TW202444409A - Composition, vaccine and method for treating influenza a - Google Patents

Abstract

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

Description

Composition, vaccine and method of treating influenza A

The present disclosure relates to a composition, a vaccine and a method for treating influenza A.

Influenza is a persistent public health problem, with annual epidemics causing significant morbidity and mortality. As a seasonal event, influenza is estimated to cause 3 to 5 million cases of severe illness and up to 650,000 respiratory deaths each year. Vaccination against mutagenic viruses is hampered by the tendency of the virus to undergo antigenic shift and antigenic drift, where reassortment of viral genes and point mutations in receptor binding proteins may weaken the protective properties of pre-existing antibodies. As a result, existing hemagglutinin (HA)-based vaccines need to be updated annually and their protective properties can easily decrease if the vaccine does not match the circulating virus strain. To address the shortcomings of current influenza vaccines, a variety of vaccine designs based on B cell and T cell immunogens have been proposed for universal influenza vaccination. Among them, peptide antigens derived from the extracellular domain of the ion channel membrane matrix protein 2 (M2e) of influenza virus present a unique target because they mediate antibody-dependent cellular cytotoxicity (ADCC) and can eradicate M2e-presenting infected cells before viral release and spread. The highly conserved nature of M2e in human seasonal influenza A viruses makes it an attractive candidate for the development of universal influenza vaccines. However, the low immunogenicity of small peptide antigens poses a major obstacle to their clinical translation, and previous clinical trial results of M2e candidate vaccines have been unsatisfactory, with decreased humoral responses despite repeated vaccinations. Recently, emerging recombinant protein strategies, vector technologies, and immunostimulatory adjuvants have rekindled enthusiasm for M2e-based universal influenza vaccines. However, despite advances in antigen and vaccine design, multiple dose regimens are still required to enhance the immunogenicity of peptides, and due to the partial protectivity of the M2e antigen, it is often relegated to a complementary role to other immunogens. As simplified vaccine regimens and formulation simplicity are of great value for improving vaccination logistics and public health strategies, a single-dose M2e peptide vaccine that can provide broad and durable influenza protection remains a highly desirable but elusive goal. Here, a nanoparticle vaccine strategy was designed to enhance antigen availability and T cell assistance in lymph node follicles to enhance the immunogenicity of the M2e antigen.

To construct M2e nanovaccines with high-density co-encapsulation of peptide antigens and immune adjuvants, the inventors demonstrated an asymmetric ionic stabilization strategy for preparing polymer nanoshells. This stabilization strategy mimics the asymmetric stabilization mechanism behind nanoscopic biological vesiculation, overcomes the energy barrier during nanoscale curvature formation, thereby preventing nanoemulsion collapse, and enables the consistent preparation of antigen-loaded nanocapsules in the absence of surfactants and stabilizers. Cyclic di-GMP (cdGMP), a putative STING (stimulator of interferon genes) agonist, was administered as the adjuvant of choice due to its role in inducing type I interferons, a cytokine that favors Th1-biased production. Anti-M2e-based ADCC against influenza-infected cells requires a specific antibody isotype. Upon evaluation of M2e nanoshells (NS(M2e+cdGMP)), the inventors found that the nanovaccine was highly effective in promoting IFNγ+ type 1 helper T cell (Th1) induction, germinal center formation, and Th1-biased anti-M2e production. At the same time, a single M2e nanoshell vaccination provided complete and durable protection against lethal influenza challenge, enabling resolution of viral titer and prevention of lung immunopathology and tissue damage. Complete protection against heterosubtypic influenza challenge was also achieved in a single-dose regimen. The special humoral response of the M2e nanoshell has aroused people's interest. The M2e nanoshell has a different design from conventional vaccines, which present immunogens on the surface of the carrier for B cell binding. The inventors studied how the nanoshell regulates the distribution of the encapsulated antigen. The nanoshell was observed to shuttle the M2e peptide to the filtration dendrite cell (FDC) network in a complement-dependent manner, allowing sustained peptide exposure in B cell filtration vesicles to induce antibody production. Notably, incorporation of a commonly used polyethylene glycol surface coating on the M2e nanovaccine abolished peptide retention in the FDC network and reduced antibody induction, highlighting the importance of surfactant-free nanoshell design in maximizing filtration vesicle targeting ( Figure 1 ). The study presents a highly efficacious and translatable candidate universal influenza vaccine and introduces FDC-targeted nanoparticle design to improve antigen immunogenicity.

In one aspect of the present disclosure, a composition is provided to improve antigen immunogenicity. The composition comprises polymer nanoparticles encapsulating antigens and adjuvants, wherein the antigen is an M2e peptide; wherein the polymer nanoparticles comprise: a water-impermeable polymer shell, and one or more aqueous cores surrounded by the polymer shell.

Preferably, the outer diameter of the polymer shell is 50-150 nm.

Preferably, the M2e peptide comprises the following 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 comprising cyclic di-GMP, cGAMP, poly (I:C) or CpG.

Preferably, the polymer shell comprises short PLGA polymers having a molecular weight between 6,000 and 18,000 Da.

Preferably, the polymer shell does not contain a surfactant.

In another aspect of the present disclosure, a method for treating a disease is provided, comprising: administering the composition of claim 1 capable of biochemically binding to cells to a subject in need thereof.

Preferably, the disease is influenza A.

Preferably, the administration comprises intravenous injection, subcutaneous injection or intraperitoneal injection.

Preferably, the subject is a human or a bird.

In yet another aspect of the present disclosure, a vaccine capable of inducing an immune response to influenza A is further provided. The vaccine comprises the above composition.

Preferably, the vaccine is a single-dose vaccine formulation.

Preferably, the outer diameter of the polymer shell is 50-150 nm.

Preferably, the M2e peptide comprises the following 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 comprising cyclic di-GMP, cGAMP, poly (I:C) or CpG.

Preferably, the polymer shell comprises short PLGA polymers having a molecular weight between 6000 and 18000 Da.

Preferably, the polymer shell does not contain a surfactant.

In yet another aspect of the present disclosure, a method for neutralizing viral infection is further provided.

The method for neutralizing viral infection comprises: vaccinating a subject in need with the above-mentioned vaccine.

Preferably, the method further comprises: enhancing the immunity of the subject with the vaccine.

Preferably, the vaccinating step and the boosting step are performed by at least one mode selected from the group consisting of: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intraperitoneal, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

Preferably, the vaccinating step and the boosting step are by subcutaneous or intranasal injection.

Preferably, the subject is a human or a bird.

The above and other aspects of the present disclosure related to other embodiments described herein will now be described in more detail. It should be understood that the present invention can be embodied in different forms and should not be construed as being limited to the embodiments described herein. Instead, these embodiments are provided so that the present disclosure will become thorough and complete and fully convey the scope of the present invention to those having ordinary knowledge in the art.

The terms herein are used for the purpose of describing specific embodiments only and are not intended to limit the present invention. As used herein and in the appended patent applications, the singular forms "a", "an" and "the" are intended to include plural forms, unless the context clearly indicates otherwise.

As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," "characterized by," or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any expressly stated limitation. For example, a composition, mixture, process, or method that comprises a list of elements is not necessarily limited to those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.

The transitional term "consisting of" excludes any element, step, or ingredient not specified. If in the claims, the term would exclude materials beyond those listed, except for impurities normally associated therewith. When the term "consisting of" appears in a claim clause, rather than immediately following the preamble, it limits only the elements listed in that clause; the claims as a whole do not exclude other elements.

The transitional term "consisting essentially of" is used to define a composition, method, including materials, steps, features, components, or elements in addition to those literally disclosed, provided that such additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristics of the claimed invention. The term "consisting essentially of" is in an intermediate position between "comprising" and "consisting of."

When the applicant defines an invention or a portion thereof with open-ended terms such as "comprising", it should be readily understood that (unless otherwise stated) the description should be read as also using the terms "consisting essentially of" or "consisting of" to describe such invention.

As used herein, the term "about" is used to indicate that a value includes inherent variations such as errors of the measuring device, the method used to determine the value, or variations in the subject matter. Typically, the term is meant to encompass variations of about or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, as the case may be.

The term "or" as used in the claims is used to mean "and/or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, although the present disclosure supports the definition referring to only alternatives and "and/or".

As used herein, "treating" or "treatment" means administering a therapeutic composition to a subject with the intent to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, a symptom of a disorder, a disease state secondary to the disorder, or a tendency toward the disorder.

As used herein, "subject" means a mammalian subject diagnosed or suspected of having or developing a disease, such as cardiovascular disease, cancer, autoimmune disease, or infection. Exemplary patients can be humans, apes, dogs, pigs, cows, cats, horses, goats, sheep, rodents, and other mammals suffering from a disease that can benefit from treatment.

"Administering" or "administration" herein means providing the treatment kit of the present application to a subject. By way of example and not limitation, administration may be performed by parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intraperitoneal, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. For example, injection can be performed by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection or intramuscular (i.m.) injection. One or more of these routes can be used. Non-oral administration can be, for example, a bolus injection or gradual perfusion over time. Alternatively or concurrently, administration can be performed by the oral route.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and/or paragraph of the reference presented.

