Compositions, vaccines, and methods for treating influenza A

Abstract

The present invention provides a composition comprising polymeric nanoparticles encapsulating an antigen and an agonist, wherein the antigen is an M2e peptide. The composition can induce an immune response to influenza A. A method for inducing an immune response to influenza A is also provided.

Description

[Technical Field]

[0001] This disclosure relates to compositions, vaccines and methods for treating influenza A. [Background technology]

[0002] Influenza is an annual epidemic that poses a persistent public health concern, resulting in serious morbidity and mortality rates. As a seasonal event, influenza is estimated to cause 3 to 5 million severe illness cases and up to 650,000 respiratory deaths annually. Vaccination efforts against the highly mutative virus are hampered by the virus's tendency to undergo antigenic shift and drift, in which viral gene recombination and point mutations in receptor-binding proteins can impair the protection provided by existing antibodies. Therefore, current hemagglutinin (HA)-based vaccines require annual updates, and their protective effect is often reduced when the vaccine does not match the circulating virus strain. To address the shortcomings of current influenza vaccines, various vaccine designs based on both B-cell and T-cell-based immunogens have been proposed for universal influenza vaccination. Among these, peptide antigens derived from the extracellular domain of influenza virus ion channel membrane matrix protein 2 (M2e) offer a unique target for mediating antibody-dependent cell-mediated cytotoxicity (ADCC), thereby eradicating infected cells presenting M2e before the virus is released and transmitted. Due to the highly conserved nature of M2e across human seasonal influenza A viruses, it is an attractive candidate for universal influenza vaccine development. However, the low immunogenicity of small peptide antigens has been a major barrier to its clinical transition, with previous clinical trials of M2e vaccine candidates showing inadequate and diminished humoral responses despite repeated dosing. More recently, the emergence of recombinant protein strategies, carrier technologies, and immunostimulatory adjuvants has rekindled enthusiasm for M2e-based universal influenza vaccines. However, despite advances in antigen and vaccine design, multi-dose regimens are still required to boost the immunogenicity of the peptides, and M2e antigens are often relegated to a complementary role to other immunogens due to their partial protective power.Given the immense value of simplified vaccine regimens and ease of formulation in improving vaccination logistics and public health strategies, a single-shot M2e peptide vaccine capable of providing broad-spectrum, sustained influenza protection remains a highly desirable but difficult-to-achieve goal. The nanoparticle vaccine strategy, as described herein, is conceived to enhance antigen availability in lymph node follicles and the assistance of T cells in order to boost the immunogenicity of M2e antigens.

[0003] To construct M2e nanovaccines in which peptide antigens and immunological adjuvants are densely encapsulated together, the inventors demonstrate an asymmetric ion stabilization strategy for preparing polymeric nanoshells. The stabilization strategy mimics the asymmetric stabilization mechanism underlying nanoscale biological vesicle formation, which overcomes the energetic barrier in the formation of strain at the nanoscale, thereby preventing the collapse of nanoemulsions and enabling the consistent preparation of antigen-loaded nanocapsules in the absence of surfactants or stabilizers. Cyclic diGMP (cdGMP), a putative STING (interferon gene stimulant) agonist, is applied as a selected adjuvant due to its role in inducing cytokines that promote Th1-biased production, which are type I interferons. Anti-M2e-based ADCCs against influenza-infected cells require specific antibody isotypes. Based on evaluation of M2e nanoshells (NS(M2e+cdGMP)), the inventors demonstrate that the nanovaccine is highly effective in promoting the induction of IFNγ+1 type helper T cells (Th1), germinal center formation, and Th1-biased anti-M2e production. Furthermore, a single dose of M2e nanoshell inoculation provided thorough and long-lasting protection against lethal influenza attacks, enabling the elimination of viral titers and the prevention of pulmonary immunological pathology and tissue damage. Complete protection against heterosubtype influenza attacks was also achieved with a single-shot regimen. Intrigued by the unusual humoral response of M2e nanoshells, which have a differential design compared to conventional vaccines that present immunogens on a carrier surface for B cell engagement, the inventors investigated how the nanoshells modulate the distribution of the encapsulated antigen. It was observed that nanoshells enable sustained peptide exposure in B cell follicles for antibody induction by traversing the network from M2e peptides to follicular dendritic cells (FDCs) in a complement-dependent manner.In particular, the incorporation of commonly used polyethylene glycol surface coatings onto M2e nanovaccines invalidated peptide retention in the FDC network and reduced antibody induction, highlighting the importance of surfactant-free nanoshell design to maximize follicular targeting (Figure 1). This study provides a highly effective and translationally viable universal influenza vaccine candidate and introduces FDC-targeted nanoparticle design to improve antigenic immunogenicity. [Overview of the project]

[0004] In one aspect of the disclosure of the present invention, a composition is provided for improving antigenic immunogenicity. The composition comprises polymeric nanoparticles encapsulating an antigen and an adjuvant, wherein the antigen is an M2e peptide; and the polymeric nanoparticles comprise a water-impermeable polymeric shell and one or more aqueous cores encapsulated by the polymeric shell. Preferably, the polymeric shell has an outer diameter of 50 to 150 nm. Preferably, the M2e peptide contains the sequence SLLTEVETPIRNEWGCRCNGSSD, SEQ ID NO: 1, SLLTEVETPIRNEWGCRCNDSSD, SEQ ID NO: 2, or SLLTEVETPTRSEWECRCSDSSD, SEQ ID NO: 3. Preferably, the adjuvant is an agonist. Preferably, the agonist is a STING agonist containing cyclic diGMP, cGAMP, poly(I:C), or CpG.

[0005] Preferably, the polymeric shell comprises a short PLGA polymer having a molecular weight of 6,000 to 18,000 Da. Preferably, the polymeric shell is surfactant-free. In another aspect of the disclosure of the present invention, a method for treating a disease is provided. The method comprises the step of administering the composition according to claim 1, which is capable of physically associating with cells, to a subject in need thereof. Preferably, the disease is influenza A. Preferably, administration includes intravenous injection, subcutaneous injection, or intraperitoneal injection. Preferably, the subject is a human or a bird.

[0006] In yet another aspect of the disclosure of the present invention, a vaccine capable of inducing an immune response to influenza A is further provided. The vaccine comprises the composition described above. Preferably, the vaccine is a single-dose vaccine formulation. Preferably, the polymeric shell has an outer diameter of 50 to 150 nm. Preferably, the M2e peptide contains the sequence SLLTEVETPIRNEWGCRCNGSSD, SEQ ID NO: 1, SLLTEVETPIRNEWGCRCNDSSD, SEQ ID NO: 2, or SLLTEVETPTRSEWECRCSDSSD, SEQ ID NO: 3. Preferably, the adjuvant is an agonist.

[0007] Preferably, the agonist is a STING agonist containing cyclic diGMP, cGAMP, poly(I:C), or CpG. Preferably, the polymeric shell comprises a short PLGA polymer having a molecular weight of 6,000 to 18,000 Da. Preferably, the polymeric shell is surfactant-free.

[0008] In yet another aspect of the disclosure of the present invention, a method for neutralizing a viral infection is further provided. Methods for neutralizing viral infections include the step of priming the aforementioned vaccine to the target group that needs it. Preferably, the method further includes a step of boosting the target with a vaccine.

[0009] Preferably, the priming step and the boosting step are by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intra-bronchial, intraperitoneal, intra-capsular, intra-cartilaginous, intra-cavity, intracerebellar, intraventricular, intra-colonic, intra-cervical, intra-gastric, intra-hepatic, intra-myocardial, intra-osseous, intra-pelvic, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intra-rectal, intra-renal, intra-retinal, intra-spinal, intra-synovial, intra-thoracic, intra-uterine, intra-vesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. Preferably, the priming step and the boosting step are by subcutaneous or intranasal. Preferably, the subject is a human or a bird.

