WO2024191777A2

WO2024191777A2 - Influenza virus immunogenic composition and method using the same

Info

Publication number: WO2024191777A2
Application number: PCT/US2024/019020
Authority: WO
Inventors: Chiou-Hwa Yuh; Tsai-Teng TZENG; Ray-Yuan CHANG; Shih-Jen Liu
Original assignee: National Health Research Institutes
Priority date: 2023-03-10
Filing date: 2024-03-08
Publication date: 2024-09-19
Also published as: TW202442674A; TWI880664B; WO2024191777A3

Abstract

An influenza virus immunogenic composition comprises: a hemagglutinin antigen; a first adjuvant which is an aluminum salt; and a second adjuvant which is CpG oligonucleotides having a sequence of 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 1).

Description

Influenza virus immunogenic composition and method using the same

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of filing date of U. S. Provisional Application Serial Number 63/451,354, filed March 10, 2023 under 35 USC § 119(e)(1).

BACKGROUND OF THE INVENTION

[0002] Field

[0003] The present invention relates to an influenza virus immunogenic composition and a method using the same. More specifically, the present invention relates to an influenza virus immunogenic composition comprising two adjuvants and a method using the same.

[0004] Description of Related Art

[0005] Global equity in vaccination coverage is essential for limiting pandemic outbreaks and preventing the transmission of new strains, according to the experience of the coronavirus disease 2019 (CO VID-19) pandemic. To achieve this goal, more investment in vaccine manufacturing capacity, novel vaccine product development, and new adjuvant formulation creation is necessary for future pandemics. Additionally, efforts to improve existing vaccine platforms are also possible approaches to maintain the availability of multiple vaccine platforms, thereby increasing manufacturing capacity. To date, several existing vaccine types, including inactivated whole-virion (WV), split-virion (SV), subunit, and recombinant hemagglutinin (HA) vaccines, with safe profiles have been used against both seasonal and pandemic influenza. Therefore, further improving and refining these existing vaccine platforms will strengthen the response to future influenza pandemics.

[0006] Aluminum salts are common adjuvants used in various licensed vaccines. Although aluminum salts are also used in the development of avian influenza vaccines, their efficacy and dose-sparing effect remain controversial. Some clinical trials have shown that the addition of aluminum to whole- or split-virion influenza vaccines does not sufficiently enhance antibody responses to meet licensing criteria and even reduces immunogenicity. Similarly, animal experiment results showed that the Th2- polarized immunity induced by aluminum-formulated vaccines has no beneficial role in viral clearance. Moreover, aluminum has no or a lower adjuvant effect on influenza vaccines at low HA doses than the MF59 adjuvant. Therefore, further advances in adjuvant technology are required to improve the induction of immune responses by aluminum-adjuvanted influenza vaccines.

SUMMARY OF THE INVENTION

[0007] The present invention provides an influenza virus immunogenic composition, which comprises: a hemagglutinin antigen; a first adjuvant which is an aluminum salt; and a second adjuvant which is CpG oligonucleotides having a sequence of 5'- TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 1) (hereinafter, CpG 1018 or CpG).

[0008] The present invention also provides a method for immunizing a subject against influenza virus infection, which comprises the following steps: administering an effective amount of any of the aforesaid influenza virus immunogenic composition to a subject in need thereof. [0009] Other novel features of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

[0010] FIG. 1 A to FIG. ID are diagrams showing the effects of adjuvants on the H7N9 WV vaccine-induced antibody responses.

[0011] FIG. IE is a diagram showing antibody responses to quadrivalent split-virion influenza vaccine with or without adjuvant.

[0012] FIG. 2 A to FIG. 2B are diagrams showing effects of adjuvants on the H7N9 WV vaccine-induced IgG isotype antibody titres.

[0013] FIG. 3 A to FIG. 3F are diagrams showing T-cell responses induced by the adjuvanted H7N9 WV vaccine.

[0014] FIG. 4 A and FIG. 4B are diagrams showing protective efficacy of the Alum-adjuvanted H7N9 vaccine with or without CpG against H7N9 challenge.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The following embodiments when read with the accompanying drawings are made to clearly exhibit the above-mentioned and other technical contents, features and/or effects of the present disclosure. Through the exposition by means of the specific embodiments, people would further understand the technical means and effects the present disclosure adopts to achieve the above-indicated objectives. Moreover, as the contents disclosed herein should be readily understood and can be implemented by a person skilled in the art, all equivalent changes or modifications which do not depart from the concept of the present disclosure should be encompassed by the appended claims. [0016] Furthermore, the ordinals recited in the specification and the claims such as ’’first”, ’’second” and so on are intended only to describe the elements claimed and imply or represent neither that the claimed elements have any proceeding ordinals, nor that sequence between one claimed element and another claimed element or between steps of a manufacturing method. The use of these ordinals is merely to differentiate one claimed element having a certain designation from another claimed element having the same designation.