Experimental part

Ethical Statement

All animal experiments were performed under the Institutional Animal Care and Use Committee (IACUC) protocol (#15-12-893) approved by Academia Sinica, Taiwan.

Cells and viruses

Madin-Darby canine kidney (MDCK) was 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/amphiphilin B (PSA) (Invitrogen) at 37°C and 5% CO2. When MDCK cells were infected with influenza virus, 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 Hsin-Ru Shih, Chang Gung University. Influenza A virus strain A/HKx31 (H3N2) was kindly provided by Professor Hong-Chih Yang, National Taiwan University College of Medicine. A/California/7/2009 (pdmH1N1) was kindly provided by Professor Li-Min Huang, National Taiwan University Hospital. All viruses were propagated in the allantoic cavity of 10-day-old specific pathogen-free (SPF) chicken embryos (JD-SPF Biotech, Miaoli, Taiwan). Virus titers were determined by plaque lytic assay as described previously.

M2e Nanoshell preparation

For this study, the consensus M2e peptide sequence [TEVETPIRNEWGCRCNDSSD] (Genescript; purity > 95%) was used. Nanoshells were prepared using an optimized water-oil-water double emulsion following a previously reported protocol. The internal aqueous phase was prepared by dissolving the desired encapsulant in 200 mM NaHCO ₃ buffer. The polymer solution was prepared by dissolving 75 mg/mL carboxyl-terminated 50:50 poly(DL-lactide-co-glycolide) (PLGA; Mw 7,000-17,000; Sigma-Aldrich) in ethyl acetate. The typical preparation method for the M2e nanoshell vaccine is to emulsify 20 μL of an aqueous solution containing 40 mg/mL M2e peptide and 5 mg/mL cdGMP (InvivoGen) in an ice-cold 200 μL polymer solution using an Ultrasonic Probe Sonicator, with a pulse mode of 40% amplitude and a switching duration of 1 and 2 seconds for 1 minute. The first emulsion is then added to 5 mL of 10 mM NaHCO ₃ and then sonicated for 2 minutes with a probe at 30% amplitude and a switching time of 1 and 2 seconds, respectively. The emulsion is then poured into 8 mL of water and heated at 40°C under gentle stirring in a fume hood to evaporate the solvent. After evaporation of the solvent for 1 hour, the nanoparticles were collected using a 100 kDa molecular weight cutoff (MWCO) Amicon filter (Sigma-Aldrich) to remove unencapsulated material. For non-asymmetric stable nanoemulsions, ester-terminated PLGA (lactide:glycolide 50:50, Mw 7,000-17,000; Sigma-Aldrich) was used as the non-charged polymer. Conditions without differential ion buffer were generated by replacing the internal aqueous buffer with 10 mM NaHCO ₃ . For NS(M2e+CpG-ODN) and NS(M2e), the internal aqueous phase was replaced with 20 μL of 40 mg/mL M2e peptide with 5 mg/mL CpG-ODN 1826 (InvivoGen) and 40 mg/mL M2e peptide, respectively. For the preparation of PEG-coated M2e nanoshells, the oil phase was replaced with 200 μL of ethyl acetate containing 50 mg/mL carboxyl-terminated PLGA and 10 mg/mL 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-conjugated polyethylene glycol (DSPE-PEG(2000)-OH; Nanocs). For nanoshells encapsulating fluorescently labeled M2e peptide antigens, M2e peptides conjugated to the N-terminus with Alexa Fluor 647 were synthesized (Creative peptides). The physicochemical properties, particle concentration, particle morphology, peptide encapsulation efficiency, and cdGMP encapsulation of the collected nanoparticles were evaluated by dynamic light scattering, nanoparticle tracking analysis, cryoEM, and Micro BCA assay (Micro BCA Protein Assay Kit; ThermoFisher Scientific), and HPLC, respectively. The lyophilized M2e nanoshell vaccine was prepared by suspending the nanoshells at a concentration of 50 mg/mL in 10 mM sodium phosphate and 25% sucrose before freezing and lyophilization. Before each immunization study, the nanoparticles were reconstituted, diluted with water to the desired concentration, and the osmotic pressure was adjusted with sucrose solution.

Antigen and adjuvant release kinetics studies

Dialysis experiments at two different pH environments (pH 5 and 7) were used to characterize the antigen and adjuvant release kinetics under physiologically relevant conditions, where M2e nanoshells were loaded into dialysis tubing (10 kDa MWCO, Slide-A-Lyzer MINI dialysis device, Thermo Fisher Scientific) and collected at predetermined time points for M2e peptide and cdGMP quantification.

Animal immunization and serum collection

All animal studies were performed with BALB/c mice, except for the comparison between C57BL/6 and AGB6 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. Yi-Ling Lin, Institute of Biomedical Sciences, Academia Sinica, Taiwan. Mice were maintained in the animal facility maintained by the Institute of Biomedical Sciences, Academia Sinica. For nanoshell vaccination, mice were immunized subcutaneously (sc) at the base of the tail with 100 μL of a solution containing 250 μg of nanoshell, 10 μg/dose of M2e peptide and 1.25 μg/dose of cdGMP nanoparticles. Free M2e peptide was administered as 10 μg of M2e peptide dissolved in 100 μL PBS. For alum-adjuvanted M2e peptide, 10 μg of M2e peptide was mixed in 100 μL of commercial alum salt adjuvant (approximately 400 μg aluminum hydroxide) for administration. For MF59-adjuvanted vaccines, 10 μg of M2e dissolved in 50 μL PBS was mixed with 50 μL MF59 (AddaVax ^™ ; InvivoGen) for administration. A prime-boost regimen was given with a 21-day interval between the primary and booster vaccinations. For serum collection, facial venous blood was collected into BD SST microcontainers (BD Biosciences) at designated time points. Serum was obtained after centrifugation at 3,000 × g for 10 min and stored at -20°C.

ELISA ( ELISA ）

Flat-bottom microwell plates (Nunc, Denmark) were coated with M2e peptide antigen (100 ng/well) overnight at room temperature. After washing, 5% (w/v) skim milk (BD Difco, Sparks, MD) in PBST (with 0.05% Tween 80) was added to each well for blocking for 1 hour. After blocking, mouse sera from predetermined time points were added to each well and incubated for 1 hour at room temperature. After repeated washing, secondary antibodies including 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 dispensed into each well and incubated in the dark for 10 min. Finally, the reaction was terminated using 100 μl of TMB stop solution (KPL, Gaithersburg, MD). The optical density was read at 450 nm using a spectrophotometer (ThermoFisher Scientific). The M2e-specific titer was calculated based on the endpoint titer.

Immunofluorescent antibody test ( IFA ）

MDCK cells were seeded at a density of 1.2×10 ⁴ cells per well in 96-well tissue culture plates. 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 with 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 each well and incubated at room temperature for 1 hour. Subsequently, each well was washed three times with PBST, and 50 μl of FITC-labeled anti-mouse secondary antibody (1:400 dilution) was added to each well and incubated at room temperature for 1 hour. 50 μl of DAPI (1:400 dilution) was added directly to each well and incubated with secondary antibody at room temperature for 15 minutes. Subsequently, each plate was washed three times with PBST (5 minutes) and covered with glycerol. Subsequently, fluorescence was observed by a fluorescence microscope (Olympus IX-83).

Antibody-dependent cytotoxicity surrogate test

The ADCC reporter bioassay was performed according to the manufacturer's protocol (Catalog No. G7015, Promega). Briefly, MDCK cells were infected with PR8 H1N1 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) at 37°C and 5% CO2. H1N1-infected MDCK cells were harvested 24 hours before the assay and plated into sterile white 96-well plates (Corning). Serum samples from mice were heat-killed at 56°C for 30 minutes and then serially diluted 5-fold in assay buffer (RPMI 1640 containing 4% ultra-low IgG FBS). Serum dilutions and a stable Jurkat cell line expressing mouse FcγR (Catalog No. G7015, Promega) were added to each well of the infected MDCK cells and incubated at 37°C for 6 hours at a 5:1 effector:target ratio. Cells were equilibrated to room temperature for 15 minutes before adding Bio-Glo luciferase assay substrate (Promega). After 10 minutes of incubation, luminescence was quantified using GloMax (Promega). Data are expressed as luminescence RLU of the signal in the absence of serum.

STING and TLR9 Activated reporter cell assay

293-Dual™ hSTING-R232 cells (InvivoGen) and HEK-Dual™ hTLR9 cells (Invivogen) were used to quantify the activity of human STING or TLR9 genes. The survival rate of reporter cells was first verified using the TOOLS Cell Count (CCK-8) Kit (BIOTOOLS Co., Ltd., Taipei, Taiwan). For reporter assays, 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 comparison NS) were prepared in 20 μl PBS. Serial dilutions of sample solutions were prepared in parallel. Sample solutions were added to 96-well plates, each well containing 180 μl of medium containing 1 × 10 ⁵ 293-Dual™ hSTING-R232 cells or HEK-Dual™ hTLR9 cells. The plates were incubated at 37°C in a _CO2 incubator for 24 h, and the supernatants were collected to assess secreted embryonic alkaline phosphatase (SEAP) activity after addition of QUANTI-Blue™ solution (InvivoGen). The absorbance at 650 nm was measured using a spectrophotometer (ThermoFisher Scientific).

