WO2021085650A1 - Vaccine - Google Patents

Abstract

The objective is to provide a novel, highly effective vaccine.　The present invention relates to a vaccine comprising a virus-like particle containing an immunomodulator molecule, wherein: the immunomodulator molecule includes interleukin-12 protein, a neuraminidase (NA) domain region originating from NA protein, and an M2 protein domain region originating from an influenza virus; the NA domain region includes an extramembrane domain, a transmembrane domain, and an intracellular domain; the M2 protein domain region includes an extramembrane domain, a transmembrane domain, and an intracellular domain; the interleukin-12 protein is bound to the extramembrane domain in the NA domain region; and the intracellular domain in the M2 protein domain region is bound to the intracellular domain in the NA domain region via a linker.

Description

vaccine

The present invention relates to a novel vaccine.

There is no doubt that the practical path to influenza vaccine development comes from the isolation of the causative virus of influenza A, which was identified as a Spanish-type pandemic respiratory disease in 1933. In fact, this causative virus was used for the first trial of an influenza vaccine. As a result, the first inactivated vaccine was created against the backdrop of urgent development in the United States and the devastating damage to US military personnel. As we all know, the Spanish influenza pandemic has recorded the worst damage in history worldwide, causing 20-40 million deaths worldwide and 80% of US military personnel who served in the late European front. More than two military personnel were reported dead in the war, a background that led to the urgent development of influenza vaccines in the United States. Since then, the development of full-scale inactivated vaccines has increased efficacy and recorded 70-80% efficacy. Against this background, the antigenicity of the virus used in vaccine development could be very similar to the antigenicity of epidemic viruses derived from the Spanish influenza virus.

However, since then, the vital efficacy of vaccines has been shaken violently due to antigen shift and antigen drift. In fact, the antigenic changes caused by the above two mechanisms, consisting of antigen shift and antigen drift, have resulted in a large number of human deaths, which was clearly represented by public health authorities as excess death. .. For example, the H1N1 virus derived from the Spanish influenza virus was constantly maintained in the human region until the mid-1950s, during which time there were three significant excess deaths caused by antigen drift of the H1N1 virus. Was confirmed. For example, nine excess deaths associated with the H1N1 virus have been recorded during the H1N1 virus epidemic since 1934. In this record, a major peak of mortality was the newly emerged Asian H2N2 influenza virus, which suddenly appeared in 1957 due to the first virus detected as an antigen shift. After that, the second antigen-shifted virus derived from the first H1N1 virus, which was derived from the avian influenza virus, spread again to the human region. In the United States, there were numerous excess mortality, but more than 10 significant excess mortality were estimated by one of the authors in Japan.

Since 1980, the number of years that vaccines have been reduced has been four in 10 years in Japan. This evidence was clearly recorded as mortality. The damage caused by the antigenic variation is not only the damage caused by influenza A virus, but also the symbolic damage caused by the antigenic variation of influenza B virus has appeared in Japan. As is known, since 1962, a herd immunity program for school children has been implemented in Japan to control the influenza pandemic throughout the society, but in April and May 1987, it became abnormal. Case occurred in a school in western Japan. 100% of the school children were vaccinated with a commercially available bivalent vaccine containing an influenza B virus strain and an A / Hong Kong (H3N2) virus strain. In fact, in mid-April 1987, an outbreak of influenza pandemic in a junior high school in western Japan (Shikoku region) was reported to the National Influenza Center. Type B causative virus was characterized in terms of antigenicity and genome. The homology of the nucleotide sequence and amino acid sequence between B / Ibaraki / 2/85 (vaccine strain) and B / Yamagata / 16/88 (new mutant strain) was 93.2 and 90.7%, respectively. there were. Based on serotyping, 386 of the 515 enrolled students were epidemiologically searched for typical influenza disease. Considering vaccine lapse and mortality, which is a measure of influenza, it is no exaggeration to say that humans are fighting antigenic variation. Numerous efforts have been made to overcome this problem, focusing on improving the quality and quantity of vaccines.

The quantitative transition of influenza vaccines is symbolically summarized as an increase in live attenuated vaccines or live low temperature adaptive vaccines. Their primary purpose is regional immunity, which can play an important role in the prevention of respiratory infections through mucosal immunity. However, we have exposed humans to a wide variety of epidemic virus variants in the past, and as a result of reflecting these epidemic profiles, all age groups have a wide variety of antibodies. I understand that I have. Given the above background, epidemic virus mutants have the potential to reject immobilization of live attenuated viruses.

Against this background, an influenza virus-like particle (VLP) vaccine containing the H5 type HA protein of influenza virus produced by Bombyx mori was developed (see Patent Document 1).

Japanese Patent No. 6205359

As a quantitative method, inactivated influenza vaccines have been developed using various techniques such as cell culture-derived vaccines; DNA vaccines; influenza virus-like particle (VLP) vaccines. In fact, in recent years, we have found that the silkworm frogs for the H5 subtype VLP vaccine and the H7 subtype VLP vaccine of triinfluenza are the production levels of the H5 VLP vaccine or the H7HA VLP vaccine, and per frog. We have developed a silkworm worm that exhibits a production level exceeding millions of HA titers (see Patent Document 1). These silk moth pupae can be used in chickens via partially purified products, but in the case of human vaccines, we must confirm their safety and efficacy in detail. In any case, inactivated vaccines in recent years are required to provide high HI antibody titers through appropriate techniques.

In recent years, the present inventors have attempted to use 12 molecules of interleukin as a vaccine in combination with an influenza VLP vaccine, and this vaccine has shown a tendency to increase the protective efficacy against avian influenza virus. Therefore, in order to improve the function of interleukin 12 (IL12) as an adjuvant, the present inventors have conducted intensive studies, and interleukin 12, the M2 protein of influenza A virus associated with the interleukin 12, and the neurominidase. To develop a virus-like particle having an immunomodulator molecule (hereinafter, also referred to as M2 / N / IL12 molecule or IL12 / N / M2 molecule) containing a protein, and to apply the virus-like particle having this immunomodulator molecule to a vaccine. I found. The present inventors disclose the production and protective efficacy of HI antibody by a vaccine containing virus-like particles containing M2, N, and IL12 molecules.

The design drawing of M2 ・ N ・ IL12 molecule is shown. The M2, N, and IL12 molecules are Interleukin-12 (interleukin 12), NA Starks (NA Stark), NA Transmembrane (TM) Domein (NA transmembrane domain), NA Cytoplasmic Domein (NA cytoplasmic domain), and Flaglinker. (Flag linker), M2 Cytoplasmic Domain (M2 cytoplasmic domain), M2 Transmembrane Domain (TM) Domein (M2 cytoplasmic domain), and M2 Small Ectodomein (M2 small extracellular domain). A schematic diagram of IL / N / M2 molecules is shown. It is a schematic diagram which shows an example of the gene manipulation process for the production of the virus-like particle which concerns on this invention. A schematic diagram of an example of a virus-like particle according to the present invention is shown. In the figure, 1 indicates an H5HA protein, 2 indicates an M2 / N / IL12 molecule, and 3 indicates an H7HA protein. It is a figure which shows that M2, N, and IL12 molecules are expressed in Sf9 cells. It is a figure which shows that FkH5 protein is expressed in Sf9 cell. It is a figure which shows that AnH7 protein is expressed in Sf9 cell. It is a figure which shows the result of the FA test which confirmed the expression of M2, N, and IL12 molecules in Sf9 cells by treating with an anti-IL12 monoclonal antibody. It is a figure which shows the result of the FA test which confirmed the expression of M2, N, and IL12 molecules in Sf9 cells by treating with the anti-mouse IL12 antibody. It is a figure which shows the result of the FA test which confirmed the co-expression of VLP containing M2, N, IL12 molecule and FkH5 protein in Sf9 cell by treating with anti-IL12 monoclonal antibody and anti-H5HA antibody. It is a figure which shows the result of the FA test which confirmed the co-expression of VLP containing M2, N, IL12 molecule and AnH7 protein in Sf9 cell by treating with anti-IL12 monoclonal antibody and anti-H7HA antibody. It is a figure which shows the result of the FA test which confirmed the co-expression of VLP containing M2, N, IL12 molecule, FkH5 protein and AnH7 protein in Sf9 cell by treating with the anti-IL12 monoclonal antibody. It is a figure which shows the result of the FA test which confirmed the co-expression of VLP containing M2, N, IL12 molecule, FkH5 protein and AnH7 protein in Sf9 cell by treating with anti-H5HA antibody and anti-H7HA antibody. It is a figure which shows the result of the blood cell adsorption test of the Sf9 cell infected with the Ac M2 / N / IL12 recombinant. It is a figure which shows the result of the blood cell adsorption test of the Sf9 cell infected with the Ac M2 / N / IL12 recombinant and the Ac FkH5 recombinant. It is a figure which shows the result of the blood cell adsorption test of the Sf9 cell infected with the Ac M2 / N / IL12 recombinant and the Ac AnH7 recombinant. As a vaccine, PBS (control), M2 ・ N ・ IL12 molecule (abbreviated as “M2 ・ IL12” in the figure), VLP containing M2 ・ IL12 and FkH5 protein, or VLP containing M2 ・ IL12 and AnH7 protein. It is a figure which shows the survival rate at the time of infecting the administered mouse with HKH5 challenge virus with time. As a vaccine, PBS (control), M2 ・ N ・ IL12 molecule (abbreviated as “M2 ・ IL12” in the figure), VLP containing M2 ・ IL12 and FkH5 protein, or VLP containing M2 ・ IL12 and AnH7 protein. It is a figure which shows the survival rate at the time of infecting the administered mouse with PR8 challenge virus with time. Is a diagram showing the measurement result of PFU (plaque-forming unit). It is a figure which shows the time-dependent change of the body weight when the challenge virus (PR8H1, HKH5, AnH7) was infected to the mouse which administered PBS (control) as a vaccine. It is a figure which shows the weight change at the time of infecting the challenge virus (PR8H1, HKH5, AnH7) in the mouse which administered VLP containing an immune modulator molecule (M2, N, IL12) and FkH5 protein as a vaccine with time. It is a figure which shows the survival rate at the time of infecting the challenge virus (PR8H1, HKH5, AnH7) in the mouse which administered PBS (control) as a vaccine with time. It is a figure which shows the survival rate at the time of infecting the challenge virus (PR8H1, HKH5, AnH7) in the mouse which administered the inactivated PR8H1 vaccine as a vaccine with time. It is a figure which shows the survival rate at the time of infecting a mouse which administered VLP containing an immune modulator molecule (M2, N, IL12) as a vaccine with a challenge virus (PR8H1, HKH5, AnH7) with time. It is a figure which shows the survival rate when the challenge virus (PR8H1, HKH5, AnH7) was infected to the mouse which administered VLP containing an immune modulator molecule (M2, N, IL12) and FkH5 protein as a vaccine with time. .. It is a figure which shows the survival rate at the time of infecting a mouse which administered VLP containing an immune modulator molecule (M2, N, IL12) and AnH7 protein as a vaccine with a challenge virus (PR8H1, HKH5, AnH7) with time. .. It is a figure which summarized the sequence information about the M2, N, IL12 molecule which concerns on this invention.

