KR101888751B1

KR101888751B1 - Vaccine against type B Influenza

Info

Publication number: KR101888751B1
Application number: KR1020160121556A
Authority: KR
Inventors: 장준; 강정옥
Original assignee: 이화여자대학교 산학협력단
Priority date: 2016-09-22
Filing date: 2016-09-22
Publication date: 2018-09-21
Also published as: WO2018056528A3; KR20180032721A; WO2018056528A2

Abstract

The present invention relates to a vaccine composition comprising a gene encoding a nucleoprotein of B / Yamagata family virus among influenza B viruses and capable of simultaneously inducing immunity against influenza B virus B / Yamagata family virus and B / Victoria family virus in an infected object A recombinant adenovirus and a recombinant adenovirus or a virus particle thereof as an active ingredient. Although the recombinant adenovirus provided in the present invention contains only a gene coding for the nucleoprotein of B / Yamagata family virus, the recombinant adenovirus can induce immunity against infection of B / Yamagata family virus as well as B / Victoria family virus Therefore, the recombinant adenovirus can be widely used as a general vaccine against influenza B virus and can be widely used for prevention and treatment of various diseases caused by infection with influenza B virus.

Description

Vaccine against type B Influenza < RTI ID = 0.0 >

The present invention relates to an influenza B vaccine, and more particularly, the present invention relates to an influenza B virus comprising a gene encoding a nucleoprotein of B / Yamagata family virus, wherein the influenza B virus, B / Yamagata family virus and B / Victoria viruses, and an influenza B vaccine composition comprising the recombinant adenovirus or the virus particle thereof as an active ingredient.

Influenza virus (Influenza virus) belongs to the family Orthomyxoviridae, and its serotype is divided into three types, A, B and C. Among them, influenza A and B viruses are two types of influenza viruses that cause epidemic human diseases, and they are generally spread through human contact, mainly through respiratory droplet propagation. The H1N1 and H3N2 subtypes of influenza A and influenza B are known to be the major causes of seasonal infections. Influenza viruses can avoid the humoral immune system through antigenic variation caused by changes in the amino acids of surface HA (haemagglutinin) and NA (neuraminidase). For this reason, influenza can cause an epidemic every year despite continued vaccine development. Most patients recover from fever and symptoms due to influenza without medical treatment, but even death from severe influenza virus infection can occur.

In particular, influenza A is known to cause epidemics, for example, pandemics such as pandemics in 1918, 1957, 1968 and 2009. This is due to the lack of pre-formed immunity against the major virus antigen hemagglutinin (HA), pandemic influenza can infect more than 50% of the population in a year and often causes more severe illness than infectious influenza You can. Accordingly, current influenza vaccines are mainly focused on preventing influenza A infection (Korean Patent No. 1582490, No. 1525180, No. 898845, etc.). However, most of these vaccines use hemagglutinin (HA) and neuraminidase (NA), two proteins on the surface of the virus as antigens, The vaccine developed in this way has the drawback that it does not show versatility. In addition, most influenza vaccines have been developed for influenza type A, and vaccine against influenza type B has not been actively developed.

Under these circumstances, the present inventors have made extensive efforts to develop an effective vaccine against influenza B. As a result, a vaccine using nucleoprotein (B-NP) as an antigen can be used as a general vaccine And completed the present invention.

One object of the present invention is to provide transformed recombinant adenoviruses capable of expressing nuclear proteins of influenza B virus.

Another object of the present invention is to provide an influenza B vaccine composition comprising the recombinant adenovirus or a virus particle thereof as an active ingredient.

While performing various studies to develop an effective vaccine against influenza B virus, the present inventors have focused on a protein that exists inside a virus that can not be easily mutated in order not to be affected by various virus strains. Unlike the surface antigens of viruses, which can easily be mutated, the proteins present inside the viruses are expected to not mutate easily because they play an important role in the survival of viruses. However, It is not easy to select a protein that can be used as a vaccine because it shows structures and functions similar to those in vivo. The present inventors have confirmed through various studies that nucleoprotein (B-NP) of influenza B virus can be used as an antigen against influenza B virus and can effectively prevent the infection of influenza B virus by using the nuclear protein To develop a vaccine. As a result, when a recombinant adenovirus containing a gene encoding a nucleoprotein of the B / Yamagata family virus is used, the recombinant adenovirus is infected with the B / Yamagata family virus and the B / Victoria family virus belonging to the influenza B virus It is possible to induce immunity against infection at the same time.

