Novel nucleic acid-based influenza vaccine composition

Abstract

The present invention relates to an influenza vaccine composition based on a novel RNA having the dual functions of an immunopotentiator and antigen capture.

Description

Technical Field

[0001] The present invention relates to an influenza vaccine composition based on a novel ribonucleic acid having the dual functions of adjuvant and antigen capture.

[0002] Particularly, the present invention is directed to the vaccine composition containing influenza antigen in the adjuvant of novel mixed structure ribonucleic acid (hsRNA) and the purposes of preventing or treating influenza virus infection and is described.Described novel mixed structure ribonucleic acid (hsRNA) is selected by spleen dendritic cell activation test in vivo, and it can be defined as specific length or sequence.When influenza antigen, for example influenza full virus antigen or surface antigen are loaded into the vaccine composition of described mixed structure ribonucleic acid and are applied in vivo (for example, by nasal cavity transmission), can effectively prevent the individual death that causes because of fatal infection. Background Art

[0003] A) Influenza virus

[0004] Influenza virus (IV) belongs to the Orthomyxoviridae family and is a single-stranded, negative-sense RNA virus composed of eight segments of RNA expressing 10 to 13 proteins. It is divided into types A (A), B (B), and C (C) based on the antigenicity caused by structural proteins attached to the viral surface, namely nuclear antigen (NP) and matrix (M) proteins. Types A (A) and B (B) are known to be primarily pathogenic to humans. Type A (A) can infect not only humans, but also pigs and birds. Type B (B) has humans as its sole reservoir.

[0005] Influenza virus is made up of two surface glycoproteins (surface antigens), i.e. hemagglutinin and neuraminidase. According to the antigenic protrusions that are combined with the host cell's receptor (sialic acid, Sialic acid), i.e. hemagglutinin, and the type and combination of neuraminidase that cuts sialic acid in order to make the virus bound to sialic acid free into the cell, influenza virus is divided into each subtype. The subtype is mainly distinguished with influenza A (A) as the center. 18 kinds of hemagglutinins from H1 to H18 and 11 kinds of neuraminidase from N1 to N11 have been found. When combined, 198 kinds of subtype influenza A (A) can be combined in theory. Influenza B virus is divided into two systems, Victoria (Victoria) and Yamagata (Yamagata), according to the antigenic type.

[0006] As a typical characteristic of RNA viruses, influenza has the characteristic that antigenic variation occurs slightly or violently almost every year. Due to the fact that influenza A (A) and B (B) are the main types, antigenic drift occurs almost every year, which is the cause of seasonal influenza epidemics. Therefore, the World Health Organization (WHO) determines the recommended vaccine strains for that season around February of each year after integrating virus epidemic information and publishes them. Antigenic drift is a point mutation within the same subtype, which is a phenomenon in which a new hemagglutinin or neuraminidase with slightly changed antigenicity is replaced by a slight antigenic variation. Antigenic shift is the same as H3N2 → H2N2, which produces a new virus by changing the subtype. In addition to the two variations described above, variations may occur when cultured in embryonated eggs, which may lead to antigenic differences. However, such variations have not been found in cell culture, so vaccines using viruses cultured in mammalian cells are preferred.

[0007] Of the two surface antigens, hemagglutinin and neuraminidase, immunity to hemagglutinin is particularly associated with influenza prevention and disease severity. Therefore, hemagglutinin is the most important component of the influenza vaccine, and neutralizing antibodies produced in the body against it play a crucial role in preventing influenza virus infection.

[0008] In 1918, human influenza A (H1N1) virus spread from birds to humans and pigs, causing a pandemic. Human influenza A (H2N2) virus emerged in 1957. Human influenza A (H1N1) virus reappeared in 1977 and, along with human influenza A (H3N2) virus, which emerged in 1968, became the primary cause of seasonal influenza. The novel influenza virus (pandemic influenza H1N1 2009) that has been circulating worldwide since April 2009 is a newly generated influenza virus that underwent antigenic shift through genetic recombination.

