (1) of this specification that the monoclonal antibody has an infection-enhancing activity and no neutralizing activity. The antigen used for obtaining the monoclonal antibody may include an envelope protein and a prM protein.

In step (2), a monoclonal antibody having neutralizing activity is obtained by changing the class or subclass of the monoclonal antibody obtained in step (1). The class or subclass of the antibody can be changed by a method using, for example, pFUSE-CHIg series (H chain) and pFUSE-CLIg series (L chain) for mice manufactured by Invivogen. Specifically, the Fab part gene is extracted from the hybridoma cell producing the monoclonal antibody obtained in step (1) and incorporated into the pFUSE plasmid vector. At this time, the pFUSE of the H chain is selected so as to be the target subclass. For example, if the target subclass is IgG2a, the heavy chain uses “pFUSE-CHIg-mG2a”. A light chain plasmid is prepared in the same manner, and two types of plasmids are cotransfected into cultured cells (eg, 293T, CHO, etc.), and the culture supernatant is collected to obtain an antibody with a changed subclass. be able to. It can be confirmed, for example, by the method described in Example 1 (2) of the present specification that the monoclonal antibody having a changed class or subclass has neutralizing activity.

In step (3), a dengue virus-infected cell that is not neutralized by the monoclonal antibody is obtained by culturing dengue virus-infected cells in the presence of the monoclonal antibody having neutralizing activity obtained in step (2). Specifically, for example, Vero cells are seeded in a ^6- well cell culture microplate (approximately 0.3 × 10 ⁶ cells / well), and after 1 to 2 days, dengue virus is applied to the cells under the condition of MOI = 0.1. Infect. After the adsorption operation for 1 hour, the cells are cultured in a culture solution adjusted so that the final concentration of the monoclonal antibody having neutralizing activity obtained in step (2) is 1 μg / mL. One week later, the newly prepared Vero cells are infected with the culture supernatant and similarly cultured in the presence of the monoclonal antibody. Passaging is preferably continued for 5 or more generations. Examples of the method for confirming the appearance of escape mutants include a method for conducting neutralization tests of the monoclonal antibodies using the escape mutant candidate virus strain as an antigen. If the neutralizing activity has disappeared, the candidate virus strain is determined to be the target escape mutant strain. Another method for confirming the appearance of escape mutants is, for example, a method of performing an infection enhancement test of the monoclonal antibody using an escape mutant candidate virus strain as an antigen. If the infection-enhancing activity has disappeared, the candidate virus strain is determined to be the target escape mutant strain.

In step (4), mutations occurring in the envelope protein of the mutant dengue virus obtained in step (3) are confirmed. Specifically, for example, the gene in the envelope region of the mutant dengue virus is amplified by RT-PCR, and the base sequence is analyzed using a DNA sequencer (for example, Applied Biosystems 3730xl DNA analyzer). Based on the revealed base sequence, it can be translated into an amino acid sequence, and the mutation site and the type of amino acid at that position can be specified.

In step (5), it is confirmed that the envelope protein of the mutant dengue virus obtained in step (3) suppresses the induction of infection-enhancing antibodies. Specifically, the following method is mentioned, for example. That is, a plasmid DNA expressing the E antigen introduced with the amino acid mutation confirmed in step (4) was prepared, co-transfected with Japanese encephalitis virus replicon plasmid DNA into 293T cells, and single-infectious particles (Single- round infectious particle (hereinafter “SRIP”). Using this SRIP as an antigen, the infection enhancing activity of the monoclonal antibody having the infection enhancing activity obtained in step (1) and having no neutralizing activity is confirmed. As a result, if the infection enhancing activity is not detected, it is determined that the dengue virus envelope protein having the amino acid mutation confirmed in step (4) can suppress the infection enhancing activity.