The recombinant adenovirus provided in the present invention includes a gene encoding a nucleoprotein of B / Yamagata series virus, unlike the virus vaccine developed so far, which is specifically immunologically specific to the infection of the virus used as an antigen Despite the fact that B / Yamagata family viruses and B / Victoria family viruses are infectious, it can be said that they have completely different characteristics from the vaccines developed so far.

As described above, the gene encoding the B / Yamagata family virus nuclear protein is included, and the B / Yamagata family virus as well as the B / Victoria family virus are also immunized against the infection, so that it can be used as a vaccine against general influenza B virus The recombinant adenovirus of the present invention, which can be used as a recombinant adenovirus, has not been reported at all and has been developed for the first time by the present inventors.

According to one embodiment of the present invention, there is provided an influenza B virus comprising a gene encoding a nucleoprotein of B / Yamagata family virus, wherein the influenza B virus, B / Yamagata family virus and B / Victoria < / RTI > family viruses.

The term " type B Influenza virus "in the present invention refers to a type of influenza virus, also referred to as influenza B virus, which shows human-specific sensitivity. The influenza B virus is rarely found in comparison with influenza A virus, has a mutation rate of 2 to 3 times lower, contains only one genotype, and includes two families of B / Victoria series and B / Yamagata series . Pathologically, it can be a cause of various respiratory failure, specifically cough, sneezing, high fever, wheezing, bronchitis, bronchiolitis, bronchitis, pneumonia, asthma and acute obstructive laryngitis.

The term "nucleoprotein" of the present invention means a nucleoprotein present in influenza B virus. The nuclear protein binds to the RNA of influenza B virus to form a core complex inside the virus particle. It is known that it plays an essential role together with PB1, PB2 and PA when the virus genome is replicated.

For the purpose of the present invention, the nucleoprotein can be used as an antigen that is expressed in an infected individual of the recombinant adenovirus of the present invention and induces or enhances immunity against influenza B virus. The nucleoprotein can be an entire wild type protein, a fragment thereof, Amino acid sequence variants, and the like. The sequence of the nucleoprotein may be, for example, a nucleoprotein derived from a B / Yamagata family virus, and, as another example, a nucleoprotein derived from a B / Yamagata / 16/1988 virus And, as yet another example, a nuclear protein consisting of the amino acid sequence of SEQ ID NO: 1 derived from B / Yamagata / 16/1988 virus.

The term "amino acid sequence variant " of the present invention means a protein having a sequence which differs from the native amino acid sequence of the protein by deletion, insertion, non-conservative or conservative substitution or a combination thereof.

For purposes of the present invention, the amino acid sequence variant refers to an amino acid sequence variant of a nuclear protein, which enhances the glycosylated form, lipidated form, and antigen presentation of the nuclear protein, A derivative form such as to include a molecule that improves the target to the target, and the like, but is not particularly limited thereto.

The term "adenovirus" of the present invention is a type of virus that causes diseases in the upper airway and conjunctiva and is present in a latent infectious state even in normal people. Most of them are infectious viruses, It is characterized by the fact that it leaves enemy immunity. The viral particles mainly consist of hexon and fentone proteins, and have a regular icosahedral shape with a diameter of 70 nm, with protrusions at each apex and no coating. Its genome consists of about 36,000 base pairs of double stranded DNA. It is known that the terminal protein (TP) is covalently bonded to the 5 'end of DNA, and the TP acts as a starting point of DNA replication. Recently, a polynucleotide encoding a protein derived from a eukaryotic cell has been used as a gene vector, which is a mediator for introducing and expressing a host eukaryotic cell by a gene recombination method.

The term "recombinant adenovirus" of the present invention means an adenovirus transformed into a genomic DNA of an adenovirus so that a polynucleotide coding for the nucleoprotein of influenza B virus is operably inserted. Conventionally, recombinant adenovirus has been used as a gene vector for introducing a polynucleotide encoding a eukaryotic cell-derived protein into a host eukaryotic cell by a gene recombination method. However, the recombinant adenovirus provided in the present invention can be used as an influenza B virus As a gene vector for delivering a polynucleotide encoding a nuclear protein of the influenza B virus as well as a role as a host for assisting the expression of the nucleoprotein after infection with the influenza B virus. Therefore, the recombinant adenovirus of the present invention itself or the virus particle thereof can be used as an active ingredient of a vaccine for preventing or treating infection of influenza B virus.