[0017] Moreover, in the present specification, a value may be interpreted to cover a range within ±10% of the value, and in particular, a range within ±5% of the value, except otherwise specified; a range may be interpreted to be composed of a plurality of subranges defined by a smaller endpoint, a smaller quartile, a median, a greater quartile, and a greater endpoint, except otherwise specified.

[0018] “An effective amount” refers to the amount of the antigen which is required to induce immune response on the subject. Effective amounts vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other agents.

[0019] The present invention provides an influenza virus immunogenic composition, which comprises: a hemagglutinin antigen; a first adjuvant which is an aluminum salt; and a second adjuvant which is CpG 1018.

[0020] In one embodiment, the hemagglutinin antigen may be an inactivated whole-virion vaccine, a split-virion vaccine, a subunit vaccine, or a recombinant vaccine. In one embodiment, the hemagglutinin antigen may be the inactivated whole-virion vaccine. In one embodiment, tire inactivated whole-virion vaccine is an inactivated whole-virion H7N9 vaccine. However, the types or subtypes or the influenza virus targeted by the influenza virus immunogenic composition is not limited to H7N9, other types or subtypes of influenza viruses, such as H1N1, H3N2 or others, may also be the influenza virus targeted by the influenza virus immunogenic composition of the present invention when the hemagglutinin antigen is modified according to the types or subtypes of the influenza virus to be targeted.

[0021] In one embodiment, the aluminum salt may be aluminum hydroxide (A1(OH)₃), aluminum phosphate or a combination thereof. In one embodiment, the aluminum salt may be A1(OH)₃.

[0022] In one embodiment, a weight ratio of the aluminum salt (the first adjuvant) to CpG 1018 (the second adjuvant) may range from 10: 1 to 1 :10, for example, about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5 or about 1 :10. In one embodiment, the weight ratio of the aluminum salt to CpG 1018 may range from 5: 1 to 1 :5. In one embodiment, the weight ratio of the aluminum salt to CpG 1018 may range from 5:1 to 1 : 1. In one embodiment, the weight ratio of the aluminum salt to CpG 1018 may be about 3:1.

[0023] In one embodiment, a human dose of the immunogenic composition may comprise, for example, 0.1 pg to 60.0 pg, 0.5 pg to 60.0 pg, 1.0 pg to

60.0 pg, 1.0 pg to 55.0 pg, 1.0 pg to 50.0 pg, 1.0 pg to 45.0 pg, 1.0 pg to

40.0 pg, 1.0 pg to 35.0 pg, 1.0 pg to 30.0 pg, 1.0 pg to 25.0 pg, 1.0 pg to

20.0 pg, 1.0 pg to 15.0 pg, 1.0 pg to 10.0 pg, 1.0 pg to 9.0 pg, 1 .0 pg to 8.0 pg, 1.0 pg to 7.0 pg, 1.0 pg to 6.0 pg, or 1.5 pg to 6.0 pg of HA protein.

[0024] In one embodiment, a human dose of the immunogenic composition may comprise, for example, 100 pg to 800 pg, 100 pg to 750 pg, 100 pg to 700 pg, 100 pg to 650 pg, 100 pg to 600 pg, 100 pg to 550 pg, 100 pg to 500 jig, 120 jig to 480 jig, 140 fig to 460 pg, 160 pg to 440 pg, 180 pg to 420 pg, 200 fig to 400 fig, 220 pg to 380 pg, 240 pg to 360 pg, 260 pg to 340 pg or 280 jig to 320 fig of the aluminum salt.

[0025] In one embodiment, the influenza virus immunogenic composition does not comprise other adjuvants. In other words, no other adjuvant is present in the influenza virus immunogenic composition of the present invention.

[0026] In one embodiment, the influenza virus immunogenic composition may comprise other carriers, such as dispersants, wetting agents or suspending agents, but the present invention is not limited thereto.

[0027] In addition to the aforesaid influenza virus immunogenic composition, the present invention further provides a method using the same.

[0028] In one embodiment, the present invention provides a method for immunizing a subject against influenza virus infection, which comprises the following steps: administering an effective amount of any of the aforesaid influenza virus immunogenic compositions to a subject in need thereof.

[0029] In one embodiment, the present invention provides a method for preventing or ameliorating an influenza virus infection, which comprises the following steps: administering an effective amount of any of the aforesaid influenza virus immunogenic compositions to a subject in need thereof.

[0030] In one embodiment, the present invention provides a method for inducing an immune response against an influenza virus in a subject, which comprises the following steps: administering an effective amount of any of the aforesaid influenza virus immunogenic compositions to a subject in need thereof.

[0031] In one embodiment, the subject may be a mammalian subject, such as a human subject. [0032] In one embodiment, the influenza vims immunogenic composition may be administered by intramuscular injection.

[0033] In addition to the aforesaid influenza vims immunogenic composition and aforesaid method, the present invention further provides a use thereof.

[0034] In one embodiment, the present invention provides a use of any of the aforesaid influenza virus immunogenic compositions for manufacturing a vaccine composition for immunizing a subject against influenza vims infection.

