INFLUENZA VACCINE COMPOSITION BASED ON NOVEL NUCLEIC ACID

Abstract

Provided is an influenza vaccine composition based on a novel ribonucleic acid having a dual function of serving as an immunity-boosting agent and capturing antigens.

Claims

1. A vaccine composition for preventing or treating influenza virus infection, the vaccine composition comprising a hetero-structured ribonucleic acid (hsRNA) comprising a double-stranded ribonucleic acid (dsRNA) and single-stranded ribonucleic acids (ssRNAs); and a human influenza virus antigen, wherein the dsRNA is formed by complementary binding between a first single-stranded RNA and a second single-stranded RNA which are complementary to each other, and the ssRNAs are linked at both the 3′-ends of the dsRNA, respectively, wherein the hsRNA has 140 nt to 1682 nt in length, wherein the dsRNA region has 106 nt to 1648 nt in length, and wherein the ssRNA region has 1 to 10 nt in length.

2. The vaccine composition for preventing or treating influenza virus infection of claim 1, wherein the ssRNA region has a UAUAG sequence at the 3′-end.

3. The vaccine composition for preventing or treating influenza virus infection of claim 1, wherein the hsRNA is obtained by complementary binding between a nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2, or has a UAUAG sequence at both the 3′-ends of dsRNA which is obtained by complementary binding between a nucleotide sequence of SEQ ID NO: 3 and a complementary sequence thereof.

4. The vaccine composition for preventing or treating influenza virus infection of claim 1, wherein the influenza virus is influenza virus A, B, or C type.

5. The vaccine composition for preventing or treating influenza virus infection of claim 1, wherein the influenza antigen is an inactivated or live attenuated influenza whole virus, a subvirion, or a subunit.

6. The vaccine composition for preventing or treating influenza virus infection of claim 1, wherein the vaccine composition has a formulation for intranasal or mucosal administration.

7. The vaccine composition for preventing or treating influenza virus infection of claim 1, wherein the vaccine composition provides a protective immunity against the human influenza virus, different subtypes of the homotypic viruses of the human influenza virus, or subtypes of the heterosubtypic viruses of the human influenza virus.

8. The vaccine composition for preventing or treating influenza virus infection of claim 1, wherein the human influenza virus antigen comprises one subtype virus antigen of type A influenza virus, one subtype virus antigen of type B influenza virus, or a combination thereof.

9. The vaccine composition for preventing or treating influenza virus infection of claim 1, wherein the human influenza virus antigen is a subtype antigen of type A influenza virus H1N1 without comprising other subtype antigens of type A influenza virus, or an H3N2 subtype antigen of type A influenza virus without comprising other subtype antigens of type A influenza virus.

10. The vaccine composition for preventing or treating influenza virus infection of claim 1, wherein the human influenza virus antigen is a BV subtype antigen of type B influenza virus without comprising other subtype antigens of type B influenza virus, or a BY subtype antigen of type B influenza virus without comprising other subtype antigens of type B influenza virus.

11. A method of immunizing a subject against an influenza virus, the method comprising administering the vaccine composition of claim 1 to the subject.

12. The method of claim 11, wherein the ssRNA region has a UAUAG sequence at the 3′-end.

13. The method of claim 11, wherein hsRNA is obtained by complementary binding between a nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2, or has a UAUAG sequence at both the 3′-ends of dsRNA which is obtained by complementary binding between a nucleotide sequence of SEQ ID NO: 3 and a complementary sequence thereof.

14. The method of claim 11, wherein the influenza virus is influenza virus type A, B, or C.

15. The method of claim 11, wherein the influenza virus antigen is an inactivated or live attenuated influenza whole virus, a subvirion, or a subunit.

16. The method of claim 11, wherein the administering is nasal or mucosal administering.

17. The method of claim 11, wherein the method provides a protective immunity against the human influenza virus, different subtypes of the homotypic viruses of the human influenza virus, or subtypes of the heterosubtypic viruses of the human influenza virus.

18. The method of claim 11, wherein the human influenza virus antigen comprises one subtype virus antigen of type A influenza virus, one subtype virus antigen of type B influenza virus, or a combination thereof.

19. The method of claim 11, wherein the human influenza virus antigen is a subtype antigen of type A influenza virus H1N1 without comprising other subtype antigens of type A influenza virus, or an H3N2 subtype antigen of type A influenza virus without comprising other subtype antigens of type A influenza virus.

20. The method of claim 11, wherein the human influenza virus antigen is a BV subtype antigen of type B influenza virus without comprising other subtype antigens of type B influenza virus, or a BY subtype antigen of type B influenza virus without comprising other subtype antigens of type B influenza virus.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0081] FIGS. 1A to 1C show a 100% protective effect of an NVT-adjuvanted inactivated A/H1N1 influenza virus (IIV) (iPR8) vaccine according to Example 1, which was confirmed upon challenge with a lethal dose of a homotypic live influenza virus (LIV) (PR8), after single intranasal administration (i.n.) of the vaccine,

[0082] wherein FIG. 1A shows the experimental schedule,

[0083] FIG. 1B shows changes in the body weight of mice after viral challenge,

[0084] FIG. 1C shows survival rates (%) up to 11 days after virus challenge; and

[0085] FIGS. 2A and 2B show a protective effect of an NVT-adjuvanted inactivated A/H1N1 influenza virus (IIV) (iPR8) vaccine according to Example 2, which was confirmed upon challenge with a lethal dose of a heterosubtypic (A/H3N2) live influenza virus, after single intranasal administration (i.n.) of the vaccine,

[0086] wherein FIG. 2A shows the experimental schedule, and

[0087] FIG. 2B shows survival rates (%) up to 14 days after virus challenge;

[0088] FIG. 3 shows results of measuring IgA levels in the bronchoalveolar lavage fluid (BALF), the nasal lavage fluid, and the serum, after administration of a vaccine including hsRNA and iPR8 antigen;