[0009] B) Flu vaccine

[0010] Due to antigenic drift and a duration of effectiveness of less than one year, influenza vaccination needs to be administered annually. Taking into account the influenza epidemic period (December to April of the following year) and the duration of the vaccination effect (an average of 6 months), the recommended vaccination time is designated as October to December each year.

[0011] Current influenza vaccines include inactivated influenza vaccines and live attenuated influenza vaccines (LAIV). Inactivated influenza vaccines are made by inactivating viruses cultured in fertilized eggs (hatching eggs) with formalin, or by growing and inactivating viruses in cell cultures to obtain surface antigens, which are then injected intramuscularly (IM) as vaccine antigens. Live influenza vaccines are administered by spraying into the nasal cavity.

[0012] Inactivated vaccines include whole virus vaccines, which use the entire virus; split vaccines (subvirion vaccines), which separate the viral envelope using ether or other methods; and subunit vaccines, which purify the hemagglutinin and neuraminidase components. Hemagglutinin and neuraminidase are antigens that directly induce neutralizing antibody responses, with hemagglutinin being the primary neutralizing antigen. Due to the side effects of whole virus vaccines in children, they are currently rarely used worldwide, including in South Korea, and are only used in a few countries. In contrast, component vaccines such as split vaccines and subunit vaccines are highly safe and proven to be effective, resulting in their highest usage rate.

[0013] In addition, vaccines containing immunopotentiators (adjuvants) such as MF-59 and virosome vaccines, which form vesicles similar to the shape of viruses, have been developed and used in some countries to enhance immune responses. In particular, to provide sufficient protective immunity against influenza virus infection for preventive or therapeutic purposes, the inclusion of a potent and safe adjuvant in the vaccine is necessary.

[0014] Most antibodies against specific subtypes or influenza viruses obtained through natural infection or vaccines are unable to form protective antibodies against other types or subtypes of influenza viruses, and there is also the problem of not showing sufficient immunogenicity against new variants within an antigen.

[0015] Because influenza viruses cause large and small mutations every year and the prevalent strains change every year, it is difficult to expect the protection provided by the vaccine administered last year, so vaccination is required every year.

[0016] For example, inactivated influenza vaccines are used to evaluate new vaccine candidates during the development phase. Survival rates are then used to assess the candidate's effectiveness and potency. However, among the various vaccine protein antigens included in a vaccine, potency is primarily assessed by the HA (hemagglutinin) content. Currently, vaccines are typically formulated with 15 μg of HA per subtype. Vaccine efficacy data are obtained through challenge tests in susceptible animals following immunization. Submission of data confirming protective efficacy, or equivalent, is required.

[0017] In the non-clinical immunogenicity study of the vaccine, appropriate immune responses such as humoral immunity and cell-mediated immunity induced in the vaccinated experimental animals should be evaluated. The evaluation is based on the induced immune response through seroconversion, geometric mean titer (GMT) of antibodies or cell-mediated immunity. The non-clinical immunogenicity test of influenza vaccine is mainly evaluated in experimental animals by hemagglutinination inhibition (HI) antibody (titer). That is, it is known that if the HI antibody titer of the serum after vaccination with killed vaccine is greater than or equal to 1:40, the risk of influenza infection will decrease, but this is not absolute. It has been proposed that when the HI antibody titer is 1:15 to 1:65, 50% of subjects can be protected from disease, and the protection rate increases with increasing titer (Hobson D et al, Journal of Hygiene 70:767-777, 1972) (de Jong JC et al, Developmental Biology. 115:63-73, 2003). The conversion rate and GMT are used as methods to measure vaccine potency (Committee for Proprietary Medicinal Products (CPMP). Note for guidance on harmonization of requirements for influenza vaccines. CPMP / BWP / 214 / 96. The European Agency for the Evaluation of Medicinal Products (EMEA), March 1997) (Treanor J et al, Vaccine. 20:1099-1105, 2002). Furthermore, neutralizing antibody titers, which are associated with functional immune responses, should be assessed. For example, for live attenuated vaccines, mucosal secretory antibodies and cell-mediated immunity also need to be evaluated. The immune responses generated after vaccination with live influenza vaccines can differ significantly from those generated with killed vaccines. This is because live vaccines produce lower serum HI antibody titers than killed vaccines, but induce greater secretory antibodies and cell-mediated immunity in the mucosa. Therefore, the HI antibody titer generated after vaccination cannot be used to predict vaccine efficacy.