The mutant dengue virus obtained by the method of the present invention and the mutant dengue virus envelope protein or those containing the envelope protein and the prM protein can be used as the vaccine component of the dengue vaccine of the present invention. Since the mutant dengue virus obtained by the method of the present invention and the mutant dengue virus envelope protein or those containing the envelope protein and the prM protein can induce neutralizing antibodies but suppress the induction of infection-enhancing antibodies, It is very useful as a vaccine component.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[Example 1: Determination of epitope site of D1-V-3H12 antibody]
(1) Confirmation of infection-enhancing and neutralizing activity of monoclonal antibodies against Dengue type 1 virus Mochizuki strains Yamanaka et al. (Yamanaka et al., J Virol. 2008 Jan; 82 (2) : 927-937), and obtained a plurality of monoclonal antibodies having serotype cross-reactivity with the envelope (E) protein of Dengue virus (Yamanaka et al., J Virol. 2013 Dec; 87 (23) : 12828-37). The infection enhancement activity and neutralization activity of the obtained monoclonal antibody were confirmed by the following method.

Monoclonal antibody diluted with 10-fold serial dilution and Dengue type 1 virus Mochizuki strain were mixed and incubated at 37 ° C. for 2 hours, and then K562 cells, which are human-derived Fcγ-containing monocytic cells, were added and further cultured for 2 days. Two systems were provided: a system in which complement with a final concentration of 5% was added to the antibody / virus mixture, and a system in which no complement was added. After fixing the cells, immunostaining was performed using a dengue virus-specific antibody, and positive cells (infected cells) were counted. Six negative controls to which no monoclonal antibody was added were placed, and when the average number of positive cells (infected cells) was less than -3SD, it was determined that the monoclonal antibody had neutralizing activity, and positive control positive cells ( When the average number of infected cells) was greater than + 3SD, the monoclonal antibody was determined to have infection-enhancing activity.

The results are shown in FIG. In the figure, C (-) represents a system without addition of complement, and C (+) represents a system with addition of complement. The left of FIG. 1 shows a representative example of a monoclonal antibody (D1-I-15C12) showing neutralizing activity at a high concentration and showing infection-increasing activity at a low concentration, and the center is a representative of a monoclonal antibody showing only neutralizing activity. As a result of the example (D1-IV-7F4), the right is the result of a representative example (D1-V-3H12) of a monoclonal antibody showing only infection-enhancing activity.
Many of the obtained monoclonal antibodies showed neutralizing activity at a high concentration and infection-increasing activity at a low concentration (similar to the left in FIG. 1), but antibodies showing only neutralizing activity (as shown in the center of FIG. 1) Similarly, a small number of antibodies (similar to the right in FIG. 1) exhibiting only infection-enhancing activity were also present. In the subsequent experiments, the D1-V-3H12 antibody (hereinafter referred to as “3H12 antibody”) that showed only the infection-enhancing activity was used, and the experiment was performed in a system in which complement was added.

(2) Subclass substitution of 3H12 antibody When examined using a commercially available isotyping kit, the subclass of 3H12 antibody was IgG1. By inserting the gene of the Fab part of the 3H12 antibody into a commercially available pFUSE vector, antibodies whose subclass was replaced with IgG2a or IgG2b without changing the antibody binding site were obtained (referred to as 3H12-IgG2a and 3H12-IgG2b, respectively). .) The neutralizing activity of these antibodies was measured using Vero cells (African green monkey kidney-derived cell line) according to the method described in Konishi et al. (Konishi et al., Vaccine 2006 24: 2200-2207). Specifically, the antibody diluted with 2-fold serial dilution and Dengue type 1 virus Mochizuki strain were mixed and incubated at 4 ° C. overnight to infect monolayer Vero cells. Counted. The number of plaques obtained in the control to which no antibody was added was defined as 100%, and the neutralization activity was expressed as a percentage reduction (% Plaque reduction).

The results are shown in FIG. Both subclass-substituted 3H12-IgG2a and 3H12-IgG2b had neutralizing activity. Therefore, 3H12-IgG2b was selected as an antibody for obtaining escape mutant virus.

(3) Acquisition of escape mutant virus As a result of subculture of Vero cells infected with Dengue type 1 virus Mochizuki strain in a culture solution to which 3H12-IgG2b was added at a concentration of 3 μg / mL, Viruses with comparable proliferative potential and not neutralized with 3H12-IgG2b were included. The infection-enhancing activity of the 3H12 antibody against the obtained escape mutant virus at a concentration of 10 μg / mL was examined by the same method as in (1) above.