Intracellular cytokine staining and flow cytometric analysis

Spleen cells derived 7 days after vaccination were stained for intracellular cytokines to identify M2e-specific CD4 ⁺ IFN ⁺ helper T cells. Single cells were prepared from spleens and plated at 1 × 10 ⁶ cells/well in round-bottom 96-well plates. CD4 ⁺ T cells were stimulated by adding 2 µg of M2e peptide antigen at 37°C and 5% CO _2. After 4 hours, GolgiPlug protein transport inhibitor (BD Biosciences, San Jose, CA) was added. Plates were incubated for an additional 4 hours and then spun down at 4°C to remove the medium. Cells were resuspended in 40 µl of a 1:400 dilution of anti-CD4-PE-Cy7 (clone RM4-5; BD Biosciences) antibody. After incubation on ice for 30 min, cells were washed, resuspended in 100 µl of Cytofix/Cytoperm solution, and incubated on ice for 20 min. After two washes, cells were stained with 40 µl of 1:200 dilution of anti-IFNγ APC antibody (clone XMG1.2; BD Biosciences) at 4°C overnight. Cells were washed three times before data acquisition using a FACS LSR II (Institute of Biomedical Sciences, Academia Sinica, Taiwan). FlowJo software was used for analysis. The values presented for the test samples were subtracted from the background values of samples without peptide stimulation.

Assessment of follicular helper T cells and germinal center B cells was performed 14 days after the primary vaccination. Inguinal lymph nodes were collected from euthanized mice, and the tissues were digested into a single cell suspension, and 1 × 10 ⁶ cells were transferred to each well of a round-bottom 96-well plate. Cells were first incubated with anti-mouse CD16/CD32 (clone 2.4G2; BD Biosciences) for 30 minutes to block Fc receptors. After Fc blocking, cells were incubated with antibodies against specific surface markers. For evaluation of follicular helper T cells, staining antibodies included anti-CD3-APC (strain 17A2; eBiosciences), anti-CD4-PE-Cy7 (strain RM4-5; BD Biosciences), anti-PD1-PerCP-eF710 (strain J43; eBiosciences), and anti-CXCR5-PE-CF594 (strain 2G8; BD Biosciences). For evaluation of GL7+ germinal center B cells, staining antibodies included anti-B220 (human/mouse) PE (strain RA3-6B2; BioLegend), and anti-GL7-Pacific Blue (strain GL7; BioLegend). After two washes, cells were resuspended in PBS containing 2% FBS and data were subsequently acquired using a FACS LSR II (BD Biosciences). Analyses were performed using FlowJo software (Flowjo LLC, Ashland, OR).

Histology and Immunohistology

Popliteal lymph nodes and lungs of mice were sectioned and fixed with 10% formalin. For histological analysis, samples were stained with hematoxylin and eosin. For examination of GL7 ⁺ B cells in lymph nodes, paraffin-embedded tissues were sectioned and dewaxed with xylene and then rehydrated with aqueous ethanol. Subsequently, samples were incubated in hot citrate buffer for 20 minutes and then cooled to room temperature. Subsequently, samples were peroxidase quenched with 3% hydrogen peroxide and avidin/biotin blocked by a commercial blocking kit (Vector Laboratories). After blocking, tissues were stained with anti-GL7 antibody (2.5 μg/mL, BioLegend, catalog number 144601). Antibody-coated tissue samples were then processed using a commercial immunohistochemistry kit (DAB 2-component kit; C09-100; OriGene) according to the manufacturer's protocol.

Hemagglutination inhibition ( HAI ) Test

The serum was mixed with PR8 H1N1 virus for 30 minutes, and then 1% chicken red blood cells were added to the mixture and incubated at room temperature for 45 minutes. The HAI titer was defined as the highest serum dilution factor that resulted in HAI; samples without any detectable HAI activity were assigned a titer of <10.

Flu virus attack

All viral challenges were performed by intranasal inoculation. Mice were first anesthetized with isoflurane anesthesia and then inoculated with 25 μL of viral solution. For lethal viral challenge, the dose of PR8 was 3×10 ⁵ PFU, the dose of A/HKx31 (H3N2) was 3×10 ⁶ PFU, and the dose of A/California/7/2009 (pdmH1N1) was 2.5×10 ⁶ PFU.

Viral load assessment

Lung tissues from mice were collected 3 days after viral challenge and homogenized by sonication in infection medium. After sonication, supernatants were collected by centrifugation at 3000×g for 30 minutes. Viral load was assessed using a 50% tissue culture infectious dose (TCID50) assay. Briefly, homogenized lung supernatants were first serially diluted with infection medium. MDCK cells were inoculated in 96-well microtiter plates (1×10 ⁴ cells/well) and incubated at 37°C for 24 hours. Subsequently, 50 μL of supernatant was added to the cells and incubated at 37°C for 1 hour. After infection, the medium was removed and the cells were washed with PBS. Subsequently, 100 μL of fresh infection medium was added to the cells and cultured at 37°C for 4 days. Afterwards, the medium was harvested and then mixed with 1% chicken red blood cells at a 1:1 ratio for hemagglutination plaque assay to assess viral titer.

Supplement activation test

Complement activation was assessed by quantification of C3a production. Mouse serum was first incubated with the indicated samples (zymosan, M2e NS, or M2ePEG-NS) at 37°C for 2 h. For detection of murine C3a, flat-bottom 96-well plates (Nunc, Denmark) were incubated with 5 µg/ml of the capture antibody rat anti-mouse C3a (strain I87-1162; BD Pharmagen) at 23°C overnight. After blocking, serum samples or mouse C3a protein (BD Pharmagen) at 1:200 were added to each well and incubated for 1 h. C3a levels were measured by sequential incubation with biotinylated rat anti-mouse C3a (strain I87-419; BD Pharmagen), 1 µg/ml streptavidin-horseradish peroxidase (Pierce), OptiEIA 3,3',5,5'-tetramethylbenzidine (TMB) substrate (BD Pharmagen), and 2 MH ₂ SO ₄ .

Immunogen tracking in lymph node follicles

To examine the distribution of M2e antigen in lymph nodes, mice were inoculated with 50 μg of PEG-free or PEG-coated nanoshells containing 2 μg of Alexa-Fluor 647-labeled M2e peptide via footpad injection. 18 hours before lymph node excision, mice were subcutaneously injected with 4 μg of BV421-labeled anti-CD35 (BD Biosciences 740029) in the footpad for in situ labeling of lymph node blebs. Subsequently, popliteal lymph nodes were processed by tissue clearing. Lymph nodes were fixed in 4% paraformaldehyde overnight at 4°C and then washed with 1X PBS at 4°C for 24 hours to remove formaldehyde residues. Subsequently, the samples were immersed in a solution containing 25% (w/v) X-CLARITY™ polymerization initiator and X-CLARITY™ hydrogel solution (Logos Biosystems) at a ratio of 1:100 for 24 hours at 4°C. After polymerization with the X-CLARITY™ polymerization system, the hydrogel-embedded tissue was placed in the Electrophoretic Tissue Clearing Solution for passive tissue clearing. The resulting lymph nodes were imaged by confocal microscopy.

Statistical analysis

Data analysis was performed using GraphPad Prism using student’s t test or ANOVA followed by Dunnett’s multiple comparison test. P values less than 0.05 were considered significant.

Hereinafter, the methods and compositions of the present disclosure are described in the following topics.

1. Asymmetric ion stabilization makes M2e Peptides and STING The agonist can be co-encapsulated in the polymer nanoshell at a high density

The inventors have previously shown that the use of short PLGA polymers with low viscosity can reduce the interfacial tension during double emulsions of hollow nanoparticles. To promote high-density co-encapsulation of M2e peptide antigens and hydrophilic cdGMP, the inventors further improved the stabilization strategy, which was inspired by the budding and vesicle formation mechanism of biological nanovesicles, which utilize asymmetric forces across the endoplasmic and exoplasmic surfaces to stabilize membrane curvature. The inventors adopted an asymmetric ionic stabilization strategy for water-in-oil-in-water (W/O/W) emulsions, which subjected anion-carrying polymers (carboxyl-terminated poly(lactic-co-glycolic acid) (PLGA-COOH)) to a high ionic strength buffer in the inner aqueous phase and a low ionic strength buffer in the outer aqueous phase. Differential buffer content exerted asymmetric ionic screening on exposed anions. Compared with the external buffer, the internal buffer exerts a higher ion shielding effect, and the anions at the endoplasmic interface experience reduced long-range electrostatic forces and repulsive forces compared with the exoplasmic side, which produces an asymmetric strain that bends toward the encapsulant phase and promotes the stability of the nanocapsule. The highly polar carboxyl groups of the amphiphilic polymer also facilitate the polarity-driven polymer alignment during the evaporation of the bisolvent, thereby forming a uniform shell shape after the polymer hardens. The simple yet complex emulsion protocol resulted in the formation of monodisperse, surfactant-free nanoshells that readily partitioned the desired encapsulating agent into their internal aqueous core ( Figure 2A , B ) and displayed a consistent efficiency of approximately 55% for nanoshell encapsulation of the M2e peptide, with input peptide concentrations ranging from 2.5 to 40 mg/mL and peptide concentrations encapsulated in each nanoshell ranging from 475 to 7,600 peptides, depending on the input peptide concentration ( Figure 3 ). Notably, in the absence of anionic polymers or differential buffers between the inner and outer aqueous phases, double emulsions resulted in capsule structure disintegration and poor antigen encapsulation due to the nanoparticles presenting a more positively favorable solid conformation and reduced interfacial area ( Figure 2A , B ). Controlling the ionic strength of the inner aqueous phase facilitates the formation of negative curvature around the encapsulant during the emulsion process, providing a robust and versatile approach for modular object encapsulation.

