KR101921384B1 - Antigen composition against orthomyxoviruses comprising influenza virus surface protein hemagglutinin monomer - Google Patents

Abstract

The present invention relates to a recombinant hemagglutinin protein antigen composition derived from an influenza virus, and more particularly to a recombinant hemagglutinin protein antigen composition comprising an influenza virus recombinant hemagglutinin monomeric protein double mutant protein as an active ingredient, The present invention relates to an antigen composition for preventing or treating a virus comprising an influenza virus recombinant hemaglutinin protein monomer double mutant protein as an active ingredient, The present invention can be used not only as an effective preventive or therapeutic agent for influenza viruses including avian influenza viruses but also as a recombinant hemaglutinin monomer protein It is possible to reduce influenza diseases including avian and human organisms by providing a material containing the antigen composition of the present invention in view of availability and safety, and thus it can be widely used in a variety of industrial fields such as medicine field and life science field It is expected that this will be a very meaningful development for society and economy.

Description

[0001] The present invention relates to an antigen composition for an organism virus comprising an influenza virus surface protein hemagglutinin monomer. [0001] This invention relates to an antigen composition for an organism virus comprising an influenza virus surface protein hemagglutinin monomer,

The present invention relates to a recombinant hemagglutinin protein antigen composition derived from an influenza virus, and more particularly to a recombinant hemagglutinin protein antigen composition comprising an influenza virus recombinant hemagglutinin monomeric protein double mutant protein as an active ingredient, To an antigen composition for preventing or treating a virus.

Humanoid and re-emergence Infectious disease is a serious problem in human health welfare in the 21st century and is a global issue with climate, environment and aging. Virus infections, ranging from SARS, bird flu, foot-and-mouth disease, food poisoning, Ebola virus and MERS (Middle respiratory syndrome), have serious socioeconomic impacts. HIV, which has not been properly addressed in the late 1990s, to be.

The outbreak of the new virus infectious diseases is bringing new disasters to the global village due to global environmental and ecological changes, poor sanitation and health system of developing countries. Due to the increase in international exchanges, the number of travelers has surged, and infectious diseases in specific areas are threatening our lives and health only in 1-2 days.

Influenza virus infection

Influenza viruses globally take 25-50 million lives every year, 10-40% of children are infected, and cause serious global diseases that cause national economic losses. After the pandemic, which killed 40 million people in 1918, 1.5 million people died of the flu in 1957, 1 million people in the Hong Kong influenza in 1968, and 20,000 new cases of swine influenza in 2009, resulting in a decade-long pandemic And, according to the worst hypothetical scenario, has the potential to infect 30% of the population in a short period of time.

Influenza is an acute respiratory disease accompanied by chills, fever, muscular pain, and cough. It spreads in the form of aerosols that are released into the air through coughing or sneezing of the infected person. In addition, it can be transmitted to animals by the excretion of algae and is generally compared with the cold. Cold is caused by infection with adenovirus, rhinovirus, coxsackievirus, coronavirus, etc. It is a respiratory disease that differs from a virus and does not accompany muscle pain or rapid fever.

Influenza infections are caused by influenza viruses belonging to the orthomyxoviridae, and because of the relatively easy antigenic variation as an RNA virus, repeated infections in the infected population are possible. Influenza viruses causing human infection are types A and B, and type A virus is divided into many virus subtypes by hemagglutinin (HA) and neuraminidase (NA) on the virus surface. Influenza virus subtypes are composed of a combination of H1-H18 and N1-N11 according to the hemagglutinin and neuraminidase surface proteins, respectively, with the recently discovered bat-derived H17N10 and H18N11 viruses. These subtypes are divided into groups 1 and 2 according to the genome sequence, and H1, H2, H5 and H6 belong to group 1 and H3, H4, H7 and H9 belong to group 2.

It is very difficult to find an antigenic determinant having a common denominator of antibody viral proteins because the antigenic determinants of each subtype are different as well as the intergroup epitope. Among these, subtypes of influenza A virus type A are determined by three different combinations of H1, H2, H3, and N1 and N2. Unlike type A, type B infects only humans, so it does not get genes from animal viruses.

Development trend of influenza virus universal vaccine

Studies on the development of a universal vaccine for influenza virus have focused on the use of highly conserved regions of amino acid residues present on virus surface proteins. Since the stem region of the hemagglutinin protein has a highly conserved antigenic determinant, active research is needed in order to produce a specific antibody that can be controlled even if the virus is changed by a seasonal mutant every year It is progressing.