According to another aspect of the present invention, there is provided a recombinant adenovirus or a viral particle thereof comprising the recombinant adenovirus or a viral particle thereof as an active ingredient, wherein the influenza B virus B / Yamagata virus and B / The present invention provides an influenza B vaccine composition capable of simultaneously inducing immunity against a Victoria virus and preventing or treating a disease caused by infection with an influenza B virus.

Since the influenza B vaccine composition provided in the present invention contains the recombinant adenovirus as an active ingredient, the advantage of the recombinant adenovirus can be expressed as it is. As described above, although the recombinant adenovirus provided by the present invention contains only genes coding for the nuclear protein of B / Yamagata family virus among influenza B virus, the influenza B virus, B / Yamagata family virus, B / Victoria family viruses. Therefore, the immunogenicity against influenza B viruses is excellent, and when used as a vaccine, the immunity against influenza B virus infection can be stably induced or enhanced Therefore, it can be used for effectively preventing or treating a disease caused by infection with influenza B virus.

The term "a disease caused by an infection of an influenza B virus" in the present invention means a respiratory disease caused by infection of an influenza B virus in an animal including a person having a respiratory system, A variety of respiratory insufficiencies can result, such as coughing, sneezing, high fever, wheezing, bronchitis, bronchiolitis, bronchitis, pneumonia, asthma and acute obstructive laryngitis.

The term "prevention" of the present invention means all actions that inhibit or delay the onset of the disease caused by influenza B virus infection by administering the influenza B vaccine composition provided by the present invention.

The term "treatment" of the present invention means any action that alleviates or alleviates symptoms of a disease already caused by infection with influenza B virus due to administration of the influenza B vaccine composition provided in the present invention.

The recombinant adenovirus or viral particle of the present invention may be administered as a vaccine to induce immunity against respiratory diseases caused by infection with influenza B virus. The recombinant adenovirus or its viral particle may be administered with a pharmaceutically acceptable carrier, excipient Or may be suitably formulated with a diluent. At this time, the carrier used may be an unnatural carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a pigment and a flavoring agent in the case of oral administration. In the case of an injection, A non-aqueous solvent such as an aqueous solvent such as water and a ring gel liquid, a vegetable oil, a higher fatty acid ester (e.g., oleic acid), and an alcohol (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) A buffering agent, a preservative, an anhydrous agent, a solubilizing agent, an isotonic agent and a stabilizer may be mixed and used. In the case of a propellant, it can be conveniently delivered in the form of an aerosol sprayer body from a pressurized pack or sprayer using a suitable propellant, such as compressed air, nitrogen, carbon dioxide, or a hydrocarbon-based low boiling point solvent.

Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like in the case of oral administration, and in the case of injections, unit dosage ampoules or a plurality of dosage forms.

The route of administration of the vaccine composition can be administered via any conventional route so long as it can reach the target tissue.

The term "administration" of the present invention means introduction of a predetermined substance into a patient by any suitable method, and is formulated into human or veterinary and administered by various routes. The recombinant adenovirus or viral particle of the present invention may be administered by a parenteral route such as intravenous, intravenous, intraarterial, intramuscular or subcutaneous routes and may be administered by oral, nasal, rectal, transdermal or aerosol inhalation Or may be administered by bolus or by slow infusion, but may be administered by way of example as a nasal route.

The vaccine composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means an amount sufficient to exhibit a vaccine effect and an amount not causing side effects or serious or excessive immune response, The severity, the activity of a particular compound, the route of administration, the rate of removal of the recombinant adenovirus or its viral particles, the duration of treatment, the drug in combination with or concurrently with the recombinant adenovirus or viral particle thereof, the age, weight, sex, , General health status, and factors well known in the pharmaceutical and medical arts. Various general considerations to be considered in determining the "therapeutically effective amount" are known to those skilled in the art and are described, for example, in Gilman et al., Eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990 , And Remington ' s Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990. In the case of administering the recombinant adenovirus or virus particle of the present invention, the number administered per administration is usually about 1 × 10 ⁷ to 1 × 10 ¹¹ , more preferably 1 × 10 ⁸ to 5 × 10 ¹⁰ And most preferably in the range of 5 × 10 ⁸ to 2 × 10 ¹⁰ .