M2e nanoshell vaccines were prepared using an inner aqueous solution containing 40 mg/mL M2e peptide and 5 mg/mL cdGMP. Despite the high encapsulant content, the resulting nanoshells retained a well-defined core-shell structure ( Figure 2C ), high encapsulation efficiency ( Figure 2D ), and physicochemical properties identical to those of empty nanoshells ( Figure 2E ). M2e nanoshells had a unimodal size distribution with an average diameter of 98.7 nm and a zeta potential of -42.7 mV. Quantification of antigen, adjuvant, and nanoparticles using the 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-labile biodegradability of the nanoshells imparts pH-responsive release kinetics to the encapsulated peptides and adjuvants. At physiological pH (approximately pH 7.4), the M2e nanoshells exhibited a sustained release profile of the M2e peptide and cdGMP, with approximately 50% of the encapsulated agent retained in the particles at day 7. At pH 5, accelerated ester hydrolysis of the PLGA shell accelerated the release of the substance, with 54.5% of the peptide and 66% of the cdGMP escaping within one day ( Figure 2F ). In preferred embodiments, the molecular weight may be between 6,000 and 18,000, and more preferably between 7,000 and 17,000. The M2e nanoshells were further demonstrated to be highly stable after freeze-drying. After storage in powder form at room temperature for 1 month and reconstitution, NS (M2e+cdGMP) retained the same size, surface charge, and antigen encapsulation as freshly prepared samples ( Figure 2G , H ; Figure 5 ). These features highlight several desirable characteristics of M2e nanoshells for clinical translation, including ease of preparation, biocompatibility, and storage.

2. Single dose M2e STING The agonist nanoshell induces a robust M2e IgG2a To develop antibodies Dependent cell-mediated cytotoxicity

To evaluate the immunogenicity of the M2e nanoshell vaccine, freeze-dried and reconstituted NS (M2e+cdGMP) were administered to mice in a single-dose vaccination regimen. For mouse vaccination, 375 μg of nanoshells containing 10 μg of M2e peptide and 1.25 μg of cdGMP dissolved 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 alum adjuvant (approximately 400 μg of aluminum hydroxide) were administered in parallel. At 42 days after the first vaccination, serum was obtained from immunized mice for peptide-specific ELISA analysis ( Figure 6A ). Compared with the control group, single-dose NS (M2e+cdGMP) induced significantly higher titers of M2e peptide-specific antibodies. Evaluation of the relative ratio of IgG1 to IgG2a showed that the M2e nanoshell exhibited a balanced Th1 and Th2 response and a high concentration of IgG2a antibodies, while no IgG2a titers were observed in the control group ( Figure 6B , Figure 7 ). Since ADCC is the main protective mechanism against M2e, which mediates the secretion of lytic enzymes by effector cells when bridging with influenza-infected cells, the inventors next evaluated the ability of vaccine-induced anti-M2e antibodies to bind to Madin-Darby canine kidney (MDCK) cells infected with two different influenza A viruses, including A/Puerto Rico/8/1934 (PR8; H1N1) and A/HKx31 (H3N2). Immunostaining using sera from mice immunized with M2e nanoshells or M2e/alum revealed that, while sera from the M2e/alum group showed negligible antibody binding to infected cells, strong and broadly reactive antibody binding was observed with sera from the M2e nanoshell group ( Figure 6C ). The ability of antibody binding to induce ADCC was assessed by a reporter cell-based bioluminescence assay, and robust activation of the luciferase reporter gene after co-culture of reporter cells with H1N1-infected MDCK cells in the presence of M2e nanocapsid serum confirmed antibody-mediated cell bridging ( Figure 6D ).

Given the robust resistance of NS(M2e+cdGMP) to M2e induction, we questioned whether particulate adjuvancy alone in the absence of a co-encapsulated molecular adjuvant would be sufficient to improve M2e immunogenicity. To evaluate the contribution of co-encapsulated STING agonists, M2e nanoshells without adjuvant (NS(M2e)) or with an equivalent dose of co-encapsulated CpG-ODN (NS(M2e+CpG-ODN)) were prepared for comparison ( Figure 6E ). The immunogenicity of nanoshell-encapsulated adjuvants was first evaluated using SEAP (secreted embryonic alkaline phosphatase) reporter cells overexpressing the R232 isoform of human STING or the human TLR9 gene, and NS(cdGMP) and NS(CpG) showed superior and comparable immunostimulation to their respective free adjuvant counterparts ( Figure 6F , G) . Although NS(M2e) and NS(M2e+CpG-ODN) showed comparable cargo encapsulation and physicochemical properties to NS(M2e+cdGMP) ( Figure 6E , Table 1 ), these alternative formulations produced significantly lower anti-M2e potencies than their STING agonist-loaded counterparts ( Figure 6H) . ). Notably, no significant improvement in immunogenicity was observed with NS(M2e) adjuvanted with free cdGMP at doses equivalent to those of NS(M2e+cdGMP), which could be attributed to the low delivery efficiency of free cyclic dinucleotides. Further evaluation of C57BL/6 mice and AGB6 mice (a mouse strain with a C57BL/6 background but lacking IFNα/β and IFNγ receptors) vaccinated with NS(M2e+cdGMP) showed that the immunogenicity of M2e nanoshells required appropriate interferon signaling ( Figure 6I ). These results highlight that the co-incorporation of STING agonists into M2e nanoshells is an essential component to enhance the immunogenicity of the M2e peptide.

Table 1 : Physicochemical properties of nanoshells encapsulating M2e antigen only, M2e + CpG-ODN 1826, and M2e + cdGMP. DLS characterization and peptide and CpG-ODN quantification showed that the morphology, physicochemical properties, and encapsulation efficiency of the three different nanoshells were comparable.

3. M2e Nanoshell Vaccine Immunization Stimulate Th1 Assistance T Cells, follicle-assisted T Cells and germinal center formation

To gain a deeper understanding of the humoral response of M2e, the inventors evaluated the Th1 helper T cells, follicular helper T cells ( _TFH ), and germinal center formation induced by vaccination with M2e peptide, M2e peptide adjuvanted with alum, and NS (M2e+cdGMP). Effector cell-mediated ADCC is primarily driven by binding to Fc receptor (FcgR) IV (related to human FcgR) IIIa), which recognizes IgG2a in BALB/c mice. Since Th1-dependent IFNγ secretion contributes to the production of IgG2a, the inventors first studied the M2e-specific CD4 ⁺ T cell response induced by different vaccine formulations. T cell responses were assessed using spleen cells harvested 7 days after vaccination by stimulation with M2e peptide. The NS (M2e+cdGMP) vaccine group showed the highest frequency of IFNγ ⁺ subsets after intracellular cytokine staining and flow cytometric analysis ( Figure 8A , B ). In contrast, no significant differences were observed in CD4 ⁺ IFNγ ⁺ T cells between the M2e+alum and control groups. The enhancement of CD4 ⁺ IFNγ ⁺ T cells by alum adjuvant was negligible, which is consistent with the lack of IgG2a in the serum titers of the M2e+alum group and highlights the role of Th1-biased adjuvants in inducing humoral responses that favor ADCC. Next, the inventors examined the presence of CD4 ⁺ CXCR5 ⁺ PD1 ⁺ _TFH in the draining lymph nodes 14 days after vaccination. Although vaccination with M2e peptide in the presence and absence of alum resulted in T _FH concentrations comparable to those of the PBS control group, vaccination with NS (M2e+cdGMP) significantly increased the T _FH population ( Figure 8C , D ). Similarly, analysis of the B lymphocyte population with GL7 activation markers (which are germinal center B cells that undergo rapid proliferation for antibody development and production) showed that vaccination with M2e nanoshells induced the highest concentration of B220 ⁺ GL7 ⁺ B lymphocytes ( Figure 8E , F ). Germinal center formation was further examined by histological analysis of sectioned lymph nodes 14 days after vaccination. Paracortical hyperplasia, characterized by infiltration of dendritic cells in the paracortex, was observed in the lymph nodes of the M2e+ alum-vaccinated group and the M2e nanoshell-vaccinated group ( Figure 8G ). Lymph nodes of the M2e nanoshell-vaccinated group also showed obvious follicular hyperplasia, indicating B cell hyperplasia and the progressive development of germinal centers. Immunohistochemistry further compared the distribution of GL-7+ B cells between different vaccine-vaccinated groups ( Figure 8H ). The significant accumulation of GL-7+ B cells in the germinal centers of M2e nanoshells indicated rapid B cell activation, which favored subsequent plasma cell development. These results suggest that the favorable lymph node environment and enhanced T cell helper function induced by STING agonist nanoshells promote anti-M2e humoral responses.