However, the stem region is close to the surface of the influenza virus membrane and is relatively difficult to externally exposed. Therefore, it is known that the antibody generated in response to the stem region is less likely to induce an immune response than the antibody binding to the head region (head region) come. In order to develop these stem-specific antibodies, a headless HA consisting of only the stem region except for the head site proposed by Professor P. Palese of the USA in 2010, or a chimera hemaglass having different head regions Chimeric HA antigens have been presented (Table 1).

Next Generation Universal Vaccine Development Status division Basic skeleton Antigenic property Antigens and references Developed vaccine using highly conserved epitope (Conserved epitope) Stem-based Headless HA (Steel J et al., Mbio, 18: e00018-10, 2010) Chimeric HA (Kramer F et al., J Virol, 87: 6542-50, 2013) Stem binding Ab CR6261 & F10 for group 1 (Ekiert, DC et al., Science, 324: 246-51, 2009)
CR8020 & CR8043 for group 2 (Ekiert, DC et al., Science, 333: 843-50, 2011)
FI6 for groups 1 & 2 (Corti, D. et al., Science, 333: 850-856, 2011) Receptor-based Receptor binding region CH65 (Whittle, JRR et al., PNAS, 108: 14216-21, 2011)
C05 (Ekiert, DC et al., Nature, 489: 526-532, 2012) M2e-based (M2e-based) M2e-HBc, M2e-M1 VLP-M2e (Kim, M.C. et al., Antiviral Res. 99 (3): 328-35, 2013) Vaccine development using other methods Molecular scaffold-based < RTI ID = 0.0 > VLP Virosome (Huckriede, A. et al., Vaccine, 23, Suppl 1: S26-38, 2005) Ferritin Hpf-HA (Kanekiyo M et al., Nature, 499: 102-06, 2013) Viral vector-based < RTI ID = 0.0 > DNA viruses 3, 1443, 2013; Brewoo, J. N. et al., Vaccine, 31: 1848-1855, 2013) DNA vaccine-based DNA vaccines mRNA-based (Zhang, H. et al., Viruses, 6: 1974-1991, 2014) Adjuvant Alum, MF59, AS03 4xM2e-tFliC (Wang BZ et al., J Control Release, 178: 1-7, 2014)

 In addition, Dr. Wilson, MD, of the United States announced the wide-ranging antibody CR6261 specific to Group 1, and then in collaboration with the Dutch team, published the specific antibody CR8020 in Group 2, using both antibody cocktails to prevent influenza A virus I opened the way to. In collaboration with the Swiss team, Dr. J. Skehel and his team developed the FI6 antibody, which was confirmed to bind to all H1-H16 subtypes of group 1 and 2 at the same time. In addition, as a result of antibody test in plasmablast of a patient infected with pandemic virus, stem specific antibody exhibiting high affinity maturation showed that stem antibody-based universal vaccine Although it should be developed, it is opening the possibility of high-risk therapeutic antibody development.

In addition, S. Harrison and colleagues reported new antibodies, C05 and CH65, that respond to receptor binding sites in the hemagglutinin protein head region, respectively. The development of a broadly neutralizing antibody is under way through priming-boosting, and extensive neutralizing antibodies using the headless hemagglutinin and chimeric hemagglutinin of Dr. P. Palese, Dr. A. Lanzavecchia, Ph.D., in Switzerland, developed a hemagglutinin-stymed antibody. These antibodies bind to receptor-binding sites that are strongly bound to the receptor binding site using heavy chain CDR3, among the six complementarity determining region (CDR) loop structures that constitute the antigen recognition site. mimic < / RTI > Since the receptor binding site is one of the highly conserved regions, the occurrence of resistant mutants is low and the therapeutic antibody is highly likely to be developed.

Recently, the so-called "mini-HA" using hemagglutinin stems has been developed and demonstrated versatility by showing the viral efficacy in an experiment in which an H1 subtype protein was used as an antigen in an animal experiment (Impagliazzo A et al., Science, 349 : 1301-6, 2015), self-assembled molecular scaffolds have been used to enable application of various protein molecules. Representative scaffold molecules include virus capsid proteins, bacterial carboxysome, and ferritin proteins. Accordingly, development of a vaccine in which ferritin is exposed to influenza hemagglutinin to improve immunogenicity has been reported (Yassine HM et al., Nat Med, 21: 1065-70, 2015).