The vaccine 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. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

According to another aspect of the present invention, there is provided a method for preventing or treating an infection of an influenza B virus, comprising administering the influenza B vaccine composition to a subject suffering from an infection with an influenza B virus, The present invention provides a method for preventing or treating a disease caused by an infection of an influenza B virus.

The term "individual" of the present invention means a living organism capable of infecting influenza B virus and capable of causing respiratory disease due to infected influenza B virus. Preferably, the respiratory organism may develop, , More preferably a mammal, and most preferably a primate, but is not particularly limited thereto.

Although the recombinant adenovirus provided in the present invention contains only a gene coding for the nucleoprotein of B / Yamagata family virus, the recombinant adenovirus can induce immunity against infection of B / Yamagata family virus as well as B / Victoria family virus Therefore, the recombinant adenovirus can be widely used as a general vaccine against influenza B virus and can be widely used for prevention and treatment of various diseases caused by infection with influenza B virus.

FIG. 1 is a Western blot analysis photograph showing the result of confirming the expression of B-NP in a host cell infected with the recombinant adenovirus (rAd / B-NP) of the present invention. In the lane 1, , Lane 2 represents Hep2 cells infected with adenovirus (rAd / mock), and lane 3 represents Hep2 cells infected with recombinant adenovirus (rAd / B-NP).
FIG. 2 is a graph showing anti-B-NP antibody titer in serum at 2 weeks after the administration of the vaccine in a mouse to which the recombinant adenovirus (rAd / B-NP) of the present invention was administered.
FIG. 3A is a graph showing the results of measurement of body weight change after infecting a mouse administered with recombinant adenovirus (rAd / B-NP) of the present invention with B / Florida / 04/06.
FIG. 3B is a graph showing the survival rate of a mouse administered with recombinant adenovirus (rAd / B-NP) of the present invention after infection with B / Florida / 04/06.
FIG. 4A is a graph showing the results of measurement of body weight change after infecting a mouse administered with the recombinant adenovirus (rAd / B-NP) of the present invention with B / Malaysia / 2056/04.
FIG. 4B is a graph showing the survival rate of a mouse administered with the recombinant adenovirus (rAd / B-NP) of the present invention after infecting with B / Malaysia / 2056/04.
FIG. 4C is a graph showing the results of measuring the titer of virus in lung cells after infecting a mouse administered with the recombinant adenovirus (rAd / B-NP) of the present invention with B / Malaysia / 2056/04.
FIG. 5A is a graph showing the results of flow cytometry after binding of Dd / B-NP166-174 tetramer to lung cells of rAd / Gcf or rAd / B-NP-administered mice.
FIG. 5B is a graph showing the results of flow cytometry analysis of FIG. 5A.
FIG. 5c shows the results of stimulating lung cells of rAd / Gcf or rAd / B-NP mice with B-NP166-174 peptide or T cell activator and then staining with CD8, CD44, IFN- The upper right section shows cells expressing CD44 or IFN-gamma among CD8-expressing cells.
FIG. 5d shows the results of stimulation of the lung cells of rAd / Gcf or rAd / B-NP mice with B-NP166-174 peptide or T cell activator and then expressing CD44 and IFN-gamma in CD8- Cell ratio of the cells.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Recombinant expression of nucleoprotein (B-NP) influenza virus Adenovirus Produce

The influenza B virus was transfected with the RNA genomic gene of B / Yamagata16 / 88 family virus by reverse transcription PCR to synthesize cDNA, and PCR was performed using the synthesized cDNA as a template to obtain nucleoprotein B -NP). The obtained B-NP gene was cloned into a T vector and the nucleotide sequence was analyzed. NP gene was selected using the analyzed nucleotide sequence, and the selected B-NP gene was introduced into a pAd vector to prepare a B-NP expression vector. The recombinant adenovirus (rAd / B-NP) capable of expressing B-NP by infecting the transformant with the adenovirus is obtained by introducing the prepared B-NP expression vector into HEK293 cells to obtain a transformant. Respectively. The recombinant adenovirus (rAd / B-NP) was infected with HEK293 cells, and the recombinant adenovirus (rAd / B-NP) Respectively.