4. Single dose M2e Nanoshell virus Seedling inoculation Grant lethality H1N1 Effective and lasting protection against attacks

To evaluate the protection conferred by the M2e nanoshell vaccine, the inventors subjected immunized mice to a lethal PR8 influenza challenge. In addition to the single-dose M2e nanoshell group and the alum-adjuvanted M2e vaccine group, the inventors also evaluated two other prime-boost vaccine regimens, including M2e nanoshells and M2e peptides adjuvanted with MF59, an oil-in-water emulsion adjuvant licensed for use in pandemic and seasonal influenza vaccines ( FIG. 9A ). M2e potency assessment showed that boosted M2e nanoshell vaccination increased M2e antibody concentrations by approximately 2 orders of magnitude ( FIG. 9B ). In contrast, the prime-boost vaccination with M2e peptide adjuvanted with MF59 produced lower anti-M2e antibody titers than the single-dose M2e nanoshell vaccination. Under PR8 challenge, one dose of alum-adjuvanted M2e vaccination conferred no protection, while two doses of MF59-adjuvanted M2e vaccination conferred partial protection, with 60% of immunized mice dying from the virus challenge. In sharp contrast, a single dose of M2e nanoshell completely protected the vaccinated mice ( Fig. 9C , D ). Notably, although two doses of nanoshell vaccine increased overall humoral responses and serum antibody binding to influenza virus-infected MDCK cells ( Figure 10A ), equivalent ADCC activity, antiviral protection, and weight recovery were observed between the prime-boost regimen and the single-dose nanoshell regimen ( Figure 9C , D ; Figure 10B ), indicating that anti-M2e-mediated protection reaches a plateau in the absence of booster vaccination.

Since a single dose of M2e nanoshell vaccination conferred protection comparable to the booster regimen, the inventors further examined the degree of protection provided by a single dose of nanoshell vaccination. The inventors collected lung tissues from the PBS control group, the single dose of M2e+alum group, and the single dose of M2e nanoshell group 3 days after influenza challenge. Assessment of viral load using a 50% tissue culture infectious dose (TCID50) assay showed that no viral titers were detected in the lungs of the M2e nanoshell group, indicating complete inhibition of viral replication, while the control group and the M2e+alum group showed high lung viral titers ( Figure 9E ). Histopathological analysis of lung tissues further supported the significant antiviral protection of the nanoshell. No lung lesions were observed in mice receiving a single dose of nanoshell vaccine. In sharp contrast, influenza challenge caused significant lung damage in the PBS control group and the M2e/alum group, which showed severe histopathological features accompanied by significant lymphocytic infiltration and perivascular inflammation ( Figure 9F ). In addition, the bronchi of the control group showed epithelial cell necrosis and wall thickening, which were associated with impaired respiratory function. The bronchi of the M2e nanoshell vaccine-vaccinated group did not have these pathological signs and showed normal histological features of thin airway walls and columnar epithelium. These results confirm that the M2e nanovaccine has an excellent protective effect in inhibiting the virus and reducing virus-induced lung damage. Notably, the M2e nanoshell-loaded antibodies showed no neutralizing ability against influenza virus ( Figure 11 ), which highlights that ADCC can effectively protect against lung infectious diseases.

Since declining titers have been identified as a major translational barrier for M2e-based vaccine formulations, we further examined the humoral responses and durability of protection conferred by a single dose of M2e nanoshells over a 40-week period. Notably, anti-M2e IgG concentrations remained stable over the observation period ( Figure 9G ). The prolonged humoral response indicated the induction of long-lived plasma cells, whose development is highly dependent on germinal center formation and helper T cell function. We assessed the protective properties of the nanoshell vaccine by challenging aged mice with a lethal dose of PR8 virus at day 273 after nanoshell vaccination and compared them with similar challenges of age-matched controls. Unlike young mice, which showed 100% mortality after lethal challenge, aged mice showed 50% mortality at the same lethal dose. This decrease in influenza susceptibility can be attributed to the reduced inflammation and immunopathology exhibited by elderly subjects. Despite the decreased mortality, the control group still had a significant decrease in weight ( Figure 9H ). In contrast, the M2e nanoshell vaccine group had a 100% survival rate, with a peak mean weight loss of less than 10% ( Figure 9H , I ), which confirmed the long-lasting protection conferred by a single dose of M2e nanoshell vaccination. The longevity of nanoshell-induced antibodies contrasts with previous work with M2e vaccination, likely due to a reduced humoral response that makes it difficult for small peptide antigens to bind to cognate B cells in lymph node follicles.

5. Nanoshell To extend M2e Peptides In lymph node follicles Retention and exposure for antibody induction

The strong humoral response and protective properties of the M2e nanoshell prompted the inventors to question how the shielded peptide within the nanocarrier could present B cell binding and antibody stimulation effects. Since conventional nanocarrier-based strategies rely on surface antigen presentation to enhance antigen binding to cognate B cells, the counterintuitive nanoshell design and its efficacy sparked curiosity. The inventors hypothesized that the nanoshell could release the M2e peptide for a long time in the lymph node follicles for sustained immune stimulation ( Figure 12A ). The FDC network in the lymph node follicles facilitates the induction of humoral immunity, which presents antigens to B cells for affinity maturation. Since the immune complex formed by activated complement products and nanoparticles can be transmitted to FDCs rich in complement receptors after being captured by subcapsular macrophages, the inventors hypothesized that the surface of the surfactant-free, anion-rich nanoshell plays a key role in complement-dependent FDC targeting. To verify this hypothesis, the inventors prepared polyethylene glycol (PEG)-coated nanoshells and compared complement activation, follicle targeting, and immunogenicity between PEG-coated nanoshells and PEG-free nanoshells. During the preparation of the nanoshells, PEG was easily incorporated into the nanoshells by adding DSPE-PEG in the oil phase. Compared to nanoshells without PEG, PEG-modified nanoshells (M2e PEG-NS) containing equivalent amounts of M2e peptide and cdGMP encapsulation had a slightly larger particle size (121 nm) but a lower cationic surface zeta potential of -24.9 mV ( Fig. 12B ; Table 2 ). To compare complement activation between M2e NS and M2e PEG-NS, we measured the concentration 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 concentrations comparable to those of zymosan-positive controls. In contrast, PEG modification completely inhibited complement activation, with PEG-NS producing C3a concentrations similar to those of control sera ( Figure 12C ). Assessment of anti-M2e titers in mice 28 and 35 days after immunization with both M2e nanoshells showed a direct correlation between complement activation of the nanoshells and their vaccine efficacy, with PEG coating reducing overall anti-M2e titers by more than an order of magnitude ( Figure 12D ). These results suggest that the surface properties of the antigen carrier can significantly alter M2e immunogenicity.

Table 2 : Physicochemical properties and encapsulation content of M2e nanoshells without PEG and with PEG.

To examine the distribution of M2e after encapsulation and delivery by two different nanoshells, the inventors performed whole-tissue fluorescence measurements of fluorescently labeled M2e peptides in draining lymph nodes (dLNs). Alexa Fluor 647 dye-conjugated M2e peptides encapsulated in nanoshells without PEG (NS(M2e-A647)) or PEG-coated nanoshells (PEG-NS(M2e-A647)) were delivered into mice via footpad injection, and popliteal lymph nodes excised at different time points were treated with the X-CLARITY™ Tissue Clearing System to obtain optically transparent lymph nodes before fluorescence examination ( Figure 13 ). Examination 4 hours after nanoshell administration showed that both NS (M2e-A647) and PEG-NS (M2e-A647) resulted in M2e localization in the border region of the lymph nodes, indicating that the nanoparticles were captured by subcapsular sinus macrophages ( Figure 12E ). In contrast, no detectable fluorescent signal was observed in the lymph nodes after administration of free M2e-A647 peptide ( Figure 14 ). Although both M2eNS and M2e PEG-NS were effective in targeting lymph nodes, significant differences were observed when antigen retention was examined 3 days after nanoshell administration. NS(M2e-A647) administration resulted in high concentrations of M2e antigen colocalization at the lymph node follicles, whereas PEG-NS(M2e-A647) was clearly distinct with no detectable antigen signal in the lymph nodes. Closer examination of the M2e distribution in the NS(M2e-647) group revealed an interesting shift in the distribution pattern at day 7. With the reduction of follicle-bound antigen signal, venule-like fluorescent outlines emerged. The venule-like outlines are reminiscent of the vascular reticular structure of the lymph node, which is an interconnected network that allows low molecular weight molecules (<70 kDa) to flow between afferent lymphatics, follicles, and high endothelial venules. Because 100-nm nanoparticles are too large to enter and exit these channels, the antigen distribution in these ductal channels reflects that small peptide antigens have been released from the nanocarriers and exported from the follicles via the ductal system. The timing of this profile is consistent with the release kinetics of the nanoshells, which have a sustained antigen release profile over several days ( Figure 12F ). At day 14, trace amounts of antigen signals were still detectable in the lymph node follicles of the NS (M2e-A647) group. The contrasting lymph node retention kinetics between PEG-coated and unmodified nanoshells provide mechanistic insights into the differences in immunogenicity between the two nanoparticles ( Figure 12F , G ). To further evaluate the distribution of nanoshells in lymph node follicles, the inventors carefully examined the follicles 3 days after nanoshell administration. NS (M2e-A647) showed polarized distribution in follicles ( Figure 12H ), which exhibited a localization profile consistent with the distribution of FDCs in germinal centers after immune activation. On the other hand, no antigen retention in follicles was observed in the PEG-NS (M2e-A647) group ( Figure 12H , I ). The inventors further showed that injection of cobra venom factor (CVF), a snake venom that depletes complement factors, significantly impaired the follicle-targeting ability of nanoshells in mice ( Figure 15 ). Together, these results demonstrate that nanoshell-encapsulated M2e antigens can be retained and released within the FDC network for prolonged 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 the nanovaccine.