[0089] FIG. 4A shows results of measuring cell and lymphocyte levels in BALF, after administration of the vaccine including hsRNA and iPR8 antigen;

[0090] FIG. 4B shows CD4+ T cell and IFNγ+T cell levels in BALF, after administration of the vaccine including hsRNA and iPR8 antigen, and IFNγ+T cell levels after 72 hours;

[0091] FIG. 5 shows results of measuring CD4+ IFNv+ T cell levels in the lung, IgG levels in the serum, and IgA levels in the nasal lavage fluid, after administration of a vaccine including hsRNA and inactivated H3N2 or B virus;

[0092] FIG. 6 shows results of measuring the body weight according to days after intramuscular and intranasal administration of a vaccine including H3N2 antigen, or a vaccine including hsRNA and H3N2 antigen, respectively and survival rates according to challenges of a different subtype of H1N1 virus;

[0093] FIG. 7 shows results of measuring CD4+ IFNv+ T cell levels in the lung and spleen, after intramuscular injection and nasal spraying of a vaccine including inactivated H3N2 antigen, or a vaccine including inactivated H3N2 antigen or hsRNA, followed by challenge administration of H3N2 or H1N1 virus;

[0094] FIG. 8 shows results of measuring the body weight according to days after intramuscular administration and nasal spraying of a vaccine including BV antigen, a vaccine including BY antigen, or a vaccine including hsRNA and BY antigen, respectively and survival rates according to challenges of a different subtype of BV virus;

[0095] FIG. 9 shows results of measuring CD4+ IFNv+ T cell levels in the lung, after intramuscular injection and nasal spraying of a vaccine including inactivated BV antigen, a vaccine including inactivated BY antigen, a vaccine including inactivated BV and hsRNA, or a vaccine including inactivated BY antigen and hsRNA, respectively, followed by challenge administration of a different subtype of BV or BY virus;

[0096] FIG. 10A shows results of measuring CD4+ T cell levels in the lung and spleen upon challenge administration of H1N1 virus after removing CD4 T cells from mice which had been subjected to nasal spraying of the vaccine including hsRNA and H3N2 antigen;

[0097] FIG. 10B shows percent survivals according to days upon challenge administration of H1N1 virus after removing CD4 T cells from mice which had been subjected to nasal spraying of the vaccine including hsRNA and H3N2 antigen;

[0098] FIG. 11A shows results of measuring antibody titers by measuring HAI titers after immunization with each vaccine composition;

[0099] FIG. 11B shows results of measuring CD4+ T cell levels in the lung and spleen upon challenge administration of H1N1 virus, after intramuscular administration of a vaccine including monovalent H3N2 in PBS, after nasal spraying administration, to a subject, of a vaccine prepared by mixing hsRNA and monovalent H3N2 in PBS, after intramuscular injection, to a subject, of a vaccine prepared by mixing H3N2 antigen, PolyIC, and AddaVax in PBS;

[0100] FIG. 11C shows percent survivals according to days upon challenge administration of H1N1 virus to mice which had been subjected to nasal spraying administration of the vaccine including hsRNA and H3N2 antigen;

[0101] FIG. 12A shows results of measuring HAI titers after intramuscular and intranasal spraying administration of a Teratect prefilled syringe (Influenza split vaccine) vaccine, or a vaccine including hsRNA and the Teratect prefilled syringe (Influenza split vaccine) vaccine, respectively; and

[0102] FIG. 12B shows results of measuring CD4+ IFNv+ T cell levels in the lung after intramuscular and nasal spraying administration of the Teratect prefilled syringe (Influenza split vaccine) vaccine, or the vaccine including hsRNA and the Teratect prefilled syringe (Influenza split vaccine) vaccine, respectively.

BEST MODE

[0103] Hereinafter, the present disclosure will be described in detail with reference to exemplary embodiments for better understanding. However, the following exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to the following exemplary embodiments. It is apparent to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present disclosure, and it is also obvious that these changes and modifications belong to the accompanying claims.

Example 1

[0104] Preparation of hsRNA and Examination of protective immunity effect by intranasal administration of hsRNA-adjuvanted inactivated influenza (iPR8) vaccine

[0105] 1. Preparation of hsRNAs Having Two 3′-Overhangs

[0106] The hsRNAs having dsRNA region and two 3′-overhangs at each strand of the dsRNA region are prepared as follows. The dsRNA region has the nucleotide sequence of SEQ ID NO: 3, and the complementary sequence thereof and each of 3′-overhang has 5 nt sequence UAUAG. The hsRNA is also referred as NexVant or NVT.

[0107] A template DNA molecule comprising a template DNA region and T7 promoter sequences linked to both 5′-ends of the template DNA region was prepared. The template DNA region has the nucleotide sequence of SEQ ID NO: 4, which corresponds to the nucleotide sequence of Sacbrood virus VP1 gene and T7 promoter has the nucleotide sequence of SEQ ID NO: 5. The template DNA molecule was ligated to PUC19 vector (Thermo Fisher, Cat SD0061) digested with SmaI, to form a recombinant vector comprising the template DNA molecule. The recombinant vector was introduced into E. coli DH5a by a transformation. The transformed cells were grown in LB/Amp medium. The cells were isolated from the supernatant of the culture. The cells were lysed by using an alkaline solution, and the recombinant vector was isolated by using Qiagen midi prep kit.

[0108] The template DNA molecule was amplified by a PCR using the recombinant vector as a template. The obtained template DNA molecule was used in in vitro transcription (IVT) using T7 polymerase as a template. In the PCR, an oligonucleotide set, wherein each oligonucleotide has a complementary sequence to a target to be amplified and the T7 promoter sequence at its 5′-end was used as primer set. PCR was conducted at 95° C., and 5 minutes, and 35 thermal cycles of 95° C., and 30 seconds and 60° C., 30 seconds and 72° C., 1 minute. The reaction mixture was incubated at 72° C. and 5 minutes.