<Vaccine>
Interleukin-12 (Interleukin-12, hereinafter abbreviated as IL-12 or IL12) is a protein identified in 1989 as a factor that activates NK cells from EBV-transformed cells. It consists of subunits of p40 and p35, and p40 has a subunit in common with IL-23. IL is thought to coordinate various actions by combining subunits in this way. By the way, the immune system of mammals includes acquired immunity by B cells and T cells and innate immunity centered on natural killer cells (Natural Ki11er Tce11, hereinafter abbreviated as NK cells). It is known that axons have only innate immunity, and acquired immunity has been achieved since becoming a vertebrate, but acquired immunity has a very short history in evolution, and organisms other than vertebrates are innate immunized. Has been responsible for immunity. In recent years, NH cells have been discovered by Shigeo Koyasu's group as cells involved in the innate immunity of the lymphocyte system, and three types of innate immune cells, NK cells, lymphoid tissue inducer (LTi) cells, and natural helper (NH) cells, have been identified. It has become clear. In this way, the acquired immune system consists of Th1 cells corresponding to NK cells, Th2 cells corresponding to NH cells, and Th17 cells corresponding to Lti cells, and innate immunity is differentiated and developed to be acquired immunity. I have come to understand.

In addition, the existence of NKT cells (mouse) and MAIT cells (human), which are intermediate cells between innate immunity and acquired immunity, has been clarified, and innate immunity and acquired immunity are closely proliferating and responsible for immunity. Has become clear. However, the research so far has focused on acquired immunity, and the research on innate immunity that supports them has been delayed due to the difficulty of research. Speaking of immunity, research on acquired immunity by T cells and B cells is the main focus. It has been considered that how to activate antibody production by B cells and how to activate cytotoxic T cells are the key to immunity.
However, in reality, there is a foundation of innate immunity, and it is considered that activating acquired immunity as well as activating innate immunity is closer to the actual immune response.

Therefore, the present inventors considered activating NK cells, which are the key to innate immunity. NK cells are known to be enhanced by 1FN-α / β, 1FN-1, 1L-2, 1L-4, lL-12, 1L-15, 1L-18, but the most NK cells among them. It is IL-12 and IL-18 that are activated, and in particular, IL-12 is believed to be the first key cytokine in the activation of NK cells.
There are three types of antigen-presenting cells (APCs): dendritic cells, macrophages / monomorphic cells, and B cells, and only dendritic cells turn naive T cells into activated T cells (effector T cells). And can show the strongest antigen presenting ability. However, conventional inactivated vaccines have been developed with the expectation that they activate Th2-type B cells of acquired immunity and stimulate antibody production. However, due to weak cell-mediated immunity, Th1-type CTLs have been developed. Although adjuvants have been developed to activate cells, development has been difficult due to strong side effects such as swelling. On the other hand, since live vaccines are close to natural infections, cell-mediated immunity can be expected, but on the contrary, if there is already immunity, the live vaccine virus itself cannot propagate, so there are problems such as the vaccine effect not being obtained. It was. On the other hand, the vaccine using IL-12 aims at activation of NK cells and activates antigen presentation by dendritic cells to activate the comprehensive immune action of innate immunity and acquired immunity. There is a merit that you can aim for.

However, local immune activation is important for cytokines, and side effects increase when administered as a single product. Therefore, we decided to develop a vaccine that combines the antigen and IL-12 with a single molecule. The vaccine containing M2, NA, and IL12 as one molecule aims at the vaccine effect by presenting the antigen of the M2 protein of influenza virus in dendritic cells. While the 23 amino acids in the N-terminal region of the M2 protein are exposed on the surface of the virus as antigens, they are well preserved and because they are the entrance to the ion channels of the virus, the antibody against this part has a neutralizing activity of the virus. It is believed that. Furthermore, the 23 amino acids in the N-terminal region of the M2 protein are attracting attention as a universal vaccine that is effective against all influenza viruses regardless of subtype because of their good storage stability. By using the entire M2 containing 23 amino acids in the N-terminal region of the M2 protein, a 19-amino acid transmembrane region following the region exposed on the surface of the 23-amino acid lipid bilayer of M2, and a 54-amino acid lipid two The IL-12 protein was placed behind the transmembrane region of the NA protein and the stalk region of the NA protein by binding to the inner region of the layer and the N-terminal region inside the lipid bilayer of the NA protein via the FLAG sequence. .. This allowed the IL-12 protein to float freely on top of the stalk and outside the lipid bilayer in a stalk-bound form.

Due to this structure, the M2 protein of influenza virus and IL-12 are always in a close positional relationship, and NK cell phagocytosis of M2 protein and antigen presentation by dendritic cells can be expected. Furthermore, in the case of membrane glycoproteins such as HA proteins, HA vaccines prepared by the same baculovirus expression system are mixed and mixed with the lipid double layer of baculovirus or an oil layer such as artificially added sesame oil by ultrasonic treatment or the like. By doing so, IL-12 and the antigen protein can be coordinated on the same artificial membrane, and the effects of both innate immunity and acquired immunity activated based on innate immunity are linked to all antigens. It can be used as a vaccine antigen.

From the above, the vaccine in the present invention is a vaccine characterized by containing an immune modulator molecule. The immune modulator molecule comprises an IL-12 protein, an NA domain region derived from a neuraminidase (NA) protein, and an M2 protein domain region derived from an influenza virus. The NA domain region includes an extracellular domain, a transmembrane domain, and an intracytoplasmic domain, and the M2 protein domain region includes an extracellular domain, a transmembrane domain, and an intracytoplasmic domain.
Then, in the immunomodulator molecule, the IL-12 protein is bound to the extracellular domain in the NA domain region, and the cytoplasmic domain in the M2 protein domain region is bound to the cytoplasmic domain in the NA domain region by a linker.

Further, the vaccine according to the present invention further comprises (i) influenza virus-like particles containing a hemagglutinin (HA) protein derived from a virus classified into influenza virus H5 type, and / or (ii) influenza virus H7 type. It may contain influenza virus-like particles containing hemagglutinin (HA) protein from the classified virus.
The influenza virus-like particles contained in the vaccine according to the present invention are classified into influenza virus H5 type hemagglutinin (HA) protein (hereinafter, also referred to as H5 protein) and / or influenza virus H7 type. In addition to the virus-derived hemagglutinin (HA) protein (hereinafter, also referred to as H7 protein), M2, N, and IL12 molecules are co-expressed.
In addition, the vaccine of the present invention can provide a survival rate equivalent to that of an animal that has been vaccinated with an inactivated influenza virus H1N1 type and infected with the influenza virus H1N1 type.
In addition, at least one selected from virus-like particles containing immune modulator molecules, influenza virus-like particles (i), and influenza virus-like particles (ii) is produced by Sf9 cells or Eri silkworms by the method described later. It is preferable to let it.
Further, it is preferable that the virus classified into influenza virus H5 type is H5N1 type virus and / or the virus classified into influenza virus H7 type is H7N9 type virus.

