TWI880664B - Influenza virus immunogenic composition and method using the same - Google Patents

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 compositions and methods of using the same

The present invention relates to an influenza virus immunogenic composition and methods of using the same. More specifically, the present invention relates to an influenza virus immunogenic composition comprising two adjuvants and methods of using the same.

Based on the experience of the 2019 coronavirus disease (COVID-19) pandemic, global equitable distribution of vaccination coverage is critical to limiting pandemic outbreaks and preventing the spread of new virus strains. To achieve this goal, more investment is needed in vaccine production capacity, development of new vaccine products, and innovation of new adjuvant formulations to cope with future pandemics. In addition, improving existing vaccine platforms is also a feasible way to maintain the availability of multiple vaccine platforms, thereby increasing production capacity. To date, several types of vaccines with existing safety have been used to combat seasonal and pandemic influenza, including inactivated whole-virion (WV), split-virion (SV), subunit, and recombinant hemagglutinin (HA) vaccines. Therefore, further improvement and refinement of these existing vaccine platforms will enhance preparedness against future influenza pandemics.

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 effects remain controversial. Some clinical trials have shown that adding aluminum to whole-virus or split-virus influenza vaccines is not sufficient to enhance antibody responses to meet licensing standards and may even reduce immunogenicity. Similarly, animal experimental results have shown that the Th2 polarized immunity induced by aluminum-formulated vaccines is not effective for viral clearance. In addition, compared with MF59 adjuvant, aluminum has no adjuvant effect or a lower adjuvant effect on low HA dose influenza vaccines. Therefore, further advancements in adjuvant technology are needed to improve the induction of immune responses by aluminum-adjuvanted influenza vaccines.

The present invention provides an influenza virus immunogenic composition, comprising: a hemagglutinin antigen; a first adjuvant, which is an aluminum salt; and a second adjuvant, which is a CpG oligonucleotide having a sequence of 5′-TGACTGTGAACGTTCGAGATGA-3′ (SEQ ID NO: 1) (hereinafter referred to as CpG 1018 or CpG).

The present invention also provides a method for immunizing a subject against influenza virus infection, comprising the following steps: administering an effective amount of any one of the aforementioned influenza virus immunogenic compositions to a subject in need thereof.

The following will be described in detail with reference to the drawings to make other novel features of the present invention more apparent.

In order to clearly demonstrate the aforementioned and other technical contents, features and/or effects of the present disclosure, the following embodiments are read in conjunction with the attached drawings. Through the description of the specific embodiments, people will further understand the technical means and effects adopted by the present disclosure to achieve the aforementioned purposes. In addition, since the contents disclosed herein should be easy to understand and implement for those skilled in the art, all equivalent changes or modifications without departing from the concept of the present disclosure should be included in the scope of the attached patent application.

In addition, the ordinal numbers listed in the specification and patent claims, such as "first", "second", etc., are intended only to describe the claimed elements and do not imply or indicate any procedural ordinal number of the claimed elements, nor do they imply or indicate the order between one claimed element and another claimed element or the order of steps in the manufacturing method. The use of these ordinal numbers is only used to make a claimed element with a specific name clearly distinguishable from another claimed element with the same name.

In addition, in the specification, unless otherwise stated, a value may be interpreted as covering a range within ±10% of the value, and specifically, covering a range within ±5% of the value; unless otherwise stated, a range may be interpreted as consisting of multiple subranges defined by a lower endpoint, a lower quartile, a median, an upper quartile, and an upper endpoint.

"Effective amount" refers to the amount of an antigen required to induce an immune response in a subject. As known to those skilled in the art, the effective amount will vary depending on the route of administration, the formulation used, and the possibility of co-use with other agents.

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

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

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

In one embodiment, the weight ratio of the aluminum salt (first adjuvant) to CpG 1018 (second adjuvant) may be between 10:1 and 1:10, such as 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 be between 5:1 and 1:5. In one embodiment, the weight ratio of the aluminum salt to CpG 1018 may be between 5:1 and 1:1. In one embodiment, the weight ratio of the aluminum salt to CpG 1018 may be about 3:1.