Western blot analysis was performed to confirm whether B-NP could be expressed in infected Hep2 cells when the purified recombinant adenovirus (rAd / B-NP) was infected to the host Hep2 cells. Specifically, Hep2 cells infected with uninfected Hep2 cells, adenovirus (rAd / mock) or Hep2 cells infected with recombinant adenovirus (rAd / B-NP) were disrupted, and a protein fraction was obtained, Western blot analysis using antibodies against B-NP was performed (Figure 1).

FIG. 1 is a Western blot analysis photograph showing the result of confirming the expression of B-NP in a host cell infected with the recombinant adenovirus (rAd / B-NP) of the present invention. In the lane 1, , Lane 2 represents Hep2 cells infected with adenovirus (rAd / mock), and lane 3 represents Hep2 cells infected with recombinant adenovirus (rAd / B-NP). As shown in FIG. 1, it was confirmed that B-NP was expressed only in the host cells infected with the recombinant adenovirus (rAd / B-NP) of the present invention.

Example 2: Preventive effect of rAd / B-NP vaccine against Influenza B infection

Example 2-1: Yamagata Effect on influenza infection

In order to investigate the vaccine effect, 1x10 ⁷ PFU and 1x10 ⁸ PFU of rAd / B-NP were administered as a vaccine, and rAd / Gcf 1x10 ⁸ PFU was administered once as a control. Of PBS were nasally administered. Blood was collected from the experimental animals at 2 weeks after the vaccination, and the antibody titer to B-NP was measured by an ELISA (FIG. 2).

FIG. 2 is a graph showing anti-B-NP antibody titer in serum at 2 weeks after the administration of the vaccine in a mouse to which the recombinant adenovirus (rAd / B-NP) of the present invention was administered. As shown in FIG. 2, it was confirmed that the anti-B-NP antibody titer in serum was increased in the mice to which the recombinant adenovirus (rAd / B-NP) of the present invention was administered.

Then, B / Yamagata lineage virus B / Florida / 04/06 was infected with 10 LD50 at 3 weeks after vaccination.

The mice consisted of an experimental group for examining the CTL (cytotoxic T lymphocytes) response in the lungs and an experimental group for observing the survival rate and the body weight change. The experimental group for the CTL response was sacrificed at the 7th day after the virus infection and the survival rate and body weight change And observed for 12 days after virus infection (Figures 3a and 3b).

FIG. 3A is a graph showing the results of measurement of body weight change after infecting a mouse administered with recombinant adenovirus (rAd / B-NP) of the present invention with B / Florida / 04/06, This is a graph showing the results of the survival rate of mice infected with recombinant adenovirus (rAd / B-NP) infected with B / Florida / 04/06.

As shown in FIGS. 3A and 3B, in the case of PBS or rAd / Gcf, the control group showed rapid weight loss after viral infection. On the 6th day, the mice died, whereas the rAd / B-NP- B-NP showed a lower survival rate compared with rAd / B-NP. In addition, the degree of defense against virus infection was found to be proportional to the dose of vaccine.

From the above results, mice administered with recombinant adenovirus (rAd / B-NP) showed improvement in weight loss and survival rate against influenza B infection of Yamagata lineage, so that the recombinant adenovirus (rAd / B- Can be used as a vaccine against influenza B virus infection.

Example 2-2: Effect on Infection with Victoria Influenza

On the other hand, since the influenza B virus includes the B / Yamagata family and the B / Victoria family virus, both seasonal influenza viruses have the same level of influence on both viruses, Of recombinant adenovirus (rAd / B-NP) could be used as a vaccine. Approximately, after administration of the recombinant adenovirus (rAd / B-NP), B / Malaysia lineage virus B / Malaysia / 2056/04 was infected with 10 LD50 at week 3 and maintained for 14 days, The activity of the virus in the lung cells was analyzed (Figs. 4A to 4C).

FIG. 4A is a graph showing the results of measurement of changes in body weight after infecting a mouse administered with the recombinant adenovirus (rAd / B-NP) of the present invention with B / Malaysia / 2056/04, FIG. 4C is a graph showing the results of measurement of survival rate after infecting mice with recombinant adenovirus (rAd / B-NP) with B / Malaysia / 2056/04, -NP) was infected with B / Malaysia / 2056/04, and the activity of virus was measured in lung cells.