Brief Description of the Drawings

[0010] [Figure 1] Figure 1 illustrates the design (left), application (upper right), and mechanism (lower right) of a single-shot M2e-based influenza vaccine for broad influenza protection. [Figure 2]Figure 2 illustrates the preparation and characterization of the M2e nanoshell vaccine. (A) Schematic diagram of the asymmetrically stabilized nanoemulsion process for cryoEM imaging of the nanoshell vaccine preparation and polymeric nanoshells. The absence of charged polymer or differential ionic buffer caused emulsion collapse. (B) Quantification of M2e peptide encapsulation after different emulsion processes for nanoparticle preparation. (C) CryoEM visualization of the M2e nanoshell vaccine encapsulating both M2e peptide antigen and cyclic cdGMP. Scale bar = 100 nm. (D) Encapsulation efficiency of M2e peptide and cdGMP by the M2e nanoshell vaccine. (E) Size and zeta potential of empty nanoshells (NS(empty)) and M2e nanoshell vaccine (NS(M2e+cdGMP)) were measured by dynamic light scattering. (F) Dynamics of dissociation of M2e peptide and cdGMP from the nanoshell vaccine at pH 5 and pH 7. (G) Image of the M2e nanoshell vaccine after freeze-drying and reconstitution. (H) The size and zeta potential of the M2e nanoshells, as measured by DLS, show comparable physicochemical properties before and after freeze-drying. [Figure 3] Figure 3 illustrates the quantification of M2e peptide and cdGMP encapsulation in nanoshells by micro-BCA assay and HPLC. (A) Standard curve and representative image of M2e peptide quantification using micro-BCA protein quantification assay. (B) M2e peptide encapsulation efficiency in nanoshells prepared using internal aqueous phases containing various concentrations of M2e peptide. (C) Quantification of cyclic cdGMP encapsulated in nanoshells by HPLC. [Figure 4] Figure 4 illustrates the values ​​for nanoshells obtained from nanoparticle tracking analysis. For a sample containing 15 μg / mL of nanoparticles, the NTA indicates approximately 1.2 × 10⁹ nanoparticles. This value corresponds to approximately 8 × 10¹¹ nanoshells per 1 mg of PLGA. Each line in Figure 4 represents an independent nanoparticle tracking analysis of the same M2e vaccine sample. The average particle size is calculated based on the average of three independent measurements. [Figure 5] Figure 5 illustrates the encapsulation of the M2e peptide in the nanoshells before and after lyophilization. The nanoshells retained their encapsulated material, and peptide loss after lyophilization and reconstitution was minimal. [Figure 6]Figure 6 illustrates anti-M2e induction and ADCC activity after M2e nanoshell vaccination in mice. (A) Titer of M2e-specific IgG after single-shot immunization with PBS, M2e peptide, M2e peptide containing Alum adjuvant, and M2e nanoshell vaccine in mice. (B) Ratio of M2e-specific IgG2a to IgG1 titer in immunized Balb / C mice at 35 days post-vaccination. Error bars represent mean ± SEM (N=5). (C) Acetone-fixed MDCK cells infected with atypical influenza to evaluate antibody binding to M2e bound to cells by anti-M2e from mouse serum. Immunofluorescence assays were performed using mouse serum obtained at 42 days. Nuclei were stained with DAPI. H1N1:A / Puerto Rico / 8 / 1934(H1N1); H3N2:A / HKx31(H3N2). Scale bar = 100 μm. (D) Antibody-dependent cell-mediated cytotoxicity (ADCC) surrogate assay using mouse serum obtained 42 days after vaccination of H1N1-infected MDCK cells. Data are presented as mean ± SEM. (N=3). (E) CryoEM images showing the morphology of nanoshells encapsulating either the M2e+cdGMP combination or the M2e+CpG-ODN1826 combination. (F) Evaluation of human STING activation by SEAP reporter cells using free cdGMP, NS(cdGMP), or empty NS after 24-hour incubation. (G) Evaluation of human TLR9 activation by SEAP reporter cells using free CpG-ODN2395, NS(CpG-ODN2395), or empty NS after 24-hour incubation. (H) M2e-specific IgG antibodies in BALB / C mice immunized via the subcutaneous pathway with NS(M2e+cdGMP), NS(M2e+CpG), NS(M2e)+free cdGMP, or NS(M2e). Error bars represent mean ± SEM (N=5). (I) M2e-specific IgG antibodies in C57BL / 6 or AGB6 mice immunized with NS (M2e + cdGMP). Error bars represent mean ± SEM (N=3). Statistical analysis was performed by one-way ANOVA or Student's t-test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 7] Figure 7 illustrates the titers of M2e-specific IgG1 and IgG2a after single-shot immunization with PBS, M2e peptide, M2e peptide containing Alum adjuvant, and M2e nanoshell vaccine in mice 35 days post-vaccination. Error bars represent mean ± SEM (N=5). [Figure 8] Figure 8 illustrates how the M2e STING agonist nanoshell promotes a lymph node environment favorable for Th1-biased antibody production. (A, B) M2e-specific IFNγ+CD4+ T cell response in immunized mice as determined by intracellular cytokine staining 7 days after primary immunization. Error bars represent mean ± SEM (N=3). (C, D) Frequency of follicular helper T cells and (E, F) GL7+ germinal center B cells in the aspiration area lymph nodes of immunized mice 14 days after immunization. Error bars represent mean ± SEM (N=3). Statistical analysis was performed by one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). (G) Perfused popliteal lymph nodes of immunized mice 14 days after vaccination were fixed and embedded in paraffin. Hematoxylin / eosin staining (H&E) was performed to identify follicular hyperplasia (white arrows) and paracortical hyperplasia (black arrows) in the lymph nodes. Scale bar = 500 μm. (H) Sections of popliteal lymph nodes were stained with anti-GL-7 antibody (brown) for germinal center identification. Scale bar = 200 μm. [Figure 9]Figure 9 illustrates the evaluation of antiviral protection by single-shot M2e nanoshell vaccination. (A) Vaccination schedule and viral attack for single-shot and prime-boost immunization regimens. (B) Evaluation of anti-M2e titers from mouse serum collected 35 days after primary vaccination. (C) Body weight change and (D) survival rate of mice after A / Puerto Rico / 8 / 1934(H1N1) infection during attack at 42 days with a viral dose of 3 × 10⁵ PFU via the intranasal route. (N=5). (E) Lung viral titers were evaluated 3 days after viral infection. (N=3). (F) Hematoxylin / eosin staining (H&E) was performed to identify lymphocyte infiltration and perivasculitis (upper panel). Scale bar = 200 μm. Lung tissue was also monitored for bronchiolar injury (lower panel), including the presence of necrotic epithelial cells (white arrows) and airway wall thickening (black arrows). Scale bar = 100 μm. (G) Anti-M2e titers in mice after primary M2e nanoshell vaccination over a period of 273 days. (N=5). (H) On day 273, the nasal cavity of mice was attacked with 3 × 10⁵ PFU of A / Puerto Rico / 8 / 1934 (H1N1), and post-infection (H) body weight change and (I) survival rate were evaluated. (N=5). Error bars represent mean ± SEM. Statistical analysis was performed by one-way ANOVA. Survival rates were analyzed using the log-rank test (**p<0.01, ***p<0.001, ****p<0.0001; ns, not significant). [Figure 10] Figure 10 illustrates the serum titers for binding to influenza virus-infected MDCK cells and serum ADCC activity in mice vaccinated with either one or two doses of the M2e nanoshell vaccine. (A) Immunofluorescence assays show that serum from mice vaccinated with the two-dose nanoshell vaccine regimen produced more antibody binding and immunofluorescence against both H1N1 and H3N2-infected MDCK cells compared to serum from mice vaccinated with one dose of nanoshell. (B) ADCC surrogate assays show comparable ADCC activity between serum from mice vaccinated with one or two doses of nanoshell. [Figure 11]Figure 11 illustrates a hemagglutination inhibition assay to test the neutralizing ability of anti-M2e antibodies against influenza virus. Anti-M2e antibodies did not show observable neutralizing ability against the influenza pathogen. [Figure 12] Figure 12 illustrates the spatiotemporal control of M2e peptide antigen distribution in lymph node follicles by nanoshell carriers. (A) Schematic diagram illustrating the effect of nanoshell surface properties on complement activation and lymph node distribution. (B) Dynamic light scattering characterization of size and zeta potential of PEG-free M2e STING agonist nanoshells (M2e NS) and PEG-coated M2e STING agonist nanoshells (M2e PEG-NS). (N=3). (C) Concentration of activated complement protein C3a in BALB / c mouse serum (control) and after incubation with zymosan, PEG-free nanoshells (M2e NS), or pegylated nanoshells (M2e PEG-NS). (N=3). (D) Titer of M2e-specific IgG in mice 35 days after immunization with M2e NS or M2e PEG-NS. Error bars represent mean ± SEM (N=5). Statistical analysis was performed by unpaired t-tests (**p<0.01). (E) To track M2e antigen distribution over a 14-day period, BALB / c mice were inoculated with nanoshells containing M2e peptide (M2e-A647) conjugated with fluorescent A647. Follicular dendritic cells (FDCs) were labeled with anti-CD35 antibody in situ, excised dLNs were cleaned, and imaged by confocal microscopy (CD35 in blue; M2e-A647 in red). Scale bar = 200 μm. (F, G) Co-localization of M2e with subcapsular macrophages and lymph node follicles was evaluated by image analysis of the coordination of M2e-A647 signaling with anti-CD35 signaling and lymph node boundary, respectively. (H) Visualization of the expanded distribution of M2e in lymph node follicles 3 days after subcutaneous administration of either M2e NS or M2e PEG-NS. Scale bar = 200 μm. (I) Quantification of M2e NS and M2e PEG-NS in lymph node follicles 3 days after inoculation. (N=3). (****p<0.0001) [Figure 13]Figure 13 illustrates an image of a clear lymph node after X-clarity treatment. Popliteal lymph nodes were excised and tissue cleaning was performed to track the distribution of M2e peptides. [Figure 14] Figure 14 illustrates fluorescence images of lymph nodes in the lymphatic drainage region after inoculation of mice with Alexa Fluorophore 647-conjugated M2e peptide. Images were obtained 4 hours after injection of the fluorescently labeled M2e peptide into the sole of the foot. [Figure 15] Figure 15 illustrates a comparison of M2e antigen retention in mouse lymph node follicles after M2e nanoshell inoculation. Due to complement factor depletion, mice received intravenous injection of cobra venom factor (CVF) 3 hours prior to nanoshell injection into the paw. Then, on day 3, lymph nodes in the inflow region were excised for evaluation. (A) Representative image of lymph node follicles showing reduced M2e-A647 retention in CVF-treated mice. (B) Quantification of M2e-A647 retention in control and CVF-treated mice. N=3. [Figure 16] Figure 16 illustrates the evaluation of the M2e nanoshell vaccine against atypical influenza attacks. (A) Vaccination and viral attack schedule. Free M2e peptides containing PBS, M2e NS, or Alum adjuvant were inoculated subcutaneously into mice. On day 42, the nasal cavity of mice was attacked with 3 × 10⁶ PFU of influenza A / HKx31(H3N2) or 2.5 × 10⁶ PFU of pandemic 2009H1N1(pdmH1N1). (B) Change in mouse body weight and (C) survival rate after influenza A / HKx31(H3N2) infection. (D) Change in mouse body weight and (E) survival rate after influenza pandemic 2009H1N1(pdmH1N1) infection. Error bars represent mean ± SEM. (N=5). Survival rates were analyzed using the log-rank test (***p<0.001). [Figure 17]Figure 17 illustrates an experimental design to investigate the immune response in chickens induced by M2e peptide combined with cyclic GMP-AMP (cGAMP) adjuvant administered via ocular and nasal immunization. Specific pathogen-free (SPF) chickens were divided into three groups: a treatment group receiving nanoshells containing M2e peptide (40 μg per chicken) and cGAMP (5 μg per chicken), a control group receiving free M2e peptide and cGAMP, and a mock group receiving phosphate-buffered saline (PBS). Chickens (n=5 per group) were immunized on day 0, and tissue collection was scheduled for post-immunization day 21. Immunization was performed via ocular and nasal administration to ensure the vaccine reached the upper respiratory tract, the primary site for initiating a mucosal immune response. Tissues were collected from the Harderian glands, lungs, cecal tonsils, and spleen for immunohistochemical (IHC) analysis. Within these tissues, the focus was on detecting cells producing IgG, IgA, and MHC II, which are indicators of both humoral and cellular immune responses. IHC staining was performed at 100× and 200× magnifications, used to identify IgA and IgG-producing cells, for each antibody and tissue type, at specific dilutions and incubation times. MHC-II-producing cells were also identified using a similar method. [Figure 18] Figure 18 shows example photographs of IgA-producing cells at 100× and 200× magnification. [Figure 19] Figure 19 shows example photographs of IgA-producing cells at 100× and 200× magnification. [Figure 20] Figure 20 shows example photographs of IgG-producing cells at 100× and 200× magnification. [Figure 21] Figure 21 shows example photographs of IgG-producing cells at 100× and 200× magnification. [Figure 22] Figure 22 illustrates the quantification of the area covered by IgG-producing cells, which showed a significant increase in the nanoshell group. Significant differences were observed in the Harderian glands and lungs compared to the control and mock groups. [Figure 23]Figure 23 illustrates photographs of MHC-II-producing cells at 100× and 200× magnification, respectively. [Figure 24] Figure 24 illustrates photographs of MHC-II-producing cells at 100× and 200× magnification, respectively. [Modes for carrying out the invention]