In one embodiment, a human dose of an immunogenic composition may include, for example, 0.1 μg to 60.0 μg of HA protein, 0.5 μg to 60.0 μg of HA protein, 1.0 μg to 60.0 μg of HA protein, 1.0 μg to 55.0 μg of HA protein, 1.0 μg to 50.0 μg of HA protein, 1.0 μg to 45.0 μg of HA protein, 1.0 μg to 40.0 μg of HA protein, 1.0 μg to 35.0 μg of HA protein, 1.0 μg to 30.0 μg of HA protein, 1.0 μg to 25.0 μg of HA protein, 1.0 μg to 20.0 μg of HA protein, 1.0 μg to 15.0 μg of HA protein, 1.0 μg to 10.0 μg of HA protein, 1.0 μg to 9.0 μg of HA protein, 1.0 1.0 μg to 8.0 μg of HA protein, 1.0 μg to 7.0 μg of HA protein, 1.0 μg to 6.0 μg of HA protein, or 1.5 μg to 6.0 μg of HA protein.

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

In one embodiment, the influenza virus immunogenic composition does not include other adjuvants. In other words, no other adjuvants are present in the influenza virus immunogenic composition of the present invention.

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

In addition to the aforementioned influenza virus immunogenic compositions, the present invention also provides methods of using the same.

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

In one embodiment, the present invention provides a method for preventing or improving influenza virus infection, comprising the following steps: administering an effective amount of any one of the aforementioned influenza virus immunogenic compositions to a subject in need thereof.

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

In one embodiment, the subject may be a mammalian subject, such as a human subject.

In one embodiment, the influenza virus immunogenic composition can be administered by intramuscular injection.

In addition to the aforementioned influenza virus immunogenic composition and the aforementioned method, the present invention also provides uses thereof.

In one embodiment, the present invention provides use of any of the aforementioned influenza virus immunogenic compositions in the preparation of a vaccine composition for immunizing a subject against influenza virus infection.

In one embodiment, the present invention provides use of any of the aforementioned influenza virus immunogenic compositions in preparing a vaccine composition for preventing or ameliorating influenza virus infection.

In one embodiment, the present invention provides use of any of the aforementioned influenza virus immunogenic compositions in the preparation of a vaccine composition for inducing an immune response against influenza virus.

Embodiment

Materials and methods

H7N9 virus and vaccine bulk

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

Quadrivalent split-virus influenza vaccine

Egg-derived quadrivalent split virus influenza vaccine stock consisting of virus 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) was obtained from Adimmune Corporation, Taiwan. H7N9 reference antisera (NIBSC numbers: 18/112 and 15/248) were obtained from the National Institute for Biological Products Control.

Overall experimental design

To evaluate the potential of aluminum salts or aluminum salts/CpG 1018 to reduce the required dose of H7N9 WV vaccine, we administered different amounts of vaccine to BALB/c mice. Specifically, mice were vaccinated with H7N9 WV vaccines containing 0.015 μg HA protein, 0.15 μg HA protein, or 1.5 μg HA protein, combined with 30 μg aluminum salts, 10 μg CpG 1018, or no other adjuvants. We selected doses of antigen and adjuvant suitable for mice, which are one-tenth of the human dose. Therefore, we used one-tenth of the aluminum salt adjuvant H7N9 WV vaccine (containing 1.5 μg HA and 30 μg aluminum salts) as the reference dose for this study.

BALB/c mice were immunized intramuscularly twice with H7N9 WV vaccines conjugated with aluminum salt or CpG 1018. Antigen-specific antibody responses and immunoglobulin G subclasses were determined using enzyme immunoassays (ELISA). Vaccine-induced protective antibody responses were analyzed using hemagglutination inhibition (HI) and microneutralization assays. Finally, to assess vaccine-induced immune responses against influenza, immunized mice were exposed to medium or high lethal doses of CBER-RG7D virus four weeks after the last vaccination.