[0109] In vitro transcription was conducted using the MEGAscript™ T7 Transcription Kit (Thermo Fisher, cat AMB13345) according to the manufacturer's protocols. The amplified template DNA molecule was used as a template. 1 ml reaction mixture comprising 10 ug linearized Template DNA, 75 mM NTP, 90 mM Tris base, 90 mM Boric acid, 2 mM EDTA, and T7 RNA polymerase 50 ul was incubated at 37° C. for 4 hours. The obtained reaction mixture was incubated at 80° C. for 20 minutes and cooled to room temperature for 30 minutes. The first single-strand RNA and the second single-strand RNA are simultaneously synthesized from each of the template DNA strands and hybridizes to form double-stranded RNAs having two 3′-overhangs.

[0110] The reaction mixture was centrifuged at 20° C., 4000 rpm for 3 minutes to remove white precipitation and obtain a supernatant solution. DNase I was added to the supernatant solution, and the resultant solution was incubated at 37° C. for 13 hours. The hsRNA was isolated from the reaction solution. As a result, the hsRNAs shown having 533 nt in length, i.e., 424 nt of dsRNA region, and 51 nt of first 3′-overhang, and 58 nt of second 3′-overhang.

[0111] Further, RNase T1 was added to the DNase I-treated reaction mixture, and the resultant mixture was incubated at 37° C. for 2 hours. Then, the reaction mixture was incubated at 80° C. for 10 minutes and cooled to room temperature for 30 minutes. Then, the hsRNA having 3′-overhangs with reduced length was isolated by a nucleic acid precipitation method using isopropanol.

[0112] The isolated hsRNA has 3′-overhangs with 5 nt in length, which is shorter than that of the DNase I-treated hsRNA. The hsRNA have UAUAG sequence at its 3′-end of the 3′-overhang and 424 nt of dsRNA region of SEQ ID NO:3. The hsRNA may have phosphate at its 5′-end and a hydroxyl group at its 3′-end. This hsRNA hereinafter referred to as “NA” hsRNA. The phosphate may be triphosphate, diphosphate, or monophosphate. The hsRNA may not have 5′-cap.

[0113] 2. Examination of protective immunity effect by intranasal administration of hsRNA NVT-adjuvanted inactivated influenza (iPR8) vaccine

[0114] In this section, a vaccine including the hsRNA NVT)-adjuvanted inactivated A/H1N1 influenza virus (IIV) iPR8 prepared in section 1 was intranasally administered, and a protective immunity effect of the vaccine on lethality of homotypic virus infection was examined.

[0115] In this example, the hsRNA and inactivated whole iPR8 were solubilized in PBS (pH 7.2), and the hsRNA in PBS was injected into the mouse via nasal injection, i.e., spraying in the nasal cavity. PolyIC (InvivoGen, Poly(I:C) (HMW) VacciGrade™ cat #, vac-pic) was used as a positive control.

[0116] Each substance was administered to mice according to dosing schedules shown in Table 1 and FIG. 1A. In detail, female Balb/c mice (7 week-old, 5 each) were anesthetized and intranasally administered once with PBS (50 μl), 0.5 μg of iPR8±5 μg of NVT, or 0.5 μg of iPR8+5 μg of Poly(I:C). Three weeks later, mice were challenged with 100 times (100MLD.sub.50) the lethal dose of PR8 (A/H1N1), which is a mouse-adapted live influenza virus. Changes in the body weight (%) and survival rates (%) of the mice were monitored until 11 days after challenge. The results are shown in FIGS. 1B and 1C.

TABLE-US-00001 TABLE 1 PR8 Survival rate Dosing substance challenge (on day 11) Normal control PBS (50 μl) — 100% group Experimental iPR8 (0.5 μg) + Challenge 100% group NA (5 μg) Control group 1 PBS (50 μl) Challenge 0% (all dead on day 7) Control group 2 iPR8 (0.5 μg) Challenge  40% Control group 3 iPR8 (0.5 μg) + Challenge  80% Poly(I:C) (5 μg)

[0117] As shown in FIGS. 1B and 1C, when only PBS was pre-administered and then PR8 was challenged (Control group 1), 100% died until day 7 after challenge, whereas an experimental group according to the present disclosure challenged with PR8 after pre-administration with iPR8+NVT showed 100% survival, and its weight gain was also recovered to nearly 100% on day 11. When only iPR8 was pre-administered and then PR8 was challenged (Control group 2), only 40% survived, and the weight gain of the surviving mice was also reduced. When iPR8+Poly(I:C) was pre-administered and then PR8 was challenged (Control group 3), only 80% survived, and the weight gain of the surviving mice was also slightly reduced.

[0118] The above results confirmed that a single intranasal administration of the hsRNA, e.g., hsRNA NVT-adjuvanted inactivated influenza (iPR8) vaccine protected all, i.e., 100% mice even when a lethal dose of the homotypic live influenza virus was challenged, indicating that the hsRNA, e.g., hsRNA NVT-adjuvanted vaccine of the present disclosure provides complete protective immunity against influenza virus infection. Furthermore, it was confirmed that NVT provides better protective immunity than Poly(I:C).

Example 2

[0119] Cross-protective immunity effect on heterosubtypic virus by intranasal administration of NVT-adjuvanted inactivated influenza (iPR8) vaccine of the present disclosure

[0120] In the present exemplary embodiment, it was tested whether the vaccine including the hsRNA, e.g., hsRNA NVT-adjuvanted inactivated A/H1N1 influenza virus (IIV) iPR8 of the present disclosure is able to provide a cross-protective immunity against heterosubtypic strains.

[0121] Each substance was administered to mice according to dosing schedules shown in Table 2 and FIG. 2A. In detail, female Balb/c mice (7 week-old, 5 each) were anesthetized and intranasally administered once with PBS (50 μl) or 20 μl of 0.1 μg to 5 μg of NVT+0.5 μg of iPR8. Three weeks later, mice were challenged with a dose of 50MLD.sub.50 of A/Hong Kong/8/68 (A/H3N2) which is a mouse-adapted live influenza virus. Survival rates (%) were monitored until 14 days after challenge. The results are shown in Table 2 and FIG. 2B.