[0018] Since the seasonal influenza vaccines currently in use only provide protective immunity against the strains used in the vaccine, there is a need to develop an economical and effective influenza vaccine that can provide sufficient cross-protective immunity against various subtypes. In order to develop a universal vaccine with cross-immunity, it is necessary to use antigens with minimal antigenic variation or methods to stimulate mucosal immunity. There are also cases where one or more HA2 domains of HA with low antigenic variation are overlapped and used as vaccine antigens (Korean registered patent 10-1637955).

[0019] C) Influenza mucosal vaccine

[0020] Vaccines administered parenterally or intranasally penetrate the lower respiratory tract and work by inducing anti-hemagglutinin IgG antibodies. Both IgA, a key antibody in mucosal immunity, and serum IgG are involved in immunity to influenza viruses. In particular, nasal stimulation of mucosal immunity can effectively prevent upper airway infections (Clements ML et al, J. Clinical Microbiology 24, 157-160, 1986). In mice, respiratory IgA plays an important role in protecting against influenza infection. The advantage of stimulating local respiratory IgA responses to influenza is that local respiratory IgA responses exhibit a broader range of protective immunity than serum responses, thus providing cross-protection against viruses containing vaccine antigens and other hemagglutinin molecules. Therefore, an influenza vaccine that induces anti-hemagglutinin responses in both local secretory organs and serum would provide superior immunity compared to existing vaccines. In contrast, parenteral vaccine injections (intramuscular, subcutaneous, etc.) are ineffective in inducing local antibody production without additional mucosal exposure (i.e., infection). That is, in order to stimulate the mucosal immune system, the vaccine must be applied topically to the mucosal surface.

[0021] Compared to the intramuscular, subcutaneous or intravenous administration of traditional parenteral methods, the most noteworthy advantage of administering influenza vaccines through the mucosa, such as intranasal spray or drip, is that it more effectively stimulates the local mucosal immune system of the respiratory system and is not limited by the rejection and uneasiness of needles, thereby increasing the vaccination rate. In fact, compared to administering live or attenuated vaccines into the muscle, when inactivated vaccines with low immunogenicity are administered into the human mucosa, a strong antibody response is induced and protective immunity is provided (Kuno-sakai et al, Vaccine 12: 1303-1310, 1994). However, the intramucosal administration method proposed in the above paper has the disadvantage that the dosage required for the patient is three times that of intramuscular administration, and therefore has not been commercially utilized.

[0022] To overcome the above-mentioned problems, other attempts have been made to enhance the immunogenicity of influenza vaccines by oral or intranasal administration, for example, by using the cholera toxin (CTB) B subunit (Tamura S. et al, Vaccine 6:409, 1988), encapsulating vaccine antigens in various microparticles (Moldoveanu Z et al, J. Inf. Dis. 167:85-90, 1993), or using attenuated live strains (Maassab HF et al, Vaccine, Plotkin SA and Mortimer FA Jr. (eds) WB Saunder Philadelphia p435, 1993). However, a practical method for enhancing the immunogenicity of influenza vaccines by administering them to the mucous membranes of the infection route has not yet been developed.

[0023] D) Vaccines and adjuvants

[0024] Vaccines or immunizations are the most effective means of preventing or treating various diseases, but there are still challenges to overcome. For example, most vaccines sometimes fail to effectively provide protective immunity, require multiple doses, and vaccine effectiveness decreases over time, necessitating additional revaccinations.