As shown in FIGS. 4a to 4c, the PBS or rAd / Mock control group showed a rapid weight loss after viral infection. On the 6th day, the mice were killed completely, whereas the rAd / B-NP vaccinated mice showed less weight loss than the control group , Showing higher survival rate and low level of viral titer in lung cells.

Therefore, it was found that the recombinant adenovirus (rAd / B-NP) of the present invention can also be used as a vaccine against influenza B infection of Victoria strain.

Example 3: of B-NP cytotoxic T lymphocyte ( CTL ) Epitope quest

To find the mouse CTL epitope of B-NP, the following peptide candidates were selected using the online prediction program (IEDB, SYFPEITHI, bimas) (Table 1).

Mouse CTL epitope candidate designation location order H-2K ^d 363-371
276-284
389-398 YNMATPVSI (SEQ ID NO: 2)
AYEKILLNL (SEQ ID NO: 3)
CFGAAYEDL (SEQ ID NO: 4) H-2L ^d 30-38
334-342
536-545
305-314 RPIIRPATL (SEQ ID NO: 5)
LPISIYAKI (SEQ ID NO: 6)
IPIKQTIPNF (SEQ ID NO: 7)
NPGIADIEDL (SEQ ID NO: 8) H-2D ^d ^{/ b} 166-174 FSPIRITFL (SEQ ID NO: 9) H-2K ^b 162-169
275-282 KTIYFSPI (SEQ ID NO: 10)
TAYEKILL (SEQ ID NO: 11) H-2D ^b 191-199
106-114
242-250 FSGLNHIMI (SEQ ID NO: 12)
NLIQNAHAV (SEQ ID NO: 13)
VAIKGGGTL (SEQ ID NO: 14)

As shown in Table 1, eight CTL epitopes were screened for the eight peptides expected to be presented in the mouse haplotype class I MHC and the six peptides expected to be presented in the b haplotype class I MHC Respectively.

First, the rAd / B-NP provided in the present invention was administered and infected with B / Yamagata / 16/88 influenza virus. Lung cells of the mice were collected on day 5, Gt; < / RTI > IFN-g + CD8 T cells when stimulated with peptide. As a result, it was confirmed that the B-NP (166-174) peptide increases the frequency of IFN-g + CD8 T cells in d haplotype mice.

In order to verify the effect of the selected B-NP (166-174) peptide, MHC class I complex (Dd / B-NP166-174 tetramer) was prepared using the peptide and H-2Dd, CD8 T cell detection is possible. Specifically, infected with B / Florida / 04/06 at week 3 after administration of rAd / Gcf (1 × ^{10 8} PFU) or rAd / B-NP (1 × 10 ⁷ PFU or 1 × ^{10 8} PFU) One cell was stained with Dd / B-NP166-174 tetramer followed by flow cytometry (Figs. 5a and 5b).

FIG. 5A is a graph showing the results of performing flow cytometry after binding of Dd / B-NP166-174 tetramer to lung cells of rAd / Gcf or rAd / B-NP-administered mice, Which is a graph showing the results of quantitative analysis of the flow cytometry analysis results.

As shown in FIGS. 5A and 5B, the Dd / B-NP166-174 tetramer was not bound to the lung cells of the virus-infected mice after rAd / Gcf administration, but the rAd / B- It was confirmed that the Dd / B-NP166-174 tetramer was bound to the lung cells.

In addition, the mouse was rAd / Gcf (1x108 PFU) or rAd / B-NP administration of (1x10 ⁷ PFU or 1x10 ⁸ PFU), and the extraction, pulmonary After breeding for 7 days and to separate the lung cells therefrom. Brefeldin A was added to the separated lung cells, treated with B-NP166-174 peptide, and immunofluorescent staining with CD8, CD44 and IFN-gamma fluorescent antibodies was performed and analyzed for flow cytometry (Fig. 5C). At this time, non-stimulated lung cells were used as a control group and lung cells treated with a T cell activator (PMA / ionomycin, PI) were used as a comparative group.