TABLE-US-00002 TABLE 2 A/H3N2 Survival rate Dosing material challenge (on day 14) Normal control PBS (50 μl) — 100% group Control group PBS (50 μl) Challenge  30% Experimental iPR8 (0.5 μg) + Challenge  60% group 1 NA (0.5 μg) Experimental iPR8 (0.5 μg) + Challenge  60% group 2 NA (2.5 μg) Experimental iPR8 (0.5 μg) + Challenge 100% group 3 NA (5 μg)

[0122] As shown in Table 2 and FIG. 2B, iPR8+NVT combination also exhibited a cross-protective immunity effect against the heterosubtypic influenza virus A/H3N2. In particular, the vaccine including 5 μg of NA exhibited a 100% protective effect.

Example 3: Efficacy of Vaccine Including hsRNA and Influenza Virus Antigen in Mouse

[0123] 1. Vaccine Formulation and Administration

[0124] In this section, the hsRNA and inactivated A/H1N1 influenza virus (IIV) iPR8 antigen prepared in Example 2 were solubilized in PBS (pH 7.2) to prepare a vaccine composition including hsRNA. In this regard, the iPR8 antigen and hsRNA were mixed at a ratio of 1:1 to 1:10, based on the weight. The total weight of the iPR8 antigen was 10 μg to 60 μg per person. The prepared vaccine composition including hsRNA was stored at 4° C. to 8° C. Immunization was conducted by spraying half of a total of 100 μl to 500 μl of the vaccine composition including hsRNA into each nostril.

[0125] 2. Effect on IgA Production

[0126] As in the section 1 above, the vaccine was administered to mice by nasal spraying, and the booster was administered in the same manner once more for two weeks. After two weeks, bronchoalveolar lavage fluid (BALF), nasal lavage fluid, and serum were collected, and IgA antibody levels were measured using ELISA method.

[0127] FIG. 3 shows results of measuring IgA levels in the BALF, nasal lavage fluid, and serum, after administration of the vaccine including hsRNA and iPR8 antigen. In FIG. 3, A and B represent IgA levels in the BALF and nasal lavage fluid, respectively. In A and D, the IgA levels were determined by measuring optical density (OD) at a wavelength of 450 nm to 590 nm in the ELISA method. A control group was subjected to intramuscular (i.m.) or intranasal (i.n.) administration of PBS.

[0128] As shown in FIG. 3, high levels of IgA were measured in the BALF and nasal lavage fluid, as compared with the control group, indicating that IgA levels are increased by intranasal administration of the vaccine composition, i.e., indicating that IgA levels are increased by mucosal immunization of the vaccine composition.

[0129] 3. Effect on T Cell Response

[0130] As in the section 1 above, the vaccine was administered to mice by nasal spraying, and after two weeks, the booster was administered in the same manner. After two weeks, BALF was collected, and CD4+ T cell levels in BALF were measured using an intracellular cytokine staining method.

[0131] For intracellular cytokine staining, cells were restimulated with indicated antigens at 37° C./5% CO2 for 20 hours. Brefeldin A (Golgi Plug, BD Biosciences) was added and cells cultured for a further 4 hours. Cells were stained with Fixable Viability Dye eFluor 780 (eBioscience), then stained for surface markers for 30 min at 4° C. Following surface staining, cells were fixed and permeabilized by incubating in BD CytoFix-CytoPerm (BD Fixation/Permealization Kit™) for 20 min in the dark at 4° C. The cells were then washed BD Perm/wash buffer (BD Fixation/Permeablization Kit™) and re-suspended in Perm/wash buffer with fluorochrome conjugated monoclonal antibody and incubated for 30 min in the dark at 4° C. Cells were then washed in Perm/wash buffer prior to flow cytometry analyses. Samples were acquired and analyzed using an ACEA Novocyte.

[0132] FIG. 4A shows results of measuring cell and lymphocyte levels in BALF, after administration of the vaccine including hsRNA and iPR8 antigen. In FIG. 4, A and B represent cell and lymphocyte levels in BALF, respectively. The lymphocyte represents the total lymphocytes including CD4+ T cells.

[0133] FIG. 4B shows CD4+ T cell and IFNγ+ T cell levels in BALF, after administration of the vaccine including hsRNA and iPR8 antigen, and IFNγ+ T cell levels after 72 hours. In FIG. 4B, A represent CD4+ T cells in BALF, and B and C represent IFNγ+CD4+ T cells in BALF and IFNγ+CD4+ T cells in the lung, respectively.

[0134] As shown in FIGS. 4A and 4B, when the vaccine including hsRNA and iPR8 antigen was administered, lymphocytes, e.g., antigen-specific CD4+T and IFNγ+CD4+ T cells increased in the BALF, as compared with the control group. Therefore, when the vaccine including hsRNA and iPR8 antigen was intranasally administered, immune responses were induced in the BALF and lung.

[0135] Further, a vaccine including inactivated H3N2 or inactivated B influenza virus, which was prepared by replacing the inactivated A/H1N1 influenza virus iPR8 in the vaccine descried in the section 1 by inactivated split H3N2 (Inactivated, split A/Cambodia/E0826360/2020, IVR-224(H3N2)) (Teratect prefilled syringe (Influenza split vaccine), Ilyang Pharmaceutical Co. Ltd.)) or inactivated split B (Inactivated, split B/Maryland/15/2016 and B/Phuket/3073/2013) (Teratect prefilled syringe (Influenza split vaccine), Ilyang Pharmaceutical Co. Ltd.) influenza virus was immunized in the same manner, and CD4+ IFNv+ T cell levels in the lung, IgG levels in the serum, and IgA levels in the nasal lavage fluid were measured. The number of CD4+ IFNv+ T cells in the lung was measured by the above-described intracellular cytokine staining method. The IgG levels in the serum, and IgA levels in the nasal lavage fluid were measured by the ELISA method. Further, immunization by intramuscular injection was also included, in addition to the nasal spraying.