[0025] Adjuvants or immunopotentiators are generally substances that are insufficient on their own to induce antibody production or cellular responses, including single or mixed compounds or agents that enhance the immunogenicity of antigens. In contrast, some adjuvants are formulated to reduce immunogenicity and side effects. In general, adjuvants provide moderately potent and sustained antigen-specific immune responses through various means, for example, by promoting antigen presentation to the immune system, reducing the amount of antigen required, and providing the added advantage of avoiding multiple injections.

[0026] Innate immune cells recognize abnormal patterns, or danger signals, present in invading infectious pathogens or vaccination antigens and transmit them to the adaptive immune system. The level and specificity of these patterns amplify the qualitative and quantitative signals inherent to the adaptive response, demonstrating the importance of innate immunity in the adaptive immune response. Adjuvanted vaccine compositions are one of the most effective and cost-effective approaches for preventing or treating disease.

[0027] To effectively develop targeted vaccines, more potent and safe adjuvants should be included in vaccine compositions. These novel adjuvants offer numerous advantages, such as broad responses to a wide range of antigens, effective induction of humoral and cellular immune responses, and neutralization and, in particular, killing of pathogens. Furthermore, adjuvants in adjuvanted vaccines can help reduce antigen requirements and provide cross-protection and long-lasting immune responses during aging.

[0028] E) Double-stranded RNA as a TLR3 ligand in innate immunity

[0029] Dendritic cells (DCs) originate from hematopoietic myeloid progenitor cells and initially differentiate into immature DCs, characterized by high endocytic activity and low T cell activation capacity. Immature DCs continuously monitor their surroundings for the presence of viruses and bacteria, using pattern recognition receptors (PRRs) similar to TLRs. Double-stranded RNA (dsRNA) signals viral infection and single-stranded RNA (ssRNA) signals pathogenicity or abnormal RNA can serve as danger signals. TLRs recognize specific chemical markers found in pathogenic organisms. When DCs encounter antigens, they activate into mature DCs and begin migrating to lymph nodes. Immature DCs phagocytose pathogens and, upon maturation, present fragments of these pathogens using major histocompatibility antigen (MHC) molecules on the surface of autologous cells. Simultaneously, the expression of T cell-activating co-stimulatory factors, such as CD80 (B7.1), CD86 (B7.2), and CD40, is greatly enhanced on autologous cells. Dendritic cells also upregulate CCR7, a substance that induces dendritic cells to migrate to the spleen or lymph nodes. At this point, the dendritic cells act as antigen-presenting cells, presenting antigens and activating helper T cells, killer T cells, and B cells.

[0030] As mentioned above, dendritic cells play a crucial role in both innate and adaptive immune responses. This is due to the maturation of dendritic cells (DCs), which are characterized by increased expression of costimulatory factors, production of proinflammatory cytokines, and antigen presentation. Other subsets of DCs exhibit distinct specialized functions. CD8α-positive classical dendritic cells (cDCs) have the selective ability to cross-present intracellular antigens via MHC class I. This function is important for the generation of CTLs targeting viral antigens or nuclear antigens from necrotic cells. In contrast, extracellular antigens are captured and transferred to the endosomes / lysosomes of CD8α-negative cDCs, where they are broken down into antigenic peptides, complexed with MHC class II molecules, and recognized by CD4 T cells. During DC maturation, antigen-loaded DCs spontaneously migrate to secondary lymph nodes and acquire the ability to stimulate T cells. These DCs produce proinflammatory cytokines, which crucially influence the induction of CD4 helper T cells and CD8 CTLs that produce specific cytokine types.