The results are shown in FIG. In FIG. 3, P # 0 is the parental virus prior to subculture, P # 11 is the escape mutant virus obtained, and P # 11Cont is obtained by subculture for 11 generations in a culture solution to which 3H12-IgG2b is not added. Dengue virus. The vertical axis in FIG. 3 represents the number of infected cells with respect to a control to which no antibody was added (Fold 数 enhancement). As apparent from FIG. 3, P # 0 and P # 11Cont were enhanced about 100 times by 3H12 antibody, whereas P # 11 was hardly enhanced by 3H12 antibody.

(4) Confirmation of mutation of escape mutant virus When the amino acid sequence of the envelope region of the obtained escape mutant virus was confirmed according to a standard method, mutations were found at positions 87 (E87) and 107 (E107) of the envelope region ( D87N and L107F respectively). That is, it was suggested that the 3H12 antibody targets an epitope including E87 and E107.

Next, in order to confirm this epitope site by reverse genetics, a plasmid DNA expressing the E gene in which both E87 and E107 or any one of the mutations is expressed was prepared, and the Japanese encephalitis virus already established SRIP was prepared by co-transfecting 293T cells with a replicon plasmid (Suzuki et al., J. Gen. Virol, 2014 Jan; 95 (Pt 1): 60-5.). The mutation of E87 was D87N, and the mutation of E107 was L107F. The infection enhancing activity of 3H12 antibody against SRIP antigen was examined by the same method as in (1) above, and compared with the infection enhancing activity of 3H12 antibody against SRIP antigen without mutation.

The results are shown in FIG. The dotted line in the figure shows the mean value ± 3SD determined from 6 wells of a negative control to which no antibody was added. 3H12 antibody showed high infection-enhancing activity against SRIP antigen (Control) without mutation, but 3H12 antibody against SRIP antigen with mutation at both E87 and E107, and SRIP antigen with mutation at E107 It did not show infection enhancing activity. On the other hand, for the SRIP antigen having a mutation in E87, the 3H12 antibody showed the same high infection enhancing activity as Control. This result suggests that E87 and E107 were mutated during the generation of escape mutant virus, but the substantial change in the epitope escaping from the neutralizing antibody is considered to be caused by the E107 mutation, and E87 is related to the epitope. However, it was considered to be a mutation that entered in order to compensate for the decrease in virus growth caused by the E107 mutation.

[Example 2: Immunogenicity of mutagenized antigen]
6-week-old male BALB / c mice (using plasmid DNA expressing the E gene of Dengue type 1 virus Mochizuki strain with both E87 and E107, or any one mutation (D87N and / or L107F) ( 3 mice per group) were immunized. Immunization was performed three times at a dose of 100 μg, and blood was collected 2 weeks after the second immunization and 1 week after the third immunization. As a control, plasmid DNA expressing the E gene without mutation was used. Using the pooled sera obtained, the neutralizing activity of antibodies induced against these immunogens against the dengue type 1 virus Mochizuki strain was examined by the method described in Example 1 (2), and the infection enhancing activity was examined. It investigated by the method as described in Example 1 (1).

The results are shown in FIG. A is the result of examining the neutralizing activity, and B is the result of examining the infection enhancing activity. In addition, 2doses is a result of serum collected after the second immunization, and 3doses is a result of serum collected after the third immunization. The dotted line B shows the mean value ± 3SD determined from 6 wells of a negative control to which no antibody was added. As is clear from A, both plasmids induced antibodies with comparable levels of neutralizing activity. On the other hand, a large difference was observed in the level of infection enhancing activity. That is, an antibody induced by a plasmid having a mutation in either E87 or E107 did not show infection-increasing activity compared to the control, whereas an antibody induced by a plasmid having a mutation in both E87 and E107 was Although it was lower than the control, it showed infection-enhancing activity. This result indicates that the reaction between the antigen epitope obtained in in vitro and the antibody does not necessarily match the immunogenicity of the antigen in in vivo, but matches for the epitope containing E107.

[Example 3: Neutralizing activity and infection enhancing activity against other serotypes]
In Example 2, the neutralizing activity and the infection enhancing activity of the induced antibody against dengue type 1 virus were examined using serum collected from mice immunized twice or three times with a mutant antigen derived from dengue type 1 virus. However, in Example 3, using antibodies collected from a mouse immunized three times with a mutant antigen derived from dengue type 1 virus, the induced antibody dengue type 2 virus (New Guinea C strain), type 3 virus (H87) Strain) or type 4 virus (H241 strain) was examined by the method described in Example 1 (2), and the infection enhancing activity was examined by the method described in Example 1 (1).