[0136] FIG. 5 shows results of measuring CD4+ IFNv+ T cell levels in the lung, IgG levels in the serum, and IgA levels in the nasal lavage fluid, after administration of the vaccine including hsRNA and inactivated H3N2 or B virus.

[0137] 4. Enhanced Protective Immunity

[0138] It was examined whether intranasal administration showed a superior protective immunity, compared to intramuscular injection, when viral infection actually occurred.

[0139] Teratect prefilled syringe (Influenza split vaccine) (Ilyang Pharmaceutical Co. Ltd.) which is a commercially available quadrivalent vaccine was intramuscularly injected into a subject, or a vaccine was prepared by mixing with hsRNA as in the above section 1 and sprayed into the subject's nasal cavity. The subject was a mouse. The Teratect prefilled syringe (Influenza split vaccine) is a quadrivalent antigen vaccine including A Influenza H1N1, i.e., A/Victoria/2570/2019, IVR-215(H1N1) and H3N2, i.e., A/Cambodia/E0826360/2020, IVR-224(H3N2), B Influenza BV, i.e., B/Maryland/15/2016 and BY, i.e., B/Phuket/3073/2013 antigen.

[0140] FIG. 5 shows results of measuring body weight according to days, after intramuscular and nasal spraying administration of the vaccine including Teratect prefilled syringe (Influenza split vaccine), or hsRNA and Teratect prefilled syringe (Influenza split vaccine). 2 weeks after administration of a predetermined amount of the antigen, the booster was administered once with the equal amount thereof. 2 weeks after booster administration, Influenza A/Korea/2785/2009 (2009 pandemic strain) balb/c adapted (provided by KCDC, National Culture Collection for Pathogens NCCP43021, Korea Disease Control and Prevention Agency) virus was challenged in an amount of 5LD50 via nasal spraying. In FIG. 5, “Flu” represents Teratect prefilled syringe (Influenza split vaccine).

[0141] In FIG. 5, A and B represent administration of 2 ug and 4 ug of the antigen per subject, respectively. As shown in FIG. 5, when the vaccine including hsRNA and Teratect prefilled syringe (Influenza split vaccine) was administered via nasal spraying, the body weight was maintained remarkably high, as compared with intramuscular administration of Teratect prefilled syringe (Influenza split vaccine) alone. The vaccine including hsRNA and Teratect prefilled syringe (Influenza split vaccine) showed improved protective effects when a large amount of virus, rather than a small amount thereof, was challenged, as compared with administration of Teratect prefilled syringe (Influenza split vaccine) alone. Through these results, a correlation between improved T cell response and the vaccine titer may be predicted.

[0142] 5. Cross-Protective Immunity

[0143] (1) Cross-Protective Immunity Between Influenza a Viruses

[0144] The commercially available Teratect prefilled syringe (Influenza split vaccine) is a quadrivalent antigen vaccine including A Influenza H1N1 and H3N2, B Influenza BV and BY antigens. B Influenza BV and BY are Victoria lineage and Yamagata lineage, respectively. The reason for using the quadrivalent antigen vaccine is that even though four antigens are individually administered, individual antigens do not provide a cross-protective immunity against other viruses among the four types of viruses.

[0145] In this experiment, H3N2 antigen in the commercially available Terateect prefilled syringe (Influenza split vaccine) was provided by the manufacturer, and intramuscularly injected into subjects, or a vaccine was prepared by mixing with hsRNA as in the section 1, and sprayed into the nasal cavity of the subjects. The subjects were Balb/c mice. 2 weeks after administration with a predetermined amount of the antigen, the booster was administered in the same manner. 2 weeks after booster administration, the different subtype H1N1 virus was challenged in an amount of 3LD50 via nasal spraying.

[0146] FIG. 6 shows results of measuring body weight according to days, after intramuscular and nasal spraying administration of the vaccine including H3N2 antigen, or the vaccine including hsRNA and H3N2 antigen, respectively and survival rates according to challenges of the different subtype H1N1 virus.

[0147] As shown in FIG. 6A, nasal spraying administration of the vaccine including hsRNA and H3N2 antigen showed high body weight, as compared with intramuscular administration of the H3N2 antigen.

[0148] As shown in FIG. 6B, when intramuscular injection of H3N2 antigen and nasal spraying administration of the vaccine including hsRNA and H3N2 antigen were performed on day 6 of the challenge, the survival rates according to challenge of the different subtype H1N1 virus were 0% and 100%, respectively, indicating that when the influenza A virus H3N2 antigen in combination with hsRNA is administered, subjects have a protective immunity against influenza A virus H1N1, and also indicating that when influenza A virus H1N1 antigen in combination with hsRNA is administered, subjects have a protective immunity against influenza A virus H3N2. In other words, when the influenza A virus antigen in combination with hsRNA is administered, subjects have a protective immunity against different subtypes of influenza A virus.

[0149] FIG. 7 shows results of measuring CD4+ IFNv+ T cell levels in the lung and spleen, after intramuscular injection and nasal spraying of a vaccine including inactivated H3N2 antigen, or a vaccine including inactivated H3N2 antigen or hsRNA, followed by challenge administration of H3N2 or H1N1 virus. The number of the CD4+ IFNv+ T cells in the lung and spleen was measured by the intracellular cytokine staining method.

[0150] As shown in FIG. 7A, when the vaccine including inactivated H3N2 antigen and hsRNA was administered via nasal spraying, high levels of CD4+ IFNv+ T cells were measured both in the H3N2 and H1N1 virus challenge. In contrast, when the vaccine including inactivated H3N2 antigen was intramuscularly administered, low levels of CD4+ IFNv+ T cells were measured both in the H3N2 and H1N1 virus challenge.