The urgency of universal vaccine development and the characterization of recombinant proteins

First, despite the choice of vaccine strains that change every 2-3 years, the occurrence of new mutant strains causes the vaccine efficacy to drop sharply and to improve the vaccine efficacy due to the vulnerability of the elderly due to the reduction of immunization Urgent.

Second, although vaccines are the most effective means of preventing viruses with high mutation, vaccine development takes 4-6 months, and vaccine efficacy is improved due to the securing of eggs for the production of large amounts of viruses and safety problems, This is desperate.

Third, because it is possible to reinfect the human body with new antigen according to the virus mutation, it has a burden to manufacture a new season vaccine by developing a sub-type suitable for the season. In addition, the development of new antiviral agents due to the spread of antiviral resistant mutants is very slow and the method to overcome them is very limited.

Fourth, it is recognized that it is a matter of time before a new mutant virus that causes common infectious diseases due to interspecies of viruses is born and causes another pandemic. In particular, the swine flu, avian influenza virus and mers virus are good examples. When avian influenza is popular, the main reason for disposing of poultry is direct control of the spread of the infection. However, direct infection of avian influenza virus, And genetic rearrangement to prevent the arrival of new pandemics.

Fifth, it is best to use the broad spectrum characteristics of the virus as a way to overcome this, and this is why the focus is on the development of a universal vaccine to achieve complete prevention through vaccination once. Therefore, the use of the versatile nature of viruses has been known to be the best method, so studies on versatile vaccines that can control the virus and identification of the target mechanism have become the focus of attention. It is important to look for shortcuts for achieving complete prevention with a single vaccination, in which the virus antigen determinants have a high conservation region, that is, a common denominator.

In addition, recombinant proteins can be used much faster than existing virus-based vaccines, which can take 4-6 months to develop, and scale-up to produce new seasonal vaccines every year, This has an easy advantage. In particular, there is no problem of securing eggs for the production of a large amount of viruses, safety and egg white allergy, and when a gene sequence of a new mutant virus is obtained, recombinant proteins can be easily produced based on the gene sequence.

FluBlok and FlucelVax, which are based on hemagglutinin trimer, have already been developed as recombinant protein vaccines. In particular, in the case of the flu block, clinical studies have shown that adults 50-64 years old (Baxter R. et al., Vaccine, 29: 2272-8, 2011) compared with the trivalent influenza vaccine.

Therefore, the inventors of the present invention have made intensive researches to overcome the problems of the prior arts. As a result of studying the production and characterization of recombinant hemaglutinin monomer protein and monitoring changes in animal-based body weight and survival rate and studying the concentration of influenza virus, It was confirmed that the hemagglutinin recombinant protein monomer had an antigen effect similar to that of the previously developed trimer. Based on the structural advantages of the monomer having the monomer site and the monomer site which are difficult to be exposed in the conventional trimer, .

The present invention provides an antigen composition comprising an influenza virus recombinant hemaglutinin monomer double mutant protein having no fear of side effects or toxicity and having an excellent preventive effect against omomycin virus including influenza virus as an active ingredient The purpose.

Since the development of a universal vaccine using an influenza virus antigen has a main target for enhancing the induction effect of an antibody recognizing a highly conserved protein region, in order to achieve the purpose of the present invention, the inventors of the present invention have found that the influenza virus-derived hemagglutinin monomer Based expression and an animal test result using a mouse, it was confirmed that the recombinant hemagglutinin protein monomer has an excellent antiviral effect on influenza virus, and thus the present invention has been completed.

The present invention provides an antigen composition for preventing or treating an influenza virus recombinant hemaglutinin protein monomer double mutant protein as an active ingredient.

The antigens composition for preventing or treating omomycosis comprising an influenza virus recombinant hemaglutinin protein monomer double mutant protein as an active ingredient according to the present invention is excellent in preventing the proliferation and replication of osmoreviruses , Avian influenza and other influenza viruses as well as a recombinant hemaglutinin monomer protein. Therefore, it is possible to provide a material containing the antigen composition of the present invention in view of its recyclability and safety. It is expected to be a very socioeconomic development because it can reduce the influenza diseases of birds and animals including humans and can be widely used in various fields such as medicine field and life science field.