The results are shown in FIG. A is the result of examining the neutralizing activity, and B is the result of examining the infection enhancing activity. The dotted line B shows the mean value ± 3SD determined from 6 wells of a negative control to which no antibody was added. As is clear from A, the induction of cross-neutralizing antibodies against dengue type 4 virus was lower compared to type 2 or type 3 virus, but for either serotype, The mutagenized antigen induced a cross neutralizing antibody equivalent to the control antigen. On the other hand, a large difference was observed in the level of infection enhancing activity. That is, the control antigen induced an antibody having infection enhancing activity against all serotypes, whereas the E107 mutant antigen did not induce an antibody having infection enhancing activity against all serotypes. Antigens with mutations in both E87 and E107 induced antibodies with infection-enhancing activity against all serotypes, but were lower than those induced by the control antigen. The antigen having a mutation only in E87 was also lower than the control antigen, and in particular, an antibody having an infection enhancing activity against type 2 virus was not induced. From this result, it is clarified that the antibody induced by the dengue type 1 virus antigen introduced with a mutation in E107 does not show the enhancing activity not only against the dengue type 1 virus but also against other serotypes. It was. It was also shown that the E87 mutation has some effect.

[Example 4: Infection enhancing activity of 3H12 antibody against other serotype E antigens mutated]
The amino acid sequences near E87 and E107 are relatively conserved across all serotypes. The 3H12 antibody is an antibody prepared using Dengue type 1 virus Mochizuki strain as an antigen, and has cross-reactivity with all other serotypes. Therefore, for the dengue type 2 virus, dengue type 3 virus, and dengue type 4 virus used in Example 3, SRIP was prepared by introducing a mutation (D87N or L107F) into E87 or E107 of each E antigen. Infection enhancing activity of the 3H12 antibody against the above was examined by the method described in Example 1 (1) and compared with the infection enhancing activity of the 3H12 antibody against the SRIP antigen having no mutation.

The results are shown in FIG. In all serotypes, the 3H12 antibody showed an infection enhancing activity against the control antigen without mutation, but did not show an infection enhancing activity against the E107 mutant antigen. The infection enhancing activity of the 3H12 antibody against the E87 mutant antigen was different depending on the serotype, and did not show the infection enhancing activity against the type 2 virus, but showed the infection enhancing activity against the type 3 and type 4 viruses. This result shows that E107 is an epitope site of 3H12 also in the E antigen of dengue type 2 virus, dengue type 3 virus, and dengue type 4 virus.

[Example 5: Immunogenicity of other serotype E antigens mutated]
In order to examine whether the reaction between the antigen epitope induced by mutation in E107 obtained in vitro and the antibody is consistent with the immunogenicity of the antigen in vivo, even in serotypes other than dengue type 1 virus, E87 or A 6-week-old female BALB / c mouse (group 1) using plasmid DNA expressing an E antigen of another serotype (type 2, type 3 or type 4) introduced with a mutation (D87N or L107F) in the amino acid of E107 3 animals) were immunized. Immunization was performed three times at a dose of 100 μg, and blood was collected 1 week after the third immunization. As a control, plasmid DNA having an E gene without mutation was used. Using the pooled sera obtained, the infection enhancing activity of antibodies induced against these immunogens against the corresponding serotype virus was examined by the same method as in Example 1 (1).

The results are shown in FIG. The dotted line in the figure shows the mean value ± 3SD determined from 6 wells of a negative control to which no antibody was added. C (+) represents a system to which complement is added, and C (−) represents a system to which no complement is added. In the system in which complement was added (left column), in the dengue type 3 virus, the antibody induced with the control antigen showed an infection-enhancing activity, whereas the antibody induced with the E107 mutant antigen did not show the infection-enhancing activity. It was. In the dengue type 2 virus and the dengue type 4 virus, the control antigen also did not show an enhancing activity under the present conditions, so the experiment was performed without adding complement to the assay system (right column). As a result, in both the dengue type 2 virus and the dengue type 4 virus, the antibody induced with the control antigen showed a high infection enhancing activity, whereas the antibody induced with the E107 mutant antigen decreased the infection enhancing activity. In the dengue type 3 virus as well, in the system in which no complement was added, the antibody induced with the E107 mutant antigen tended to have a decreased tendency to enhance infection. On the other hand, the E87 mutant antigen was also found to have an effect of suppressing the infection-enhancing activity in the dengue type 3 virus of the system to which complement was added. In addition, the dengue type 2 virus and the dengue type 4 virus that do not contain complement were also found to have an effect of suppressing the infection enhancing activity. This result shows that mutation introduction into E107 also suppresses induction of an antibody having infection-enhancing activity in dengue type 2 virus, dengue type 3 virus, and dengue type 4 virus. Moreover, although it is an effect weaker than the mutation introduction into E107, it shows that the mutation introduction into E87 has the same effect.