Immunization of mice

Female BALB/c mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). All animals were maintained at the Animal Center of the National Institutes of Health, which is accredited by AAALAC International. Female BALB/c mice aged six to eight weeks were immunized intramuscularly twice with 50 μL of H7N9 WV (whole virus particles) vaccine, 2 weeks apart. For vaccine formulation, H7N9 WV antigen was mixed with PBS buffer with or without aluminum adjuvant (AlHydrogel) (2% w/v aluminum hydroxide suspension; Brenntag AG) and CpG 1018 in the same final volume. CpG 1018 oligonucleotide (5'-TGACTGTGAACGTTCGAGATGA-3') was synthesized by GeneDerix and resuspended in double distilled water (ddH ₂ O). Unadjuvanted and adjuvanted vaccines were continuously mixed on a rotator at room temperature for 2 hours prior to administration. All animal experiments were performed in accordance with IACUC-approved protocols (Protocol No.: NHRI-IACUC-110044).

Immunoassay

ELISA was used to measure specific antibody responses to H7N9. Briefly, 50 μL of 0.5 μg/mL HA protein (H7N9 WV vaccine stock) in 0.1 M carbonate buffer (pH 9.5) was incubated overnight at 4 ^° C and then coated on a 96-well microtiter plate. The coated plate was washed twice with PBS solution containing 0.05% Tween 20 and then blocked with PBS solution containing 3% BSA at room temperature for 1 hour. Diluted sera from immunized animals were added to the wells and incubated at room temperature for 2 hours. HRP-conjugated goat anti-mouse IgG (1:10000; Thermo Scientific), HRP-conjugated rabbit anti-mouse IgG1 (1:5000; Invitrogen), and HRP-conjugated rabbit anti-mouse IgG2a (1:5000; Invitrogen) were used as secondary antibodies. The assay was developed using TMB substrate set (BioLegend). Absorbance was measured at 450 nm using a SpectraMax M2 microplate reader (Molecular Device).

Hemagglutination inhibition (HI) assay

HI titers in mouse sera were determined according to standard protocols. Briefly, mouse sera were pretreated with receptor-destroying enzyme (RDE II, Denka Seiken) and preabsorbed with turkey red blood cells (TRBC). After centrifugation to remove TRBC, sera were serially diluted two-fold from the initial 1:10 dilution and mixed with 50 μL of 4 hemagglutinating units of formaldehyde-inactivated H7N9 virus for 1 h at room temperature. Next, 50 μL of 0.5% TRBC suspension was added and incubated at room temperature for 40 min. Finally, the assay plate was tilted and the TRBC flow pattern was read. The HI titer is proportional to the highest serum dilution that completely inhibits hemagglutination. The HI value of serum that fails to inhibit hemagglutination at an initial dilution of 1:10 is 5.

Microneutralization test

MDCK cells were inoculated (3×10 ⁴ cells/well) in 96-well plates for 24 hours to form a monolayer. Pre-immune sera and antisera to H7N9 were pre-treated at 56 ^℃ for 30 minutes to destroy heat-labile non-specific viral inhibitors. The sera were diluted to an initial dilution of 1/10 in DMEM, 200 TCID ₅₀ of H7N9 were added to the wells, and then incubated at 35 ^℃ for 2 hours. Subsequently, the virus-serum mixture was inoculated onto the MDCK monolayer cells, and the cells were incubated at 35 ^℃ . Each serum dilution was prepared in quadruplicate. The cytopathic effect of each well was recorded after 4-5 days of culture. The 50% neutralization (NT ₅₀ ) titer was calculated using the Reed-Muench formula. For calculation purposes, a neutralization titer below the 1:10 starting dilution was assigned a value of 5.

Cytokine production assay

Seven days after the last vaccination, mice were sacrificed, spleen cells were collected and plated at a density of 5×10 ⁶ cells per well in 24-well plates. Cells were stimulated with 5 μg/mL recombinant H7 ectodomain (A/Guangdong/17SF003/2016) produced by ExpiCHO Expression System (Thermo-Fisher). After 3 days of stimulation at 37 ^o C, supernatants were collected and cytokine production was measured. The amount of secreted mouse IFN-γ, IL-5, IL-13, and IL-2 was assessed by ELISA using a matching antibody set (Invitrogen) according to the manufacturer's instructions.

Animal challenge

Four weeks after the last vaccination, mice were challenged intranasally with 2 or 10 times the 50% minimum lethal dose (MLD ₅₀ ) of CBER-RG7D in a 20 μL volume under isoflurane anesthesia. Subsequently, mice were monitored daily for weight loss and survival, and mice were euthanized and scored as dead if weight loss exceeded 20%. To determine the amount of virus in the lungs, lung tissues were homogenized in 2 mL PBS containing 200 U/mL penicillin and 200 μg/mL streptomycin using a gentleMACS® Dissociator (Miltenyi Biotec). After centrifugation at 600 × g for 10 min at 4°C, the clear supernatant was collected for viral RNA quantification.