Figure 1 shows the 2009 Pandemic Influenza A / Korea / 01/2009 (KR01) hemagglutinin tertiary structure and hemagglutinin monomer structure model. In the 2009 Pandemic virus hemagglutinin structure, the head sites interact with each other. The monomers and the multimers interact with each other through the head region (above), and the hemagglutinin monomer structure (Below). The hemagglutinin was labeled purple at the head and cyan at the stem.
Figure 2 shows the characteristics of the hemagglutinin protein. Comparing the results of gel filtration chromatography of hemagglutinin protein, it is a comparison between seasonal mutant CU44 and 2009 Pandemic KR01 (above). The results of the centrifugation and mass spectrometry for the analysis are shown in comparison (below).
Figure 3 compares the binding affinities of KR01 (left) and CU44 (right) hemagglutinin to the receptor.
FIG. 4 shows the positions of the six amino acid residues present in the primary structure of the hemagglutinin protein and the monomer interface (above), showing the differences in the amino acid sequences of various virus variants (middle) The six positions indicate the positions of the amino acid residues on the tertiary structure (below).
Figure 5 shows the sequence of the hemagglutinin mutant protein.
FIG. 6 shows the results of His-tag affinity, ion exchange and gel filtration chromatography of Hemagglutinin F88E / V91W double mutant proteins and non-denaturing electrophoresis results at each step.
Fig. 7 shows the characteristics of the hemagglutinin F88E / V91W double mutant protein. Size exclusion chromatography (A), non-denaturing electrophoresis (B), multi-angle laser scattering (C), and fluorescence scan change analysis (D).
FIG. 8 shows a schedule of mouse animal test time-infected with influenza virus PR8 and mKR01 using hemagglutinin protein monomer antigen.
FIG. 9 shows the results of animal experiments of CU44 wild-trimeric hemoglutinin antigen and CU44 F88E / V91W monomer double mutant protein antigen in PR8 and KR01 virus-infected mice, and PR8 (upper) and mKR01 Weight change.
FIG. 10 shows the results of animal experiments of the CU44 wild-trimeric hemoglutinin antigen and the CU44 F88E / V91W monomer double mutant protein antigen in PR8 and KR01 virus-infected mice, and PR8 (upper) and mKR01 , Respectively.
FIG. 11 shows the virus concentration in the lung tissue as a result of animal experiments of the CU44 wild-trimeric hemoglutinin antigen and the CU44 F88E / V91W monomer double mutant protein antigen in PR8 and KR01 virus-infected mice, and mKR01 PR8 (right) virus-infected mice.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to an antigen composition for preventing or treating an influenza virus recombinant hemagglutinin protein monomer double mutant protein as an active ingredient.

As used herein, " prophylactic " or " treatment " refers to a reduction or amelioration of a symptom of a disease, a reduction in the extent of a disease, a delay or alleviation of disease progression, an improvement, alleviation or stabilization of a disease state, Beneficial treatment results, and the like.

As used herein, a "composition" is considered to include any product that comprises a particular ingredient, as well as any product that is made directly or indirectly by the combination of the particular ingredient.

The antiviral antigen composition may be in the form of a pharmaceutical composition to be administered to a mammal including a vertebrate animal, preferably a human, or it may be applied to any commonly known form such as a food composition, an ordinary antiviral agent, and the like.

In the present invention, an antigen composition comprising an influenza hemagglutinin monomer double mutant protein as an active ingredient was firstly shown to have an excellent virus-preventing effect against influenza virus belonging to orthomyxoviridae.

In the present invention, the influenza virus recombinant hemagglutinin protein monomer may be a double mutant protein of SEQ ID NO: 1.

In the present invention, the influenza virus recombinant hemagglutinin protein monomer may be characterized by exhibiting a preventive or therapeutic effect on an animal and human influenza by inhibiting the proliferation and replication of an omycinovirus.

In the present invention, the Osmical virus may be characterized as being influenza virus, but is not limited thereto.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating an orthomyxovirus infection disease, which comprises an influenza hemagglutinin monomer double mutant protein as an active ingredient.

Influenza hemagglutinin monomers can easily be mutated using the Bac-to-bac system, and can be easily expressed using insect cells sf9 and hi5 cells, purified by column chromatography to a purity of 95% or more It is a purified protein antigen that is easy to characterize.

The antigen protein mutation can be preferably performed by selecting a bacmid into which a target gene has been inserted by selecting a bac-to-bac system in bacteria and transfecting the cell. More preferably, the antigen protein can be made by other genetic engineering methods.

The hemagglutinin protein may more preferably be a protein expressed in animals other than sf9 and hi5 insect cells and other cells including bacteria.