6. Single dose M2e Nanoshell vaccine Grant Different subtypes Influenza virus Broad Effect Protection

Since the collective molecules and particulate adjuvants of the M2e STING agonist nanoshell contribute to the robust and persistent anti-M2e potency of the nanovaccine, the inventors then examined the protective efficacy of the single-dose nanoshell regimen against heterosubtypic influenza viruses ( Figure 16A ). The inventors first examined the vaccine's protectiveness against the HKx31 strain, an H3N2 variant virus that has a retained M2e sequence compared to the previously examined H1N1 virus. Under viral challenge, M2e nanoshell vaccination completely protected mice from death, while the alum-adjuvanted vaccine control conferred no significant protection or survival benefit, with all mice dying within four days after challenge ( Figure 16B , C ). Further antiviral evaluation was performed using the 2009 pandemic H1N1 strain (pdmH1N1), which clearly has up to 4 M2e amino acid residues that differ from the 23-peptide-long 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 still had complete protection against pdmH1N1 ( Figure 16D , E ), which highlights the broad applicability of the nanoshell vaccine against heterosubtypic influenza viruses.

Table 3: M2e peptide sequences of consensus M2e antigens for vaccine development, A/Puerto Rico/8/1934, A/Aichi/2/1968, and A/California/7/2009.

Discuss

To overcome the low immunogenicity of the M2e antigen used for influenza vaccination, various antigen modification strategies have generated M2e-based fusion proteins and carrier proteins, potent immunogens, and immune cell targeting ligands. To the best of the inventors' knowledge, single-dose vaccine formulations that confer complete protection against heterotypic influenza challenges have not been achieved previously. The inventors demonstrated the rational integration of molecular and particulate adjuvants in vaccine design to enhance the ADCC activity of the M2e antigen using asymmetric stable polymer nanoshells. Under a single-dose vaccine regimen, high-density M2e antigens and STING agonists co-encapsulated in anionic nanoshells achieved broad and long-lasting protection against influenza, which has enormous public health impact and value. Compared to the main fusion protein design strategy used to enhance the immunogenicity of M2e, the 23-amino acid-long M2e peptides used in this study offer a scalability advantage, as such peptides can be easily synthesized by solid-phase peptide synthesis. Importantly, the inventors showed that the peak of anti-M2e antibody-mediated protection was achieved after a single nanoshell vaccination. Despite a 2-order-of-magnitude increase in anti-M2e potency, booster injections of nanoshell vaccines did not confer a significant protective benefit. The plateau of protection can be explained by the mechanism of action of anti-M2e, which can act as a bridge between infected cells and effector cells to stimulate ADCC. Unlike neutralizing antibodies that rely on pathogen binding to neutralize viruses, ADCC-inducing antibodies intercept viral replication by stimulating the perforin and granzyme of effector cells to break down infected cells. The activity of ADCC-inducing antibodies usually forms an S-shaped relationship with the lytic function of effector cells, which show saturated maximum cytotoxicity when a specific antibody concentration is reached. This concentration is inversely proportional to the antibody affinity and reflects the antibody coating density on the target cell required to fully activate the effector cells. The plateau of protection against M2e observed in mice suggests that the potency achieved by the single-dose regimen provides sufficient coverage of infected cells under lethal challenge, thereby effectively recruiting effector cells to clear the virus. Achieving such concentrations with peptide antigens under a single-dose regimen demonstrates the extraordinary adjuvant effect of the follicle-targeted STING agonist nanoshell. This robust ADCC-inducing ability may have therapeutic implications for other respiratory pathogens and cancers.

The inventors' studies have also highlighted the efficacy of STING agonist adjuvants in enhancing Th1-biased humoral responses induced by ADCC. Activation of STING by cyclic dinucleotides directly phosphorylates IRF3, which in turn stimulates the expression of type I interferons, which have profound effects in shaping acquired immune responses. Although STING agonist adjuvants have attracted great interest in the development of vaccines against infectious pathogens and cancer, the use of cyclic dinucleotides and the assessment of their adjuvant effects against alternative adjuvants have been challenging due to the poor intracellular delivery efficiency of these compounds. Similar to other nanocarriers used to enhance the delivery of STING agonists, the polymer nanoshells in this study can enhance lymph node targeting and immune cell uptake of hydrophilic molecules. The inventors further confirmed here a nanoshell-based comparison between cdGMP and CpG-ODN 1826, an alternative adjuvant that functions as an adjuvant by activating TLR9. For M2e nanoshell preparation, after unifying the dosage and delivery profiles of cdGMP and CpG-ODN, it was demonstrated that cdGMP was significantly superior to CpG-ODN in enhancing the production of anti-M2e titers. The reduced humoral responses by class B CpG-ODN may be due to its lower ability to stimulate type I IFN and its tendency to induce low-affinity, short-lived plasma cells. The inventors' observations are consistent with a recent M2e vaccine study showing that CpG-ODN adjuvants performed poorly compared to poly(I:C), a TLR3 agonist that, like cdGMP, can activate IRF3 to induce type I IFN. Since type I IFNs can enhance humoral responses through multiple mechanisms, including promoting CD4 ⁺ T cell activation, stimulating follicular helper T cells, and enhancing germinal center formation, the inventors verified the relevant properties of these type I IFNs in mice vaccinated with STING agonist nanoshells. Lymph nodes of nanoshell-vaccinated mice showed an increase in Th1, T _FH , and GL7 ⁺ germinal center B cell populations. These cell populations collectively facilitate the development and maturation of long-lived plasma cells, which help to establish a lasting humoral response.

Another factor that highlights the remarkable immunogenicity of the nanoshells is their ability to prolong the exposure of the M2e peptide antigen in lymph node follicles. The sustained antigen retention in the germinal center helps to guide the affinity maturation and survival of cognate B cells during the rapid proliferation of germinal center B cells, and the resultant enhancement of humoral responses has led to the emergence of vaccination strategies based on slow delivery immunization injection strategies and designed delivery systems. The inventors have demonstrated that shielding the peptide antigen in a biodegradable nanoshell, rather than coating it on the surface of the nanoparticles, confers unexpected spatiotemporal control over the antigen distribution in the lymph node follicles. The anionic polymers used for asymmetric emulsion stabilization and the surfactant-free nature of the nanoshells endow the particles with highly anionic surfaces that can activate complement systems via the classical pathway in the presence of calcium ions. Anionic nanoshells can effectively target FDCs in a complement-dependent manner, while the addition of the commonly used PEG stabilizer abolishes the complement activity and follicle targeting ability. After delivery to the FDC network, degradation of the nanoshells allows for continued exposure of the germinal center to the peptide antigen for B cell stimulation. The presentation of an antigen shuttling mechanism adds a new design principle to the vaccine paradigm, which typically anchors antigens on the particle surface to bind B cells. Compared to typical nanoparticle vaccines, whose surface-bound antigens and moieties can affect their isomeric activation and follicular targeting capabilities, encapsulation of antigens in degradable anionic nanocapsules presents a versatile alternative for directing antigens to lymph node follicles. Given the recent discovery that antigens encounter extracellular proteases prior to reaching lymph node follicles, leading to epitope degradation, nanoshell-encapsulated antigens may offer additional advantages in antigen protection compared to antigens presented on the surface of nanoparticle vaccines. Further tuning of the capsule degradation capacity may provide broader control over antigen persistence in germinal centers, which could add another dimension in vaccine design to enhance acquired immunity.