Quantification of viral RNA

Clarified supernatants from homogenized lung tissues of H7N9-infected mice were collected for viral load detection. RNA was extracted from tissue supernatants using the QIAamp Viral RNA Mini Kit (Qiagen). RNA extracts were reverse transcribed using the FIREScript RT cDNA synthesis kit (Solis Biodyne) and Uni12 primers. Viral RNA was quantified by real-time PCR in the QuantStudio 6 Flex Real-Time PCR System (ABI) using Power SYBR® Green PCR Master Mix (ABI). The viral copy number was estimated by the standard curve method using primers specific for the H7N9 HA gene (Forward: TGAAAATGGATGGGAAGGCC (SEQ ID NO: 2), Reverse: TGCCGATTGAGTGCTTTTGT (SEQ ID NO: 3)).

Statistical analysis

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

result

Aluminum salt/CpG 1018 strongly enhances the immunogenicity of H7N9 WV vaccine.

Figures 1A to 1D are graphs showing the effects of adjuvants on antibody responses induced by H7N9 WV vaccines. BALB/c mice (n=5 per group) were immunized intramuscularly twice with H7N9 WV vaccines combined with aluminum hydroxide (aluminum salt) or CpG 1018. Serum samples were collected at designated time points after the first immunization for evaluation of humoral immune responses. In Figures 1A and 1B, the amount of total IgG antibodies to H7N9 WV was assessed by ELISA. In Figure 1C, H7N9-specific hemagglutination inhibition (HI) antibodies were quantified by hemagglutination inhibition assay. The lower dotted line represents a serum sample at 10 times the initial dilution. The upper dotted line represents a ≥4-fold increase in HI titer, also known as a 4-fold seroconversion. In FIG. 1D , vaccine-induced neutralization activity against H7N9 was assessed by microneutralization assay. The dashed line indicates serum samples at 20-fold initial dilution. The log ₁₀ -converted IgG titers and log ₂ -converted HI and NT titers of sera collected at week 6 after the first immunization were analyzed by two-way ANOVA with Tukey's posttest. * indicates comparison of adjuvant effect between groups with the same antigen dose. # indicates comparison between various antigen doses with aluminum salt/CpG 1018 adjuvant. @ indicates comparison between the 1.5 μg HA group with aluminum salt, the 0.15 μg HA group with aluminum salt, and the 0.15 μg HA group with aluminum salt/CpG 1018. * ^/# P < 0.05, ** ^/##/@@ P < 0.01, *** P < 0.001, **** ^/#### P < 0.0001.

FIG. 1E is a graph showing antibody responses to adjuvanted or non-adjuvanted quadrivalent split virus influenza vaccines. BALB/c mice (n=5 per group) were immunized twice intramuscularly with quadrivalent split virus influenza vaccines containing 0.15 μg HA protein per strain in combination with 30 μg aluminum salt, 10 μg CpG 1018, or without other vaccine adjuvants. Serum samples were collected at week 6 after the first immunization for evaluation of humoral immune responses. In FIG. 1E , total IgG antibody levels against each strain were evaluated by ELISA. Log ₁₀ -converted IgG titers were analyzed by Student's t test. *P<0.05, **P<0.01, ***P<0.001.

Administration of the H7N9 WV vaccine alone induced the production of H7N9-specific total IgG antibodies in a dose-dependent manner (Fig. 1A). Aluminum salt-formulated vaccines significantly increased antibody titers compared with H7N9 WV vaccine alone, regardless of the antigen dose. In contrast, CpG 1018 alone had no significant immunostimulatory effect on the H7N9 WV vaccine at a dose of 0.15 μg HA (data not shown). Importantly, the aluminum salt/CpG 1018-formulated vaccine synergistically increased antibody titers compared with either the aluminum salt-adjuvanted or CpG 1018-adjuvanted vaccines. We also successfully demonstrated the synergistic effect of the aluminum salt/CpG 1018 adjuvant system on quadrivalent split virus (SV) influenza vaccine (Figure 1E). After the first immunization, the IgG antibody titer gradually increased to a peak at week 8, slightly decreased to a level similar to that at week 4, and then continued to week 20 (Figure 1B). When CpG 1018 was combined with the MF59 squalene oil-in-water emulsion (SWE) adjuvant, no synergistic effect was found.