[Example 6: Immunogenicity of mutated amino acids in mutated antigen]
In the plasmid encoding the E protein derived from dengue type 1 virus, the amino acid E107 is changed to various amino acids (phenylalanine, isoleucine, proline, lysine, glutamine or tryptophan), or the amino acid E87 is changed to various amino acids (asparagine, arginine, glutamic acid). , Glutamine, phenylalanine or valine), and plasmids obtained by mutating one point each were used, and 6-week-old male BALB / c mice (3 mice per group) were immunized using the obtained plasmids. Immunization was performed 3 times at a dose of 100 μg, and blood was collected 2 weeks after the third immunization. The method described in Example 1 (1) shows that the obtained pooled serum is used to increase the infection-inducing activity of antibodies induced against these immunogens against

Dengue

1, 2, 3, or 4 viruses. I examined it. Serum dilution conditions were 1: 160 dilution. As a control, immunization with a plasmid that does not introduce a mutation into the E protein was also evaluated.

The results are shown in FIG. Fold Enhancement was calculated from the degree of infection enhancement relative to the negative control and expressed in a graph. According to this result, it can be seen that even if leucine of E107 is substituted with any amino acid, although the degree of difference is recognized depending on the type of serotype, induction of infection enhancing activity can be suppressed. Similarly, it can be seen that even if aspartic acid of E87 is substituted with any amino acid, although the degree of difference is recognized depending on the type of serotype, induction of infection enhancing activity can be suppressed.

[Example 7: Infection enhancing activity of 3H12 antibody against mutant antigens of different dengue virus strains]
The above was a study using a prototype, but then a study was performed using 4 serotype strains that were relatively newly isolated (see Table 3 below). First, one strain was selected from each serotype, and a plasmid encoding the E region was prepared. Next, mutations (D87N or L107F) were introduced into E87 or E107 using the control plasmid of each serotype without mutation as a template to prepare each SRIP. The infection enhancing activity of the 3H12 antibody against the SRIP antigen was examined by the method described in Example 1 (1), and compared with the infection enhancing activity of the 3H12 antibody against the SRIP antigen having no mutation.

The results are shown in FIG. Fold enhancement indicating how many times the number of infected cells increased from the negative control was evaluated. From FIG. 10, the 3H12 antibody did not show suppression of infection enhancing activity against the SRIP antigen having a mutation in E87 and the SRIP antigen having no mutation (Control), but against the SRIP antigen having a mutation in E107. It was found that the infection-enhancing activity was significantly suppressed. This suggests that the target site of the 3H12 antibody is at position E107 relative to all serotype viruses regardless of the strain type.

[Example 8: Immunogenicity of mutant antigens of different dengue virus strains (Part 1)]
Next, 6-week-old male BALB / c mice (6 mice per group) were prepared using plasmid DNA in which mutation (D87N or L107F) was introduced into any one of E87 and E107 prepared with reference to Example 7. ) (BDV-1 to BDV-4). Immunization was performed 3 times at a dose of 100 μg, and blood was collected 2 weeks after the third immunization. As a control, plasmid DNA expressing the E gene without mutation was used. Using the pooled serum obtained, the neutralizing activity of antibodies induced against these immunogens against dengue type 1, type 2, type 3 or type 4 virus is the method described in Example 1 (2) The infection enhancing activity was examined by the method described in Example 1 (1).