FIG. 5c shows the results of stimulating lung cells of rAd / Gcf or rAd / B-NP mice with B-NP166-174 peptide or T cell activator and then staining with CD8, CD44, IFN- The upper right section shows cells expressing CD44 or IFN-gamma among CD8-expressing cells. As shown in FIG. 5C, when the lung cells of rAd / B-NP-treated mice were not stimulated, CD44 or IFN-gamma-expressing cells were not found in CD8-expressing cells, but B-NP166-174 peptide In the case of stimulation, it was confirmed that CD44 or IFN-gamma-expressing cells appeared in CD8-expressing cells.

In addition, cells immunostained with the fluorescent antibody for CD8 were isolated, and the ratio of cells expressing CD44 and IFN-gamma was analyzed and compared (FIG. 5D).

FIG. 5d shows the results of stimulation of the lung cells of rAd / Gcf or rAd / B-NP mice with B-NP166-174 peptide or T cell activator and then expressing CD44 and IFN-gamma in CD8- Cell ratio of the cells. As shown in FIG. 5D, in the cells stimulated with T cell activator (P / I), CD8 and CDNA-expressing cells expressing both CD44 and IFN-gamma in all cells regardless of rAd / B- In the cells stimulated with B-NP166-174 peptide, the percentage of cells expressing CD44 and IFN-gamma increased in the CD8-expressing cells only in the rAd / B-NP immunoreactive cells Respectively.

Therefore, it was confirmed that B-NP166-174 peptide can promote the immune response in mice immunized with rAd / B-NP, and B-NP166-174 peptide in rAd / B-NP was found to stimulate immunity It is analyzed that it will play an important role.