In addition, the protective antibody responses induced by the vaccines were analyzed by hemagglutination inhibition (HI) and microneutralization assays. After vaccination with H7N9 WV vaccine alone, all animals in the 0.015 μg HA group had HI titers below or equal to the detection limit, and 40% of the animals in the 0.15 μg HA group and the 1.5 μg HA group achieved a 4-fold serum antibody conversion at week 6 (Figure 1C). In contrast, the group containing aluminum salt adjuvant showed higher HI titers, with 4-fold serum antibody conversion and neutralization (NT ₅₀ ) titers compared with the group treated without adjuvant (Figure 1C and Figure 1D). In the aluminum salt/CpG 1018 group, the addition of CpG 1018 further increased the HI titer and NT _{50 titer in the 0.15 μg HA group and the 1.5 μg HA group, but not in the 0.015 μg HA group. It is worth noting that at week 6 after vaccination, the specific antibody amount, HI titer, and NT 50 titer} of the 0.15 μg HA group containing the aluminum salt/CpG 1018 combination adjuvant were significantly higher than those of the control group. These results support the dose-saving effect of the aluminum salt and CpG 1018 combination adjuvant on the H7N9 WV vaccine, with a 10-fold reduction in the amount of antigen used. Importantly, the combination of aluminum salt and CpG 1018 more effectively increased the specific antibody quantity, HI titer, and _NT50 titer.

Aluminum salt/CpG 1018 shifted the antibody response to IgG2a dominance.

Figures 2A-2B are graphs showing the effect of adjuvants on IgG isotype antibody titers induced by H7N9 WV vaccines. BALB/c mice (n=5 per group) were immunized intramuscularly twice with H7N9 WV vaccines combined with aluminum hydroxide (aluminum salt) or CpG 1018. Serum samples were collected at week 6 after the first immunization for evaluation of humoral immune responses. In Figures 2A and 2B, antigen-specific immunoglobulin G subclasses IgG1 and IgG2a in mouse sera were quantified by ELISA. The log ₁₀ -converted IgG1 titers and IgG2a titers were analyzed by two-way analysis of variance with Tukey's posttest. *P＜0.05, **P＜0.01, ***P＜0.001, ****P＜0.0001.

IgG2a plays an important role in viral clearance during infection. To evaluate the effect of CpG 1018 on IgG isotype switching, the amounts of IgG1 and IgG2a isotypes were measured by ELISA. After two doses of vaccination, the vaccine containing aluminum salt adjuvant significantly increased the antigen-specific IgG1 titer in the 0.015 μg HA group and the 0.15 μg HA group, and this increased IgG1 titer was offset in the group with the addition of CpG 1018 (Figure 2A). However, this result was not observed in the 1.5 μg HA group. On the other hand, the antigen-specific IgG2a titer was significantly increased in the H7N9 WV group containing aluminum salt adjuvant, regardless of the dose of antigen (Figure 2B). The combination of aluminum salt and CpG 1018 further increased the titer, which was similar to the observation of total IgG titer (Figure 1A). The results showed that CpG 1018 coadjuvantation re-converted the humoral immunity induced by aluminum salt to an IgG2a-dominated antibody response.

Aluminum salt/CpG 1018 induces Th1 polarized immune response

Figures 3A to 3F are graphs showing T cell responses induced by adjuvanted H7N9 WV vaccines. BALB/c mice (n=4 per group) were immunized intramuscularly twice with H7N9 WV vaccines combined with aluminum hydroxide (aluminum salt) or CpG 1018. Spleen cells were collected on day 7 after the second immunization, and the amount of secreted IFN-γ, IL-2, IL-5, and IL-13 (Figures 3A to 3D) was evaluated after restimulation with recombinant H7 protein. In Figures 3E and 3F, the ratio of IFN-γ to IL-5 and the ratio of IFN-γ to IL-13 were calculated. The log ₁₀ -transformed cytokine levels and the IFN-γ/IL-5 and IFN-γ/IL-13 ratios were analyzed by two-way variance with Tukey's post hoc test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