The results are shown in FIG. 11 and FIG. FIG. 11 shows the result of examining the neutralizing activity, and FIG. 12 shows the result of examining the infection enhancing activity. The dotted line in FIG. 12 shows the mean value ± 3SD determined from 6 wells of a negative control to which no antibody was added. As is clear from FIG. 11, all plasmids induced antibodies having the same level of neutralizing activity except for plasmids prepared using dengue type 2 virus (genotype American, BDV-2). On the other hand, a large difference was observed in the level of infection enhancing activity. That is, in any strain, the antibody induced by a plasmid having a mutation in either E87 or E107 did not show any infection-stimulating activity against all serotype viruses, whereas the control plasmid DNA had an infection-enhancing activity. showed that. This suggests that mutations in E87 or E107 are involved in the induction of infection-enhancing antibodies even if the strains are different.

[Example 9: Immunogenicity of mutant antigens of different dengue virus strains (2)]
Four types of plasmids used in Example 8 were mixed, and 6-week-old male BALB / c mice (6 per group) were immunized as a 4-valent vaccine. The following three groups were established for the 4-valent vaccine: E107 mutated BDV-1 to BDV-4, E87 mutated BDV-1 to BDV-4, no mutation BDV-1 to BDV-4, once The immunization was performed 3 times at a total dose of 100 μg, in which 4 types of 25 μg of each plasmid were mixed, and blood was collected 2 weeks after the third immunization. Using the pooled serum obtained, the neutralizing activity of antibodies induced against these immunogens against dengue type 1, type 2, type 3 or type 4 virus is the method described in Example 1 (2) The infection enhancing activity was examined by the method described in Example 1 (1).

The results are shown in FIG. 13 and FIG. FIG. 13 shows the result of examining the neutralizing activity, and FIG. 14 shows the result of examining the infection enhancing activity. The dotted line in FIG. 14 shows the mean value ± 3SD determined from 6 wells of a negative control to which no antibody was added. As is clear from FIG. 13, all groups induced antibodies with equivalent neutralizing activity levels. On the other hand, a large difference was observed in the level of infection enhancing activity. That is, an antibody induced by a plasmid having a mutation in either E87 or E107 did not show an infection-enhancing activity, whereas the control plasmid DNA showed an infection-enhancing activity. This suggests that the action of suppressing the induction of infection-enhancing antibodies possessed by each vaccine is maintained even in a quadrivalent combination vaccine.

[Example 10: Effect of prM2 mutation on immunogenicity]
Analysis of the base sequence of the prM region of the escape mutant virus obtained in Example 1 (3) revealed that the second histidine (H) in the prM region was replaced with asparagine (N). That is, a total of three mutations (prM2, E87, E107) have been inserted into the prM / E region of the acquired escape mutant. Therefore, a total of 4 types of plasmids combining mutation and non-mutation were newly prepared for each of 3 points of prM2, E87, and E107, and 6-week-old male BALB / c mice (6 mice per group) were immunized. . Immunization was performed 3 times at a dose of 100 μg, and blood was collected 2 weeks after the third immunization. Using the pooled serum obtained, the neutralizing activity of antibodies induced against these immunogens against dengue type 1, type 2, type 3 or type 4 virus is the method described in Example 1 (2) Thus, the infection enhancing activity was examined by the method described in Example 1 (1). Table 4 shows the details of the plasmid.

The results are shown in FIG. 15 and FIG. FIG. 15 shows the result of examining the neutralizing activity, and FIG. 16 shows the result of examining the infection enhancing activity. The dotted line in FIG. 16 shows the mean value ± 3SD determined from 6 wells of a negative control to which no antibody was added. As is clear from FIG. 15, all the plasmids induced antibodies having the same level of neutralizing activity. On the other hand, from FIG. 16, a large difference was observed in the infection-enhancing activity level. That is, it was found that a plasmid having a mutation in E87 and E107 and having no mutation in prM2 (E87 + E107) does not show an infection enhancing activity. In Example 2 (FIG. 5) and Example 3 (FIG. 6), some infection-enhancing activity was observed when plasmids into which both E87 and E107 mutations were introduced were used. The plasmid was prepared by directly cloning the prM / E region of the escape mutant virus strain obtained in Example 1. At that time, in addition to both E87 and E107 mutations, prM2 was also mutated. It is thought that what was introduced was included.
Also, from the right column of FIG. 16, when a mutation is inserted into prM2, infection enhancing activity is induced even if both E87 and E107 mutations are introduced (prM2 + E87 + E107). Since no control DNA plasmid exhibits infection-increasing activity against all serotypes (Control), infection-inducing activity is induced when there is no mutation in prM2 and a mutation is introduced into both E87 and E107 It is suggested that not. It is speculated that prM2 is a site involved in the induction of infection-enhancing antibodies.