[0011] The aforementioned and other aspects of the disclosure of the present invention will be described in more detail below with respect to other embodiments described herein. It will be understood that the present invention can be embodied in various forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure is thorough and complete and so that the scope of the invention can be fully conveyed to those skilled in the art. The terms used in the description of the inventions herein are for the sole purpose of describing specific embodiments and are not intended to limit the inventions. Where used in the description of the inventions and in the appended claims, the singular forms "a," "an," and "the" are also intended to include the plural forms unless the context clearly indicates otherwise. As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “have,” “have,” “contain,” “contain,” “characterized by,” or any other variations thereof are intended to encompass non-exclusive inclusions, subject to any expressly indicated limitations. For example, a composition, mixture, process, or method comprising an enumeration of elements is not necessarily limited to those elements alone, but may include other elements not expressly enumerated or that are inherently associated with such composition, mixture, process, or method.

[0012] The transitional phrase "consisting of" excludes all unspecified elements, steps, or components. When described in the claims, such a transitional phrase is expected to exclude materials other than those enumerated, except for any impurities that typically accompany them, from being included in the claim. When the phrase "consisting of" appears in a clause of the claim body rather than immediately following the preamble, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole. The transitional phrase "essentially from" is used to define a composition or method that includes materials, steps, features, components, or elements in addition to those literally disclosed, but these additional materials, steps, features, components, or elements do not substantially affect the basis and novel features of the claimed invention. The term "essentially from" occupies an intermediate position between "includes" and "consists of." If the applicant defines an invention or part thereof using open-ended terms such as “including” or “consisting of,” it should be readily apparent that such description will be interpreted as describing such invention using the terms “essentially consisting of” or “consisting of” (unless otherwise specified).

[0013] The term "approximately," as used herein, is used to indicate that a value includes, for example, errors between measuring devices, inherent variations in the methods employed to determine the value, or variations present between the subjects of study. Typically, this term means, depending on the context, to include variations of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or less. The use of the term “or” in the claims is used to mean “and / or” unless it is explicitly indicated that it refers only to the options or that the options are mutually exclusive; however, this disclosure supports the definition that it refers only to the options and “and / or”. "To treat" or "treatment" as used herein means administering a therapeutic composition to a subject for the purpose of treating, reducing, alleviating, improving, preventing, or mitigating a disorder, symptoms of a disorder, conditions secondary to a disorder, or predisposition to a disorder.

[0014] "Subject" as used herein refers to a mammalian subject that has been diagnosed with, or is suspected of having or developing, a disease such as cardiovascular disease, cancer, autoimmune disease, or infection. Exemplary patients may be humans, apes, dogs, pigs, cattle, cattle, horses, goats, sheep, rodents, and other mammals that have a disease that may benefit from treatment. "Administering" or "dosing" in this specification means providing the treatment kit of this application to a subject. Administration can be performed, for example, but is not limited to, parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intra-bronchial, intraperitoneal, intracapsular, intracartilaginous, intrasinusial, intracavitary, intracerebellar, intraventricular, intracolon, intracervix, intrastomal, intrahepatic, intramyocardium, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal cord, intrabursal, intrathoracic, intrauterine, intrabladder, bolus, vagina, rectum, buccal, sublingual, intranasal, and percutaneously. For example, injection can be performed by intravenous (IV), subcutaneous (SC), intradermal (ID), intraperitoneal (IP), or intramuscular (IM) injection. One or more such routes may be employed. Parenteral administration may be performed, for example, by bolus injection or by prolonged, stepwise perfusion. Alternatively, or concurrently, administration may be performed via oral route.

[0015] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for teaching relating to the sentences and / or paragraphs in which the references are presented. [Examples]

[0016] Chapter on Experiments Ethical statement All animal experiments were conducted under the Institutional Animal Care and Use Committee (IACUC) protocol (#15-12-893) approved by Academia Sinica in Taiwan.

[0017] Cells and viruses Maidin-Derby canine kidneys (MDCK) were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). MDCK cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) containing 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 1% penicillin / streptomycin / amphotericin B (PSA) (Invitrogen), and cultured at 37°C and 5% CO2. When infecting MDCK cells with influenza virus, an infection medium (DMEM containing 0.075% BSA, 1% non-essential amino acids, 1% sodium pyruvate, 1% HEPES, 1% PSA, and 2 μg / mL TPCK-treated trypsin) was used. Influenza A virus strain A / Puerto Rico / 8 / 1934 (H1N1) was kindly provided by Professor Shin-Ru Shih of Chang Gung University. Influenza A virus strain A / HKx31 (H3N2) was kindly provided by Professor Hung-Chih Yang of National Taiwan University College of Medicine. Furthermore, A / California / 7 / 2009 (pdmH1N1) was kindly provided by Professor Li-Min Huang of National Taiwan University Hospital. All viruses were grown in the allantois space of 10-day-old SPF chicken embryos (JD-SPF Biotech, Miaoli, Taiwan). Viral titers were determined by plaque assay as previously described.

[0018] Preparation of M2e nanoshells For the study of this invention, the consensus M2e peptide sequence [TEVETPIRNEWGCRCNDSSD] was adopted (Genescript; purity > 95%). Nanoshells were prepared by an optimized water-oil-water biemulsion according to previously reported protocols. The internal aqueous phase was prepared by dissolving the desired encapsulation material in 200 mM NaHCO3 buffer. The polymer solution was prepared by dissolving 75 mg / mL of 50:50 poly(DL-lactide-co-glycolide) (PLGA; Mw 7,000~17,000; Sigma-Aldrich) with carboxyl groups at its terminus in ethyl acetate. For a typical preparation of the M2e nanoshell vaccine, 20 μL of an aqueous solution containing 40 mg / mL of M2e peptide and 5 mg / mL of cdGMP (InvivoGen) was emulsified in 200 μL of polymer solution on ice using an ultrasonic probe of an ultrasonic generator in a pulse mode of 40% amplitude with 1 and 2 second on-off intervals for 1 minute. The first emulsion was then added to 5 mL of 10 mM NaHCO3, which was then sonicated with the probe at 30% amplitude with 1 and 2 second on-off intervals for 2 minutes. The emulsion was then poured into 8 mL of water and heated at 40°C in a fume hood with gentle stirring for solvent evaporation. After 1 hour of solvent evaporation, nanoparticles were collected using an Amicon filter (Sigma-Aldrich) with a molecular weight cutoff (MWCO) of 100 kDa to remove any unencapsulated material. For asymmetrically stabilized nanoemulsions, PLGA (50:50 lactide:glycolide, Mw 7,000-17,000; Sigma-Aldrich) with ester terminus was used as an uncharged polymer. Replacing the internal aqueous buffer with 10 mM NaHCO3 created a condition where differential ionic buffer was absent. For NS(M2e+CpG-ODN) and NS(M2e), the internal aqueous phase was replaced by 20 μL of 40 mg / mL M2e peptide with 5 mg / mL CpG-ODN 1826 (InvivoGen) and 40 mg / mL M2e peptide, respectively.For PEG-coated M2e nanoshell preparations, the oil phase was replaced with 200 μL of ethyl acetate containing 50 mg / mL of carboxyl-terminated PLGA and 10 mg / mL of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugate polyethylene glycol (DSPE-PEG(2000)-OH; Nanocs). For nanoshells encapsulating fluorescently labeled M2e peptide antigens, the M2e peptide was synthesized using Alexa Fluor 647 (Creative peptides) conjugated to its N-terminus. The collected nanoparticles were evaluated by dynamic light scattering, nanoparticle tracking analysis, cryoEM, and microBCA assay (microBCA protein assay kit; ThermoFisher Scientific), and physicochemical properties, particle concentration, particle morphology, peptide encapsulation efficiency, and cdGMP encapsulation were evaluated by HPLC. The lyophilized M2e nanoshell vaccine was prepared by suspending the nanoshells at a concentration of 50 mg / mL in 10 mM disodium phosphate and 25% sucrose prior to freezing and lyophilization. Before each immunization study, the nanoparticles were reconstituted, diluted to the desired concentration with water, and the osmotic pressure was adjusted with sucrose solution.

[0019] Dynamics of antigen and adjuvant release The kinetics of antigen-adjuvant dissociation under physiologically appropriate conditions were characterized using dialysis experiments in two different pH environments (pH 5 and 7). In these experiments, M2e nanoshells were loaded into dialysis tubes (10 kDa MWCO, Slide-A-Lyzer MINI Dialysis Device, Thermo Fisher Scientific), and samples were collected at predetermined time points for quantification of M2e peptides and cdGMP.