Conclusion

In summary, the inventors' research demonstrated a highly effective M2e nanovaccine that achieved broad and long-lasting influenza protection in a single-dose regimen. The combination of the adjuvant effect of the STING agonist and the antigen retention in nanoparticle-mediated FDCs resulted in a significant improvement in the antigen immunogenicity. The nanoshell vaccine made it possible to simplify the design of the M2e antigen, enabling the preparation of a translationally feasible vaccine formulation based on a 23-amino acid-long peptide antigen. This study further provides mechanistic insights and design inspiration for peptide antigen delivery, adding firepower to the nanotechnology toolkit for pandemic prevention.

In addition to the above experiments, the M2e nano vaccine disclosed in this disclosure is also applicable to birds. Subsequently, the experiments and results are disclosed as follows:

7. Depend on M2e Peptide binding ring GMP-AMP （ cGAMP ) Adjuvant-induced immune response in chickens

Experimental design

This study aimed to investigate the immune response induced by M2e peptide combined with cyclic GMP-AMP (cGAMP) adjuvant in chickens via ocular and nasal immunization. Specific pathogen-free (SPF) chickens were divided into three groups: a nanoshell-treated group receiving encapsulated 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 buffer ( Figure 17 ).

Immunization Program

Chickens (n=5 per group) were immunized on day 0, and tissues were collected on day 21 after immunization. Immunizations were performed via oculonasal administration to ensure that the vaccine reached the upper respiratory tract, a key site for initiating mucosal immune responses.

Tissue collection and analysis

Tissues were collected from Harderian glands, lungs, cecal tonsils, and spleen for immunohistochemistry (IHC) analysis. The focus was on detecting cells producing IgA, IgG, and MHC II in these tissues to indicate humoral and cellular immune responses.

IHC staining was performed at specific dilutions and incubation times for each antibody and tissue type, with magnifications of 100x and 200x to identify IgA and IgG producing cells. A similar approach was used to identify MHC-II producing cells.

result

IgA and IgG production

As shown in Figures 18 to 21 , IHC analysis showed that the Harderian gland, lung, and cecal tonsils of the nanoshell group significantly induced IgA and IgG-producing cells compared to the control and mock groups. The spleen also showed an increased concentration of IgG-producing cells, indicating the presence of a systemic immune response.

Quantification of the area covered by IgG-producing cells showed a significant increase in the nanoshell group compared to the control and mock groups, with significant differences noted in the Harderian glands and lungs ( Figure 22 ). The results showed a robust humoral immune response, especially in the mucosal tissues.

MHC II expression

Cells producing MHC II, which indicates antigen presentation and T cell activation, were found primarily in the bursa and spleen ( Figures 23 and 24 ). The nanoshell group showed enhanced MHC II expression, indicating effective activation of cellular immunity in response to the nanoshell vaccine.

Conclusion

Immunization of chickens with the nanoformulations using the M2e peptide combined with cGAMP adjuvant elicited a significant immune response characterized by increased production of IgA and IgG antibodies and activation of MHC II-producing cells. These findings highlight the potential of nanoshells encapsulating the M2e peptide and cGAMP as a promising vaccine candidate for inducing robust immunity in chickens, which is important in controlling viral infections in poultry.

It is obvious to those skilled in the art that various modifications and variations of the disclosed embodiments are possible. The specification and examples are intended to be considered as exemplary only, with the true scope of the present disclosure being indicated by the following claims and their equivalents.

without

Figure 1 illustrates the design (left), application (upper right), and mechanism (lower right) of a single-dose M2e-based influenza vaccine for broad influenza protection.

Figure 2 shows the preparation and characterization of the M2e nanoshell vaccine. (A) Schematic diagram of the asymmetric stable nanoemulsion process used for nanoshell vaccine preparation and cryoEM image of the polymer nanoshell. The lack of charged polymer or differential ionic buffer will lead to emulsion collapse. (B) Quantitative results of M2e peptide encapsulation after different emulsion processes for nanoparticle preparation. (C) CryoEM image of M2e nanoshell vaccine co-encapsulating M2e peptide antigen and cyclic di-GMP. Scale bar = 100 nm. (D) Encapsulation efficiency of M2e peptide and cdGMP by M2e nanoshell vaccine. (E) Size and zeta potential of empty nanoshells (NS(empty)) and M2e nanoshell vaccines (NS(M2e+cdGMP)) measured by dynamic light scattering. (F) Release kinetics of M2e peptide and cdGMP in nanoshell vaccines at pH 5 and pH 7. (G) Images of M2e nanoshell vaccines after freeze-drying and reconstitution. (H) Size and zeta potential of M2e nanoshells measured by DLS show that the physicochemical properties before and after freeze-drying are equivalent.

Figure 3 shows the quantification of M2e peptide and cdGMP encapsulated in nanoshells using the Micro BCA assay and HPLC. (A) Standard curve and representative images of M2e peptide quantification using the Micro BCA protein quantification assay. (B) M2e peptide encapsulation efficiency of nanoshells prepared with different M2e peptide concentrations in the internal aqueous phase. (C) Quantification of cyclodi-GMP encapsulated in nanoshells using HPLC.

Figure 4 shows the nanoshell counts using nanoparticle tracking analysis. For the sample containing 15 µg/mL of nanoparticles, NTA showed approximately 1.2×10 ⁹ nanoparticles. This number is equivalent to approximately 8×10 ¹¹ 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 was calculated based on the average of 3 independent measurements.

Figure 5 shows the encapsulation of the M2e peptide in the nanoshell before and after freeze-drying. After freeze-drying and reconstitution, the nanoshell retained its encapsulant and the loss of the peptide was negligible.

Figure 6 shows anti-M2e induction and ADCC activity in mice after vaccination with M2e nanoshell vaccine. (A) M2e-specific IgG titers in mice after single-dose immunization with PBS, M2e peptide, M2e peptide with alum adjuvant, and M2e nanoshell vaccine. (B) Ratio of M2e-specific IgG2a to IgG1 titers in immunized Balb/C mice on day 35 after vaccination. Error bars represent mean ± SEM (N = 5). (C) Acetone-fixed heterotypic influenza-infected MDCK cells to assess the binding of anti-M2e antibodies from mouse sera to cell-bound M2e. Immunofluorescence assay was performed using sera from mice on day 42. Cell nuclei were stained with DAPI. H1N1: A/Puerto Rico/8/1934 (H1N1); H3N2: A/HKx31 (H3N2). Scale bar = 100 μm. (D) Antibody-dependent cytotoxicity (ADCC) surrogate assay was performed on H1N1-infected MDCK cells using mouse sera derived on day 42 after vaccination. Data are presented as mean ± SEM (N = 3). (E) CryoEM images show the morphology of nanoshells encapsulating combinations of M2e + cdGMP or M2e + CpG-ODN 1826. (F) Human STING activation was assessed using SEAP reporter cells containing free cdGMP, NS (cdGMP), or empty NS after 24 h of culture. (G) Human TLR9 activation was assessed using SEAP reporter cells containing free CpG-ODN2395, NS (CpG-ODN2395), or empty NS after 24 h of culture. (H) M2e-specific IgG antibodies in BALB/C mice immunized subcutaneously 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 mice or AGB6 mice immunized with NS (M2e + cdGMP). Error bars represent mean ± SEM (N = 3). Statistical analysis was performed using one-way analysis of variance or student's t test (*p ＜ 0.05, **p ＜ 0.01, ***p ＜ 0.001, ****p ＜ 0.0001).

Figure 7 shows the M2e-specific IgG1 and IgG2a titers of mice immunized with PBS, M2e peptide, alum-adjuvanted M2e peptide, and M2e nanoshell vaccine on day 35 after vaccination. Error bars represent mean ± SEM (N = 5).

FIG8 shows that the M2e STING agonist nanoshells promote a lymph node environment that is favorable for Th1-biased antibody production. (A, B) M2e-specific IFNγ ⁺ CD4 ⁺ T cell responses in immunized mice were measured by intracellular cytokine staining 7 days after the primary immunization. Error bars represent mean ± SEM (N = 3). (C, D) The frequency of follicular helper T cells and (E, F) GL7+ germinal center B cells in the draining lymph nodes of immunized mice 14 days after immunization. Error bars represent mean ± SEM (N = 3). Statistical analysis was performed using one-way analysis of variance (*p < 0.05, **p < 0.01, ***p < 0.001). (G) Perfused popliteal lymph nodes of immunized mice were fixed and embedded in paraffin 14 days after vaccination. Hematoxylin/eosin (H&E) staining was performed to identify follicular hyperplasia (white arrows) and paracortical hyperplasia (black arrows) in the lymph nodes. Scale bar = 500 μm. (H) Popliteal lymph node sections were stained with anti-GL-7 antibody (brown) to identify germinal centers. Scale bar = 200 μm.