IgG subclass switching is associated with Th1- and Th2-polarized immune responses. To examine the effect of CpG 1018 on aluminum-induced T cell responses, we evaluated T cell responses after immunization with H7N9 WV vaccines with or without aluminum or CpG 1018. Spleen cells from immunized mice were stimulated with recombinant H7 protein. The secretion of Th1-type cytokines (IL-2 and IFN-γ) and Th2-type cytokines (IL-5 and IL-13) was measured by ELISA. The group containing CpG adjuvant produced the lowest amounts of IL-2, IFN-γ, and IL-13 among all groups, and even the amount of IL-5 was undetectable in this group (Figures 3A to 3D). In contrast, vaccination with the WV/aluminum salt-containing vaccine induced higher amounts of IL-2, IFN-γ, IL-5, and IL-13. The addition of CpG 1018 to aluminum salt significantly suppressed the amounts of IL-5 and IL-13 (Fig. 3C and 3D), while the amounts of IL-2 and IFN-γ remained or slightly decreased (Fig. 3A and 3B). The results suggest that the activities of aluminum salt and CpG1018 counteract each other, especially in regulating Th2-mediated immune responses. Trends in the amounts of these cytokines induced by the vaccine were observed in mice receiving 0.15 μg of HA antigen or 1.5 μg of HA antigen. In addition, analysis of the Th1/Th2 ratio showed that H7N9 WV vaccines with CpG 1018 or aluminum salt/CpG 1018 as adjuvants produced a Th1-biased response (Figure 3E and Figure 3F). The IFN-γ/IL-5 ratio and IFN-γ/IL-13 ratio in the 0.15 μg WV/aluminum salt/CpG 1018 group increased by approximately 21.2 times and 6.3 times, respectively, compared with the 0.15 μg WV/aluminum salt group. Therefore, these results indicate that the addition of CpG 1018 biases the immune response toward Th1.

Aluminum salt/CpG 1018 confers strong protection against H7N9 challenge

Figures 4A and 4B are graphs showing the protective efficacy of H7N9 vaccines with or without aluminum salt adjuvants against H7N9 challenge. BALB/c mice (n=8 per group) were immunized intramuscularly twice with PBS or adjuvanted H7N9 WV vaccine. Four weeks after the final immunization, mice were challenged intranasally with CBER-RG7D H7N9 virus at 2 times MLD ₅₀ or 10 times MLD _50. In Figure 4A, survival rate was monitored every day after H7N9 challenge. The significant difference between the PBS group and other groups was calculated using the log-rank test, **p<0.01, ***p<0.001. In Figure 4B, the weight change (%) of mice was monitored every day after H7N9 challenge as an indicator of disease progression. Only the weight results of surviving mice are shown as mean ± SD. Due to lack of data due to death, the significant differences in Figure 4B (days 3 to 9 after infection) were calculated using Student's t test. * indicates comparison between the 1.5 μg HA group containing aluminum salt and the 0.15 μg HA group containing aluminum salt/CpG. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

To evaluate the role of vaccine-induced immune responses in preventing influenza, mice were first exposed to a lethal dose (2 MLD ₅₀ ) of H7N9 virus. The weight of mice was monitored daily after challenge as an indicator of disease progression. After 2 MLD ₅₀ virus challenge, the survival rate of the PBS (control) group was 25%. In contrast, all mice in the H7N9 vaccine groups with aluminum salt adjuvant and aluminum salt/CpG 1018 adjuvant survived, regardless of the dose of antigen. In addition, vaccinated mice also lost less weight compared with the control group, indicating that the H7N9 vaccines containing aluminum salt adjuvant and aluminum salt/CpG 1018 adjuvant were sufficient to provide protection against a moderate dose (2 MLD ₅₀ ) of H7N9 virus challenge. Furthermore, two-way ANOVA showed that the protection against H7N9-induced weight loss was an adjuvant-dependent effect (two-way ANOVA (within the 1.5 μg HA group): 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, P<0.0001; two-way ANOVA (within the 0.15 μg HA group): 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). Although not shown in the figure, Sidak post hoc test showed that there was no significant difference in weight change between groups using the same adjuvant each time, even if the antigen dose was different. It is worth noting that on days 3 to 4 after challenge, in the groups administered with 0.15 μg HA antigen or 1.5 μg HA antigen, the aluminum salt/CpG 1018 combination adjuvant could significantly prevent weight loss compared with the aluminum salt adjuvant.