[0020] Animal immunization and serum collection Except for comparisons between C57BL / 6 and AGB6 mice, all animal experiments were performed using BALB / c mice. SPF BALB / c 7-week-old female mice and C57BL / 6 mice were purchased from the National Laboratory Animal Center, Taipei, Taiwan. AGB6 mice were kindly provided by Dr. Lin, Yi-Ling of the Institute of Biomedical Sciences, Academia Sinica, Taiwan. The mice were housed in an animal facility maintained by the Institute of Biomedical Sciences, Academia Sinica. For nanoshell vaccination, mice were subcutaneously (sc) immunized via the base of the tail with 10 μg / dose of M2e peptide and 1.25 μg / dose of cdGMP nanoparticles in 100 μL of a solution containing 250 μg of nanoshells. Free M2e peptide was administered together with 10 μg of M2e peptide solubilized in 100 μL of PBS. For M2e peptides containing an alum adjuvant, 10 μg of M2e peptide was mixed with 100 μL of commercial alum salt adjuvant (approximately 400 μg of aluminum hydroxide) for administration. For vaccines containing an MF59 adjuvant, 10 μg of M2e in 50 μL of PBS was mixed with 50 μL of MF59 (AddaVax®; InvivoGen) for administration. Prime-boost regimens were administered with a 21-day interval between primary kuching and booster vaccination. For serum collection, blood was collected from the facial vein at the indicated time points into a BD SST microtina (BD Biosciences). After centrifugation at 3,000 × g for 10 minutes, serum was obtained and stored at -20°C.

[0021] Enzyme-linked immunosorbent assay (ELISA) Flat-bottom microplates (Nunc, Denmark) were coated overnight at room temperature with M2e peptide antigen (100 ng / well). After washing, 5% (w / v) skim milk (BD Difco, Sparks, MD) in PBST (containing 0.05% Tween80) was added to the wells for 1 hour for blocking. After blocking, mouse serum obtained at predetermined time points was added to the wells and incubated at room temperature for 1 hour. After repeated washing, secondary antibodies containing goat anti-mouse IgG HRP conjugate (Jackson ImmunoResearch), goat anti-mouse IgG1 HRP conjugate (Abcam), or goat anti-mouse IgG2a HRP conjugate (Abcam) were added and incubated for 1 hour. After further washing, 100 μl of TMB microwell peroxidase substrate (KPL, Gaithersburg, MD) was distributed to each well and incubated in the dark for 10 minutes. Finally, the reaction was stopped using 100 μl of TMB stop solution (KPL, Gaithersburg, MD). The optical density at 450 nm was read using a spectrophotometer (ThermoFisher Scientific). The specific titer of M2e was calculated based on the titer of the endpoint.

[0022] Immunofluorescence antibody assay (IFA) MDCK cells were placed in a 96-well tissue culture plate, with 1.2 × 10⁶ cells per well. 4Cells were implanted at a density of 100 cells. After 24 hours of incubation, MDCK cells were washed twice with infectious medium and then infected with H1N1 or H3N2 virus at MOI=1. After 24 hours of incubation, infected MDCK cells were washed twice with PBST and then fixed by adding cold 80% acetone. After incubation with acetone at -20°C for 20 minutes, acetone-fixed, infected, and uninfected MDCK cells were blocked in 100 ml of 1% BSA-PBS at room temperature for 2 hours. After blocking, 50 μl of pooled serum (1:200 dilution in 1% BSA-PBST) was added to the wells and incubated at room temperature for 1 hour. The wells were then washed three times with PBST, and 50 μl of FITC-tagged anti-mouse secondary antibody (1:400 dilution) was added to the wells and incubated at room temperature for 1 hour. 50 μl of DAPI (1:400 dilution) was added directly to the wells and incubated with the secondary antibody at room temperature for 15 minutes. The plate was then washed three times with PBST (5 minutes each) and covered with glycerol. Fluorescence was then visualized using fluorescence microscopy (Olympus Corporation, IX-83).

[0023] Antibody-dependent cell-mediated cytotoxicity surrogate assay The ADCC reporter bioassay was performed according to the manufacturer's protocol (catalog number G7015, Promega). Briefly, MDCK cells were infected with PR8H1N1 at 37°C and 5% CO2 using infection medium (DMEM containing 0.075% BSA, 1% non-essential amino acids, 1% sodium pyruvate, 1% HEPES, 1% PSA, and 2 μg / mL TPCK-treated trypsin). The H1N1-infected MDCK cells were harvested and inoculated into sterile white 96-well plates (Corning) 24 hours prior to the assay. Mouse-derived serum samples were heat-inactivated at 56°C for 30 minutes and then sequentially diluted 5-fold with assay buffer (RPMI1640 containing 4% ultra-low IgG FBS). Serum dilutions and a stable Jurkat cell line expressing mouse FcγR (catalog number G7015, Promega) were added to wells planted with infected MDCK cells, and incubated at 37°C for 6 hours in a 5:1 effector:target cell ratio. Cells were equilibrated to room temperature for 15 minutes before adding the Bio-Glo luciferase assay substrate (Promega). After a 10-minute incubation, luminescence was quantified using GloMax (Promega). Data are expressed as RLU (Radioluminescence Units) of the signal in the absence of serum.

[0024] Reporter cell assay for STING and TLR9 activation The activity of human STING or TLR9 genes was quantified using 293-Dual® hSTING-R232 cells (InvivoGen) and HEK-Dual® hTLR9 cells (InvivoGen), respectively. First, reporter cell viability was verified using the TOOLS Cell Counting (CCK-8) kit (BIOTOOLS Co., Ltd., Taipei, Taiwan). To perform the reporter assay, free cdGMP (6 μg), NS(cdGMP) (6 μg cdGMP, 0.75 mg PLGA), free CpG-ODN2395 (2 μg), NS(CpG) (2 μg CpG, 0.25 mg PLGA), and empty NS (0.75 mg PLGA or 0.25 mg PLGA, equivalent to the NS being compared) were prepared in 20 μl of PBS. Serially diluted sample solutions were prepared in parallel. The sample solution is placed in a 96-well plate, 1 × 10⁶ 5 The cells were added to 180 μl of culture medium containing either 293-Dual® hSTING-R232 cells or HEK-Dual® hTLR9 cells. The plates were incubated in a CO2 incubator at 37°C for 24 hours, and the supernatant was collected to evaluate secretory placental alkaline phosphatase (SEAP) activity after addition of QUANTI-Blue® solution (InvivoGen). The absorbance at 650 nm was measured using a spectrophotometer (ThermoFisher Scientific).

[0025] Intracellular cytokine staining and flow cytometry analysis M2e-specific CD4 + IFNγ + Intracellular cytokine staining to identify helper T cells was performed using splenocytes obtained 7 days after vaccination. Single-cell preparations were made from the spleen, and 1 × 10⁶ cells were placed in a round-bottom 96-well plate. 6 Plated in individual cells / well. 2 μg of M2e peptide antigen was added at 37°C with 5% CO2 to form CD4 +T cells were stimulated. After 4 hours, GolgiPlug protein transport inhibitor (BD Biosciences, Jose, CA) was added. The plate was incubated for another 4 hours, then rotated at 4°C to remove the medium. The cells were resuspended in 40 μl of 1:400 dilution of anti-CD4-PE-Cy7 (clone RM4-5; BD Biosciences) antibody. After incubation on ice for 30 minutes, the cells were washed and resuspended in 100 μl of Cytofix / Cytoperm solution, and incubated on ice for 20 minutes. After two washes, the cells were stained overnight at 4°C with 40 μl of 1:200 dilution of anti-IFNγAPC antibody (clone XMG1.2; BD Biosciences). The cells were washed three times before acquisition using FACS LSR II (Institute of Biomedical Sciences, Academia Sinica). Analysis was performed using FlowJo software. The background value determined for the unstimulated sample was subtracted from the value obtained from the test sample.

[0026] Follicular helper T cells and germinal center B cells were evaluated 14 days after primary vaccination. Inguinal lymph nodes were collected from euthanized mice, followed by tissue digestion to obtain single-cell suspensions, and 1 × 10⁶ cells were collected. 6The cells were transferred to each well of a round-bottom 96-well plate. First, the cells were incubated with anti-mouse CD16 / CD32 (clone 2.4G2; BD Biosciences) for 30 minutes to block the Fc receptor. After blocking Fc, the cells were incubated with antibodies corresponding to specific surface markers. For evaluation of follicular helper T cells, staining antibodies included anti-CD3-APC (clone 17A2; eBiosciences), anti-CD4-PE-Cy7 (clone RM4-5; BD Biosciences), anti-PD1-PerCP-eF710 (clone J43; eBiosciences), and anti-CXCR5-PE-CF594 (clone 2G8; BD Biosciences). For evaluation of GL7+ germinal center B cells, staining antibodies included anti-B220 (human / mouse)PE (clone RA3-6B2; BioLegend), and anti-GL7-Pacific Blue (clone GL7; BioLegend). After two washes, the cells were resuspended in PBS containing 2% FBS and subsequently acquired using FACS LSR II (BD Biosciences). Analysis was performed using FlowJo software (FlowJo LLC, Ashland, OR).

[0027] Histology and immunohistochemistry Popliteal lymph nodes and lungs were removed from mice and fixed with 10% formalin. For histological analysis, the samples were stained with hematoxylin and eosin. GL7 in the lymph nodes. +To examine B cells, paraffin-embedded tissues were sectioned, deparaffinized with xylene, and rehydrated with ethanol in water. The samples were then incubated in hot citrate buffer for 20 minutes and then cooled to room temperature. The samples were then peroxidase quenched with 3% hydrogen peroxide and avidin / biotin blocked with a commercial blocking kit (Vector Laboratories). After blocking, the tissues were stained with an antibody against GL7 (2.5 μg / mL, BioLegend catalog number 144601). The tissue samples coated with the antibody were then treated using a commercial immunohistochemistry kit (DAB 2-component kit; C09-100; OriGene) according to the manufacturer's protocol.