[0035] In one embodiment, the present invention provides a use of any of the aforesaid influenza virus immunogenic compositions for manufacturing a vaccine composition for preventing or ameliorating an influenza vims infection.

[0036] In one embodiment, the present invention provides a use of any of the aforesaid influenza virus immunogenic compositions for manufacturing a vaccine composition for inducing an immune response against an influenza vims.

[0037] Embodiments

[0038] Materials and methods

[0039] H7N9 vims and vaccine bulk

[0040] H7N9 A/Anhui/1/2013 (NIBRG-268), A/Guangdong/17SF003/2016 reassortant virus (CBER-RG7D), and A/Gansu/23277/2019 (IDCDC- RG64A) reassortant vimses, all generated using reverse genetics, were obtained from the National Institute of Biological Standard and Control (NIB SC), the Centre for Bio-logics Evaluation and Research (CBER), and the Centres for Disease Control and Prevention (CDC), respectively. All H7N9 reassortant vimses were propagated in Madin-Darby canine kidney (MDCK) cells with OptiPRO SFM medium supplemented with 4 mM glutamine and 2 pg/mL TPCK-treated trypsin (Sigma). All experiments with H7N9 reassortant viruses were conducted in a biosafety level 2 (BSL-2) laboratory. The bulk of the inactivated H7N9 whole-virion vaccine made from CBER-RG7D virus was produced using an MDCK cell-based manufacturing system by a PIC/S GMP Bioproduction Plant at the National Health Research Institutes (NHRI), Taiwan. HA antigen content in the vaccine bulk was measured by single radial immunodiffusion assay using reference antiserum and antigen from NIB SC, codes 18/112 and 18/196.

[0041] Quadrivalent split-virion influenza vaccine

[0042] Egg-derived quadrivalent split-virion influenza vaccine bulk consisting of the strains A/Victoria/2570/2019 IVR-215 (H1N1), A/Cambodia/e0826360/2020 IVR-224 (H3N2), B/Victoria/705/2018 BVR- 11 (Victoria lineage), and B/Phuket/3073/2013 Wild type (Yamagata lineage) were obtained from Adimmune Corporation, Taiwan. H7N9 reference antisera (NIBSC code: 18/112 and 15/248) were obtained from the National Institute of Biological Standard and Control.

[0043] Overall experimental design

[0044] To evaluate the potential of Alum or Alum/CpG 1018 to reduce the required dosage of the H7N9 WV vaccine, we administered varying amounts of the vaccine to BALB/c mice. Specifically, the mice received the H7N9 WV vaccine containing 0.015, 0.15, or 1.5 pg of HA protein, in combination with 30 pg of Alum, 10 pg of CpG 1018, or no additional adjuvant. We selected a mouse-appropriate dosage that was one-tenth of the human dose for both the antigen and adjuvant. Hence, we utilized one-tenth of Alum- adjuvanted H7N9 WV vaccine, which contained 1.5 pg of HA and 30 pg of Alum, as a reference dose in this study. [0045] BALB/c mice were intramuscularly immunized twice with the H7N9 WV vaccine in combination with Alum or CpG 1018. ELISA was used to determine the antigen-specific antibody response and immuno-globulin G subclasses. Hemagglutination inhibition (HI) and microneutralization assays were used to analyze vaccine-induced protective antibody responses. Finally, to assess the vaccine-induced immune response in protection against influenza, immunized mice were exposed to modest or high lethal doses of CBER-RG7D virus four weeks after the last vaccination.

[0046] Immunization of mice

[0047] Female BALB/c mice were obtained from the National Laboratory Animal Centre (Taipei, Taiwan). All animals were housed at the Animal Centre of the NHRI, which has been accredited by AAALAC International. Six- to eight-week-old female BALB/c mice were immunized intramuscularly with 50 pL of the H7N9 WV (whole- virion) vaccine twice at 2 weeks apart. For vaccine formulation, the H7N9 WV antigen was mixed with or without Alhydrogel (2% w/v suspension of aluminum hydroxide; Brenntag AG) and CpG 1018 in PBS buffer in an equal final volume. The CpG 1018 oligonucleotide (5’-TGACTGTGAACGTTCGAGATGA-3’) was synthesized by GeneDerix and resuspended in ddlLO. Unadjuvanted and adjuvanted vaccines were rotated under constant mixing on a rotating mixer for 2 h at room temperature before administration. All animal experiments were conducted according to an lACUC-approved protocol (protocol number: NHRI-IACUC-110044).

[0048] Immunoassay

[0049] The specific antibody response against H7N9 was detennined by ELISA. In brief, 50 pL of 0.5 pg/mL HA protein (H7N9 WV vaccine bulk) in 0.1 M carbonate buffer (pH 9.5) was coated onto 96-well micro-plates by overnight incubation at 4°C. The coated plates were washed twice with 0.05% Tween 20 in PBS and then blocked with 3% BSA in PBS at room temperature for 1 h. Diluted sera from immunized animals were added to the wells and incubated for 2 h at room temperature. HRP-conjugated goat antimouse IgG (1:10000; Thermo Scientific), HRP-conjugated rabbit anti-mouse IgGl (1:5000; Invitrogen), and HRP-conjugated rabbit anti-mouse IgG2a (1 : 5000; Invitrogen) were used as the secondary anti-bodies. The assay was developed by using the TMB substrate set (BioLegend). The absorbance was measured using a SpectraMax M2 microplate reader (Molecular Device) at 450 nm.