Next, we used a higher challenge dose (10 MLD ₅₀ ) to more rigorously evaluate the effects of vaccine-induced immunity on survival and weight change. In addition, to evaluate the potential of the aluminum salt/CpG 1018 combination adjuvant, the HA antigen dose was further reduced to 0.15 μg. After virus challenge with a higher dose, all mice in the control group died, and an 87.5% survival rate was observed after administration of 1.5 μg HA/aluminum salt vaccine (Figure 4A). In contrast, all mice in the 0.15 μg HA/aluminum salt/CpG 1018 group survived during the observation period. There were no significant differences between the vaccinated groups based on log-rank comparisons. As shown in Figure 4B, 0.15 μg HA/aluminum salt/CpG showed the best weight loss protection effect in the vaccine-vaccinated group. Interestingly, mice in the 1.5 μg HA/aluminum salt group lost significantly more weight than mice in the 0.15 μg HA/aluminum salt/CpG 1018 group on days 3 and 4 after challenge. These findings emphasize the importance of adding CpG 1018 to vaccines containing aluminum salt adjuvants to improve protection against H7N9 challenge. Notably, after challenge with 10 MLD ₅₀ , mice receiving 0.15 μg HA/aluminum salt/CpG 1018 showed significantly improved disease progression compared with those receiving 1.5 μg HA/aluminum salt ( Figure 4B ), suggesting that the combination of aluminum salt and CpG 1018 can not only achieve a dose-saving effect of H7N9 WV vaccine but also confer better protective immunity against challenge.

To evaluate the role of the aluminum salt/CpG 1018 combination adjuvant in viral clearance, viral RNA in lung tissue was analyzed. The amount of viral RNA in the lungs of H7N9-infected mice was quantified by RT-qPCR on day 3 after challenge (n=6 per group). When challenged with 10 MLD ₅₀ virus, up to 7.2 log (copy number/mL) of viral RNA was detected in the lungs on day 3 after challenge. Although not shown in the figure, consistent with the survival rate in Figure 4A, the amount of viral RNA in the 0.15 μg HA/aluminum salt/CpG 1018 group was similar to that in the 1.5 μg HA/aluminum salt group, and the amount of viral RNA showed at least a 2.6 log reduction compared with the control group.

Aluminum salt/CpG 1018 increases cross-reactivity of H7N9 vaccine

To investigate the potential cross-protective immunity of adjuvanted WV vaccines against various H7N9 strains, we evaluated the WHO-selected H7N9 candidate vaccines, including A/Anhui/1/2013 (NIBRG-268) H7N9 recombinant virus and A/Gansu/23277/2019 (IDCDC-RG64A) H7N9 recombinant virus, which were derived from the first wave of the outbreak or infected cases in March 2019. Even though not shown in the figure, consistent with the results shown in Figure 1C, the homologous HI titers generated by the vaccine containing aluminum salt/CpG 1018 adjuvant were higher than those induced by the vaccine containing aluminum salt adjuvant. Overall, the geometric mean HI titers against Anhui virus were approximately 2-3 times lower than those against homologous strains, regardless of the type of adjuvant. Notably, only the aluminum salt/CpG 1018 adjuvant system induced heterologous HI titers against Anhui virus that reached a seroprotective threshold HI titer of ≥40, while approximately 50% of the animals in the aluminum salt adjuvant group did not reach this level. In addition, cross-reactivity against the Gansu strain was significantly lower than that against the Anhui strain, with all animals in the aluminum salt adjuvant group and the aluminum salt/CpG 1018 adjuvant group showing HI titers below the detection limit.

Antigen conservation is an important strategy for pandemic vaccine development due to global vaccine production constraints during disease outbreaks. However, multiple clinical studies have shown that current influenza vaccines adjuvanted with aluminum (aluminum salts) fail to adequately enhance immune responses and fail to meet licensing standards. In addition, previous studies have shown that the effect of CpG on antigen binding to aluminum salts depends on buffer conditions and antigen properties, and not all aluminum salt/CpG combination adjuvants can produce synergistic effects on all types of vaccines. Therefore, the effect of clinically approved CpG 1018 on the immune response induced by aluminum salt-adjuvanted killed whole virus (WV) vaccines was unexpected.