[0151] Further, as shown in FIG. 7B, when the vaccine including inactivated H3N2 antigen and hsRNA was administered via nasal spraying, high levels of CD4+ IFNv+ T cells were measured both in the H3N2 and H1N1 virus challenge. In contrast, when the vaccine including inactivated H3N2 antigen was intramuscularly administered, low levels of CD4+ IFNv+ T cells were measured both in the H3N2 and H1N1 virus challenge.

[0152] These results indicate that the vaccine including influenza A virus antigen in combination with hsRNA increased production of CD4+ IFNv+ T cells, thereby providing a protective immunity against different types or subtypes of virus.

[0153] In addition, these results indicate that when the influenza A virus H3N2 antigen in combination with hsRNA is intranasally administered, subjects have a protective immunity against influenza A virus H1N1. These results also indicate that when the influenza A virus H1N1 antigen in combination with hsRNA is intranasally administered, subjects have a protective immunity against influenza A virus H3N2. In other words, when the influenza A virus antigen in combination with hsRNA is intranasally administered, subjects have a protective immunity against different subtypes of influenza A virus.

[0154] (2) Cross-Protective Immunity Between Influenza B Viruses

[0155] The commercially available Teratect prefilled syringe (Influenza split vaccine) is a quadrivalent antigen vaccine including A Influenza H1N1 and H3N2, B Influenza BV and BY antigens. B Influenza BV and BY are Victoria lineage and Yamagata lineage, respectively. The reason for using the quadrivalent antigen vaccine is that even though four antigens are individually administered, individual antigens do not provide a cross-protective immunity against other viruses among the four types of viruses.

[0156] In this experiment, BV antigen in the commercially available Terateect prefilled syringe (Influenza split vaccine) was provided by the manufacturer, and intramuscularly injected into subjects, or BY antigen in the commercially available Terateect prefilled syringe (Influenza split vaccine) was provided by the manufacturer, and intramuscularly injected into subjects, or a vaccine was prepared by mixing BY antigen with hsRNA as in the section 1, and sprayed into the nasal cavity of the subjects. The BV virus used in the vaccine was a B/Maryland/15/2016 strain. The BY virus used in the vaccine was a B/Phuket/3073/2013 strain.

[0157] The subjects were Balb/c mice. 2 weeks after administration of a predetermined amount of 2 ug of the antigen, the booster was administered once with the equal amount thereof. 2 weeks after booster administration, the different subtype BV virus B/Shangdong/7/97 was challenged in an amount of 3LD50 via nasal spraying.

[0158] FIG. 8 shows results of measuring body weight according to days, after intramuscular and nasal spraying administration of the vaccine including BV antigen, the vaccine including BY antigen, or the vaccine including hsRNA and BY antigen, respectively, and survival rates according to challenges of the different subtype of BV virus.

[0159] As shown in FIG. 8A, when the vaccine including hsRNA and BY antigen was administered via nasal spraying, the survival rates according to challenge of the different subtype BV virus were high, as compared with intramuscular administration of BY antigen or BV antigen, indicating that when the influenza B virus BY antigen in combination with hsRNA is administered, subjects have a protective immunity against B virus BV, and also indicating that when influenza B virus BV antigen in combination with hsRNA is administered, subjects have a protective immunity against influenza B virus BY. In other words, when the influenza B virus antigen in combination with hsRNA is administered, subjects have a protective immunity against different subtypes of influenza B virus.

[0160] When the BV antigen was administered via intramuscular injection, the survival rates according to challenge of BV virus were about 60%, indicating that the BV strains used in the vaccine and challenge were B/Phuket/3073/2013 and B/Shangdong/7/97, respectively, which are different from each other, and the cross-protective immunity therebetween is not achieved when intramuscular injection is performed without hsRNA.

[0161] As shown in FIG. 8B, nasal spraying administration of the vaccine including hsRNA and BY antigen showed high body weight, as compared with intramuscular administration of the BY antigen or BV antigen.

[0162] FIG. 9 shows results of measuring CD4+ IFNv+ T cell levels in the lung, after intramuscular injection and nasal spraying of the vaccine including inactivated BV antigen, the vaccine including inactivated BY antigen, the vaccine including inactivated BV and hsRNA, or the vaccine including inactivated BY antigen and hsRNA, followed by challenge administration of BV or BY virus. The number of CD4+ IFNv+ T cells in the lung was measured by the intracellular cytokine staining.

[0163] As shown in FIG. 9A, high levels of CD4+ IFNv+ T cells were observed both in nasal spraying administrations of the vaccine including inactivated BV and hsRNA and the vaccine including inactivated BY and hsRNA, when challenged with BV virus. In contrast, low levels of CD4+ IFNv+ T cells were observed both in intramuscular administrations of the vaccine including inactivated BV and the vaccine including inactivated BY, when challenged with BV virus.

[0164] As shown in FIG. 9B, high levels of CD4+ IFNv+ T cells were observed both in nasal spraying administrations of the vaccine including inactivated BV and hsRNA and the vaccine including inactivated BY and hsRNA, when challenged with BY virus. In contrast, low levels of CD4+ IFNv+ T cells were observed both in intramuscular administrations of the vaccine including inactivated BV and the vaccine including inactivated BY, when challenged with BY virus.

[0165] These results indicate that intramuscular injection of the vaccine including inactivated BV or BY does not induce T cell responses, i.e., an increase in the number of CD4+ IFNv+ T cells. In other words, intramuscular injection of the vaccine including inactivated BV or BY does not induce T cell responses, thereby providing no cross-protective immunity against different subtypes of the same B virus.

[0166] In contrast, nasal spraying administration of the vaccine including inactivated BV and hsRNA or the vaccine including inactivated BY and hsRNA induces T cell responses, i.e., an increase in the number of CD4+ IFNv+ T cells. In other words, nasal spraying administration of the vaccine including inactivated BV and hsRNA or the vaccine including inactivated BY and hsRNA induces T cell responses. Since T cell responses are induced, the vaccine including inactivated BV and hsRNA induces T cell responses, i.e., an increase in the number of CD4+ IFNv+ T cells, when challenged with BV virus which is the same subtype, as well as when challenged with BY virus which is the different subtype. In addition, since T cell responses are induced, the vaccine including inactivated BY and hsRNA induces T cell responses, i.e., an increase in the number of CD4+ IFNv+ T cells, when challenged with BY virus which is the same subtype, as well as when challenged with BV virus which is the different subtype.