[0050] Hemagglutination inhibition (HI) assay

[0051] HI titres in mouse sera were determined according to a harmonized protocol. Briefly, mouse sera were pretreated with receptor-destroying enzyme (RDE II, Denka Seiken) and preabsorbed with turkey red blood cells (TRBCs). After removing the TRBCs by centrifugation, the sera were twofold serially diluted from the initial 1:10 dilution and mixed with 4 hemagglutinating units of fonnaldehyde-inactivated H7N9 virus in a volume of 50 pL at room temperature for 1 h. Next, 50 pL of 0.5% TRBC suspension was added and incubated at room temperature for 40 min. Finally, assay plates were tilted, and the TRBC flow pattern was read. The HI titre was proportional to the highest serum dilution that completely inhibited hemagglutination. Sera that failed to inhibit hemagglutination at the initial dilution of 1:10 were assigned an HI value of 5.

[0052] Microneutralization assay

[0053] MDCK cells were seeded (3 * 10⁴ cells/well) in 96-well plates for 24 h to form a monolayer. Preimmunization sera and antisera against H7N9 were pretreated at 56°C for 30 min to destroy heat-labile nonspecific viral inhibitory substances. The sera were diluted to an initial dilution of 1/10 with DMEM, added to a well containing 200 TCID₅₀ of H7N9 in a volume of 0.2 mL, and then incubated at 35°C for 2 h. Subsequently, the virusserum mixture was inoculated onto MDCK cell monolayers, and the cells were incubated at 35 °C. Quadruplicates were prepared for each serum dilution. The cytopathic effect in each well was recorded after 4-5 days of incubation. The 50% neutralization (NT₅₀) titre was calculated using the Reed-Muench formula. Neutralization titres below the starting dilution of 1:10 were assigned a value of 5 for calculation purposes.

[0054] Cytokine production assay

[0055] Seven days after the final vaccination, the mice were sacrificed, and splenocytes were collected and plated at a density of 5 x 10⁶ cells per well in 24-well plates. The cells were stimulated with 5 pg/mL recombinant H7 ectodomain (A/Guangdong/17SF003/2016) produced in the ExpiCHO expression system (Thermo-Fisher). After stimulation for 3 days at 37°C, the supernatant was harvested and assayed for cytokine production. The levels of secreted mouse IFN-y, IL-5, IL- 13 and IL-2 were evaluated by ELISA using the matching antibody set (Invitrogen) in accordance with the manufacturer’s instructions.

[0056] Animal challenge

[0057] Four weeks after the last vaccination, the mice were challenged intranasally with a 2- or 10-fold 50% minimum lethal dose (MLD₅₀) of CBER-RG7D in a 20 pL volume under isoflurane anesthesia. Subsequently, the mice were monitored daily for weight loss and survival and were euthanized and scored as dead if more than 20% of their body weight was lost. To determine the viral load in the lung, lung tissues were homogenized in 2 mL of PBS containing 200 U/mL penicillin and 200 pg/mL streptomycin using a gentleMACS® Dissociator (Miltenyi Biotec). After centrifugation at 600 x g for 10 min at 4 °C, the clarified supernatant was harvested for viral RNA quantification.

[0058] Quantification of viral RNA load

[0059] Clarified supernatant of homogenized lung tissue from H7N9-infected mice was harvested for viral load detection. RNA extraction was carried out on tissue supernatant with QIAamp Viral RNA Mini Kit (Qiagen). RNA extracts were reverse-transcrbed using FIREScript RT cDNA synthesis kit (Solis Biodyne) and Uni 12 primer. Viral RNA was quantified by real-time PCR in a QuantStudio 6 Flex Real-Time PCR System (ABI) using the Power SYBR® Green PCR Master Mix (ABI). The viral copy number was estimated by the standard curve method with primers specific for H7N9 HA gene (Forward: TGAAAATGGATGGGAAGGCC (SEQ ID NO: 2), Reverse: TGCCGATTGAGTGCTTTTGT (SEQ ID NO: 3)).

[0060] Statistical analysis

[0061] Statistical data were generated using GraphPad Prism software. The statistical significance of differential findings between experimental groups was determined by unpaired Student’s t test, one-way ANOVA, or two-way ANOVA with Tukey’s or Sidak’s posttest. Significant differences in Kaplan-Meier survival curves were analyzed with a log-rank test. Differences were considered statistically significant if the p value was < 0.05.