[0031] To induce activation of tumor antigen-specific CTLs in tumor vaccines, tumor antigens must be cross-presented by CD8α-positive dendritic cells. Furthermore, activation of CD4 T cells by mature CD8α-negative dendritic cells is required. CD8α-negative cDCs, the predominant population in the mouse spleen, possess the selective ability to present extracellular antigens directly to CD4 T cells. At the site of infection or immunization, pathogen-derived substances or immunopotentiators activate dendritic cells, inducing the expression of costimulatory factors and cytokines, which, in conjunction with MHC-antigen complexes, differentiate cognate T cells into antigen-specific CTLs and helper T cells.

[0032] TLR3 is normally expressed in endosomal compartments of myeloid dendritic cells (mDCs), B cells, monocyte-derived macrophages, and many tumor tissues. It detects viral double-stranded RNA (dsRNA) in infected cells, a hallmark of viral infection or replication. Recognition of dsRNA by TLR3, MDA-5, and NLRP3 stimulates type I interferon and proinflammatory cytokines. When mDCs are activated into mature antigen-presenting cells (mAPCs), antigenic epitopes are loaded onto MHC-I molecules for presentation to naive T cells. Dendritic cells activated by TLR3 not only contribute to the induction of innate and adaptive immune responses against microbial pathogens but also stimulate antitumor CD8+ T cells, thereby promoting natural killer (NK) cell activation and tumor cell death.

[0033] F) Single-stranded RNA as a TLR7 ligand in innate immunity

[0034] Single-stranded RNA oligonucleotides comprising (preferably rich in) guanosine (G) and uridine (U) can be derived from invading pathogens, and these nucleotides stimulate dendritic cells and macrophages to secrete interferon α, proinflammatory cytokines, and regulatory factors. They are found to be recognized by TLR7 and TLR8. TLR7 detects single-stranded RNA and induces inflammatory responses through MYD88 and TRAF6 in NF-kappa-B activation, cytokine secretion, and innate immunity. Very similar to TLR3 and TLR9, TLR7 and TLR8 are also typically expressed on the membrane of endosomes. In addition, TLR7 responds to chemical ligands (e.g., imidazoquinoline). Crystal structure studies have shown that TLR7 is a dual receptor for single-stranded RNA containing guanosine and uridine. The ligand binding first region conserved in TLR7 is used for small ligand binding, and the second region is used for single-stranded RNA binding. It is known that the first region preferentially detects guanosine, and the second region specifically binds to the uridine portion of single-stranded RNA.

[0035] Because current seasonal influenza vaccines only provide protective immunity against the strains used in the vaccine, there is a need to develop an economical and effective influenza vaccine that can provide a sufficient degree of cross-protective immunity against various subtypes.

[0036] To develop a universal vaccine with cross-immunity, it is necessary to use antigens with minimal antigenic variation or methods to stimulate mucosal immunity. There are also cases where one or more HA2 domains of HA with low antigenic variation are overlapped and used as vaccine antigens (Korean Patent No. 10-1637955).

[0037]

Prior art literature

[0038] [Patent Literature]

[0039] (Patent Document 1) Korean registered patent 10-1637955 Summary of the Invention

[0040] Technical issues

[0041] The object of the present invention is to provide an influenza vaccine with enhanced versatility.

[0042] Because current seasonal influenza vaccines only provide protective immunity against the strains used in the vaccine, there is a need to develop an economical and effective influenza vaccine that can provide a sufficient degree of cross-protective immunity against various subtypes.

[0043] The purpose of the present invention is to provide a new universal influenza vaccine that can provide protective immunity not only against the subtype with the same antigen as the vaccine, but also against various other subtypes of influenza A (A) virus, thereby being able to cope with the emergence of new variant influenza.

[0044] Technical Solution

[0045] The present invention provides a vaccine composition for preventing or treating influenza virus infection based on a novel hybrid structure RNA (hsRNA) as an adjuvant.

[0046] Specifically, the present invention provides a vaccine and pharmaceutical composition comprising whole or surface inactivated influenza virus antigens loaded with hsRNA having a novel structure, thereby providing protective immunity not only against homologous viruses but also against heterologous subtypes. In particular, the vaccine composition of the present invention exhibits a potent and safe protective effect, particularly when administered intranasally.