<Virus-like particles>
The virus-like particle of the present invention may have a virus-like particle structure containing only the chimeric cytokine M2, N, IL12, or may have a virus-like particle structure containing HA protein and M2, N, IL12. .. The virus-like particles contain the HA protein of the virus, which does not contain RNA derived from influenza virus, and the structure of the virus-like particles is preferably particles having a diameter of 50 to 150 nm, more preferably 60 nm to 120 nm. It has a structure in which distinct spikes (eg, HA spikes) are densely arranged on the surface of the virus, and is morphologically very similar to a virus particle.
Since the virus-like particles of the present invention are expressed by the silk moth, they may have lipids or sugar chain modifications derived from the silk moth.
Examples of the lipid include glyceroglycolipid, glycosphingolipid, cholesterol, phospholipid and the like.

Examples of glyceroglycolipids include sulfoxyribosylglyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.
Examples of glycosphingolipids include galactosyl cerebroside, lactosyl cerebroside, and ganglioside.
Examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylic acid, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin and the like.
From the above lipids, fatty acids and the like induced by hydrolysis and the like may be contained. Examples of the fatty acid include myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid and the like.
The vaccine containing virus-like particles of the present invention has a structure in which spikes of recombinant virus membrane proteins having immunogenicity are densely provided, so that an unprecedentedly high immunogenicity can be achieved without using an adjuvant. It becomes possible to show the sex. Since no adjuvant is used, side effects such as allergies and anaphylactic shock can be reduced.

In the vaccine of the present invention, the virus-like particles of the present invention containing the virus-like particles of the present invention have high immunogenicity. Therefore, even if a commonly used adjuvant is not used, an unprecedented high immunogenicity is obtained. It is possible to show sex. Moreover, since a commonly used adjuvant is not used, side effects such as allergies and anaphylactic shock can be reduced. Further, as a matter of course, since the nucleic acid derived from the virus does not exist inside the hollow of the spherical outer shell of the virus-like particle of the present invention, it is non-pathogenic. The vaccine of the present invention can effectively prevent viral infections in animals including humans. In addition, the vaccine of the present invention can significantly shorten the period required for production as compared with the conventional production method using chicken eggs. Therefore, in the event of an epidemic of viral infections such as influenza, vaccines can be procured in a short period of time, and it is possible to make a great contribution to protecting people from infectious diseases and maintaining their health. Furthermore, in the production of a vaccine using conventional chicken eggs, it is necessary to have a highly safe facility such as a P3 facility in order to handle the seed virus used for the vaccine, and it is necessary to produce a large amount of vaccine because the immunogenicity of the vaccine is low. While a huge manufacturing cost is required for some reason such as the above, the manufacturing method of the present invention can significantly reduce the manufacturing cost.

In one embodiment, the invention is a method of inoculating an animal with a vaccine against viral infection, the effective amount of a vaccine comprising the polypeptide according to the invention or produced according to the production method according to the invention. Is a method comprising administering to the animal.
The animal is not particularly limited as long as it is a homeothermic animal, and may be a non-human animal such as a bird, a pig, a cow, a dog, or a cat.
In one embodiment, the invention is a method of inducing an immune response against a virus to an animal, the efficacy of a vaccine comprising the polypeptide according to the invention or produced by the production method according to the invention. A method comprising administering an amount to the animal.
In another aspect, the invention relates to a vaccine composition. In addition to the virus-like particles, the vaccine composition may contain additional components and a pharmaceutically acceptable carrier. Examples of the additional component include an adjuvant and a component that enhances the anti-influenza virus action such as an anti-influenza virus agent. Examples of the adjuvant include aluminum hydroxide, aluminum phosphate, saponin, water-in-oil emulsion, oil-in-water emulsion, emulsion in water-in-oil, acrylic or methacrylic acid polymer, maleic anhydride, alkenyl derivative, carbomer, and block copolymer. .. Examples of the antiviral agent include neuromitase inhibitors such as zanamivir, oseltamivir, peramivir, and laninamivir, amantadine, rimantadine, and RNA polymerase inhibitors. The pharmaceutically acceptable carriers contained in the vaccine composition of the present invention include solvents, dispersants, coating agents, stabilizers (albumin, ethylenediamine tetraacetate alkali salt), and diluents that are permitted to be used as pharmaceuticals. (Water, saline, dextrose, ethanol, glycerol, etc.), preservatives, excipients, antibacterial agents, antifungal agents, isotonic agents (sodium chloride, dextrose, mannitol, sorbitol, lactose, etc.), absorption retarders Including.

For example, the vaccine composition of the present invention can be in a dosage form suitable for parenteral administration or oral administration. Examples of the composition for parenteral administration include injections, nasal drops and the like, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. Includes the dosage form of. Such injections can be prepared according to known methods, for example, by dissolving, suspending or emulsifying the antigenic protein in a sterile aqueous or oily solution usually used for injections. The prepared injection solution is usually filled in a suitable ampoule, vial or syringe. In addition, the parenteral pharmaceutical composition is prepared in a dosage form of a dosage unit suitable for the dose of the active ingredient.
An effective amount of a vaccine is an amount sufficient to achieve a biological effect such as inducing sufficient humoral or cell-mediated immunity against the virus. In addition, administration methods include inhalation, intranasal, oral, parenteral (eg, intradermal, intramuscular, intravenous, intraperitoneal, and subcutaneous administration). The effective amount and method of administration may depend on the age, sex, condition and body weight of the person being administered. For example, in the case of influenza vaccine, in general, a vaccine containing 15 μg or more of HA protein per strain in 1 ml, 0.25 ml subcutaneously for those aged 6 months or more and less than 3 years old, and those aged 3 to 13 years old 0.5 ml is injected subcutaneously twice at intervals of approximately 2-4 weeks. For persons 13 years and older, 0.5 ml is injected subcutaneously once or twice at intervals of approximately 1 to 4 weeks.

<Method for producing virus-like particles containing immunomodulator molecules>
In the present invention, it is preferable to produce virus-like particles using a protein expression system using Sf9 cells or Eri silkworm. In one aspect of the method for producing a vaccine of the present invention, in a DNA fragment encoding an immunomodulator molecule, the amino acid sequence of the protein of the encoded immunomodulator molecule does not change as compared with the corresponding mammalian amino acid sequence. As described above, a step of modifying the codon of the DNA fragment to obtain a codon-optimized DNA fragment for expression, a step of inserting the obtained codon-optimized DNA fragment into a vector, the obtained vector, and a baculovirus-derived DNA. The step of co-transfecting Sf9 cells, the step of obtaining a baculovirus recombinant containing a codon-optimized DNA fragment from the obtained Sf9 cells, and infecting the eri silkworm with the baculovirus recombinant to produce the eri silkworm. It is preferable to include a step of breeding and a step of isolating the immunomodulator molecule from the Eri silkworm.

In the present invention, for the purpose of improving the protein expression level using silk moth, Nerome K, Sugita S, Kuroda K, Hirose T, Matsuda S, Majima K, et al. The large-scale production of an artificial influenza virus-like Codon optimization can be performed using a technique similar to that described in particle vaccine in silkworm pupae. Vaccine 2015; 33: 117-25.
For example, an amino acid sequence corresponding to the NA domain region, IL-12 protein, and NA domain region used in the present invention is obtained from an amino acid sequence registered in Genbank or the like in a mouse, and is a basis for nucleic acid design. And. Based on the obtained amino acid sequence, the virus-like particle gene sequence is determined, and the virus-like particle gene sequence is modified in consideration of optimizing the codon usage frequency of the silk moth. By such a procedure, a codon-optimized DNA fragment for expression of Eri silkworm of M2, N, IL12 molecule is obtained.

Then, the obtained codon-optimized DNA fragment is inserted into a known vector suitable for baculovirus by using any known method, and the obtained vector and the linearized baculovirus-derived DNA are obtained. , A step of co-transfecting Sf9 cells, a step of obtaining a baculovirus recombinant obtained by recombining a codon-optimized DNA fragment from the obtained Sf9 cells, infecting Eri silkworm with the baculovirus recombinant to produce Eri silkworm. Virus-like particles may be obtained through a step of breeding and a step of isolating virus-like particles from the Eri silkworm.