[0062] Results

[0063] Alum/CpG 1018 robustly enhanced the immunogenicity of the H7N9 WV vaccine

[0064] FIG. 1 A to FIG. ID are diagrams showing the effects of adjuvants on the H7N9 WV vaccine-induced antibody responses. BAEB/c mice (n = 5 per group) were intramuscularly immunized twice with the H7N9 WV vaccine in combination with aluminum hydroxide (Alum) or CpG 1018. Serum samples were collected for humoral immune response evaluation at the indicated timepoint after the first immunization. In FIG. 1 A and FIG. IB, the levels of total IgG antibodies against H7N9 WV were assessed by ELISA. In FIG. 1C, H7N9-specific hemagglutination-inhibition (HI) antibodies were quantified by hemagglutination inhibition assay. The lower dashed line indicates a 10-fold initial dilution of serum samples. The upper dashed line represents a > 4-fold rise in HI titre, also called 4-fold seroconversion. In FIG. ID, vaccine-induced neutralizing activity against H7N9 was evaluated by microneutralization assay. The dashed line indicates a 20-fold initial dilution of serum samples. The logio-transformed IgG titre and log2- transformed HI and NT titres of sera collected at week 6 after the first immunization were analyzed by two-way ANOVA with Tukey’s posttest. * indicates comparisons of the adjuvant effect among the same antigen dose groups. # indicates comparisons between the various antigen doses that were adjuvanted with Alum/CpG 1018. @ indicates comparisons between the 1.5 p g HA with Alum and 0.15 pg HA with Alum and 0.15 pg HA with Alum/CpG 1018 groups. * ^/# P < 0.05,

< 0.01, *** P < 0.001,

****/#### p < Q QQQJ

[0065] FIG. IE is a diagram showing antibody responses to quadrivalent split-virion influenza vaccine with or without adjuvant. BALB/c mice (n = 5 per group) were intramuscularly immunized twice with the quadrivalent split- virion influenza vaccine containing 0.15 pg of HA protein for each strain, in combination with 30 pg of Alum, 10 pg of CpG 1018, or no additional adjuvant. Serum samples were collected for humoral immune response evaluation at week 6 after the first immunization. In FIG. IE, the levels of total IgG antibodies against each strain were assessed by ELISA. The logio-transformed IgG titers were analyzed by Student’s t test. *P<0.05, **F<0.01, ***P<0.001.

[0066] Administration of the H7N9 WV vaccine alone induced H7N9- specific total IgG antibody production (FIG. 1 A) in a dose-dependent manner. The Alum-formulated vaccine significantly increased the antibody titre compared with the H7N9 WV alone vaccine, regardless of antigen dose. In contrast, CpG 1018 alone had no obvious immunostimulatory effect on the H7N9 WV vaccine at a dose of 0.15 pg HA (data not shown). Importantly, Alum/CpG 1018-formulated vaccines synergistically increased the antibody titres compared to those with Alum- or CpG 1018-adjuvanted vaccines. We also successfully confirmed this synergistic effect of the Alum/CpG 1018 adjuvant system on the quadrivalent split-virion (SV) influenza vaccine (FIG. IE). After the first immunization, the IgG antibody titre gradually increased to a peak at week 8, slightly decreased to a level similar to that at week 4, and then persisted until week 20 (FIG. IB). The synergistic effect of CpG 1018 addition was not found when it was combined with MF59-like squalene oil-in-water emulsion (SWE) adjuvant.

[0067] Moreover, vaccine-induced protective antibody responses were analyzed by hemagglutination inhibition (HI) and microneutralization assays. After administration of the H7N9 WV alone vaccine, all animals in the 0.015 pg HA group had HI titres below or equal to the detection limit, and 40% of animals from the 0.15 and 1.5 pg HA groups achieved 4-fold seroconversion at week 6 (FIG. 1C). In contrast, the Alum-adjuvanted groups showed higher HI titres with 4-fold sero-conversion and neutralization (NT₅₀) titres, relative to those of the groups without adjuvant treatment (FIG. 1C and FIG. ID). Within the Alum/CpG 1018 groups, the addition of CpG 1018 further increased the HI and NT₅₀ titres in the 0.15 and 1.5 pg HA groups but not in the 0.015 pg HA group. Notably, the 0.15 pg HA group with the Alum/CpG 1018 combination adjuvant had a significantly higher specific antibody level, HI titre and NT50 titre than the reference group at week 6 after vaccination. These results supported a dosesparing effect of the Alum and CpG 1018 combination adjuvant on the H7N9 WV vaccine, with a 10-fold reduction in antigen usage. Importantly, the combination of Alum and CpG 1018 more potently increased specific antibody levels, HI titres and NT₅₀ titres.