The purified hemagglutinin protein was purified by a Ni-NTA column, a Mono Q column and a superdex 200HR column chromatography to a purity of 95% or more. The purified hemagglutinin protein was then analyzed for characteristics, and hemagglutinin antigens derived from various influenza virus . In carrying out the purification process, a high-purity protein can be purified using other purification methods.

In particular, it can be prepared by purifying it in an active form using trypsin, further purifying it, and using other protease than trypsin. By performing the purification process, residues remaining in the extract solution and the like can be removed or stabilized.

The recombinant hemagglutinin protein antigen composition comprises a high purity recombinant hemaglutinin monomer protein present in an aqueous phase in a PBS buffer used as a comparative group in animal experiments and the immunogenicity is determined by the relationship of the recombinant protein content of the sample Seems to be.

The pharmaceutical composition for the prevention or treatment of diseases of the genome according to an embodiment of the present invention may contain the recombinant hemaglutinin monomer protein singly, May contain a pharmaceutically acceptable carrier, excipient, or diluent according to conventional methods used in the art.

The composition may be administered orally or parenterally according to a conventional method. For example, when the composition is orally administered, the composition may be provided in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups and the like. And in the form of pharmaceutical compositions to be administered to animals, including birds, preferably humans, birds.

In the case where the composition is provided as a mixture in which a component other than the recombinant hemaglutinin monomer protein double mutant protein which is an active ingredient of the composition for preventing or treating an Osmomycin infectious disease is added, The composition may comprise from 0.001% to 99.9%, preferably from 0.1% to 99%, more preferably from 1% to 50% by weight of the recombinant hemaglutinin monomer protein relative to the total weight of the composition have.

The preferred dosage amount may be a content suitable for the treatment or prevention of the subject and / or disease, including the age, sex, general health and weight of the subject, the kind and severity of the disease, the type of formulation, The type and amount of the other ingredients, the proportion of the composition, the route and the duration of administration, etc., and may be appropriately selected by those skilled in the art, but is preferably 50 mg / day for adults (70 kg) To 100 mg may be administered.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

According to another preferred embodiment of the present invention, there is provided a food composition for preventing or ameliorating an infectious disease of an organism comprising a recombinant hemaglutinin monomer protein as an active ingredient.

The food composition of the present invention may be in any form suitable for administration to an animal body including the human body, any form usual for oral administration, for example additives or adjuvants of food or feed, food or feed, , Solid forms such as tablets, pills, granules, capsules, and foamed formulations, or liquid forms such as solutions, suspensions, emulsions, drinks and the like. In addition, it may contain nutrients, vitamins, electrolytes, etc., and these components may be used independently or in combination.

Since the above-mentioned organism infectious diseases are caused by viruses, antibiotics are not effective unless they are secondary infection problems such as bacterial diseases. Therefore, the present inventors can effectively prevent infection by providing a composition comprising the recombinant hemaglutinin monomer protein as an active ingredient.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Example 1: Preparation of an influenza virus hemagglutinin monomer antigen

1-1: Design of Structure-Based Hemagglutinin Monomer Material

1-1-1: Origin of Hemagglutinin Monomer

The present inventors have identified the tertiary structure of pandemic influenza A / Korea / 01/2009 (KR01) hemagglutinin in 2009 (Cho KJ et al., J Gen Virol, 64: 1712-22, 2013) The revealed hemagglutinin structure was originally stable in the trimer form, whereas the revealed structure showed the structure of a monomer having a molecular arrangement showing monomer-monomer interaction (Fig. 1). Thus, in 2009, the pandemic virus hemagglutinin not only showed a relaxed structure of the head region from the stem region as a monomer form, but also exhibited a much more flexible structure, (KJ et al., J Gen. Virol., 64: 1712-22, 2013). In the present study,

1-1-2: Hemagglutinin monomer characteristics

Considering that the development of influenza universal vaccine has been made in an effort to expose high amino acid conserved regions such as headless hemagglutinin, chimeric hemagglutinin, and hemagglutinin consisting of the stem region only in the molecular scaffold, Using the characteristics of the hemagglutinin monomer, it was judged that there would be another universal vaccine. That is, while the hemagglutinin trimer is hidden within the trimer by the interface between the monomer having the monomer-monomer interaction, the monomer is exposed to this interface. Especially, trimer is difficult to expose the stem region, but monomers have relatively easy exposure to the stem region according to interface exposure.