[0028] Hemagglutination inhibition (HAI) assay Serum was mixed with PR8 H1N1 virus for 30 minutes, then 1% chicken RBC was added to the mixture and incubated at room temperature for 45 minutes. The HAI titer was defined as the maximum serum dilution factor that resulted in HAI; samples with no detectable HAI activity were assigned a titer of less than 10.

[0029] Influenza virus challenge All virus challenges were performed via intranasal inoculation. First, mice were anesthetized by isoflurane anesthesia and inoculated with 25 μL of virus solution. For lethal virus challenge, PR8 was administered at a dose of 3×10 5 PFU, A / HKx31 (H3N2) was administered at 3×10 6 PFU, and A / California / 7 / 2009 (pdmH1N1) was administered at 2.5×10 6 PFU.

[0030] Evaluation of viral load Lung tissue was collected from mice 3 days after viral attack, and the tissue was placed in infection medium for homogenization by sonication. After sonication, the supernatant was collected after centrifugation at 3000 × g for 30 minutes. Viral load was assessed by a 50% tissue culture infectious dose (TCID50) assay. Briefly, lung homogenate supernatant was first sequentially diluted with infection medium. MDCK cells were placed in a 96-well microplate (1 × 10⁶ cells). 4 The cells were inoculated into individual cells per well and incubated at 37°C for 24 hours. Then, 50 μL of supernatant was added to the cells and incubated at 37°C for 1 hour. After infection, the culture medium was removed and the cells were washed with PBS. Then, 100 μL of fresh infection medium was added to the cells and incubated at 37°C for 4 days. The culture medium was then collected and mixed with 1% chicken RBCs in a 1:1 ratio for a hemagglutination spot assay to assess viral titer.

[0031] Complement activation assay Complement activation was evaluated by quantification of C3a products. First, mouse serum was incubated with the designated sample (zymosan, M2e NS, or M2e PEG-NS) at 37°C for 2 hours. For the detection of mouse C3a, a flat-bottomed 96-well plate (Nunc Denmark) was incubated overnight at 23°C with 5 μg / ml capture antibody rat anti-mouse C3a (clone I87-1162; BD Pharmagen). After blocking, 1:200 of serum sample or mouse C3a protein (BD Pharmagen) was added to the wells and incubated for 1 hour. C3a content was determined by sequential incubation with biotin rat anti-mouse C3a (clone I87-419; BD Pharmagen), 1 μg / ml streptavidin-horseradish peroxidase (Pierce), OptiEIA 3,3',5,5'-tetramethylbenzidine (TMB) substrate (BD Pharmagen), and 2M H2SO4.

[0032] Immunogen tracking in lymph node follicles To examine the distribution of M2e antigen in lymph nodes, mice were inoculated via paw-plantar injection with nanoshells containing 50 μg of PEG-free or PEG-coated nanoshells containing 2 μg of M2e peptide with an Alexa-fluor 647 tag. Eighteen hours prior to lymph node dissection, 4 μg of BV421-labeled anti-CD35 (BD Biosciences 740029) was injected subcutaneously into the paw of the mice for in-situ labeling of lymph node follicles. Popliteal lymph nodes were then treated by tissue cleaning. The lymph nodes were fixed overnight in 4% paraformaldehyde at 4°C, and then washed in 1×PBS at 4°C for 24 hours to remove formaldehyde residue. Next, the samples were immersed for 24 hours at 4°C in a solution containing 25% (w / v) X-CLARITY® polymerization initiator and X-CLARITY® hydrogel solution (Logos Biosystems) in a 1:100 ratio. After polymerization in the X-CLARITY® polymerization system, the tissue embedded in the hydrogel was placed in an electrophoretic tissue cleaning solution for passive tissue cleaning. The resulting lymph nodes were imaged using confocal microscopy.

[0033] statistical analysis Data were analyzed using Student's t-test or ANOVA, followed by Dunnett's multiple comparison test using a GraphPad prism. P-values ​​less than 0.05 were considered statistically significant.

[0034] Hereafter, the methods and compositions disclosed in the present invention will be described in the following subject matter. 1. Asymmetric ion stabilization enables high-density co-encapsulation of M2e peptides and STING agonists in polymeric nanoshells. The inventors have previously shown that the use of short PLGA polymers with low viscosity can reduce interfacial tension between biemulsions for constructing hollow nanoparticles. To facilitate high-density co-encapsulation of M2e peptide antigens and hydrophilic cdGMP, the inventors further improved the stabilization strategy using insights from biological nanovesicle budding and vesicle formation mechanisms that utilize asymmetric forces across the internal and external surfaces for membrane bending stabilization. The inventors employed an asymmetric ion stabilization strategy for water-in-oil-in-water (W / O / W) emulsions, exposing an anionic polymer (carboxylate-terminated poly(lactic acid-co-glycolic acid) (PLGA-COOH)) to a high ionic strength buffer in the internal aqueous phase and a low ionic strength buffer in the external aqueous phase. Due to the presence of differential buffers, asymmetric ionic screening of the exposed anions is required. Because the internal buffer exhibits a higher ionic screening effect compared to the external buffer, anions at the internal interface experience a long-range reduction in electrostatic force and repulsion compared to the external side, thus creating an asymmetric strain that bends toward the encapsulating material phase, facilitating the stabilization of the nanocapsules. Furthermore, the highly polar carboxyl groups of the amphiphilic polymer facilitate polarity-driven polymer alignment during double solvent evaporation, resulting in uniform shell formation during polymer curing. The simple yet complex emulsion protocol results in the formation of monodisperse, surfactant-free nanoshells with readily distributed desired encapsulating material within their internal aqueous core (Figures 2A, B). Encapsulation of M2e peptides in the nanoshells shows a consistent efficiency of approximately 55% at input peptide concentrations ranging from 2.5 to 40 mg / mL, and the concentration of peptides encapsulated in each nanoshell can range from 475 to 7,600 peptides depending on the input peptide concentration (Figure 3). Notably, the biemulsion in the absence of anionic polymers or differential buffers across the internal and external aqueous phases resulted in capsule structure collapse and poor antigen encapsulation, as it is assumed that the nanoparticles have a more energetically favorable solid conformation and the interface area is reduced (Figure 2A, B).Controlling the ionic strength of the internal aqueous phase supports the formation of negative curvature around the encapsulating material during the emulsion process, thus providing a robust and versatile approach for modular cargo encapsulation.

[0035] M2e nanoshell vaccines were prepared using an internal aqueous phase solution containing 40 mg / mL of M2e peptide and 5 mg / mL of cdGMP. Despite the high content of encapsulating material, the resulting nanoshells retained a clear core-shell structure (Figure 2C), high encapsulation efficiency (Figure 2D), and physicochemical properties identical to empty nanoshells (Figure 2E). The M2e nanoshells had a unimodal particle size distribution, an average diameter of 98.7 nm, and a zeta potential of -42.7 mV. Quantification of antigens, adjuvants, and nanoparticles by BCA assay, high-performance liquid chromatography, and nanoparticle tracking analysis showed that each nanoshell contained approximately 7,600 peptide antigens and 3,000 cdGMP molecules (Figure 4). The acid-instability biodegradability of the nanoshells contributes to the pH-responsive dissociation kinetics of the encapsulated peptides and adjuvants. At physiological pH (approximately pH 7.4), the M2e nanoshells exhibited a sustained M2e peptide and cdGMP release profile, with approximately 50% of the encapsulation material retained within the particles by day 7. At pH 5, accelerated ester hydrolysis of the PLGA shell increased the rate of cargo release, with 54.5% of the peptide and 66% of the cdGMP being released within 1 day (Figure 2F). In preferred embodiments, the molecular weight may be 6,000–18,000, and more preferably 7,000–17,000. The M2e nanoshells were further demonstrated to be highly stable after lyophilization. When reconstituted after storage at room temperature for one month in powdered form, NS(M2e+cdGMP) retained comparable size, surface charge, and antigen encapsulation to that of a freshly prepared sample (Figures 2G, H; Figure 5). These characterizations highlight several desirable features of the M2e nanoshells for clinical transition, such as ease of preparation, biocompatibility, and storability.

[0036] 2. Single-dose M2e STING agonist nanoshells induce robust anti-M2e IgG2a against antibody-dependent cell-mediated cytotoxicity. To evaluate the immunogenicity of the M2e nanoshell vaccine, mice were administered lyophilized and reconstituted NS(M2e+cdGMP) in a single-shot vaccination regimen. For mouse inoculation, 375 μg of nanoshells containing 10 μg of M2e peptide and 1.25 μg of cdGMP in 100 μL of PBS solution were injected subcutaneously into each Balb / C mouse via the base of the tail. For comparison, free M2e peptide and M2e peptide mixed in 100 μL of commercial aluminum salt adjuvant (approximately 400 μg of aluminum hydroxide) were administered concurrently. 42 days after primary vaccination, serum was obtained from immunized mice for peptide-specific ELISA analysis (Figure 6A). Compared to the control group, the single-dose NS(M2e+cdGMP) induced significantly higher titers of M2e peptide-specific antibodies. Evaluation of the relative ratio of IgG1 and IgG2a revealed that the M2e nanoshell showed a balanced Th1 and Th2 response with high levels of IgG2a antibody, whereas no IgG2a titer was observed in the control group (Figures 6B and 7). Since ADCC is a primary anti-M2e defense mechanism that mediates the secretion of lytic enzymes from effector cells during cross-linking with influenza-infected cells, the inventors then evaluated the binding ability of vaccine-induced anti-M2e antibodies against Maidin-Derby canine kidney (MDCK) cells infected with two different influenza A viruses, A / Puerto Rico / 8 / 1934 (PR8;H1N1) and A / HKx31 (H3N2). Immunostaining using serum from mice immunized with M2e nanoshells or M2e / Alum showed that serum from the M2e / Alum group exhibited only minimal antibody binding to infected cells, while serum from the M2e nanoshell group showed strong and widespread antibody binding (Figure 6C). Evaluation of the ability to induce antibody-mediated ADCC using a reporter cell-based bioluminescence assay revealed that when reporter cells were co-incubated with H1N1-infected MDCK cells in the presence of M2e nanoshell serum, robust activation of the luciferase reporter gene led to antibody-mediated cell cross-linking (Figure 6D).