[Example 11: Infection enhancing activity of 3H12 antibody against prM2 mutant antigen]
The binding activity of 3H12 antibody to single-infectious particles (SRIP) having mutations in the 2nd amino acid of prM protein of Dengue type 1 virus (Mochizuki strain) and / or both E87 and E107 of envelope protein is used as an indicator of infection enhancing activity This was confirmed again, and Example 1 (FIG. 4) was verified. The symbols in the figure are the same as those in the tenth embodiment.

The results are shown in FIG. From FIG. 17, the 3H12 antibody does not exhibit infection-enhancing activity (prM2 + E87 + E107, E87 + E107) against SRIP having mutations in E87 and E107 regardless of the presence or absence of prM2 mutation, whereas SR87 having no mutation in E87 and E107. In contrast, the 3H12 antibody was found to exhibit infection-enhancing activity (Control, prM2). This supported the results of FIG. 4 and suggested that the recognition epitope of the 3H12 antibody has nothing to do with the mutation of prM2.

The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims

A dengue vaccine antigen that induces a neutralizing antibody but suppresses induction of an infection-enhancing antibody, comprising an envelope protein having a mutation at the 107th or 87th amino acid of the amino acid sequence of a dengue virus envelope protein, Dengue vaccine antigen.
The dengue vaccine antigen according to claim 1, wherein the leucine at position 107 is substituted with one selected from phenylalanine, tryptophan, methionine, proline, alanine, valine and isoleucine.
The dengue vaccine antigen according to claim 2, wherein the leucine at position 107 is substituted with phenylalanine.
The dengue according to any one of claims 1 to 3, wherein the envelope protein having a mutation at the 107th amino acid consists of an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4. Vaccine antigen.
The aspartic acid at position 87 is substituted with one selected from glutamic acid, tyrosine, cysteine, arginine, histidine, lysine, serine, threonine, glutamine, asparagine and glycine. Dengue vaccine antigen.
The dengue vaccine antigen according to claim 5, wherein the aspartic acid at position 87 is substituted with asparagine.
The dengue vaccine according to claim 1, 5 or 6, wherein the envelope protein having a mutation at the 87th amino acid consists of an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 5. antigen.
A dengue vaccine comprising or expressing the dengue vaccine antigen according to any one of claims 1 to 7.
An envelope protein having a mutation at the 107th or 87th amino acid in the amino acid sequence of a dengue virus envelope protein, a nucleic acid encoding the envelope protein, a vector containing a nucleic acid encoding the envelope protein, or a dengue virus expressing the envelope protein The dengue vaccine according to claim 8, comprising:
The dengue vaccine according to claim 8 or 9, comprising a vaccine component against pathogens other than dengue virus.
A method for obtaining a dengue vaccine antigen or a dengue virus that expresses the antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies, comprising the following steps (1) to (5) Method:
(1) A step of obtaining a monoclonal antibody having an infection enhancing activity but not a neutralizing activity, using a dengue virus envelope protein as an antigen,
(2) obtaining a monoclonal antibody having neutralizing activity by changing the class or subclass of the monoclonal antibody obtained in (1),
(3) obtaining a mutant dengue virus that is not neutralized by the monoclonal antibody by culturing dengue virus-infected cells in the presence of the monoclonal antibody having neutralizing activity obtained in (2);
(4) a step of confirming a mutation occurring in the envelope protein of the mutant dengue virus obtained in (3), and (5) that the envelope protein of the mutant dengue virus obtained in (3) suppresses the induction of the infection-enhancing antibody. The process of confirming.
A dengue vaccine antigen that induces neutralizing antibodies but suppresses induction of infection-enhancing antibodies, and has a dengue virus envelope protein with mutations at amino acids 107 and 87 of the amino acid sequence and a mutation at the amino acid at position 2. Dengue vaccine antigen characterized in that it contains a dengue virus precursor membrane protein.