[0047] The hybrid RNA (hsRNA) is selected through an in vivo splenic dendritic cell activation assay and can be defined as a specific length or sequence. When an influenza antigen, such as a whole influenza virus or surface antigen, is loaded onto a vaccine composition comprising the novel hybrid RNA (hsRNA), it is effectively used to prevent individual deaths from lethal infections (e.g., intranasal administration).

[0048] In one embodiment, the present invention provides a vaccine composition for preventing or treating influenza A virus infection, comprising a hybrid RNA (hsRNA) comprising double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA); and a human influenza A antigen, wherein the double-stranded RNA (dsRNA) is complementary, the single-stranded RNA (ssRNA) is located at either end of the 3'-end of the double-stranded RNA (dsRNA), and the hybrid RNA (hsRNA) is formed by complementary binding of the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2. The hybrid RNA (hsRNA) has a length of 533 bases and is also referred to herein as "NA."

[0049] The double-stranded ribonucleic acid (dsRNA) may have a base length of 424 bp and a base sequence of SEQ ID NO: 3.

[0050] The RNA selected in the present invention exhibits potent mucosal immune activation. When the mixed structure RNA of the present invention is used as a conventional administration route, such as as a vaccine adjuvant for nasal spray, a strong cross-protective immune effect can be obtained against homologous and heterologous subtypes of viruses.

[0051] The vaccine composition of the present invention can be administered to the mucosa via intranasal administration. In this case, the RNA acts as a powerful and safe immunopotentiator (adjuvant).

[0052] The human influenza A (A) antigen can be a whole influenza virus or a surface antigen. In one embodiment, the human influenza antigen can be (i) an inactivated or live attenuated whole influenza virus; or (ii) a surface antigen containing part or all of the hemagglutinin or neuraminidase, and the hemagglutinin or neuraminidase can be extracted from a whole virus propagation solution or recombinantly expressed and purified.

[0053] The vaccine composition of the present invention is characterized by providing protective immunity against not only homologous influenza viruses but also heterosubtypic viruses, particularly when delivered intranasally, achieving a potent and safe protective effect.

[0054] In one embodiment, the present invention provides a vaccine composition for preventing or treating influenza A (A) type H1N1 or H3N2 virus infection, characterized in that the human influenza A (A) type antigen is human influenza A (A) type antigen (H1N1).

[0055] In a specific embodiment, an inactivated A / H1N1 influenza virus (IV) vaccine complexed or incorporated into a mixed-structure RNA adjuvant of the present invention, when administered intranasally (in), can prevent death from infection with a lethal dose of the same A / H1N1 IV strain. In another embodiment, the same vaccine, when administered intranasally, can prevent death from infection with a lethal dose of a subtype (e.g., A / H3N2).

[0056] Beneficial effects

[0057] The vaccine composition of the present invention, which loads an antigen (Ag) onto a hybrid structured RNA (hsRNA), not only stimulates strong innate immunity but also effectively delivers the antigen complex to antigen-presenting cells, thereby effectively presenting the antigen to T cells and B cells, thereby enhancing specific adaptive immunity. Thus, the vaccine composition of the present invention can effectively protect against infection by both homologous and heterologous influenza viruses with a single intranasal spray, thereby inducing beneficial cross-defense immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 shows the 100% protective effect of the NA-conjugated inactivated A / H1N1 influenza virus (IIV) (iPR8) vaccine (RNA-conjugated vaccine) when administered once intranasally (in) according to Example 1, when a lethal dose of homologous live influenza virus (LIV) (PR8) was inoculated.

[0059] Figure 1a Show the experimental plan.

[0060] Figure 1b Shown are the changes in body weight of mice after virus inoculation.

[0061] Figure 1c The survival rate (%) on day 11 after virus inoculation is shown.