In the present invention, (i) an optimized DNA fragment for expressing influenza virus-like particles containing hemagglutinin (HA) protein derived from a virus classified into influenza virus H5 type, which is obtained by the same method as described above. And / or (ii) An optimized DNA fragment for expressing influenza virus-like particles containing a hemagglutinin (HA) protein derived from a virus classified into influenza virus H7 was prepared, and the same operation was performed to prepare the above-mentioned in Eri silkworm. (I) and / or (ii) above may be expressed, or these optimized DNA fragments may be co-expressed together with a codon-optimized DNA fragment for the expression of Eri silkworms of M2, N, IL12 molecules. Good.

The time of introduction (inoculation) of the baculovirus recombinant into the silk moth is not particularly limited as long as it is in the silk moth stage, but it is preferably early in the silk moth stage, specifically, on the day of molting (molting 1). The day) to the 4th day (5th day of molting) is preferable, and the next day (2nd day of molting) to the day after next (3rd day of molting) is more preferable.
After inoculating the baculovirus recombinant into Eri silkworm pupae, these pupae are bred for a predetermined period, and the temperature conditions at that time are preferably 20 ° C. to 30 ° C., more preferably 22 ° C. to 28 ° C., and 24 ° C. to 24 ° C. 27 ° C. is more preferable, and 26 ° C. is particularly preferable. The period of pupal breeding is not particularly limited as long as it is the period from after the above inoculation to before the pupae metamorphose into adults (moths), but 2 to 6 days after the above inoculation is preferable, and 2 to 4 days is more preferable. It is preferable, and 3 days is more preferable.
Any method well known to those skilled in the art can be used for the recovery of virus-like particles from Eri silkworm. For example, Eri silkworm can be homogenized in an isotonic buffer solution and then recovered using immobilized erythrocytes or a sialic acid column (fetin column). It is also possible to obtain a fraction contained in the form of virus-like particles by using a fractionation method such as a sucrose density gradient centrifugation method.
Next, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

1. 1. Materials and methods 1.1. Cells and virus Sf9 cells (Sigma-Aldrich, Japan, Tokyo) derived from the ovarian tissue of Sporodoptera fulgiperda (Sporodoptera fulgiperda) were added with 10% fetal bovine serum (FBS) in Grace Insect Medium (Thermo Fisher). It was cultured. Maidin-Derby canine kidney (MDCK) cells were cultured in Eagle's Minimal Essential Medium (MEM) containing 10% FBS.
Ac NPV recombinant (hereinafter, also referred to as Ac M2 / N / IL12 recombinant) in which a DNA sequence encoding M2 / N / IL12 molecule is incorporated into Autographa California nuclear polymorphic disease virus (Ac NPV), FkH5HA protein in Ac NPV. Ac NPV recombinant (hereinafter, also referred to as Ac FkH5 recombinant) incorporating a DNA sequence encoding AnH7HA protein, and Ac NPV recombinant (hereinafter, also referred to as Ac AnH7 recombinant) incorporating a DNA sequence encoding AnH7HA protein in Ac NPV. ) Was obtained by the method described later.
A / PR / 8/34 (H1N1) strain (PR8H1) of influenza virus was cultured and PR8H1 was infected with mice in order to restore the virulence in the mice. In addition, HKH5 (RG-A / BarnSwallow / HongKong / 1161/2010-A / PR / 8/34 [R] (6 + 2) (H5N1), and AnH7 (RG-A), which are recombinant (attenuated) influenza viruses. / Anhui / 1/2013-A / PR / 8/34 [R] (6 + 2) (H7N9) will be sold to Dr. RG Webster of St Jude Chicken's Research Hospital, Memphis, TN, USA. It was propagated in 10-day-old fertilized chicken eggs and MDCK cells.

1.2. Creation of Ac NPV recombinant The synthesis of FkH5 gene and AnH7 gene was performed by Japan Genewith Co., Ltd. (Saitama, Japan). Regarding the production of Ac NPV recombinants containing these HA genes and M2 ・ N ・ IL12 genes, Maegawa K, Shibata T, Yamaguchi R, Hiroike K, Izzati U. Z, et al. Overexpression of a virus-like particke influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range expansion. Arch Virol 2018; 163 (10): 2787-2797. .. Similarly, the M2, N, and IL12 genes were also synthesized by Japan Genewith Co., Ltd. The M2, N, and IL12 genes include glycine linker (L1), M2 small extracellular domain (23 amino acids), M2 transmembrane domain (19 amino acids), M2 cytoplasmic domain (54 amino acids), Flag linker (11 amino acids), and NA cytoplasm. Designed to encode a membrane protein constructed by combining two subsystems of IL12 (p35 and p40) with a domain (6 amino acids), an NA transmembrane domain (31 amino acids), and NA Stoke (41 amino acids). It was. The synthesized gene was inserted downstream of the pFast Bac1 (pFast Bact) polyhedrosis promoter, and the results and plasmid were used to introduce into DH10Bac. After that, Maegawa K, Shibata T, Yamaguchi R, Hiroike K, Izzati U. Z, et al. Overexpression of a virus-like particke influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range expansion. Arch Virol. 2018; 163 (10): 2787-2797. As described in doi: 10.1007 / s00705-018-3941-4., The Ac NPV recombinant was purified and used to introduce into Sf9 cells.
In addition, each DNA sequence and each amino acid sequence used in this Example are summarized at the end of this specification.

1.3. Preparation of VLP Vaccine in Eli silk moth The pupa of Eli silk moth was used to prepare the VLP vaccine. To address this procedure, tussah pupae were infected with the Ac NPV recombinant described above. Inoculation of baculovirus into pupae and preparation of each VLP vaccine are performed by Maegawa K, Shibata T, Yamaguchi R, Hiroike K, Izzati U.Z, et al. Overexpression of a virus-like particke influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range expansion. Arch Virol 2018; 163 (10): 2787-2797. Doi: 10.1007 / s00705-018-3941-4.
1.4. Hemagglutination test and inhibition of blood cell aggregation (HI) For the hemagglutination test and HI test, see Nerome K, Sugita S, Kuroda K, Hirose T, Matsuda S, Majima K, et al. The large-scale production of an artificial influenza. virus-like particle vaccine in silkworm pupae. Vaccine 2015; 33: 117-25.

1.5. Blood cell adsorption test Sf9 cells infected with the Ac NPV recombinant described above were washed once with PBS, and Maegawa K, Shibata T, Yamaguchi R, Hiroike K, Izzati U. Z, et al. Overexpression of a virus-like. particke influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range expansion. Arch Virol 2018; 163 (10): 2787-2797. A blood cell adsorption test was performed using 0.5% chicken erythrocytes.

1.6. Fluorescent antibody (FA) test and antibody Sf9 cells were infected with each of the above Ac NPV recombinants and maintained at 27 ° C. for 1 day in Grace insect medium supplemented with 10% FBS. Subsequent FA trials included Maegawa K, Shibata T, Yamaguchi R, Hiroike K, Izzati U. Z, et al. Overexpression of a virus-like particke influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range. expansion. Arch Virol 2018; 163 (10): 2787-2797. Doi: 10.1007 / s00705-018-3941-4. The anti-Flag mouse monoclonal antibody M2 and the anti-DYKDDDDK tag mouse monoclonal antibody were purchased from Sigma Aldrich (St. Louis, MO, USA) and Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively. Antisera against HKH5 virus, PRHA virus, and AnH7 virus were prepared in mice immunized with each purified virus (HA titer of 2,048-4,096) three times.

1.7. Protective effect against intranasal infection by influenza virus for antigen administration A series of mouse groups, including 5 mice each, were subjected to VLP, FkH5 protein and M2 containing inactivated PRH1, M2, N, IL12 molecules in PBS. A VLP containing N. IL12 molecule, a VLP containing AnH7 protein and M2.N. IL12 molecule, or a vaccine containing VLP containing FkH5 protein, AnH7 protein and M2.N. IL12 molecule, 4 Vaccines with HA titers of 000 to 16,800 were immunized via the abdomen. Two weeks later, the same immunization was repeated via the same intraperitoneal route. After 6 weeks, mice were infected with the virus by antigen administration with challenge virus via the nasal route, and the health status and survival of the mice immunized with the above three vaccines were observed daily for 9 days.
Mice that died during the course of the experiment were autopsied for lung removal and collection of whole blood. The viral load in the removed lung was measured by the plaque method for homogenates in all the removed lungs. For infection with antigen virus, undiluted virus stock solutions of pathogenic PRH1 virus and FkH5 virus were used.

2. Result 2.1. Design of Immunomodulator Molecules To boost the immune response of influenza VLP vaccines, we will incorporate IL-12 molecules and a series of structural proteins of influenza A virus into silkworm-derived VLP vaccines. I tried. To approach this strategy, we primarily selected the cytokine IL-12, a partial region of neuraminidase, and the influenza virus M2 protein. These six polypeptides were tandemly linked according to the order of M2-IL-12 molecules as described in the "Materials and Methods" section (FIGS. 1A-1C). Finally, the immune modulator molecule (FIG. 1B) was placed as presented on the surface of influenza VLPs by protein expression using Sf9 cells or Eli silkworm hosts (FIG. 1D).