In the present invention, we demonstrated that the formulation of aluminum hydroxide with CpG 1018 can not only reduce the use of antigens, but also induce better protective immunity against the H7N9 virus than the formulation using aluminum salt alone, and this knowledge can be used to solve the dilemma of insufficient vaccine production capacity during pandemics. In addition, the Th1 polarization and IgG2a-dominated immune response induced by the aluminum salt/CpG 1018 formulation vaccine may be more helpful in fighting future pandemics. Therefore, this aluminum salt/CpG 1018 adjuvant system provides a platform to enhance the vaccine efficacy and production capacity of existing aluminum-formulated vaccines against various diseases, especially avian influenza and seasonal influenza vaccines for elderly individuals.

Although the present disclosure has been described through its embodiments, it should be understood that many other possible modifications and variations may be made without departing from the spirit of the present disclosure and the scope of the claimed patent.

Figures 1A to 1D are graphs showing the effects of adjuvants on antibody responses induced by H7N9 WV vaccines. Figure 1E is a graph showing antibody responses to adjuvanted or non-adjuvanted quadrivalent split virus influenza vaccines. Figures 2A to 2B are graphs showing the effects of adjuvants on IgG isotype antibody titers induced by H7N9 WV vaccines. Figures 3A to 3F are graphs showing T cell responses induced by adjuvanted H7N9 WV vaccines. Figures 4A and 4B are graphs showing the protective efficacy of H7N9 vaccines with or without aluminum salt adjuvants against H7N9 challenge.

TW202442674A_113108494_SEQL.xml

Claims (14)

An influenza virus immunogenic composition comprises: a hemagglutinin antigen, which is an inactivated whole virus H7N9 vaccine or a quadrivalent split virus influenza vaccine; a first adjuvant, which is aluminum hydroxide; and a second adjuvant, which is a CpG oligonucleotide having a sequence of 5' - TGACTGTGAACGTTCGAGATGA-3 ' (SEQ ID NO: 1). The immunogenic composition as described in claim 1, wherein the inactivated whole virus vaccine is an inactivated whole virus H7N9 vaccine. The immunogenic composition as described in claim 1, wherein the weight ratio of the first adjuvant to the second adjuvant is between 10:1 and 1:10. The immunogenic composition as described in claim 3, wherein the weight ratio of the first adjuvant to the second adjuvant is between 5:1 and 1:5. The immunogenic composition as described in claim 4, wherein the weight ratio of the first adjuvant to the second adjuvant is between 5:1 and 1:1. An immunogenic composition as described in claim 1, wherein the human dose of the immunogenic composition comprises 0.1 μg to 60.0 μg of HA protein. The immunogenic composition as described in claim 1, wherein the human dose of the immunogenic composition comprises 100 μg to 800 μg of the first adjuvant. An influenza virus immunogenic composition is used for preparing a drug for immunizing a subject against influenza virus infection, wherein the influenza virus immunogenic composition comprises: a hemagglutinin antigen, which is an inactivated whole virus H7N9 vaccine or a quadrivalent split virus influenza vaccine; a first adjuvant, which is aluminum hydroxide; and a second adjuvant, which is a CpG oligonucleotide having a sequence of 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 1). The use as described in claim 8, wherein the inactivated whole virus vaccine is an inactivated whole virus H7N9 vaccine. The use as described in claim 8, wherein the weight ratio of the first adjuvant to the second adjuvant is between 10:1 and 1:10. The use as described in claim 10, wherein the weight ratio of the first adjuvant to the second adjuvant is between 5:1 and 1:5. The use as described in claim 11, wherein the weight ratio of the first adjuvant to the second adjuvant is between 5:1 and 1:1. The use as described in claim 8, wherein the human dose of the immunogenic composition comprises 0.1 μg to 60.0 μg of HA protein. The use as described in claim 8, wherein the human dose of the immunogenic composition comprises 100 μg to 800 μg of the first adjuvant.