[0167] In other words, nasal spraying administration of the vaccine including inactivated BV and hsRNA or the vaccine including inactivated BY and hsRNA may provide a cross-protective immunity against different subtypes of the same B virus.

[0168] Therefore, nasal spraying administration of a vaccine including an inactivated specific type of A, B, or C virus antigen and hsRNA may provide a cross-protective immunity against different subtypes of the same A, B, or C virus, indicating that nasal spraying administration of a vaccine including an inactivated specific type of influenza virus antigen and hsRNA may provide a cross-protective immunity against different subtypes of influenza virus, thereby providing a protective immunity against all types of influenza viruses regardless of the type of influenza viruses, i.e., thereby being used as a general purpose vaccine.

[0169] 6. Mechanism of Action of Cross-Protective Immunity

[0170] When the vaccine including the inactivated influenza virus antigen and hsRNA is administered to a subject by nasal spraying, as described above, whether or not it provides a protective immunity against other types of influenza virus different from the administered inactivated influenza virus, i.e., the mechanism of action that provides a cross-immunity was examined by the following experiment.

[0171] H3N2 antigen in the commercially available Terateect prefilled syringe (Influenza split vaccine) was provided by the manufacturer, and 2 ug thereof was intramuscularly injected into subjects, or a vaccine was prepared by mixing the H3N2 monovalent vaccine with hsRNA as in the section 1, and sprayed into the nasal cavity of the subjects. The subjects were Balb/c mice. 2 weeks after administration of a predetermined amount of the antigen, the booster was administered once with the equal amount thereof. 2 weeks after booster administration, 200 ug of CD4 T cell depletion antibody, i.e., CD4 depletion antibody (GK1.5 clone, (GK1.5 clone, BE0003-1) which is a product from BioXcell was intraperitoneally administered on day 28 and day 32 after the first immunization to remove CD4 T cells in the subjects.

[0172] Next, removal of the CD4 T cells from the subjects was examined by a flow cytometry method. After confirming that CD4 T cells were removed, 3LD50 of another subtype of H1N1 virus was challenged through intranasal administration. As a control group, mice, to which a H3N2 monovalent vaccine was intramuscularly administered or a vaccine including hsRNA and H3N2 antigen was administered via nasal spraying, were treated with an antibody of an isotype control and challenged with H1N1 virus. The results are shown in FIGS. 10A and 10B.

[0173] FIG. 10A shows results of measuring CD4+ T cell levels in the lung and spleen upon challenge administration of H1N1 virus after removing CD4 T cells from mice which had been subjected to nasal spraying of the vaccine including hsRNA and H3N2 antigen. As shown in FIG. 10A, upon challenge administration of H1N1 virus after removing CD4 T cells from mice which had been subjected to immunization with the vaccine including hsRNA and H3N2 antigen, CD4+ T cell levels in the lung and spleen were remarkably reduced, as compared with the control group. In contrast, the control group, i.e., upon challenge administration of H1N1 virus without removing CD4 T cells from mice which had been subjected to intramuscular injection of the H3N2 monovalent vaccine or nasal spraying of the vaccine including hsRNA and H3N2 antigen, high CD4+ T cell levels in the lung and spleen were observed.

[0174] FIG. 10B shows percent survivals according to days upon challenge administration of H1N1 virus after removing CD4 T cells from mice which had been subjected to intranasal spraying of the vaccine including hsRNA and H3N2 antigen. In FIG. 10B, the blue line represents challenge administration of H1N1 virus without removing CD4 T cells from those immunized with the vaccine including hsRNA and H3N2 antigen, and the green line represents challenge administration of H1N1 virus after removing CD4 T cells from those immunized with the vaccine including hsRNA and H3N2 antigen. In addition, the red line represents an experimental group which was intramuscularly injected with the vaccine including H32N antigen, and the pink line represents a control group administered with PBS, i.e., immunized with no vaccine.

[0175] Further, to investigate the importance of T cell responses specific to the lower respiratory organ, e.g., the lung, only when the vaccine including hsRNA and influenza virus antigen is administered by nasal spraying, the following experiment was performed.

[0176] H3N2 monovalent antigen in the commercially available Terateect prefilled syringe (Influenza split vaccine) was provided by the manufacturer, and 2 ug thereof was intramuscularly injected into subjects, or a vaccine was prepared by mixing the H3N2 monovalent vaccine with 10 ug of hsRNA as in the section 1, and sprayed into the nasal cavity of the subjects. In addition, a vaccine prepared by mixing 10 ug of hsRNA, 10 ug of PolyIC, and 25% final volume of AddaVax in PBS as in the section 1 was intramuscularly injected into subjects, which were compared with those administered with a vaccine including PolyIC (Invivogen) and/or AddaVax™ (InvivoGen) and influenza virus antigen. AddaVax™ is an adjuvant having a squalene-based oil-in-water nano-emulsion formulation that induces immune enhancement. PolyIC is known to induce strong T cell responses as a TLR3 agonist. The subjects were Balb/c mice. 2 weeks after administration of a predetermined amount of the antigen, the booster was administered once with the equal amount thereof. 2 weeks after booster administration, each 3LD50 of H1N1 and H3N2 virus was challenged via nasal spraying. A control group was intramuscularly administered with a vaccine including H3N2 in PBS.

[0177] FIG. 11A shows results of measuring antibody titers by measuring HAI titers after immunization with each vaccine composition.