2.2. Expression of recombinant DNA encoding immunomodulator molecule and VLP antigen A total DNA gene (2,130 nucleotides) encoding a total peptide of 710 amino acids was synthesized and inserted downstream of the pFast Bac1 polyhedrosis promoter. The resulting plasmid was used to transform DH10Bac. Finally, the Ac M2 / N / IL12 recombinant was infected with Sf9 and Eri silkworm (Fig. 2A). Maegawa K, Shibata T, Yamaguchi R, Hiroike K, Izzati U.Z, et al. Overexpression of a virus-like particke influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear Polyhedrosis virus and host-range expansion. Arch Virol 2018; 163 (10): 2787-2797. Doi: 10.1007 / s00705-018-3941-4. Each Ac NPV recombinant allowed Sf9 cells to be single-infected or double-infected. As shown in FIG. 2A, a molecular size of about 100,000 was confirmed, so that the M2, N, and IL12 molecules were supported to be expressed in Sf9 cells (pointed by arrows). It should be noted that this molecular weight can be influenced by the carbohydrate chain. The bands shown in FIGS. 2B and 2C (pointed by arrows) supported the expression of VLPs containing the FkH5 protein and VLPs containing the AnH7 protein, respectively, in Sf9 cells. In addition, the pupae of the silk moth were double-infected with this Ac M2 / N / IL12 recombinant in combination with the Ac FkH5 recombinant and the Ac AnH7 recombinant. In addition, the Ac M2 / N / IL12 recombinant was mixed with the Ac FkH5 recombinant or the Ac AnH7 recombinant to infect the Eri silkworm.

2.3. Analysis of double-infected insect cells by FA test We include VLPs containing M2 · N · IL12 molecules and FkH5 protein and / or M2 · N · IL12 molecules and AnH7 protein. When VLPs were prepared, it was predicted that monovalent VLP vaccines and divalent VLP vaccines associated with M2, N, and IL12 molecules could be synthesized. First, Sf9 cells were subjected to their Ac NPV recombinants. Double infected. This result is shown in the FA test of FIG. Expression of the M2, N, and IL12 molecules was confirmed in FIG. 3A (red) treated with the IL12 monoclonal antibody. In contrast, Sf9 cells infected with Ac M2 · N · IL12 were determined to be green labeled with anti-mouse IL12 antibody (FIG. 3B). On the other hand, they are different neutral colors between red and green, and co-expression of M2 ・ N ・ IL12 and FkH5 VLP antigen (Fig. 3C), or co-expression of M2 ・ N ・ IL12 and AnH7 VLP antigen. The purpose was to observe the neutral colors showing expression (FIG. 3D). Figures 3E and 3F highlighted a much more diverse neutral color, indicating co-expression between VLP antigens containing M2, N, IL12 molecules and FkH5 and AnH7 proteins.

2.4. Hemagglutinin adsorption activity of double-infected cells As shown in FIGS. 3A to 3F, the present inventors were able to confirm that a large number of cells were capable of co-expression of the above three recombinants. Next, co-expression by the above three Ac NPV recombinants was confirmed by exposure on infected cells by a blood cell adsorption test of FkH5 VLP antigen or AnH7 VLP antigen. FIG. 4A showed negative hemodsorption in cells infected with the Ac M2 ・ N ・ IL12 recombinant, and as a result, the expressed M2 ・ N ・ IL12 molecules contained hemagglutination molecules. It was shown not to. This was in sharp contrast to the typical blood cell adsorption profile observed in FIGS. 4B and 4C. When cells were infected with the Ac M2 / N / IL12 recombinant and the Ac FkH5 or Ac AnH7 recombinant, the presence of the VLP antigen on the surface of the infected cells was shown as an exposed state.

2.5. The protective efficacy of three different VLP vaccines in mice M2, N, IL12 molecules (abbreviated as "M2, IL12" in FIG. 5) are possible immunodefensive M2 proteins of influenza A virus. Since it contains an M2 protein showing a very high degree of homology between influenza A viruses, the protective activity of a vaccine containing virus-like particles containing an immune modulator molecule was examined in mice. The vaccine showed 80% and 60% survival against infection with PRbH1 and HKH5 viruses, indicating protection against the above two viruses (FIG. 5A). Since the H7 virus did not show pathogenicity in mice, no effective mortality and survival data could be obtained from this experiment. On the other hand, it should be noted that a vaccine containing a virus-like particle containing an immunomodulator molecule and an FkH5 protein or a virus-like particle containing an immunomodulator molecule and an H7 protein is effective against infection with a highly pathogenic PR8H1 virus. , 90-100%, which is extremely high protection (Fig. 5B). In contrast, immunization of mice with virus-like particles containing only immunomodulator molecules showed a 20% mortality rate, suggesting a weak but protective protection (Fig. 5B).

In addition, a follow-up study was conducted on the protective efficacy of three different VLP vaccines in mice. PBS (control), inactivated PR8H1, VLP containing M2 ・ N ・ IL12 molecule, VLP containing FkH5 protein and M2 ・ N ・ IL12 molecule, or AnH7 as described in 1.7 above. Daily changes in mouse health and body weight (%) after infection with the challenge virus (HkH5, AnH7, and PR8H1) in mice immunized with a vaccine containing VLPs containing proteins and M2, N, IL12 molecules. Examined. As shown by the arrows in FIG. 6A, mice infected with PR8 virus after PBS control treatment lost body weight 6 days after infection. In contrast, no significant changes in body weight were observed in the HkH5 and AnH7 virus-infected mice (Fig. 6A). In addition, similar weight loss was not confirmed in the vaccinated mouse group containing VLPs containing FkH5 protein infected with HkH5 and AnH7 viruses and M2, N, IL12 molecules (Fig. 6B). In contrast, a group of vaccine mice containing VLPs containing FkH5 protein and M2, N, IL12 molecules showed unexpected weight loss (see arrow in FIG. 6B). However, this result was inconsistent with that of non-immune control mice (see arrow in FIG. 6A), suggesting that further analysis of the results using the highly pathogenic avian influenza virus is needed.
In addition, control mice treated with PBS (control) showed 60% and 20% survival rates for HkH5 virus and PR8H1 virus, respectively, but 100% survival rates for AnH7 virus (). FIG. 6C). In addition, the protective activity of the simple inactivated vaccine (PR8 vaccine) against the PR8H1, HkH5, and AnH7 challenge viruses was 100%, 60%, and 100%, respectively (Fig. 6D).
In contrast, mice immunized with VLP containing M2 ・ N ・ IL12 molecule (Fig. 6E), mice immunized with VLP containing FkH5 protein and M2 ・ N ・ IL12 molecule (Fig. 6F), and AnH7 protein. All mice immunized with VLP containing M2, N, and IL12 molecules (FIG. 6G) were shown to have improved survival rates as compared to FIGS. 6C and 6D.
Each experimental group consisted of 10 mice.

2.6. Confirmation of HI antibody titer and protective effect in mice used for immunization and subsequent challenge antigen-administered infection As shown in Table 1, immunization with only virus-like particles containing immunomodulator molecules (M2, N, IL12 molecules) Mice that were subsequently infected with the antigen-administering viruses of HKH5, AnH7, and PRbH1 exhibited HI titers for the antigen-administered virus only. Interestingly, higher HI antibody titers against AnH7 and PR8H1 viruses were shown during the infection period with antigen administration over a week, suggesting an adjuvant effect of the immune modulator molecule. As can be seen in Table 1, virus-like particles containing an immunomodulator molecule (M2, N, IL12 molecule) and FkH5 protein, and a virus containing an immunomodulator molecule (M2, N, IL12 molecule) and AnH7 protein. Vaccine immunization with virus-like particles, followed by infection by antigen administration of homologous HKH5 or AnH7 strains, resulted in low levels of HI antibody titers against homologous antibodies, resulting in a HI titer of 64-1024. Shown. In contrast, immunization with the VLP vaccine, as well as the HI immune response in mice used in subsequent homoviral and heteroviral infections, was immunized with virus-like particles containing immunomodulator molecules (M2, N, IL12). Later, it was about the same as the immune response in immunization by single infection with a single viral antigen. For example, the HI antibody titer against the virus for antigen administration was relatively higher than the HI antibody titer obtained from the first vaccine immunization, suggesting an adjuvant effect.

Also, as shown in Table 1, the divalent H5 and H7 VLP vaccines inhibited mouse PR8H1 (H1 subtype), HKH5 (H5 subtype) and AnH7 (H7 subtype). We also supported that co-administration of VLP vaccines containing M2, N, IL12 molecules with H5 and H7 proteins could protect animals from completely different PR8H1 belonging to the HA subtype, and protected them from mutual infection.