In addition, the hemagglutinin monomer exhibits most of the antigenic determinants possessed by the trimer. There is no significant difference in antibody recognition from the trimer (the dissociation constant K _D exhibiting antibody binding ability does not differ greatly), and the substrate specificity is the same have. In some papers, it is described that a trimer is necessary because a part of the monomer-monomer interface of the trimer functions as an antigenic determinant. However, even though an antigen-antibody reaction having a part of an interface of an actual trimer as an antigenic determinant has no interface (Cho KJ et al., J Gen Virol, 64: 1712-22, 2013).

1-1-3: Structure-based hemaglutinin monomer design

The hemagglutinin protein has a trimer stabilized form, and amino acid variation is essential to make it a monomer. A / Thailand / CU44 / 2006 (CU44) Seasonal mutants with trimer structure Starting from the hemagglutinin gene, a major amino acid located at the intermembrane interface predicted to participate in the trimer-monomer equilibrium Six residues were selected to modify the charge or size of the amino acid residues to inhibit the formation of trimers and to induce the monomers.

1-2: Preparation of influenza virus hemagglutinin monomer antigen

The influenza virus recombinant hemagglutinin protein was expressed using the Bac-to-bac system using insect cells sf9 and hi5 cells. Hemagglutinin, foldon domain, Histek (derived from A / Thailand / CU44 / 2006 (CU44), one of the seasonal influenza virus mutants, or 2009 Panthemic A / Korea / 01 / 6xHis-tag) was prepared. Bac-to-bac system was selected from bacteria and bacmid with target gene inserted was selected and transfected into cells.

The hemagglutinin CU44 or KR01 protein was purified by a Ni-NTA column, a Mono Q column and a superdex 200HR column chromatography to a purity of 90% or more, followed by characterization. The hemagglutinin obtained from CU44 and KR01 was purified into an active form using trypsin (Fig. 2).

The CU44 hemagglutinin protein was purified by conventional trimerization and confirmed by gel filtration chromatography and centrifugation for analysis. In contrast, the 2009 Pandemic KR01 hemagglutinin protein was found to be a monomer, and the mass spectrometer (MALDI-TOF) analysis showed no difference in the viral protein base protomers. Mass spectrometry was performed by Korea Basic Science Research Institute using Voyager DE-STR MALDI-TOF (PerkinElmer, MA, USA) capable of 25,000-250,000 m / z analysis. The sedimentation coefficients were determined using an Optimal XL-I analytical ultracentrifuge (Beckman Coulter, CA, USA).

Example 2: Characterization of influenza virus hemagglutinin monomer

2-1: Characteristics of Hemagglutinin Monomer

Influenza viruses consist of eight negative RNA strand segments, each containing one or two protein genes. Influenza viruses have a high rate of mutation and are very fast to evolve. In particular, hemagglutinin, a viral surface glycoprotein, undergoes selection pressure due to the immune system of the host cell, so that virus evolution is higher than other viral proteins, and the role as a major antigen determinant providing protein is very important.

Six variants of amino acid residues at the monomer interface of the wild hemagglutinin trimer derived from CU44 were prepared and two mutant proteins, F88E and V91W, showing monomer characteristics, were selected. In order to further enhance the properties of the monomer, a double mutation protein having two mutations at the same time, that is, F88E / V91W, was prepared to obtain a stable mutant protein.

By utilizing such hemagglutinin monomers, it is possible to firstly expose parts or stems that are difficult to be exposed in the trimer, secondly, to tolerance problems caused by the characteristics of hemagglutinin, which exhibits rapid mutations, and third, And that it would be useful for the development of a universal vaccine that can overcome the low vaccine effects caused by subtypes.

It is also much more valuable than trimer in the use of various molecular scaffolds. Monomer hemagglutinin can be easily introduced into nanoparticles because it can exist stably even if it does not match the symmetry of trimer. Therefore, multigene ) Vaccine development.

2-2: Characteristics of influenza virus hemagglutinin monomer

Sialo-fetuin and asialo-fetuin (Sigma-Aldrich, MO, USA) were coated on a 96-well plate at 1 μg / ml, When demigam and seasonal mutant CU44-derived hemagglutinin, each hemagglutinin protein was treated, washed several times and then measured using anti-6xHis-tag antibody, KR01 pandemic and seasonal mutant CU44-derived hemagglutinin All of them did not bind to Alopethein in all, and did not bind very weakly in Asa Alopecthin (Fig. 3). Therefore, it was confirmed that there was no significant difference in the binding force to the receptor between the trimer and the monomer.