[0037] From the perspective of robust anti-M2e induction by NS(M2e+cdGMP), the inventors investigated whether the adjuvant activity of the microparticles alone could be sufficient to improve M2e immunogenicity in the absence of adjuvants of the molecules encapsulated together. To evaluate the contribution of the co-encapsulated STING agonist, M2e nanoshells prepared without adjuvants (NS(M2e)) or M2e nanoshells prepared with an equivalent dose of co-encapsulated CpG-ODN ((NS(M2e+CpG-ODN)) were prepared for comparison (Figure 6E). First, the immunogenicity of the nanoshell-encapsulated adjuvants was evaluated using SEAP (secreted placental alkaline phosphatase) reporter cells overexpressing either the human STING R232 isoform or the human TLR9 gene. NS(cdGMP) and NS(CpG) showed excellent immunostimulation, comparable to their respective free adjuvant counterparts (Figure 6F, G). While NS(M2e) and NS(M2e+CpG-ODN) showed comparable cargo encapsulation and physicochemical properties to NS(M2e+cdGMP) (Figure 6E, Table 1), their alternatives... The formulation produced significantly reduced anti-M2e titers compared to the STING agonist-loaded counterpart (Figure 6H). Notably, NS(M2e) containing an equivalent dose of free cdGMP as an adjuvant did not show an observable improvement in immunogenicity, which may be due to the low delivery efficiency of the free cyclic dinucleotide. Further evaluation of NS(M2e+cdGMP) vaccination in C57BL / 6 and AGB6 mice, mouse strains with a C57BL / 6 background but deficient in IFNα / β and IFNγ receptors, indicated that the immunogenicity of the M2e nanoshell required appropriate interferon signaling (Figure 6I). These results highlight the co-incorporation of the STING agonist into the M2e nanoshell as an essential component for enhancing the immunogenicity of the M2e peptide.

[0038] [Table 1]

[0039] 3. M2e nanoshell vaccination stimulates Th1 helper T cells, follicular helper T cells, and germinal center formation. To gain mechanistic insights into the humoral response to M2e, the inventors have developed a method for inducing Th1 helper T cells and follicular helper T cells (T) induced by vaccination with M2e peptide, M2e peptide containing Alum adjuvant, and NS (M2e+cdGMP). FH ), and the induction of germinal center formation were evaluated. Effector cell-mediated ADCC is initiated primarily through engagement with the Fc receptor (FcgR) IV (relating to human FcgR) IIIa) that recognizes IgG2a in Balb / C mice. Since IgG2a production is assisted by Th1-dependent IFNγ secretion, we first evaluated M2e-specific CD4 induced by different vaccine formulations. + The T cell response was investigated. Seven days after vaccination, the T cell response was evaluated by stimulating harvested splenocytes with M2e peptide. After intracellular cytokine staining and flow cytometry analysis, the group vaccinated with NS(M2e+cdGMP) showed IFNγ + The maximum frequency of the subset was shown (Figure 8A, B). In contrast, CD4 was observed between the M2e+Alum and the control group. + IFNγ + No significant difference was observed in T cells. CD4 with Alum adjuvant + IFNγ + The minimal T-cell enhancement was consistent with the absence of IgG2a in serum titers in the M2e+Alum group, highlighting the role of Th1-biased adjuvants in inducing a humoral response favorable to ADCC. We then investigated CD4 in the inflow area lymph nodes 14 days after vaccination. + CXCR5 + PD1 + T FH The presence of Alum was examined. M2e peptide vaccination in the presence and absence of Alum resulted in levels of T equivalent to the PBS control group. FHHowever, NS (M2e+cdGMP) vaccination was T FH The population increased significantly (Figure 8C, D). Similarly, analysis of B lymphocyte populations, which are germinal center B cells that undergo rapid proliferation for antibody development and production, using the GL7 activation marker, showed that M2e nanoshell vaccination resulted in the highest levels of B220. + GL7 + This demonstrated the induction of B lymphocytes (Figure 8E, F). Further examination of germinal center formation was performed by histological analysis of dissected lymph nodes 14 days after vaccination. Paracortical hyperplasia, characterized by dendritic cell infiltration in the paracortical region, was observed in the lymph nodes of both the M2e+Alum and M2e Nanoshell-vaccinated groups (Figure 8G). Lymph nodes in the M2e Nanoshell-vaccinated group also showed marked follicular hyperplasia, an indicator of B cell proliferation and progressive germinal center development. Immunohistochemical examination further revealed GL-7 differences between the different vaccination groups. + B cell distribution was contrasted (Figure 8H). The marked clustering of GL-7+ B cells in germinal centers from the M2e nanoshell group indicates rapid B cell activation favorable to subsequent plasma cell development. These results represent a favorable lymph node environment and enhanced T cell helper function induced by STING agonist nanoshells to promote the anti-M2e humoral response.

[0040] 4. A single dose of M2e nanoshell vaccine provides effective and long-lasting protection against lethal H1N1 attacks. To evaluate the protective effect mediated by the M2e nanoshell vaccine, the inventors subjected immunized mice to attacks with lethal PR8 influenza. In addition to the M2e vaccine group containing single-dose M2e nanoshells and Alum adjuvant, the inventors evaluated two additional prime-boost vaccine regimens using M2e peptides containing M2e nanoshells and MF59, an oil-in-water emulsion adjuvant approved for use in pandemic and seasonal influenza vaccines, as an adjuvant (Figure 9A). M2e titer assessments showed that M2e nanoshell booster vaccination increased M2e antibody levels by approximately two orders of magnitude (Figure 9B). In contrast, prime-boost vaccination with M2e peptides containing MF59 adjuvant resulted in lower anti-M2e antibody titers than single-dose M2e nanoshell vaccination. Under PR8 attack, M2e vaccination with one dose of Alum adjuvant did not confer observable protection, while M2e vaccine with two doses of MF59 adjuvant conferred partial protection, with 60% of immunized mice dying from the viral attack. In stark contrast, a single dose of M2e nanoshells completely protected vaccinated mice (Figure 9C, D). Notably, despite the two doses of nanoshell vaccines increasing the overall humoral response and serum antibody binding to influenza virus-infected MDCK cells (Figure 10A), comparable ADCC activity, antiviral protection, and weight recovery profiles were observed with the prime-boost and single-dose nanoshell regimens (Figure 9C, D; Figure 10B), indicating that a plateau of anti-M2e-mediated protection was achieved even without booster vaccination.

[0041] Since a single dose of M2e nanoshell vaccination provides protection equivalent to a booster regimen, the inventors further investigated the degree of protection provided by a single-shot nanoshell vaccination. Three days after an influenza attack, the inventors collected lung tissue from PBS controls, single-shot M2e+Alum, and single-shot M2e nanoshell groups. Assessment of viral loading by 50% tissue culture infectious dose (TCID50) assay showed no detectable viral titer in the lungs of the M2e nanoshell group, indicating complete suppression of viral replication, whereas the control and M2e+Alum groups exhibited high lung viral titers (Figure 9E). Histopathological analysis of lung tissue further supported the remarkable antiviral protective effect of nanoshell. Mice that received a single-dose nanoshell vaccine showed no observable lung lesions. In stark contrast, the influenza attack inflicted clear lung damage in the PBS control and M2e / Alum groups, showing severe histopathology with marked lymphocyte infiltration and perivasculitis (Figure 9F). In addition, the bronchioles of the control group showed necrotic epithelial cells and wall thickening associated with impaired respiratory function. The bronchioles of the M2e nanoshell-vaccinated group showed normal histological features with thin-walled airways and columnar epithelium, without these pathological signs. These results demonstrate the superior protective effect of the M2e nanovaccine in viral suppression and mitigation of virus-induced lung damage. Notably, the antibodies carried by the M2e nanoshell did not show neutralizing ability against the influenza virus (Figure 11), highlighting that ADCC can effectively provide protection against pulmonary infectious diseases.

[0042] Since antibody titer decay has been considered a major transitional barrier for M2e-based vaccine formulations, we further examined the persistence of humoral response and protective effects conferred by a single shot of M2e nanoshell over a period of 40 weeks. Surprisingly, anti-M2e IgG levels remained stable throughout the observation period (Figure 9G). The prolonged humoral response suggests the induction of long-lived plasma cells, whose development is highly dependent on germinal center formation and helper T cell function. On day 273 after nanoshell vaccination, we evaluated the protective capacity of the nanoshell vaccine by attacking aged mice with a lethal dose of PR8 virus, and similarly attacking a control group of the same age for comparison. Unlike the lethal attack in young mice, which showed a 100% mortality rate, aged mice showed a 50% mortality rate at the same lethal dose. This reduction in influenza susceptibility may be due to a decrease in inflammation and immunological pathology exhibited by aged subjects. Despite the reduction in mortality, weight loss was still observable in the control group (Figure 9H). In comparison, the group vaccinated with M2e nanoshells had a 100% survival rate and a peak mean weight loss of less than 10% (Figure 9H, I), demonstrating the long-lasting protective effect conferred by a single dose of M2e nanoshells. The longevity of the nanoshell-induced antibodies contrasts with previous M2e vaccination efforts, where a reduced humoral response may be attributed to difficulty in engagement between the small peptide antigen and its symbiotic B cells in lymph node follicles.