[0062] Figure 2 shows the protective effect of the NA-conjugated inactivated A / H1N1 influenza virus (IIV) (iPR8) vaccine (RNA-conjugated vaccine) when inoculated with a lethal dose of a live influenza virus of a heterologous subtype (A / H3N2) according to Example 2, after a single intranasal (in) administration.

[0063] Figure 2a Show the experimental plan.

[0064] Figure 2b The survival rate (%) on the 14th day after virus inoculation is shown.

[0065] Best Mode

[0066] Below, the present invention will be described in detail by way of examples to facilitate understanding of the present invention. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto. It is obvious to those skilled in the art that various changes and modifications can be made within the scope and technical range of the present invention, and naturally, such changes and modifications belong to the appended claims.

[0067] [Example 1]

[0068] Confirmation of protective immune effect by intranasal administration of the NA-adjuvanted inactivated influenza (iPR8) vaccine of the present invention ( FIG1 )

[0069] In this example, the inactivated A / H1N1 influenza virus (IIV) (iPR8) vaccine (RNA-conjugated vaccine) complexed with NA (ribonucleic acid antigen with a specific length and sequence proposed in the present invention) was administered into the nasal cavity, and the protective immune effect of the vaccine against lethal homologous virus infection was confirmed.

[0070] According to Table 1 and Figure 1a Each substance was administered to mice according to the administration schedule shown. Specifically, female Balb / c mice (7 weeks old, 5 mice each) were anesthetized and PBS (50 μl) was administered once to the nasal cavity, along with 0.5 μg iPR8 ± 5 μg NA and 0.5 μg iPR8 + 5 μg Poly(I:C). Three weeks later, the mice were given 100 times the lethal dose (100 MLD). 50 ), mice were inoculated with mouse-adapted live influenza virus PR8 (A / H1N1). The weight change (%) and survival rate (%) of the mice were monitored until day 11 after inoculation. The results are shown in Figure 1b and Figure 1c .

[0071]

Table 1

[0072]

[0073] like Figure 1b and Figure 1c As shown, when PBS alone was pre-administered followed by PR8 inoculation (control group 1), 100% of the mice died by day 7 after inoculation. In contrast, the experimental group according to the present invention, which was pre-administered with iPR8+NA and then PR8, survived 100%, and the weight gain rate also recovered to almost 100% by day 11. When iPR8 alone was pre-administered followed by PR8 inoculation (control group 2), only 40% of the mice survived, and the weight gain rate of the surviving mice was also reduced. When iPR8+Poly(I:C) was pre-administered followed by PR8 inoculation (control group 3), only 80% of the mice survived, and the weight gain rate of the surviving mice was also slightly reduced.

[0074] These results demonstrate that a single intranasal administration of the NA-complexed inactivated influenza (iPR8) vaccine protected all (100%) mice even after inoculation with a lethal dose of homologous live influenza virus, confirming that the NA-complexed vaccine of the present invention provides complete protective immunity. Furthermore, NA was shown to provide superior protective immunity compared to poly(I:C).

[0075] [Example 2]

[0076] Cross-protective immunity against heterologous subtype viruses provided by intranasal administration of the NA-adjuvanted inactivated influenza (iPR8) vaccine of the present invention ( FIG. 2 )

[0077] In this example, it was tested whether the NA-complexed inactivated A / H1N1 influenza virus (IIV) (iPR8) vaccine (RNA-conjugated vaccine) of the present invention can provide cross-protective immunity against heterologous subtype strains.

[0078] According to Table 2 and Figure 2a Each substance was administered to mice according to the administration schedule shown. Specifically, female Balb / c mice (7 weeks old, 5 mice each) were anesthetized and PBS (50 μl) or 0.1 to 5 μg NA + 0.5 μg iPR8 20 μl was administered once to the nasal cavity. Three weeks later, 50 MLD 50 The mice were inoculated with mouse-adapted live influenza virus A / Hong Kong / 8 / 68 (A / H3N2) in an amount of 1:1. The survival rate (%) of the mice was monitored until day 14 after inoculation. The results are shown in Tables 2 and Figure 2b .