<110> Ewha University-Industry Collaboration Foundation <120> Vaccine against type B Influenza <130> KPA160522-KR <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> recombinant viral nucleoprotein <400> 1 Met Ser Asn Met Asp Ile Asp Gly Ile Asn Thr Gly Thr Ile Asp Lys 1 5 10 15 Thr Pro Glu Glu Ile Thr Ser Gly Thr Ser Gly Thr Thr Arg Pro Ile 20 25 30 Ile Arg Pro Ala Thr Leu Ala Pro Pro Ser Asn Lys Arg Thr Arg Asn 35 40 45 Pro Ser Pro Glu Arg Ala Thr Thr Ser Ser Glu Ala Asp Val Gly Arg 50 55 60 Lys Thr Gln Lys Lys Gln Thr Pro Thr Glu Ile Lys Lys Ser Val Tyr 65 70 75 80 Asn Met Val Val Lys Leu Gly Glu Phe Tyr Asn Gln Met Met Val Lys 85 90 95 Ala Gly Leu Asn Asp Asp Met Glu Arg Asn Leu Ile Gln Asn Ala His 100 105 110 Ala Val Glu Arg Ile Leu Leu Ala Ala Thr Asp Asp Lys Lys Thr Glu 115 120 125 Phe Gln Lys Lys Lys Asn Ala Arg Asp Val Lys Glu Gly Lys Glu Glu 130 135 140 Ile Asp His Asn Lys Thr Gly Gly Thr Phe Tyr Lys Met Val Arg Asp 145 150 155 160 Asp Lys Thr Ile Tyr Phe Ser Pro Ile Arg Ile Thr Phe Leu Lys Glu 165 170 175 Glu Val Lys Thr Met Tyr Lys Thr Thr Met Gly Ser Asp Gly Phe Ser 180 185 190 Gly Leu Asn His Ile Met Ile Gly His Ser Gln Met Asn Asp Val Cys 195 200 205 Phe Gln Arg Ser Lys Ala Leu Lys Arg Val Gly Leu Asp Pro Ser Leu 210 215 220 Ile Ser Thr Phe Ala Gly Ser Thr Leu Pro Arg Arg Ser Gly Ala Thr 225 230 235 240 Gly Val Ala Ile Lys Gly Aly Ile Arg 245 250 255 Phe Ile Gly Arg Ala Met Ala Asp Arg Gly Leu Leu Arg Asp Ile Lys 260 265 270 Ala Lys Thr Ala Tyr Glu Lys Ile Leu Leu Asn Leu Lys Asn Lys Cys 275 280 285 Ser Ala Pro Gln Gln Lys Ala Leu Val Asp Gln Val Ile Gly Ser Arg 290 295 300 Asn Pro Gly Ile Ala Asp Ile Glu Asp Leu Thr Leu Leu Ala Arg Ser 305 310 315 320 Met Val Val Val Val Pro Ser Val Ala Ser Lys Val Val Leu Pro Ile 325 330 335 Ser Ile Tyr Ala Lys Ile Pro Gln Leu Gly Phe Asn Val Glu Glu Tyr 340 345 350 Ser Met Val Gly Tyr Glu Ala Met Ala Leu Tyr Asn Met Ala Thr Pro 355 360 365 Val Ser Ile Leu Arg Met Gly Asp Asp Ala Lys Asp Lys Ser Gln Leu 370 375 380 Phe Phe Met Ser Cys Phe Gly Ala Ala Tyr Glu Asp Leu Arg Val Leu 385 390 395 400 Ser Ala Leu Thr Gly Thr Glu Phe Lys Pro Arg Ser Ala Leu Lys Cys 405 410 415 Lys Gly Phe His Val Ala Lys Glu Gln Val Glu Gly Met Gly Ala 420 425 430 Ala Leu Met Ser Ile Lys Leu Gln Phe Trp Ala Pro Met Thr Arg Ser 435 440 445 Gly Gly Asn Glu Gly Gly Asp Gly Gly Ser Gly Gln Ile Ser Cys 450 455 460 Ser Pro Val Phe Ala Val Glu Arg Pro Ile Ala Leu Ser Lys Gln Ala 465 470 475 480 Val Arg Arg Met Leu Ser Met Asn Ile Glu Gly Arg Asp Ala Asp Val 485 490 495 Lys Gly Asn Leu Leu Lys Met Met As Asp Ser Met Ala Lys Lys Thr 500 505 510 Asn Gly Asn Ala Phe Ile Gly Lys Lys Met Phe Gln Ile Ser Asp Lys 515 520 525 Asn Lys Thr Asn Pro Val Glu Ile Pro Ile Lys Gln Thr Ile Pro Asn 530 535 540 Phe Phe Phe Gly Arg Asp Thr Ala Glu Asp Tyr Asp Asp Leu Asp Tyr 545 550 555 560 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 2 Tyr Asn Met Ala Thr 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 3 Ala Tyr Glu Lys Ile Leu Leu Asn Leu 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 4 Cys Phe Gly Ala Ala Tyr Glu Asp Leu 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 5 Arg Pro Ile Ile Arg Pro Ala Thr Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 6 Leu Pro Ile Ser Ile Tyr Ala Lys Ile 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 7 Ile Pro Ile Lys Gln Thr Ile Pro Asn Phe 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 8 Asn Pro Gly Ile Ala Asp Ile Glu Asp Leu 1 5 10 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 9 Phe Ser Pro Ile Arg Ile Thr Phe Leu 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 10 Lys Thr Ile Tyr Phe Ser Pro Ile 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 11 Thr Ala Tyr Glu Lys Ile Leu Leu 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 12 Phe Ser Gly Leu Asn His Ile Met Ile 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 13 Asn Leu Ile Gln Asn Ala His Ala Val 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial peptide <400> 14 Val Ala Ile Lys Gly Gly Gly Thr Leu 1 5

Claims

A gene encoding a nucleoprotein comprising as an epitope a peptide consisting of the amino acid sequence of SEQ ID NO: 9 of the B / Yamagata family virus in influenza B virus, wherein the infected B / Yamagata family virus and B / Victoria A recombinant adenovirus capable of simultaneously inducing an immune response against a series of viruses.

The method according to claim 1,
Wherein the nucleoprotein is a nucleoprotein comprising as an epitope a peptide consisting of the amino acid sequence of SEQ ID NO: 9 derived from B / Yamagata / 16/1988 virus.

delete

A recombinant adenovirus or a viral particle thereof according to claim 1 or 2 as an active ingredient and simultaneously administering to the subject an immunity against influenza B virus, B / Yamagata virus and B / Victoria virus, An influenza B vaccine composition capable of preventing or treating a disease caused by infection with a virus.

5. The method of claim 4,
Wherein the vaccine composition further comprises a pharmaceutically acceptable excipient, diluent or carrier.

5. The method of claim 4,
Wherein the disease caused by the infection of the influenza B virus is selected from the group consisting of cough, sneezing, fever, wheezing, bronchitis, bronchiolitis, pneumonia, asthma, bronchitis, croup and respiratory failure.