Example 3: Preparation of recombinant hemaglutinin protein monomer mutant protein

3-1: Mutation from recombinant hemagglutinin protein trimer to monomer

KR01 The hemagglutinin protein was a monomer which showed the head part dissociated from the stem part. In addition, six major amino acid residues predicted to be involved in the trimeric-monomer equilibrium were selected (FIG. 4) by comparing the trimeric CU44 hemagglutinin with the KR01 hemagglutinin structure, which is a monomer structure.

That is, the selected amino acid residues are those residing in the monomer-to-monomer interface in the hemoglobinin protein trimer, mutating the amino acid of CU44 hemagglutinin to the amino acid residue of KR01 hemagglutinin, The charge or the size of the monomer was changed so that the formation of the trimers was inhibited and the monomers were finally selected. These 6 amino acid residues were prepared through point mutation experiments to obtain the final 6 mutant proteins: S199F (Ser199Phe), R75L (Arg75Leu), F88E (Phe88Glu), V91W (Val91Trp), R106E (Arg106Glu) , G47E (Gly47Glu) (Figure 5).

3-2: Expression and purification of recombinant hemaglutinin protein monomer mutant protein

Six mutated protein genes were prepared, and protein oligomer forms were confirmed by expressing six mutated proteins, performing protein purification chromatography and non-denaturing electrophoresis analysis. Of the six mutated proteins, the F88E and V91W mutant proteins showed monomeric properties like KR01 hemagglutinin and the other four mutated proteins showed trimer like CU44 hemagglutinin (Fig. 6).

Mutation Protein Characterization The double mutant protein of F88E / V91W was constructed, expressed and purified to further enhance the monomer properties of each mutant protein. The amino acid sequence (SEQ ID NO: 1) of the highly purified F88E / V91W double mutant protein was confirmed. In addition, it was confirmed that the size was accurately measured by size exclusion chromatography-multi-angle laser scattering (SEC-MALS), fluorescence scan change (DSF), and native denaturing electrophoresis analysis (FIG. 7) . When the F88E / V91W double mutant protein was treated with thrombin, the polydon domain was truncated, indicating that the trimer was converted to a monomer.

In addition, the size of expression was expanded to allow mass purification in mg for use in animal experiments to confirm the antigen value, and the influenza virus immunoactivity was verified by animal experiments of the F88E / V91W mutant protein. Vaccine efficacy in mice infected with mouse-adapted PR8 virus or mouse-adapted mKR01 virus, respectively, was evaluated.

Example 4: Hemagglutinin monomer-based animal experiment

4-1: Hemagglutinin monomer-based animal experiment

The PR8 virus (mouse adapted A / Korea / 01/2009 virus, PR8) or the mKR01 virus adapted to the mouse (mouse adapted A / mKR01) was evaluated by measuring changes in body weight and survival rate in mice. As a result of inoculation of monomeric and trimer antigens into BALB C mice at 3-week intervals and infecting PR8 or mKR01 virus, the trimer and mononuclear antigen-positive mice showed PR8 and KR01 viruses, respectively, Both of the infected groups showed increased body weight and increased survival rates.

Specifically, an animal experimental scheme was established to compare the immunogenicity of antigens by measuring the body weight and survival rate in mice that were respectively infected with mouse-adapted PR8 virus and mouse-adapted mKR01 virus (FIG. 8 ).

Recombinant hemagglutinin monomer protein antigens were subcutaneously injected into 8-week-old BALB C mice at 3-week intervals, and then PR8 or mKR01 viruses adapted to mice were inoculated into 5 LD ₅₀ (5 x 10 ² PFU / mouse ). The recombinant antigen proteins were compared with the PBS buffer solution and the SAS ejuvant in the control group.

Three days after the infection, the virus in the lung tissue was extracted from the PBS buffer solution and infected with MDCK (Madin-Darby canine kidney) cells. Respectively. As a result of animal experiments, the mice that received the trichain chain CU44 wild hemagglutinin antigen or the recombinant hemagglutinin protein monomer F88E / V91W double mutant protein antigen showed an increase in body weight in both experimental groups infected with PR8 or mKR01 virus (Fig. 9).

In the case of the PR8 virus test group, mice that did not receive antigen and those that received adjuvant were observed to lose more than 20% of their body weight over 7 days. However, mice that received trimer and monomer antigens showed little change in body weight for two weeks with naive mice. In the mKR01 virus-treated group, triceps and mononergic mice showed a tendency to decrease in body weight with naive mice, but recovered within 4 days.