[0079]

Table 2

[0080] Administered substances Whether to vaccinate against A / H3N2 Survival rate (day 14) Normal control group PBS (50ul) - 100％ control group PBS (50ul) vaccination 30％ Experimental Group 1 iPR8 (0.5 μg) + NA (0.5 μg) vaccination 60％ Experimental Group 2 iPR8 (0.5 μg) + NA (2.5 μg) vaccination 60％ Experimental Group 3 iPR8 (0.5 μg) + NA (5 μg) vaccination 100％

[0081] As shown in Table 2 and Figure 2b As shown, the iPR8+NA combination also demonstrated cross-protective immunity against heterologous influenza virus A / H3N2. In particular, the vaccine containing 5 μg of NA demonstrated a 100% protective effect. <110> NA Vaccine Laboratory Co., Ltd. Korea Institute of Chemical Technology <120> Novel nucleic acid-based influenza vaccine composition <130> NAVI19P-0003-WO <150> KR 10-2019-0106158 <151> 2019-08-28 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 482 <212> RNA <213> Artificial sequence <220> <223> NA <400> 1 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacuugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gcccuauagu gagucguauu agucgacugc agaggccugc augcaagcuu ggcguaauca 480 ug 482 <210> 2 <211> 475 <212> RNA <213> Artificial sequence <220> <223> NA <400> 2 gggcgaucuc acuuucacuc ucauuuuaga uucugcucua auccccguag ccugcgcccu 60 acuaacauua ucugccuuau cauaaucagu uaccgguaaa uaggcgcuag ccguauuucu 120 ccgcauaaca guaucauaua uauaaggcac agugaaaugu acugauuuuu guucucccaa 180 guggaacguu uuaguauaag uugaguguga cugacacuca uccguauuag uagaugaucg 240 auuauacucc gcugauauaa ucacuguucc agugugaaac gcauuugaaa caaaaucaaa 300 acguaacucu auuggcccug accaaaaguu auacaaccca guuacauauu ccauuggggu 360 uauuuguccu ucauaguccu gaucauaaaa ucucacaccu gaguccaaaa cuguauuauc 420 gcccuauagu gagucguauu aggauccgau aucuagaugc auucgcgagg uaccg 475 <210> 3 <211> 424 <212> RNA <213> Artificial sequence <220> <223> VP11 <400> 3 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacugugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gccc 424

Claims

1. A vaccine composition for preventing or treating influenza A virus infection, comprising a hetero-structured RNA (hsRNA) containing double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA); and a human influenza A antigen. in The double-stranded RNA (dsRNA) has complementarity, the single-stranded RNA (ssRNA) is located at both 3'-ends of the double-stranded RNA (dsRNA), and the hybrid RNA (hsRNA) is formed by the complementary binding of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO:

2. in, Human influenza A(A) antigen is inactivated influenza virus A(A). Among them, the vaccine composition is for intranasal administration.

2. The vaccine composition for preventing or treating influenza A (A) virus infection according to claim 1, wherein the double-stranded ribonucleic acid (dsRNA) has a nucleotide length of 424 bp and a nucleotide sequence of SEQ ID NO:

3.

3. The vaccine composition for preventing or treating influenza A virus infection according to claim 1, wherein the vaccine composition provides protective immunity against homotypic or heterosubtypic viruses, wherein the human influenza A(A) antigen is a human inactivated influenza A(A) H1N1 antigen, and wherein the heterologous subtype virus is an influenza A(A) H3N2 virus.

4. The vaccine composition for preventing or treating influenza A(A) virus infection according to claim 1, wherein the influenza A(A) virus infection is influenza A(A) type H1N1 or H3N2 virus infection, and wherein the human influenza A(A) type antigen is a human inactivated influenza A(A) type H1N1 antigen.

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