In Table 1, the numbers in parentheses in the column of protective activity represent the mortality rate (%) due to challenge virus infection, and the molecule (left) is immunized with the vaccine and killed mice when infected with the virus. It shows the rate. The denominator (right) shows the mouse mortality rate when immunized with a negative control PBS and infected with the virus.

Regarding 2.6 above, the same experiment was performed again on mice. The results are shown in Table 2. From the results shown in Table 2, it was found that the vaccine according to the present invention has a protective ability against various antigens.

2.7. Cross-defense efficacy of three different interactive VLP vaccines Vaccine containing virus-like particles containing immunomodulator molecule (M2 ・ N ・ IL12 molecule) and FkH5 protein, immunomodulator molecule (M2 ・ N ・ IL12 molecule) The protective efficacy of vaccines containing virus-like particles containing AnH7 vaccine protein and vaccines containing immunomodulator molecules (M2, N, IL12 molecules) is significantly different between HA subtypes H1 and H5 or H7 hemagglutinin. , Assessed their interaction with full defense. As shown in FIG. 5C, immunization with a vaccine containing an immunomodulator molecule (M2, N, IL12 molecule) alone did not protect against FkH5 infection, but an immunomodulator molecule (M2, N, IL12 molecule) and an FkH5 protein. Vaccines containing virus-like particles containing and and vaccines containing virus-like particles containing immunomodulator molecules (M2, N, IL12 molecules) and AnH7 protein completely defended against each other. As already mentioned, the pathogenicity of the AnH7 virus is so low that this system does not allow comparison of vaccine efficacy, but in any case, all test mice showed 100% survival (Figure). 5B). In contrast, mice vaccinated with PBS as a negative control of the vaccine were immunized with the M2 ・ N ・ IL12 + HKH5 VLP vaccine when the survival rate was 20%, followed by a significantly different subtype of PR8H1 virus. It was interesting to find that the survival rate of the mice fed was 90% (Fig. 5B). Not surprisingly, the same vaccine showed 100% protective activity against each of the AnH7 and HKH5 viruses.

Table 3 below summarizes the cross-protective activity of the VLP vaccine according to the invention evaluated in mice immunized with various vaccines and infected with the challenge virus. In Table 3, the number after + or-represents the percentage of protective activity observed in mice infected with the challenge virus.

The above results can be summarized as follows.
As the structure of the immune support molecule, the present inventors succeeded in constructing the M2 peptide and the stalk peptide of the neuraminidase protein of IL-12, which is an immune reaction modulator, and the neuraminidase protein of influenza A virus, based on the molecular design of each structural gene. did. As an approach to the above strategy, the DNA encoding all the above proteins, which is 2,130 nucleotides in length, is recombinant DNA (M2 · N) by the Autographa California nuclear polyhedrosis virus (Ac NPV). -The preparation of IL12) was carried out. In this study, we also found two recombinant DNAs in the Ac NPV vector, Fukushima H5 (FkH5) and Anhui H7 (FkH5) for different influenza virus-like particle (VLP) vaccines. Recombinant DNA containing AnH7) was also prepared. Their expression was confirmed in single or double infection in Sf9 cells. Western blot analysis pointed to their expression, indicating their appropriate molecular size. Similarly, their large-scale production in Eli silkworms could also be investigated by hemagglutination (HA), hemagglutination inhibition (HI), and Western blot analysis. Double infection of the recombinant virus with M2, N, IL12, FkH5, and AnH7 resulted in co-expression of the above three antigens within the same infected Sf9 cells. These VLP antigens grown in Eli silkworm frogs were subjected to immunological experiments in mice, and this immunological test was performed after immunization with a post-immunization HI response (using three antigen-administering viruses, in mice). Infection and protective efficacy by antigen administration). As a normal result, it was clear that the HI antibody produced in mice after immunization and subsequent antigen infection reacts with each subtype-specific HA antigen. Despite the subtype-specific HI response, immunization with the influenza VLP vaccine against FkH5 or ANH7 associated with M2, N, and IL12 showed protective efficacy across the HA subtype of influenza A virus. It is suggested that this vaccine can overcome the ineffectiveness caused by antigen drift. This result could be a step towards a glimpse of future universal vaccines through the further introduction of immune molecules.
After confirming the role of broad protective activity in influenza virus, the chimeric cytokine (immune modulator molecule) system may be useful in the field of other infectious viruses, including the broad viral family. In fact, the present inventors have already developed the DPT vaccine, which is a vaccine for measles, mumps and rubella virus, and the ZIKA and mad dog disease VLP vaccines in Kaiko, and these vaccines have also been developed as chimeric cytokines. We are promoting.

Information about the sequences used in the examples is described below.
(1) Sequence of FkH5 gene
Influenza A virus (A / tufted duck / Fukushima / 16/2011 (H5N1)) viral cRNA, segment 4, complete sequence
ACCESSION AB629698

(2) DNA sequence of FkH5 gene optimized for expression (SEQ ID NO: 1)
ATGGAAAAAATCGTGCTGCTGTTCACAACAATCGGTCTGGTGAAATCAGACCACATCTGCATCGGTTACCACGCTAACAACTCAACAGAACAAGTGGACACAATCATGGAAAAAAACGTGACAGTGACACACGCTCAAGACATCCTGGAAAAAACACACAACGGTAAACTGTGCGACCTGAACGGTGTGAAACCTCTGATCCTGAAAGACTGCTCAGTGGCTGGTTGGCTGCTGGGTAACCCTCTGTGCGACGAATTCATCAACGTGCCTGAATGGTCATACATCGTGGAAAAAGCTAACCCTGCTAACGACCTGTGCTACCCTGGTAACTTCAACGACTACGAAGAACTGAAACACCTGCTGTCAAGAATCAACCACTTCGAAAAAATCCAAATCATCCCTAAAGACTCATGGTCAGACCACGAAGCTTCACTGGGTGTGTCAGCTGCTTGCTCATACCAAGGTAACTCATCATTCTTCAGAAACGTGGTGTGGCTGATCAAAAAAGACAACGCTTACCCTACAATCAAAAAAGGTTACAACAACACAAACCAAGAAGACCTGCTGGTGCTGTGGGGTATCCACCACCCTAACGACGAAGCTGAACAAACAAGACTGTACCAAAACCCTACAACATACATCTCAATCGGTACATCAACACTGAACCAAAGACTGGTGCCTAAAATCGCTACAAGATCAAAAATCAACGGTCAAAGAGGTAGAATCGACTTCTTCTGGACAATCCTGAAACCTAACGACGCTATCCACTTCGAATCAAACGGTAACTTCATCGCTCCTGAATACGCTTACAAAATCGTGAAAAAAGGTGACTCAACAATCATGAAATCAGAAGTGGAATACGGTAACTGCAACACAAGATGCCAAACACCTATCGGTGCTATCAACTCATCAATGCCTTTCCACAACATCCACCCTCTGACAATCGGTGAATGCCCTAAATACGTGAAATCAAACAAACTGGTGCTGGCTACAGGTCTGA GAAACTCACCTCAAAGAGAAACAAGAGGTCTGTTCGGTGCTATCGCTGGTTTCATCGAAGGTGGTTGGCAAGGTATGGTGGACGGTTGGTACGGTTACCACCACTCAAACGAACAAGGTTCAGGTTACGCTGCTGACAAAGAATCAACACAAAAAGCTATCGACGGTGTGACAAACAAAGTGAACTCAATCATCGACAAAATGAACACACAATTCGAAGCTGTGGGTAGAGAATTCAACAACCTGGAAAGAAGAATCGAAAACCTGAACAAAAAAATGGAAGACGGTTTCCTGGACGTGTGGACATACAACGCTGAACTGCTGGTGCTGATGGAAAACGAAAGAACACTGGACTTCCACGACTCAAACGTGAAAAACCTGTACGACAAAGTGAGACTGCAACTGAAAGACAACGCTAAAGAACTGGGTAACGGTTGCTTCGAATTCTACCACAAATGCAACAACGAATGCATGGAATCAGTGAGAAACGGTACATACGACTACCCTCAATACTCAGAAGAAGCTAGACTGAAAAGAGAAGAAATCTCAGGTGTGAAACTGGAATCAATCGGTATCTACCAAATCCTGTCAATCTACTCAACAGTGGCTTCATCACTGGTGCTGGCTATCATGATGGCTGGTCTGTCACTGTGGATGTGCTCAAACGGTTCACTGCAATGCAGAATCTGCATCGACTACAAAGACGACGACGACAAATAA