Figure 9 shows the evaluation of antiviral protection by single-dose M2e nanoshell vaccination. (A) Vaccination schedule and virus challenge for single-dose and prime-boost immunization regimens. (B) Evaluation of anti-M2e titers in mouse sera collected on day 35 after the primary vaccination. (C) Weight change and (D) survival rate of mice challenged with 3 × 10 ⁵ PFU of virus via intranasal route on day 42 after A/Puerto Rico/8/1934 (H1N1) infection (N=5). (E) Virus titers in lungs were evaluated 3 days after virus infection (N=3). (F) Hematoxylin/eosin (H&E) staining was performed to identify lymphocytic infiltration and perivascular inflammation (upper panel). Scale bar = 200 μm. Lung tissue was also monitored for bronchial injury (lower panel), including the presence of epithelial necrosis (white arrows) and airway wall thickening (black arrows). Scale bar = 100 μm. (G) Anti-M2e titers in mice 273 days after primary M2e nanoshell vaccination (N = 5). (H) Mice were challenged intranasally with 3 × 10 ⁵ PFU of A/Puerto Rico/8/1934 (H1N1) on day 273, and (H) weight change and (I) survival were assessed after infection (N = 5). Error bars represent mean ± SEM. Statistical analysis was performed using one-way analysis of variance. Survival was analyzed using the log-rank test (**p < 0.01, ***p < 0.001, ****p <0.0001; ns, not significant).

Figure 10 shows the serum titers binding to influenza virus infected MDCK cells and serum ADCC activity of mice vaccinated with one or two doses of M2e nanoshell vaccine. (A) Immunofluorescence assay showed that the serum of mice receiving the two-dose nanoshell vaccine regimen produced higher antibody binding and immunofluorescence to H1N1- and H3N2-infected MDCK cells compared to the serum of mice vaccinated with one dose of nanoshell vaccine. (B) ADCC surrogate assay showed that ADCC activity was equivalent between the serum from mice vaccinated with one or two doses of nanoshell vaccine.

Figure 11 shows a hemagglutination inhibition test to examine the ability of anti-M2e antibodies to neutralize influenza virus. Anti-M2e antibodies showed no significant neutralization ability against influenza pathogens.

Figure 12 shows the spatiotemporal control of M2e peptide antigen distribution in lymph node follicles by nanoshell carriers. (A) Schematic representation of the effect of nanoshell surface properties on complement activation and lymph node distribution. (B) Dynamic light scattering characteristics of size and zeta potential of M2e STING agonist nanoshells without PEG (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 subsequently cultured with zymosan (Zymosan), nanoshells without PEG (M2e NS), or PEGylated nanoshells (M2e PEG-NS) (N=3). (D) M2e-specific IgG titers in mice 35 days after immunization with M2e NS or M2e PEG-NS. Error bars represent mean ± SEM (N = 5). Statistical analysis was performed using unpaired t-test (**p < 0.01). (E) BALB/c mice were immunized with nanoshells containing fluorescent A647-conjugated M2e peptide (M2e-A647) to track M2e antigen distribution over a 14-day period. Filter dendrites (FDCs) were labeled in situ with anti-CD35 antibodies, and excised dLNs were cleaned and imaged using confocal microscopy (CD35 blue; M2e-A647 red). Scale bar = 200 μm. (F, G) Colocalization of M2e with subcapsular macrophages and lymph node follicles was assessed by imaging analysis of M2e-A647 signal coordinated with lymph node border signal and anti-CD35 signal, respectively. (H) Magnified image of M2e distribution in lymph node follicles 3 days after subcutaneous administration of 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 shows the image of a clear lymph node after X-clarity treatment. Popliteal lymph nodes were removed and tissue clearing was performed to track the distribution of M2e peptide.

Figure 14 shows fluorescent images of draining lymph nodes of mice after vaccination with M2e peptide conjugated with Alexa Fluor 647. The images were obtained 4 hours after injection of the fluorescently labeled M2e peptide into the paw.

Figure 15 shows a comparison of M2e antigen retention in lymph node follicles in mice after vaccination with M2e nanoshells. For complement factor elimination, mice received an intravenous injection of cobra venom factor (CVF) 3 hours prior to nanoshell injection. Subsequently, draining lymph nodes were removed on day 3 for evaluation. (A) Representative images of lymph node follicles showing reduced retention of M2e-A647 at lymph node follicles in mice treated with CVF. (B) Quantification of M2e-A647 retention in control and CVF treated mice (N=3).

Figure 16 shows the evaluation of M2e nanoshell vaccines against heterotypic influenza challenge. (A) Vaccination and virus challenge schedule. Mice were subcutaneously vaccinated with PBS, M2e NS, or free M2e peptide adjuvanted with alum. Mice were challenged intranasally with 3 × 10 ⁶ PFU influenza A/HKx31 (H3N2) or 2.5 × 10 ⁶ PFU 2009 pandemic H1N1 (pdmH1N1) on day 42. (B) Percentage weight change and (C) survival of mice after influenza A/HKx31 (H3N2) infection. (D) Percentage weight change and (E) survival of mice after 2009 pandemic H1N1 (pdmH1N1) infection. Error bars represent mean ± SEM (N=5). Survival was analyzed using the log-rank test (*** p < 0.001).

Figure 17 shows the experimental design for studying the immune response induced by the combination of M2e peptide and cyclic GMP-AMP (cGAMP) adjuvant in chickens administered via oculonasal immunization. Specific pathogen-free (SPF) chickens were divided into three groups: a nanoshell-treated group receiving encapsulated 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 tissues were collected on day 21 after immunization. Immunizations are administered via the eye and nose to ensure that the vaccine reaches the upper respiratory tract, a key site for initiating mucosal immune responses. Tissues are collected from the Harderian gland, lungs, cecal tonsils, and spleen for immunohistochemistry (IHC) analysis. The focus is on detecting cells producing IgG, IgA, and MHC II in these tissues to demonstrate humoral and cellular immune responses. IHC staining is performed at specific dilutions and incubation times for each antibody and tissue type, with magnifications of 100x and 200x to identify cells producing IgA and IgG. A similar approach is also used to identify cells producing MHC-II.

Figures 18 and 19 show photographs of IgA-producing cells at 100x and 200x, respectively.

Figures 20 and 21 show 100x and 200x photographs of IgG-producing cells, respectively.

FIG22 shows the quantification of the area covered by IgG-producing cells showing a significant increase in the nanoshell group , with significant differences in the Harderian gland and lung compared to the control and mock groups.

Figures 23 and 24 show 100x and 200x photographs of cells producing MHC-II, respectively.

TW202444409A_113113341_SEQL.xml

Claims (24)

A composition comprising a polymer nanoparticle encapsulating an antigen and an adjuvant, wherein the antigen is an M2e peptide; wherein the polymer nanoparticle comprises: a water-impermeable polymer shell, and one or more aqueous cores surrounded by the polymer shell. The composition of claim 1, wherein the outer diameter of the polymer shell is 50-150 nm. The composition of claim 1, wherein the M2e peptide comprises the following sequence: SLLTEVETPIRNEWGCRCNGSSD (SEQ ID. No. 1), SLLTEVETPIRNEWGCRCNDSSD (SEQ ID. No. 2) or SLLTEVETPTRSEWECRCSDSSD (SEQ ID. No. 3). The composition of claim 1, wherein the adjuvant is an agonist. The composition of claim 4, wherein the agonist is a STING agonist comprising cyclic di-GMP, cGAMP, poly (I:C) or CpG. The composition of claim 1, wherein the polymer shell comprises a short PLGA polymer having a molecular weight between 6000 and 18000 Da. A composition as claimed in claim 1, wherein the polymer shell does not contain a surfactant. A method for treating a disease, comprising: Administering a composition as claimed in claim 1 that is capable of physically binding to cells to a subject in need thereof. The method of claim 8, wherein the disease is influenza A. The method of claim 8, wherein the administration comprises intravenous injection, subcutaneous injection or intraperitoneal injection. The method of claim 8, wherein the subject is a human or a bird. A vaccine capable of inducing an immune response to influenza A, comprising the composition of claim 1. If the vaccine is claimed in item 12, it is a single-dose vaccine preparation. The vaccine of claim 12, wherein the outer diameter of the polymer shell is 50-150 nm. The vaccine of claim 12, wherein the M2e peptide comprises the following sequence: SLLTEVETPIRNEWGCRCNGSSD (SEQ ID. No. 1), SLLTEVETPIRNEWGCRCNDSSD (SEQ ID. No. 2) or SLLTEVETPTRSEWECRCSDSSD (SEQ ID. No. 3). The vaccine of claim 12, wherein the adjuvant is a agonist. A vaccine as claimed in claim 16, wherein the agonist is a STING agonist comprising cyclic di-GMP, cGAMP, poly (I:C) or CpG. The vaccine of claim 12, wherein the polymer shell comprises short PLGA polymers having a molecular weight between 6000 and 18000 Da. A vaccine as claimed in claim 12, wherein the polymer shell does not contain a surfactant. A method for neutralizing viral infection, comprising: Vaccinating a subject in need with the vaccine of claim 12. The method of claim 20 further comprises: Using the vaccine to enhance the immunity of the subject. The method of claim 20, wherein the vaccinating step and the boosting step are performed by at least one mode selected from the group consisting of: non-oral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intraperitoneal, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. The method of claim 22, wherein the vaccinating step and the boosting step are performed subcutaneously or intranasally. The method of claim 20, wherein the subject is a human or a bird.