[0068] Alum/CpG 1018 shifted the antibody response toward IgG2a dominance

[0069] FIG. 2A to FIG. 2B are diagrams showing effects of adjuvants on the H7N9 WV vaccine-induced IgG isotype antibody titres. BALB/c mice (n = 5 per group) were intramuscularly immunized twice with the H7N9 WV vaccine in combination with aluminum hydroxide (Alum) or CpG 1018. Serum samples were collected for humoral immune response evaluation at week 6 after the first immunization. In FIG. 2A and FIG. 2B, antigenspecific immunoglobulin G subclasses, IgGl and IgG2a, in mouse serum were quantified by ELISA. The logio -transformed IgGl and IgG2a titres were analyzed by two-way ANOVA with Tukey’s posttest. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0070] IgG2a plays an important role in viral clearance during infection. To evaluate the effect of CpG 1018 on IgG isotype switching, the levels of the IgGl and IgG2a isotypes were measured by ELISA. After two doses of vaccine, the Alum-adjuvanted vaccine markedly increased antigen-specific IgGl titres in the 0.015 and 0.15 pg HA groups, and this raised IgGl titre was offset in the groups with CpG 1018 addition (FIG. 2A). However, this finding was not observed in the 1.5 jig HA group. On the other hand, the antigen-specific IgG2a titres significantly increased in the Alum-adjuvanted H7N9 WV groups (FIG. 2B), regardless of the antigen dose. The titre was further increased after the combination of Alum with CpG 1018, which was similar to the observation for the total IgG titre (FIG. 1 A). The results suggested that CpG 1018 coadjuvantation redirected the Alum-induced humoral immunity toward the IgG2a-dominated antibody response.

[0071] Alum/CpG 1018 induced a Th 1 -polarized immune response

[0072] FIG. 3A to FIG. 3F are diagrams showing T-cell responses induced by the adjuvanted H7N9 WV vaccine. BALB/c mice (n = 4 per group) were intramuscularly immunized twice with the H7N9 WV vaccine in combination with aluminum hydroxide (Alum) or CpG 1018. Splenocytes were collected at day 7 after the second immunization, and the levels of secreted IFN-y, IL-2, IL-5 and IL- 13 (FIG. 3 A to FIG. 3D) were evaluated after restimulation with recombinant H7 protein. In FIG. 3E and FIG. 3F, the ratios of IFN- y to IL-5 and IFN- y to IL-13 were calculated. The logio- transformed cytokine level and IFN- y /IL-5 and IFN- y /IL- 13 ratios were analyzed by two-way ANOVA with Tukey’s posttest. * P <0.05, ** P < 0.01, *** P < 0.001, **** F < 0.0001.

[0073] IgG subclass switching is associated with Thl- and Th2-polarized immune responses. To examine the impact of CpG 1018 on Alum-induced T-cell responses, we assessed T-cell responses after immunization with the H7N9 WV vaccine with or without Alum or CpG 1018. Splenocytes from the immunized mice were stimulated with recombinant H7 protein. The secreted levels of Thl-type cytokines (IL-2 and IFN-y) and Th2-type cytokines (IL-5 and IL- 13) were measured by ELISA. The CpG-adjuvanted group produced the lowest amounts of IL-2, IFN-y, and IL-13 among the groups, and the level of IL-5 was even undetectable in this group (FIG. 3 A to FIG. 3D). In contrast, vaccination with WV/Alum induced higher levels of IL -2, IFN-y, IL-5 and IL-13. The addition of CpG 1018 to Alum profoundly suppressed the levels of IL-5 and IL-13 (FIG. 3C and FIG. 3D), while the levels of IL-2 and IFN-y were maintained or slightly decreased (FIG. 3 A and FIG. 3B). The results indicated that Alum and CpG1018 offset each other’s activity, especially in regulating the Th2-mediated immune response. Vaccine-induced trends in these cytokine levels were observed in mice receiving 0.15 or 1.5 pg of HA antigen. Furthermore, analysis of the Thl/Th2 ratio showed that the H7N9 WV vaccines adjuvanted with CpG 1018 or Alum/CpG 1018 produced Th 1 -biased responses (FIG. 3E and FIG. 3F). IFN-y/IL-5 and IFN-y/IL-13 ratios were increased by approximately 21.2- and 6.3-fold, respectively, in the 0.15 pg WV/Alum/CpG 1018 group compared with those in the 0.15 pg WV/Alum group. Therefore, these results indicated that the addition of CpG 1018 shifts immune responses toward a Th I bias.