The survival rate according to the weight change was 100% for the PR8 and mKR01 viruses in the control group with no trimer or monomer antigens, and 100% for the PR8 group in the adjuvant group (Fig. 10).

In the mKR01 virus test group, the trimer or monomer antigen showed a survival rate of 67%, although the survival rate was different from that of naive mice not given the virus. Nevertheless, both the trimeric and monomeric antigens showed no difference in survival rates and showed superior immunogenicity compared to the comparative group.

4-2: Animal experiments Virus analysis in mouse lung tissue

MDCK cells were cultured in Eagle's medium (DMEM, Gibco BRL, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS, Htclone Lab, Utah, USA) in a 5% CO 2 incubator at 37 ° C Respectively. L-glutamic acid (2 x L-lysine) containing 1.5% (w / v) agarose, 25 mM HEPES buffer, 2% bovine serum albumin (BSA) and 1% penicillin- streptomycin was used for plaque reduction assay. 15 medium supplemented with 2 μg / ml trypsin was added thereto, and the mixture was incubated in a 37 ° C. incubator for about 48-72 hours, and then stained with 0.5% (v / v) crystal violet.

Regardless of the type of influenza virus and the multiplicity of infection (MOI) (virus number / cell number of virus inoculated), MDCK cells showed consistent antiviral activity in relation to the inhibition of virus proliferation using MDCK cells.

As a result of the analysis of lung tissue viruses, in the test group infected with the PR8 virus, the viruses showed a decrease of about 2 log viruses in all the mice that gave the trimeric and monomeric antigens as compared with the control group that did not give any antigen. In the test group infected with the mKR01 virus, Both monomer antigens showed about 0.5 log difference (Figure 11).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea University Sejong Campus Industry & Academy Collaboration Foundation <120> ANTIGEN COMPOSITION AGAINST ORTHOMYXOVIRUSES COMPRISING INFLUENZA          VIRUS SURFACE PROTEIN HEMAGGLUTININ MONOMER <130> JKP-0618 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 514 <212> PRT <213> Artificial Sequence <220> <223> F88E / V91W double mutant protein <400> 1 Ala Asp Pro Gly Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser   1 5 10 15 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His              20 25 30 Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu Leu          35 40 45 Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp      50 55 60 Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser Trp  65 70 75 80 Ser Tyr Ile Val Glu Lys Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro                  85 90 95 Gly His Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val             100 105 110 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro         115 120 125 Asn His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys     130 135 140 Ser Ser Phe Tyr Lys Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu 145 150 155 160 Tyr Pro Asn Leu Ser Lys Ser Tyr Ala Asn Asn Lys Glu Lys Glu Val                 165 170 175 Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asp Gln Arg             180 185 190 Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Ser Val Ser Ser His         195 200 205 Tyr Ser Arg Lys Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg     210 215 220 Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly 225 230 235 240 Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Arg Tyr                 245 250 255 Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Asn Ser Asn             260 265 270 Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly Ala         275 280 285 Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile Gly     290 295 300 Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly 305 310 315 320 Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile                 325 330 335 Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr             340 345 350 Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln         355 360 365 Lys Ser Thr Gln Asn Ale Ile Asn Gly Ile Thr Asn Lys Val Asn Ser     370 375 380 Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe 385 390 395 400 Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp                 405 410 415 Gly Glu Ile Asp Trp Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu             420 425 430 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu         435 440 445 Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly     450 455 460 Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asp Glu Cys Met Glu 465 470 475 480 Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser                 485 490 495 Lys Leu Ser Arg Glu Lys Ile Asp Gly Val Ser Gly Arg Leu Val Pro             500 505 510 Arg Gly        

Claims (4)

An influenza virus recombinant hemaglutinin protein monomer double mutation protein as an active ingredient,
Wherein the influenza virus recombinant hemagglutinin protein monomer double mutant protein is represented by SEQ. ID. NO. 1.
2. The method according to claim 1, wherein the influenza virus recombinant hemagglutinin protein monomer has a prophylactic or therapeutic effect on an animal and a human influenza by inhibiting proliferation and replication of the omomycin virus, Or therapeutic antigen composition.
2. The antigens composition according to claim 1, wherein the organism virus is an influenza virus.
2. The antigens composition according to claim 1, wherein the influenza virus recombinant hemaglutinin protein monomer is contained in an amount of 0.001 to 99.9% by weight.

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