(3) DNA sequence of AnH7 gene (SEQ ID NO: 2)
(GISAID's EpiFlu ^TM Database: SegmentID: EPI439507)
ATGAACACTCAAATCCTGGTATTCGCTCTGATTGCGATCATTCCAACAAATGCAGACAAAATCTGCCTCGGACATCATGCCGTGTCAAACGGAACCAAAGTAAACACATTAACTGAAAGAGGAGTGGAAGTCGTCAATGCAACTGAAACAGTGGAACGAACAAACATCCCCAGGATCTGCTCAAAAGGGAAAAGGACAGTTGACCTCGGTCAATGTGGACTCCTGGGGACAATCACTGGACCACCTCAATGTGACCAATTCCTAGAATTTTCAGCCGATTTAATTATTGAGAGGCGAGAAGGAAGTGATGTCTGTTATCCTGGGAAATTCGTGAATGAAGAAGCTCTGAGGCAAATTCTCAGAGAATCAGGCGGAATTGACAAGGAAGCAATGGGATTCACATACAGTGGAATAAGAACTAATGGAGCAACCAGTGCATGTAGGAGATCAGGATCTTCATTCTATGCAGAAATGAAATGGCTCCTGTCAAACACAGATAATGCTGCATTCCCGCAGATGACTAAGTCATATAAAAATACAAGAAAAAGCCCAGCTCTAATAGTATGGGGGATCCATCATTCCGTATCAACTGCAGAGCAAACCAAGCTATATGGGAGTGGAAACAAACTGGTGACAGTTGGGAGTTCTAATTATCAACAATCTTTTGTACCGAGTCCAGGAGCGAGACCACAAGTTAATGGTCTATCTGGAAGAATTGACTTTCATTGGCTAATGCTAAATCCCAATGATACAGTCACTTTCAGTTTCAATGGGGCTTTCATAGCTCCAGACCGTGCAAGCTTCCTGAGAGGAAAATCTATGGGAATCCAGAGTGGAGTACAGGTTGATGCCAATTGTGAAGGGGACTGCTATCATAGTGGAGGGACAATAATAAGTAACTTGCCATTTCAGAACATAGATAGCAGGGCAGTTGGAAAATGTCCGAGATATGTTAAGCAAAGGAGTCTGCTGCTAGCAACAGGGATGAAGAATGTTCCTG AGATTCCAAAGGGAAGAGGCCTATTTGGTGCTATAGCGGGTTTCATTGAAAATGGATGGGAAGGCCTAATTGATGGTTGGTATGGTTTCAGACACCAGAATGCACAGGGAGAGGGAACTGCTGCAGATTACAAAAGCACTCAATCGGCAATTGATCAAATAACAGGAAAATTAAACCGGCTTATAGAAAAAACCAACCAACAATTTGAGTTGATAGACAATGAATTCAATGAGGTAGAGAAGCAAATCGGTAATGTGATAAATTGGACCAGAGATTCTATAACAGAAGTGTGGTCATACAATGCTGAACTCTTGGTAGCAATGGAGAACCAGCATACAATTGATCTGGCTGATTCAGAAATGGACAAACTGTACGAACGAGTGAAAAGACAGCTGAGAGAGAATGCTGAAGAAGATGGCACTGGTTGCTTTGAAATATTTCACAAGTGTGATGATGACTGTATGGCCAGTATTAGAAATAACACCTATGATCACAGCAAATACAGGGAAGAGGCAATGCAAAATAGAATACAGATTGACCCAGTCAAACTAAGCAGCGGCTACAAAGATGTGATACTTTGGTTTAGCTTCGGGGCATCATGTTTCATACTTCTAGCCATTGTAATGGGCCTTGTCTTCATATGTGTAAAGAATGGAAACATGCGGTGCACTATTTGTATATAA

(4) DNA sequence of AnH7 gene optimized for expression (SEQ ID NO: 3)
ATGAACACACAAATCCTGGTGTTCGCTCTGATCGCTATCATCCCTACAAACGCTGACAAAATCTGCCTGGGTCACCACGCTGTGTCAAACGGTACAAAAGTGAACACACTGACAGAAAGAGGTGTGGAAGTGGTGAACGCTACAGAAACAGTGGAAAGAACAAACATCCCTAGAATCTGCTCAAAAGGTAAAAGAACAGTGGACCTGGGTCAATGCGGTCTGCTGGGTACAATCACAGGTCCTCCTCAATGCGACCAATTCCTGGAATTCTCAGCTGACCTGATCATCGAAAGAAGAGAAGGTTCAGACGTGTGCTACCCTGGTAAATTCGTGAACGAAGAAGCTCTGAGACAAATCCTGAGAGAATCAGGTGGTATCGACAAAGAAGCTATGGGTTTCACATACTCAGGTATCAGAACAAACGGTGCTACATCAGCTTGCAGAAGATCAGGTTCATCATTCTACGCTGAAATGAAATGGCTGCTGTCAAACACAGACAACGCTGCTTTCCCTCAAATGACAAAATCATACAAAAACACAAGAAAATCACCTGCTCTGATCGTGTGGGGTATCCACCACTCAGTGTCAACAGCTGAACAAACAAAACTGTACGGTTCAGGTAACAAACTGGTGACAGTGGGTTCATCAAACTACCAACAATCATTCGTGCCTTCACCTGGTGCTAGACCTCAAGTGAACGGTCTGTCAGGTAGAATCGACTTCCACTGGCTGATGCTGAACCCTAACGACACAGTGACATTCTCATTCAACGGTGCTTTCATCGCTCCTGACAGAGCTTCATTCCTGAGAGGTAAATCAATGGGTATCCAATCAGGTGTGCAAGTGGACGCTAACTGCGAAGGTGACTGCTACCACTCAGGTGGTACAATCATCTCAAACCTGCCTTTCCAAAACATCGACTCAAGAGCTGTGGGTAAATGCCCTAGATACGTGAAACAAAGATCACTGCTGCTGGCTACAGGTATGAAAAACGTGCCTG AAATCCCTAAAGGTAGAGGTCTGTTCGGTGCTATCGCTGGTTTCATCGAAAACGGTTGGGAAGGTCTGATCGACGGTTGGTACGGTTTCAGACACCAAAACGCTCAAGGTGAAGGTACAGCTGCTGACTACAAATCAACACAATCAGCTATCGACCAAATCACAGGTAAACTGAACAGACTGATCGAAAAAACAAACCAACAATTCGAACTGATCGACAACGAATTCAACGAAGTGGAAAAACAAATCGGTAACGTGATCAACTGGACAAGAGACTCAATCACAGAAGTGTGGTCATACAACGCTGAACTGCTGGTGGCTATGGAAAACCAACACACAATCGACCTGGCTGACTCAGAAATGGACAAACTGTACGAAAGAGTGAAAAGACAACTGAGAGAAAACGCTGAAGAAGACGGTACAGGTTGCTTCGAAATCTTCCACAAATGCGACGACGACTGCATGGCTTCAATCAGAAACAACACATACGACCACTCAAAATACAGAGAAGAAGCTATGCAAAACAGAATCCAAATCGACCCTGTGAAACTGTCATCAGGTTACAAAGACGTGATCCTGTGGTTCTCATTCGGTGCTTCATGCTTCATCCTGCTGGCTATCGTGATGGGTCTGGTGTTCATCTGCGTGAAAAACGGTAACATGAGATGCACAATCTGCATCGACTACAAAGACGACGACGACAAATAA

-Amino acid sequence used to prepare M2, N, IL12 molecules (5) Amino acid sequence of M2 small extracellular domain (24 amino acids) (SEQ ID NO: 4)
MSLLTEVETPIRNEWGCRCNDSSD
(6) Amino acid sequence of M2 transmembrane domain (19 amino acids) (SEQ ID NO: 5)
PLVVAASIIGILHLILWIL
(7) Amino acid sequence of M2 cytoplasmic domain (54 amino acids) (SEQ ID NO: 6)
DRLFFKCIYRFFEHGLKRGPSTEGVPESMREEYRKEQQSAVDADDSHFVSIELE
(7) Amino acid sequence of the linker (1 amino acid) between M2-Flag (SEQ ID NO: 7)
G
(8) Amino acid sequence of Flag (7 amino acids) (SEQ ID NO: 8)
DYKDDDDK
(9) Amino acid sequence of linker (2 amino acids) between Flag-NA cytoplasmic domain (SEQ ID NO: 9)
GG
(10) Amino acid sequence of NA cytoplasmic domain (6 amino acids) (SEQ ID NO: 10)
MNPNQK
(11) Amino acid sequence of NA transmembrane domain (31 amino acids) (SEQ ID NO: 11)
IITIGSVSLTIATVCFLMQIAILVTTVTLHF
(12) Amino acid sequence of NA Stoke (39 amino acids) (SEQ ID NO: 12)
KQYECDSPASNQVMPCEPIIIERNITEIVYLNNTTIEKE
(13) Amino acid sequence of linker (2 amino acids) between NA Stoke-murine IL12p40 subunit (SEQ ID NO: 13)
GG
(14) Amino acid sequence of p40 subunit (313 amino acids) of murine IL12 (SEQ ID NO: 14)
MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGTASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGALLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS
(15) Linker between the p40 subunit of Murine IL12 and the p35 subunit of Murine IL12 (SEQ ID NO: 15).
GGGGTGGGGSGGGGTGGGG
(16) Amino acid sequence of p35 subunit (193 amino acids) of murine IL12 (SEQ ID NO: 16)
RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKM

-Known DNA sequence (17) DNA sequence of M2 protein
Based on the sequence of the MP gene of A / Aichi / 2/1968 (H3N2), the M2 gene is connected as follows to form the sequence of the M2 gene.
ACCESSION CY121118
CDS join (14..39,728..995)
/ gene = "M2"
(18) Amino acid sequence of M2 protein
ACCESSION AFM71859
(19) Sequence of NA gene
A / Aichi / 2/1968 (H3N2)
ACCESSION CY121119
Based on the above sequence, it was designed based on the sequence of the NA transmembrane domain (31 amino acids) and the sequence of NA stalk (41 amino acids).
(20) DNA sequence of two subunits (p35 and p40) of mouse IL12 As the p40 subunit (313 amino acids) of Murine IL12, the sequences from the 23rd to the 335th of the following sequences were used.
ACCESSION AAF22555
As the amino acid sequence of the p35 subunit (193 amino acids) of murine IL12, the sequences from the 23rd to the 215th of the following sequences were used.
ACCESSION AAA39292

(21) Expression-optimized DNA sequence used to prepare M2, NA, and IL12 molecules (because codons are optimized for Bombyx Mori, they are also optimized for Eli silk moth) ( SEQ ID NO: 17)
ATGTCACTGCTGACAGAAGTGGAAACACCTATCAGAAACGAATGGGGTTGCAGATGCAACGACTCATCAGACCCTCTGGTGGTGGCTGCTTCAATCATCGGTATCCTGCACCTGATCCTGTGGATCCTGGACAGACTGTTCTTCAAATGCATCTACAGATTCTTCGAACACGGTCTGAAAAGAGGTCCTTCAACAGAAGGTGTGCCTGAATCAATGAGAGAAGAATACAGAAAAGAACAACAATCAGCTGTGGACGCTGACGACTCACACTTCGTGTCAATCGAACTGGAAGGTGACTACAAAGACGACGACGACAAAGGTGGTATGAACCCTAACCAAAAAATCATCACAATCGGTTCAGTGTCACTGACAATCGCTACAGTGTGCTTCCTGATGCAAATCGCTATCCTGGTGACAACAGTGACACTGCACTTCAAACAATACGAATGCGACTCACCTGCTTCAAACCAAGTGATGCCTTGCGAACCTATCATCATCGAAAGAAACATCACAGAAATCGTGTACCTGAACAACACAACAATCGAAAAAGAAGGTGGTATGTGGGAACTGGAAAAAGACGTGTACGTGGTGGAAGTGGACTGGACACCTGACGCTCCTGGTGAAACAGTGAACCTGACATGCGACACACCTGAAGAAGACGACATCACATGGACATCAGACCAAAGACACGGTGTGATCGGTTCAGGTAAAACACTGACAATCACAGTGAAAGAATTCCTGGACGCTGGTCAATACACATGCCACAAAGGTGGTGAAACACTGTCACACTCACACCTGCTGCTGCACAAAAAAGAAAACGGTATCTGGTCAACAGAAATCCTGAAAAACTTCAAAAACAAAACATTCCTGAAATGCGAAGCTCCTAACTACTCAGGTAGATTCACATGCTCATGGCTGGTGCAAAGAAACATGGACCTGAAATTCAACATCAAATCATCATCATCATCACCTGACTCAAGAGCTGTGACATGCGGTACAG CTTCACTGTCAGCTGAAAAAGTGACACTGGACCAAAGAGACTACGAAAAATACTCAGTGTCATGCCAAGAAGACGTGACATGCCCTACAGCTGAAGAAACACTGCCTATCGAACTGGCTCTGGAAGCTAGACAACAAAACAAATACGAAAACTACTCAACATCATTCTTCATCAGAGACATCATCAAACCTGACCCTCCTAAAAACCTGCAAATGAAACCTCTGAAAAACTCACAAGTGGAAGTGTCATGGGAATACCCTGACTCATGGTCAACACCTCACTCATACTTCTCACTGAAATTCTTCGTGAGAATCCAAAGAAAAAAAGAAAAAATGAAAGAAACAGAAGAAGGTTGCAACCAAAAAGGTGCTCTGCTGGTGGAAAAAACATCAACAGAAGTGCAATGCAAAGGTGGTAACGTGTGCGTGCAAGCTCAAGACAGATACTACAACTCATCATGCTCAAAATGGGCTTGCGTGCCTTGCAGAGTGAGATCAGGTGGTGGTGGTACAGGTGGTGGTGGTTCAGGTGGTGGTGGTACAGGTGGTGGTGGTAGAGTGATCCCTGTGTCAGGTCCTGCTAGATGCCTGTCACAATCAAGAAACCTGCTGAAAACAACAGACGACATGGTGAAAACAGCTAGAGAAAAACTGAAACACTACTCATGCACAGCTGAAGACATCGACCACGAAGACATCACAAGAGACCAAACATCAACACTGAAAACATGCCTGCCTCTGGAACTGCACAAAAACGAATCATGCCTGGCTACAAGAGAAACATCATCAACAACAAGAGGTTCATGCCTGCCTCCTCAAAAAACATCACTGATGATGACACTGTGCCTGGGTTCAATCTACGAAGACCTGAAAATGTACCAAACAGAATTCCAAGCTATCAACGCTGCTCTGCAAAACCACAACCACCAACAAATCATCCTGGACAAAGGTATGCTGGTGGCTATCGACGAACTGATGCAATCACTGAACCACAACGGTGA AACACTGAGACAAAAAACCTCCTGTGGGTGAAGCTGACCCTTACAGAGTGAAAATGAAACTGTGCATCCTGCTGCACGCTTTCAACAAGAGTGGTGACAATCAACAGAGTGATGGGTTACCTGTCATCAAGCTtaa

Claims (7)

1. A vaccine containing virus-like particles containing an immunomodulator molecule.
   The immune modulator molecule comprises an interleukin 12 protein, an NA domain region derived from a neuraminidase (NA) protein, and an M2 protein domain region derived from an influenza virus.
   The NA domain region includes an extracellular domain, a transmembrane domain, and an intracytoplasmic domain.
   The M2 protein domain region comprises an extracellular domain, a transmembrane domain, and an intracytoplasmic domain.
   Interleukin 12 protein is bound to the epimembrane domain in the NA domain region
   A vaccine in which the intracytoplasmic domain in the M2 protein domain region is linked to the intracytoplasmic domain in the NA domain region via a linker.
2. further,
   (I) Influenza virus-like particles containing hemagglutinin (HA) protein derived from a virus classified as influenza virus H5 type and / or
   (Ii) Containing influenza virus-like particles containing hemagglutinin (HA) protein derived from a virus classified as influenza virus H7 type,
   vaccine.
3. The vaccine according to claim 1 or 2, which provides a survival rate equivalent to that of an animal infected with influenza virus H1N1 after vaccination containing inactivated influenza virus H1N1.
4. Claims 1 to 1, wherein at least one selected from a virus-like particle containing an immunomodulator molecule, an influenza virus-like particle (i), and an influenza virus-like particle (ii) is produced by Sf9 cells or Eri silkworm. The vaccine according to any one of 3.
5. The vaccine according to any one of claims 1 to 4, wherein the virus classified into influenza virus H5 type is H5N1 type virus and / or the virus classified into influenza virus H7 type is H7N9 type virus.
6. The method for producing a vaccine according to any one of claims 1 to 5.
   In the DNA fragment encoding the immunomodulator molecule, the amino acid sequence of the protein of the encoded immunomodulator molecule is not mutated as compared with the corresponding amino acid sequence in the mammal to be vaccinated. The process of modifying codons to obtain codon-optimized DNA fragments for expression,
   The step of inserting the obtained codon-optimized DNA fragment into a vector,
   A step of cotransfecting the obtained vector and baculovirus-derived DNA into Sf9 cells,
   Step of obtaining a baculovirus recombinant containing a codon-optimized DNA fragment from the obtained Sf9 cells,
   A step of infecting Eri silkworm with the baculovirus recombinant to breed Eri silkworm, and a step of isolating an immunomodulator molecule from the Eri silkworm.
   How to make a vaccine, including.
7. The method for producing a vaccine according to claim 6, wherein the sequence of the codon-optimized DNA fragment is the base sequence of SEQ ID NO: 17.

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