[0074] Alum/CpG 1018 conferred robust protection against H7N9 challenge [0075] FIG. 4 A and FIG. 4B are diagrams showing protective efficacy of the Alum-adjuvanted H7N9 vaccine with or without CpG against H7N9 challenge. BALB/c mice (n = 8 for each group) were intramuscularly immunized twice with PBS or adjuvanted H7N9 WV vaccines. Four weeks after the final immunization, mice were intranasally challenged with 2 -fold MLD₅₀ or 10-fold MLD₅₀ of CBER-RG7D H7N9 virus. In FIG. 4A, survival rate was monitored daily after H7N9 challenge. Significant differences between the PBS group and the other groups were calculated using the logrank test, **p < 0.01, *** < 0.001. In FIG. 4B, body weight change (%) of the mice was monitored daily after H7N9 challenge as an indicator of disease progression. Only surviving mice are shown in body weight results that are presented as the mean ± SD. Significant differences (on day 3 to day 9 postinfection) in FIG. 4B were calculated using Student’s t test due to missing data resulting from death. * indicates comparisons between the 1.5 pg HA with Alum and 0.15 pg HA with Alum/CpG groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0076] To assess the role of the vaccine-induced immune response in protection against influenza, mice were first exposed to a lethal dose (2 MLD₅o) of H7N9 virus. The body weight of the mice was monitored daily after challenge as an indicator of disease progression. Upon 2 MLD₅₀ virus challenge, the survival rate of the PBS (control) group was 25%. In contrast, all mice in the Alum- and Alum/CpG 1018-adjuvanted H7N9 vaccine groups survived, regardless of antigen dose. Moreover, the vaccinated mice also showed less body weight loss relative to that of the control group, suggesting that both Alum- and Alum/CpG 1018-adjuvanted H7N9 vaccines were sufficient to confer protection against challenge with a modest dose (2 MLD50) of H7N9 virus. Furthermore, two-way ANOVA indicated that the protection from H7N9-induced weight loss was an adjuvant-dependent effect (two-way ANOVA (within 1.5 pg HA groups): time effect, F (14, 196) = 36.85, P < 0.0001; adjuvant effect, F (1, 14) = 9.858, P =0.0072; interaction effect, F (14, 196) = 5.614, < 0.0001; two-way ANOVA (within 0.15 pg HA groups): time effect, F (14, 196) = 31.18, P < 0.0001; adjuvant effect, F (1, 14) = 5.355, P = 0.0364; interaction effect, F (14, 196) = 4.498, P < 0.0001). Even not shown in the figure, Sidak’s posttest showed that, even with varying antigen dosages, there was no significant difference in body weight change between groups adjuvanted with the same adjuvant at each time. Notably, on day 3 to day 4 post-challenge, the Alum/CpG 1018 combination adjuvant significantly protected against body weight loss compared with the Alum adjuvant in the groups administered either 0.15 or 1.5 pg HA antigen.

[0077] Next, we utilized a higher challenge dose (10 MLD50) to more rigorously evaluate the impact of vaccine-induced immunity on survival and body weight change. Moreover, to evaluate the potential of the Alum/CpG 1018 combination adjuvant, the HA antigen dose was further reduced to 0.15 pg. Following virus challenge with the higher dose, all control mice died, and an 87.5% survival rate was observed after administration of 1.5 pg HA/ Alum vaccines (FIG. 4A). In contrast, all mice in the 0.15 pg HA/Alum/CpG 1018 group survived during the observation period. According to log-rank comparison, the vaccinated groups did not significantly differ from each other. As shown in FIG. 4B, 0.15 pg HA/ Alum/CpG exhibited the best prophylactic efficacy on weight loss among the vaccinated groups. Interestingly, mice in the 1.5 pg HA/Alum group lost significantly more body weight on days 3 and 4 postchallenge than mice in the 0.15 pg HA/Alum/CpG 1018 group. These findings highlighted the importance of CpG 1018 addition to the Alum-adjuvanted vaccine to induce better protective efficacy against H7N9 challenge. Of note, mice receiving 0.15 pg HA/ Alum/CpG 1018 had a significant improvement in disease progression compared with those in the 1.5 pg HA/Alum group upon challenge with 10 MLD₅₀ (FIG. 4B), indicating that the combination of Alum with CpG 1018 could not only achieve a dose-sparing effect for the H7N9 WV vaccine but also confer better protective immunity against challenge.

[0078] To evaluate the role of the Alum/CpGlOl 8 combination adjuvant in viral clearance, the viral RNA in lung tissue were analyzed. Viral RNA loads (n = 6 for each group) in the lungs of H7N9-infected mice at day 3 postchallenge were quantified by RT-qPCR. Upon 10 MLD₅₀ virus challenge, up to 7.2 log (copies/mL) of viral RNA load were detected in the lung on day 3 postchallenge. Even not shown in the figure, consistent with the survival rate in FIG. 4 A, the viral RNA load in the 0.15 pg HA/Alum/CpG 1018 group was similar to that in 1.5 pg HA/Alum group, and the viral RNA load showed at least a 2.6 log reduction compared with the control group.