[0043] 5. Nanoshells enable long-term retention and exposure of M2e peptides in lymph node follicles for antibody induction. The strong humoral response and protective capabilities of the M2e nanoshells raised the question for us as to how the shielded peptides within the nanocarrier could be presented for B cell binding and antibody stimulation. The counterintuitive nanoshell design and its performance were intriguing, given that conventional nanocarrier-based strategies rely on surface antigen presentation to enhance antigen engagement with cognate B cells. We hypothesized that the nanoshells could release M2e peptides in a prolonged manner within lymph node follicles for sustained immune stimulation (Figure 12A). Induction of humoral immunity is facilitated by a network of FDCs in lymph node follicles that present antigens to B cells for affinity maturation. Since immune complexes formed by activated complement products and nanoparticles can be relayed to complement receptor-rich FDCs after their capture by subcapsular macrophages, we hypothesized that the surfactant-free, anion-rich nanoshell surface plays a crucial role in complement-dependent FDC targeting. To test this hypothesis, the inventors prepared polyethylene glycol (PEG) coated nanoshells and compared complement activation, follicular targeting, and immunogenicity between PEG-coated nanoshells and PEG-free nanoshells. PEG incorporation into the nanoshells was readily achieved by adding DSPE-PEG to the oil phase during nanoshell preparation. Compared to PEG-free nanoshells, PEG-modified nanoshells (M2e PEG-NS) contained equivalent M2e peptides and cdGMP encapsulation, had a slightly larger particle size (121 nm), but exhibited a smaller anionic surface zeta potential at -24.9 mV (Figure 12B; Table 2). To compare complement activation between M2e NS and M2e PEG-NS, the inventors measured the level of anaphylatoxin C3a, a proteolytic product of the central complement protein C3, after incubation of the particles in mouse serum. M2e NS induced significant complement activation, resulting in C3a levels comparable to those of zymosan-positive controls. In contrast, PEG modification completely suppressed complement activation in PEG-NS, resulting in C3a levels similar to those of control serum (Figure 12C).Anti-M2e titer assessments at 28 and 35 days after immunization with two types of M2e nanoshells in mice showed a direct correlation between nanoshell complement activation and its vaccine performance, with PEG coating reducing the overall anti-M2e titer by more than an order of magnitude (Figure 12D). These results indicate that M2e immunogenicity can be significantly altered by the surface properties of the antigen carrier.

[0044] [Table 2]

[0045] To examine the M2e distribution during encapsulation and delivery by two different nanoshells, the inventors performed whole-tissue fluorescence measurements of fluorescently labeled M2e peptides in inflow region lymph nodes (dLNs). Alexa Fluor 647 dye-conjugated M2e peptides, encapsulated in either PEG-free nanoshells (NS(M2e-A647)) or PEG-coated nanoshells (PEG-NS(M2e-A647)), were delivered to mice via paw-plantar injection. Popliteal lymph nodes excised at different time points were treated using the X-CLARITY® tissue cleaning system to obtain optically clear lymph nodes before fluorescence examination (Figure 13). Four hours after examination, nanoshell administration showed that both NS(M2e-A647) and PEG-NS(M2e-A647) resulted in M2e localization in the lymph node boundary region. This indicates nanoparticle capture by subcapsular sinus macrophages (Figure 12E). In contrast, no detectable fluorescence signal was observed in the lymph nodes after administration of free M2e-A647 peptide (Figure 14). Despite efficient lymph node targeting by both M2e NS and M2e PEG-NS, an observable difference emerged at antigen retention testing 3 days after nanoshell administration. NS (M2e-A647) administration resulted in high levels of M2e antigen co-localization in lymph node follicles, whereas PEG-NS (M2e-A647) was largely excluded, with no detectable antigen signal in the lymph nodes. Further examination of M2e distribution from the NS (M2e-647) group showed an interesting shift in the distribution pattern on day 7. As the follicle-bound antigen signal decayed, a venule-like fluorescence pattern emerged. The venule-like pattern is reminiscent of the mesh-like structure of lymph node conduits, which are interconnected networks that allow the passage of low molecular weight molecules (<70 kDa) between afferent lymphatic vessels, follicles, and high endothelial venules. Since 100 nm nanoparticles are too large to access these channels, the antigen distribution in these conduit channels reflects that small peptide antigens were released from nanocarriers and exported from the follicles through the conduit system.The timing of this pattern's appearance coincides with the dynamics of nanoshell dissociation, which exhibits a sustained antigen release profile over several days (Figure 12F). On day 14, trace amounts of antigen signaling remained detectable in lymph node follicles in the NS(M2e-A647) group. The contrasting dynamics of lymph node retention between PEG-coated and unmodified nanoshells provide mechanistic insight into the discrepancy between the immunogenicity of the two nanoparticles (Figures 12F, G). To further evaluate the distribution of nanoshells in lymph node follicles, we rigorously examined the follicles three days after nanoshell administration. NS(M2e-A647) showed a polar distribution in the follicles (Figure 12H), demonstrating a localization pattern consistent with the distribution of FDCs in germinal centers during immune activation. In contrast, no antigen retention in follicles was observed in the PEG-NS(M2e-A647) group (Figures 12H, I). The inventors further demonstrated that injection with cobra venom factor (CVF), a snake toxin that depletes complement factors, significantly impaired the follicle targeting ability of nanoshells in mice (Figure 15). In summary, these results demonstrate that M2e antigens encapsulated in nanoshells can be retained and released into the FDC network for long-term B cell stimulation in a complement-dependent manner, highlighting the unique nanoshell surface and antigen release properties that contribute to the single-dose efficacy of nanovaccines.

[0046] 6. A single-dose M2e nanoshell vaccine provides broad protection against heterozygous subtypes of influenza viruses. Since the collective molecular and microparticle adjuvant activity of M2e STING agonist nanoshells contributes to the robust and long-lasting anti-M2e titer of the nanovaccine, the inventors then tested the protective efficacy of a single-dose nanoshell regimen against heterosubtype influenza viruses (Figure 16A). First, the inventors tested the protective efficacy of the vaccine against the HKx31 strain, an H3N2 mutant virus with a conserved M2e sequence compared to H1N1 viruses tested previously. Under viral attack, M2e nanoshell vaccination completely protected mice from death, whereas the vaccine control containing Alum adjuvant did not confer any observable protective efficacy or survival effect, and all mice died within 4 days after attack (Figures 16B, C). Notably, further antiviral evaluations were performed using the pandemic 2009 H1N1 strain (pdmH1N1), which has four different M2e amino acid residues from the 23-length peptide M2e antigen used in the nanoshell vaccine (Table 3) (SEQ ID NO: 1: SLLTEVETPIRNEWGCRCNGSSD; SEQ ID NO: 2: SLLTEVETPIRNEWGCRCNDSSD; SEQ ID NO: 3: SLLTEVETPTRSEWECRCSDSSD). Despite the differences in peptide sequences, the single-dose nanoshell vaccine remained completely protective against pdmH1N1 (Figure 16D, E), highlighting the broad applicability of the nanoshell vaccine against hetero-subtype influenza viruses.

[0047] [Table 3]

[0048] Discussion To overcome the low immunogenicity of M2e antigens in influenza vaccination, various antigen modification strategies have been used to create M2e-based fusion proteins possessing carrier proteins, strong immunogens, and ligands that target immune cells. To the best of our knowledge, a single-dose vaccine formulation that provides complete protection against atypical influenza attacks has not yet been achieved. We demonstrate the rational integration of molecular and microparticle adjuvant activity in vaccine design to enhance the ADCC activity of M2e antigens using asymmetrically stabilized polymeric nanoshells. High-density co-encapsulation of M2e antigens and STING agonists in anionic nanoshells enabled broad and long-lasting anti-influenza protection under a single-dose vaccine regimen. This has enormous public health implications and value. In contrast to the dominant strategies for fusion protein design to enhance M2e immunogenicity, the use of a 23-amino acid M2e peptide in our research offers the advantage of scalability, as the peptide can be easily synthesized via solid-phase peptide synthesis. Importantly, the inventors demonstrated that the peak of anti-M2e antibody-mediated protection could be achieved after a single dose of nanoshell inoculation. Booster shots of the nanoshell vaccine did not provide a significant protective benefit, despite a two-order-order increase in anti-M2e titer. The plateau in protection can be explained by the mechanism of action of anti-M2e, which acts as a bridge between infected cells and effector cells for ADCC stimulation. Unlike neutralizing antibodies that rely on pathogen binding for viral neutralization, ADCC-inducing antibodies interfere with viral replication by lysing infected cells with perforin and granzyme stimulation from effector cells. The activity of ADCC-inducing antibodies typically forms an S-shaped relationship with the effector cell lytic function, exhibiting maximal cytotoxicity saturated at a certain antibody concentration. Such concentrations correlate inversely with antibody affinity and reflect the antibody coating density on target cells required to fully activate effector cells.The observed plateau in anti-M2e defense in mice indicates that the titer achieved by the single-dose regimen provided sufficient coverage of infected cells under lethal attack, enabling effective effector cell recruitment for viral elimination. Achieving such levels with peptide antigens in a single-dose regimen demonstrates the remarkable adjuvant activity of the follicle-targeting STING agonist nanoshell. This robust ability to induce ADCC may have therapeutic relevance to other respiratory pathogens and cancers.