[0178] FIG. 11B shows results of measuring CD4+ T cell levels in the lung and spleen upon challenge administration of H1N1 virus after intramuscular administration of the vaccine including monovalent H3N2 in PBS, after intranasal spraying administration, to a subject, of the vaccine including hsRNA and monovalent H3N2 in PBS, after intramuscular administration, to a subject, of the vaccine prepared by mixing H3N2 antigen, PolyIC, and AddaVax in PBS.

[0179] As shown in FIG. 11B, when the vaccine including H3N2 antigen, PolyIC and AddaVax was intramuscularly administered, T cell responses were observed in the spleen (see B), whereas T cell responses were not observed in the respiratory organ including the lung (see A). In addition, when the vaccine including H3N2 antigen and hsRNA was administered via nasal spraying, T cell responses were induced when challenged with H3N2 virus, as well as when challenged with different subtypes of H1N1 virus.

[0180] FIG. 11C shows percent survivals according to days upon challenge administration of H1N1 virus to mice which had been subjected to intranasal spraying administration of the vaccine including hsRNA and H3N2 antigen. In FIG. 11C, the blue line represents challenge administration of H1N1 virus after immunization with the vaccine including hsRNA and H3N2 antigen via nasal spraying, and the green line represents challenge administration of H1N1 virus after intramuscular immunization with the vaccine including polyIC, Addavax, and H3N2 antigen. The red line represents challenge administration of H1N1 virus after intramuscular immunization with the vaccine including H3N2 antigen and PBS. The pink line represents a mock group administered with no vaccine.

[0181] As described above, when the vaccine including influenza virus antigen and hsRNA was administered via the mucous membrane, T cell responses were induced in the respiratory organs including the lung, and the spleen. In particular, the vaccine including influenza virus antigen and hsRNA induced remarkable T cell responses in the respiratory organs including the lung. It is likely that not only antigen-specific protective immunity but also antigen nonspecific protective immunity is improved through induction of the T cell responses by the vaccine including influenza virus antigen and hsRNA. For example, through induction of T cell responses, the vaccine including the antigen and hsRNA may provide a protective immunity against the administered antigen-containing pathogen and may also provide a protective immunity against different antigens. Therefore, when the administered antigen is the influenza virus antigen, the vaccine may also exhibit a protective immunity or an enhanced protective immunity against different types or subtypes of influenza virus. For example, when the administered antigen is A influenza virus H3N2 antigen, the vaccine may exhibit a protective immunity or an enhanced protective immunity against B influenza virus or other subtypes of A influenza virus different from H3N2, such as H1N1.

[0182] 7. Examination of Cross-Reactivity of Vaccine Including hsRNA and Influenza Virus Antigen

[0183] Since influenza virus strains change every year, vaccines against influenza viruses use antigens derived from different influenza virus strains every year.

[0184] In this experiment, it was examined whether a vaccine against influenza virus epidemic in a specific year provides a protective immunity against influenza viruses epidemic in another year. Table 3 shows viruses epidemic in each year.

TABLE-US-00003 TABLE 3 Type Year virus H1N1 20-21  1. A/Guangdong-Maonan/SWL1536/2019 19-20  2. A/Brisbane/02/2018 18-19  3. A/Michigan/45/2015 H3N2 20-21  4. A/Hong Kong/2671/2019 19-20  5. A/Kansas/14 18-19  6. A/Singapore/INFIMH-16-0019/2016 BY 18-21  7. B/Phuket/3073/3/2013 20-21  8. B/Victoria/705/2018 BV 19-20  9. B/Maryland/15/2016 18-19 10. B/Brisbane/60/2008

[0185] In detail, Teratect prefilled syringe (Influenza split vaccine) which is a commercially available quadrivalent vaccine was intramuscularly injected into a subject, or a vaccine was prepared by mixing Teratect prefilled syringe (Influenza split vaccine) vaccine with hsRNA as in the above section 1 and sprayed into the subject's nasal cavity. The Teratect prefilled syringe (Influenza split vaccine) vaccine is a quadrivalent antigen vaccine including A Influenza H1N1 and H3N2, B Influenza BV and BY antigens, and includes antigens derived from virus strains 1, 4, 7, and 8 epidemic in 2020 to 2021, as shown in Table 3. The subjects were Balb/c mice. 2 weeks after administration of a predetermined amount of the antigen, the booster was administered once with the equal amount thereof. 2 weeks after booster administration, the sera were isolated from subjects, and HAI titers were measured using a hemagglutination inhibition assay to examine neutralizing antibody levels. Further, 2 weeks after challenge, the lungs were isolated from the subjects, and CD4+ IFNv+ T cell levels in the lungs were measured. The number of CD4+ IFNv+ T cells in the lungs was measured by an intracellular cytokine staining method.

[0186] FIG. 12A shows results of measuring HAI titers after intramuscular and nasal spraying administration of Teratect prefilled syringe (Influenza split vaccine) vaccine, or the vaccine including hsRNA and the Teratect prefilled syringe (Influenza split vaccine) vaccine, respectively. As shown in FIG. 12, production of neutralizing antibodies against some viruses was not induced (see the box).

[0187] FIG. 12B shows results of measuring CD4+ IFNv+ T cell levels in the lungs after intramuscular and nasal spraying administration of Teratect prefilled syringe (Influenza split vaccine) vaccine, or the vaccine including hsRNA and the Teratect prefilled syringe (Influenza split vaccine) vaccine, respectively. As shown in FIG. 12B, when the vaccine including hsRNA and the Teratect prefilled syringe (Influenza split vaccine) vaccine was administered via the mucous membrane, T cell production was increased (see the box).

[0188] As described above, when the vaccine including hsRNA and the antigen including the influenza virus antigen was administered via the mucous membrane, antigen-specific T cell responses were induced in the mucous membrane and/or respiratory organs including lungs, and spleen. Unlike antibody responses, these T cell responses showed a cross-reactivity to other antigens, for example, virus variants. Accordingly, the vaccine including hsRNA and the antigen including the influenza virus antigen may increase not only a protective immunity against the administered antigen, for example, the specific administered virus strain, but also a protective immunity against its variants or other types of virus.