[0079] Alum/CpG 1018 increased the cross-reactivity of H7N9 vaccine [0080] To investigate the potential cross-protective immunity of the adjuvanted WV vaccine against various H7N9 strains, we evaluated the cross-reactive HI titre against WHO-selected H7N9 vaccine candidates, including A/ Anhui/1/2013 (NIBRG-268) and A/Gansu/23277/2019 (IDCDC-RG64A) H7N9 reassortant viruses derived from the first epidemic wave or the infected case in March 2019, respectively. Even not shown in the figure, consistent with the findings shown in FIG. 1C, the homologous HI titres generated by the Alum/CpG 1018-adjuvanted vaccine were higher than those elicited by the Alum-adjuvanted vaccine. Overall, the geometric mean HI titres against the Anhui virus were approximately 2-3-fold lower than the titres against the homologous strain, irrespective of the adjuvant type. Notably, only the Alum/CpG 1018 adjuvant system induced heterologous HI titres against the Anhui virus that met the seroprotective threshold HI titre of >40, whereas approximately 50% of the animals in the Alum-adjuvanted groups did not achieve this level. Moreover, the crossreactivity against the Gansu strain was remarkably lower than that against the Anhui strain, with all animals in both the Alum and Alum/CpG 1018- adjuvanted groups exhibiting HI titres below the detection limit. [0081] Antigen sparing is an important strategy for pandemic vaccine development because of the limitation of worldwide vaccine production during disease outbreaks. However, several clinical studies have demonstrated that the current aluminum (Alum)-adjuvanted influenza vaccines fail to sufficiently enhance immune responses to meet licensing criteria. Furthermore, previous studies have suggested that the influence of CpG on the binding of antigens to Alum depends on buffer conditions and the nature of the antigen, and not all Alum/CpG combination adjuvants may exert a synergistic effect on all types of vaccines. Therefore, the impact of clinically approved CpG 1018 on the immune response induced by Alum- adjuvanted inactivated whole-virion (WV) vaccines is unexpected.

[0082] In the present invention, we demonstrated that the formulation of aluminum hydroxide with CpG 1018 can not only reduce the antigen used but also elicit better protective immunity against the H7N9 virus than that with Alum alone, and this information could be used to solve the dilemma of insufficient production capacity of pandemic vaccines. Moreover, the Thl- polarized and IgG2a-dominated immune responses induced by the Alum/CpG 1018-formulated vaccine may be more helpful to combat future pandemics. Therefore, this Alum/CpG 1018 adjuvant system provides a platform to strengthen the vaccine efficacy and production capacity of existing aluminum-formulated vaccines for various diseases, especially avian influenza and elderly individual -used seasonal influenza vaccines.

[0083] Although the present disclosure has been explained in relation to its embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the disclosure as hereinafter claimed.

Claims

1. An influenza virus immunogenic composition, comprising: a hemagglutinin antigen; a first adjuvant which is an aluminum salt; and a second adjuvant which is CpG oligonucleotides having a sequence of 5'- TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 1).

2. The immunogenic composition of claim 1, wherein the hemagglutinin antigen is an inactivated whole-virion vaccine, a split-virion vaccine, a subunit vaccine, or a recombinant vaccine.

3. The immunogenic composition of claim 2, wherein the hemagglutinin antigen is the inactivated whole-virion vaccine.

4. The immunogenic composition of claim 3, wherein the inactivated wholevirion vaccine is an inactivated whole-virion H7N9 vaccine.

5. The immunogenic composition of claim 1, wherein the aluminum salt is A1(OH)₃, aluminum phosphate or a combination thereof.

6. The immunogenic composition of claim 1, wherein a weight ratio of the first adjuvant to the second adjuvant ranges from 10: 1 to 1 : 10.

7. The immunogenic composition of claim 6, wherein the weight ratio of the first adjuvant to the second adjuvant ranges from 5: 1 to 1 : 5.

8. The immunogenic composition of claim 7, wherein the weight ratio of the first adjuvant to the second adjuvant ranges from 5: 1 to 1 : 1.

9. The immunogenic composition of claim 1, wherein a human dose of the immunogenic composition comprises 0.1 pg to 60.0 pg of HA protein.

10. The immunogenic composition of claim 1, wherein a human dose of the immunogenic composition comprises 100 pg to 800 pg of the first adjuvant.

11. A method for immunizing a subject against influenza virus infection, comprising the following steps: administering an effective amount of an influenza virus immunogenic composition to a subject in need thereof, wherein the influenza virus immunogenic composition comprises: a hemagglutinin antigen; a second adjuvant which is CpG oligonucleotides having a sequence of 5'- TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 1).

12. The method of claim 11, wherein the hemagglutinin antigen is an inactivated whole-virion vaccine, a split-virion vaccine, a subunit vaccine, or a recombinant vaccine.

13. The method of claim 12, wherein the hemagglutinin antigen is the inactivated whole-virion vaccine.

14. The method of claim 13, wherein the inactivated whole-virion vaccine is an inactivated whole-virion H7N9 vaccine.

15. The method of claim 11, wherein the aluminum salt is A1(OH)B, aluminum phosphate or a combination thereof.

16. The method of claim 11, wherein a weight ratio of the first adjuvant to the second adjuvant ranges from 10: 1 to 1 : 10.

17. The method of claim 16, wherein the weight ratio of the first adjuvant to the second adjuvant ranges from 5: 1 to 1 :5.

18. The method of claim 17, wherein the weight ratio of the first adjuvant to the second adjuvant ranges from 5:1 to 1 :1.

19. The method of claim 11, wherein a human dose of the immunogenic composition comprises 0.1 pg to 60.0 pg of HA protein.

20. The method of claim 11, wherein a human dose of the immunogenic composition comprises 100 pg to 800 pg of the first adjuvant.