[0049] Our research also highlights the efficacy of STING agonist adjuvants in boosting Th1-biased humoral responses for ADCC induction. Activation of STING by cyclic dinucleotides directly phosphorylates IRF3, subsequently stimulating type I interferon expression, which has a significant impact on the formation of adaptive immune responses. While STING agonist adjuvants have attracted considerable interest in vaccine development against infectious pathogens and cancer, evaluating the usefulness of cyclic dinucleotides and their adjuvant activity compared to alternative adjuvants has remained challenging due to the poor intracellular delivery efficiency of the compounds. Similar to other nanocarriers designed to enhance STING agonist delivery, the polymeric nanoshells in our research are capable of enhancing lymph node targeting and the uptake of hydrophilic molecules by immune cells. We further demonstrate, in this specification, a nanoshell-based comparison between cdGMP and CpG-ODN1826, alternative adjuvants that exert their adjuvant function via TLR9 activation. When the administration and delivery profiles of cdGMP and CpG-ODN in the case of M2e nanoshell preparations were summarized, cdGMP demonstrated significantly superiority over CpG-ODN in enhancing anti-M2e titer production. The reduction in humoral response by class B CpG-ODN may be due to its lower ability to induce type I IFN stimulation and its tendency to induce low-affinity, short-lived plasma cells. Our observations are consistent with recent M2e vaccine studies showing that CpG-ODN adjuvantization is inferior in performance compared to poly(I:C), a TLR3 agonist that activates IRF3 similarly to cdGMP in the case of type I IFN induction. Type I IFN is CD4 + Because humoral responses can be enhanced through multiple mechanisms such as promoting T cell activation, stimulating follicular helper T cells, and strengthening germinal center formation, the inventors investigated the properties of these type I IFNs in mice inoculated with the STING agonist nanoshell. Lymph nodes in mice inoculated with nanoshell showed Th1, T FH , and GL7 +This represented an increase in germinal center B cell populations. Collectively, these cell populations support the development and maturation of long-lived plasma cells, which facilitate the establishment of sustained humoral responses.

[0050] Another factor highlighting the remarkable immunogenicity of nanoshells is their ability to prolong the exposure of M2e peptide antigens in lymph node follicles. Sustained antigen retention in germinal centers aids direct affinity maturation and survival of cognate B cells during the rapid proliferation of germinal center B cells, and efforts to enhance humoral responses have spurred the emergence of slow-delivery immunization strategies and vaccination strategies based on designer delivery systems. We demonstrate that shielding peptide antigens within biodegradable nanoshell packaging materials, rather than coating them on the surface of nanoparticles, imparts unexpected spatiotemporal control over antigen distribution in lymph node follicles. The anionic polymer and surfactant-free nature employed in the asymmetric emulsion stabilization of the nanoshells imparts a highly anion-rich surface to the particles, which can activate the complement system via the classical pathway in the presence of calcium ions. The anionic nanoshells can efficiently target FDCs in a complement-dependent manner, and the inclusion of commonly used PEG stabilizers eliminates complement activity and follicle targeting ability. Upon delivery to the FDC network, the nanoshell degrades, allowing for sustained exposure of the peptide antigen in germinal centers for B cell stimulation. The elucidation of the antigen-shutting mechanism adds a novel design principle to the vaccine paradigm, which typically immobilizes antigens on the surface of microparticles for B cell engagement. Encapsulating antigens in degradable anionic nanocapsules provides a multifunctional alternative for directing antigens to lymph node follicles, compared to typical nanoparticle vaccines where the surface-bound antigen and its subgroups may influence its complement activation and follicle targeting capabilities. Given recent findings that antigens may encounter extracellular proteases causing epitope destruction before reaching lymph node follicles, nanoshell-encapsulated antigens may offer the additional benefit of antigen protection compared to surface-presented nanoparticle vaccines. Further tuning of capsule degradation could provide broader control over antigen persistence in germinal centers, adding another dimension to vaccine design aimed at enhancing adaptive immunity.

[0051] conclusion In conclusion, our research demonstrates a highly effective M2e nanovaccine that achieves broad and long-lasting influenza protection with a single-dose regimen. The collective adjuvant activity of the STING agonist and the nanoparticle-mediated antigen retention in the FDC resulted in a significant increase in antigenic immunogenicity. The nanoshell vaccine simplifies M2e antigen design and enables the preparation of translationally viable vaccine formulations based on peptide antigens with a length of 23 amino acids. The research of this invention further provides mechanistic insights and design suggestions regarding peptide antigen delivery and will be added to the stockpile of nanotechnology toolsets for pandemic preparedness. In addition to the experiments described above, the M2e nanovaccine disclosed in this invention is also suitable for birds. The experiments and results are disclosed below.

[0052] 7. Immune response in chickens induced by M2e peptide combined with cyclic GMP-AMP (cGAMP) adjuvant. Experimental Design This study aimed to investigate the immune response in chickens induced by M2e peptides combined with cyclic GMP-AMP (cGAMP) adjuvant administered via ocular and nasal immunization. Specific pathogen-free (SPF) chickens were divided into three groups: a treatment group receiving nanoshells containing M2e peptides (40 μg per chicken) and cGAMP (5 μg per chicken), a control group receiving free M2e peptides and cGAMP, and a mock group receiving phosphate-buffered saline (PBS) (Figure 17).

[0053] Immunization Procedure Chickens (n=5 per group) were immunized on day 0, and tissue collection was scheduled for day 21 post-immunization. Immunization was performed via ocular and nasal administration to ensure the vaccine reached the upper respiratory tract, which is the primary site for initiating a mucosal immune response.

[0054] Organizational data collection and analysis Tissues were collected from the Harderian glands, lungs, cecal tonsils, and spleen for immunohistochemical (IHC) analysis. The focus within these tissues was on detecting cells producing IgA, IgG, and MHC II, which are indicators of both humoral and cellular immune responses. IHC staining was performed at 100× and 200× magnifications, used to identify IgA and IgG-producing cells, for each antibody and tissue type, at specific dilutions and incubation times. MHC-II-producing cells were also identified using a similar method.

[0055] result Production of IgA and IgG As shown in Figures 18–21, IHC analysis revealed a significant induction of IgA and IgG-producing cells in the Harderian glands, lungs, and cecal tonsils in the nanoshell group compared to the control and mock groups. The spleen also showed increased levels of IgG-producing cells, indicating a systemic immune response. Quantification of the area covered by IgG-producing cells showed a significant increase in the nanoshell group, with statistically significant differences observed in the Harderian glands and lungs compared to the control and mock groups (Figure 22). This result suggests a robust humoral immune response, particularly in mucosal tissues.

[0056] MHC II expression Cells producing MHC II, an indicator of antigen presentation and T cell activation, were predominantly found in the bursa and spleen (Figures 23 and 24). The nanoshell group showed enhanced MHC II expression, suggesting effective activation of cellular immunity in response to the nanoshell vaccine.

[0057] conclusion Immunization of chickens with a nano-formulation of M2e peptides combined with the cGAMP adjuvant induced a significant immune response characterized by increased IgA and IgG antibody production and activation of MHC II-producing cells. These findings highlight the potential of M2e peptides and cGAMP-encapsulated nanoshells as promising vaccine candidates for inducing robust immunity in chickens, which means controlling viral infections in poultry. It is expected that various modifications and variations can be made to the disclosed embodiments. The specification and examples are to be considered merely illustrative, and the true scope of this disclosure is intended to be indicated by the following claims and their equivalents.

Claims

1. A composition comprising polymeric nanoparticles encapsulating an antigen and an adjuvant, The antigen is an M2e peptide; Polymeric nanoparticles are A water-impermeable polymer shell, and One or more aqueous cores encapsulated by a polymeric shell A composition containing the following:

2. The composition according to claim 1, wherein the polymeric shell has an outer diameter of 50 to 150 nm.

3. The composition according to claim 1, wherein the M2e peptide comprises the sequence of SLLTEVETPIRNEWGCRCNGSSD, SEQ ID NO: 1, SLLTEVETPIRNEWGCRCNDSSD, SEQ ID NO: 2, or SLLTEVETPTRSEWEECRCSDSSD, SEQ ID NO:

3.

4. The composition according to claim 1, wherein the adjuvant is an agonist.

5. The composition according to claim 4, wherein the agonist is a STING agonist comprising cyclic diGMP, cGAMP, poly(I:C), or CpG.

6. The composition according to claim 1, wherein the polymeric shell comprises a short PLGA polymer having a molecular weight of 6,000 to 18,000 Da.

7. The composition according to claim 1, wherein the polymeric shell is free of surfactants.

8. A method for treating a disease, comprising the step of administering a composition according to claim 1, which is capable of physically associating with cells, to a subject in need thereof.

9. The method according to claim 8, wherein the disease is influenza A.

10. The method according to claim 8, wherein the administration includes intravenous injection, subcutaneous injection, or intraperitoneal injection.

11. The method according to claim 8, wherein the subject is a human or a bird.

12. A vaccine comprising the composition described in claim 1, which can induce an immune response to influenza A.

13. The vaccine according to claim 12, which is a single-dose vaccine formulation.

14. The vaccine according to claim 12, wherein the polymeric shell has an outer diameter of 50 to 150 nm.

15. The vaccine according to claim 12, wherein the M2e peptide comprises the sequence of SLLTEVETPIRNEWGCRCNGSSD, SEQ ID NO: 1, SLLTEVETPIRNEWGCRCNDSSD, SEQ ID NO: 2, or SLLTEVETPTRSEWEECRCSDSSD, SEQ ID NO:

3.

16. The vaccine according to claim 12, wherein the adjuvant is an agonist.

17. The vaccine according to claim 16, wherein the agonist is a STING agonist comprising cyclic diGMP, cGAMP, poly(I:C), or CpG.

18. The vaccine according to claim 12, wherein the polymeric shell comprises a short PLGA polymer having a molecular weight of 6,000 to 18,000 Da.

19. The vaccine according to claim 12, wherein the polymeric shell is surfactant-free.

20. A method for neutralizing a viral infection, comprising the step of priming a target requiring the vaccine described in claim 12.

21. The method according to claim 20, further comprising the step of boosting the target with a vaccine.

22. The method according to claim 20, wherein the priming step and the boosting step are performed in at least one manner selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intra-bronchial, intraperitoneal, intracapsular, intracartilage, intracerebral, intraventricular, intracolon, intracervix, intrastomical, intrahepatic, intramyocardium, intraosseous, intrapelvic, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal cord, intrabursal, intrathoracic, intrauterine, intrabladder, bolus, vagina, rectum, buccal, sublingual, intranasal, and percutaneous.

23. The method according to claim 22, wherein the priming step and the boosting step are performed subcutaneously or intranasally.

24. The method according to claim 20, wherein the subject is a human or a bird.

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