KR20170010717A - Mucoadhesive polymer adjuvant-based influenza vaccine - Google Patents

Abstract

The present invention relates to a vaccine composition for treating influenza A comprising a combination of adjuvant and an antigen having a neutralization ability with respect to various influenza antigens. The vaccine composition may induce excellent humoral immunity, cellular immunity and mucosal immunity effects compared to existing cholera toxin with respect to five types of different influenza virus sub-types.

Description

MUCOADHESIVE POLYMER ADJUVANT-BASED INFLUENZA VACCINE [0002]

The present invention relates to a vaccine composition for the treatment of influenza A, comprising a mucosal adhesion enhancer composition and an antigen.

Although the vaccine is the most cost-effective biomedical approach to preventing influenza and related complications, current vaccine manufacturing processes are not efficient and administration of such vaccines is still inconvenient.

On the other hand, vaccine strains are required to be periodically updated to respond to viruses that are circulating due to repeated antigenic drift. Specifically, it is essential to develop an anti-influenza vaccine with cross-protection potential against seasonal drift variants as well as occasional recombinant viruses.

As a candidate candidate for development of the vaccine, the influenza matrix protein 2, specifically the ectodomain (M2e), is well conserved among the influenza strains and is considered an attractive target to induce cross-protection against the influenza A subtype. Similarly, the fusion domain of the stalk domain, specifically the hemagglutinin (HA) (HA2), is another region that is strongly conserved and has been shown to be a subtype of influenza A virus, Specific antibody response with cross-protection against < RTI ID = 0.0 > However, these M2 and HA2-based vaccines have the disadvantage of low immunogenicity and have reduced mortality but not morbidity in animal studies.

Thus, many studies have been focused on increasing the immunogenicity of M2 and HA2-based vaccines with various vaccine adjuvants. Hundreds of different very bunts have been tested over the last few generations for new vaccines, but the majority have been found to lack efficacy, unacceptable local or systemic toxicity, manufacturing difficulties, low stability, and excessive costs Due to the limitations involved, human use has not been approved. For the above reasons, so far, aluminum-based mineral salts in human vaccines have been the most widely used as very bunt. However, the aztans are not strong enough to induce sufficient antibody-specific lipid immunity against subunit protein antigens or to stimulate cell-mediated immunity. Furthermore, the zombant can induce an immunoglobulin E antibody response coupled with an allergic response in some human subjects.

The mucosal surface, specifically the respiratory mucosa, is a major access point for many infectious agents and is the first defense against infection, so that the mucosal vaccine is a powerful next generation vaccine that can induce a protective immune response in mucosal and systemic immunity, respectively It shows the form of strategy. Mucosal vaccines provide benefits in terms of production and regulation for systemic vaccines. Specifically, mucosal vaccines are practical for large-volume vaccination and do not pose a risk of blood-borne infection caused by contaminated needles. The ease of administration, the possibility of delivery by individuals without medical training, and the improvement of compliance are considered to be an advantage of mucosal vaccine strategies, particularly prevention of epidemic spread, including influenza virus infection.

However, few commercially available mucosal vaccines are presently available because effective mucosal vaccination is quite challenging due to the difficulty of producing effective mucosal immunity and the lack of a safe and effective mucosal avant-bundle and delivery system 10-2013-0044204).

The present invention provides a composition comprising an mucoadhesive adhesive avant-form composition and an antigen, wherein the mucoadhesive adhesive multi-blunt composition comprises a complex of an anionic polymer, a toll-like receptor agonist, and a saponin, Wherein said antigen is an influenza A virus comprising an sM2 antigen protein having the amino acid sequence of SEQ ID NO: 1 and a protein having an amino acid residue 15-137 sequence in the HA2 antigen protein of A / EM / Korea / W149 / 06 (H5N1) There is provided a therapeutic vaccine composition.

However, the problems to be solved by the present invention are not limited to the problems described above, and other problems not described can be clearly understood by those skilled in the art from the following description.

One aspect of the disclosure includes a mucoadhesive adhesive budent composition and an antigen, wherein the mucoadhesive adhesive composition comprises a complex of an anionic polymer, a toll-like receptor agonist, and a saponin Wherein the antigen comprises an antigenic protein having an amino acid sequence of SEQ ID NO: 1 and a protein having an amino acid residue 15-137 sequence in the HA2 antigen protein of A / EM / Korea / W149 / 06 (H5N1) And a vaccine composition for treating influenza A.

According to one embodiment of the invention, the influenza A therapeutic vaccine composition is a cross-protection against influenza A subtype selected from the group consisting of H5N1, H1N1, H5N2, H7N3 and H9N2 subtypes of the influenza A Lt; / RTI >

According to one embodiment of the invention, the influenza A therapeutic vaccine composition may be a powder of the mucoadhesive avant-born composition.

Embodiments herein are efficient mucosubstance systems for influenza vaccines based on the recombinant fusion protein sM2HA2 comprising the Stalks domains of consensus matrix proteins-2 (sM2) and (HA2), including poly-gamma -glutamic acid ) -Based novel influenza vaccine composition.

Embodiments of the present invention are directed to a novel mucosal vaccine composition based on recombinant fusion protein sM2HA2 and mucoadhesive gamma-PGA / MPLA / QS21 aztans. The novel mucosal vaccine compositions can be prepared by a simple and robust strategy, can enhance the immune response, and can ensure broad protection against various influenza subtypes. The immunogenicity and efficacy of the novel mucosal vaccine compositions was assessed with an animal model of < RTI ID = 0.0 >

The present invention relates to a recombinant fusion protein sM2HA2 comprising the Stalks domain of consensus matrix proteins-2 (sM2) and HA (HA2), and poly-gamma -glutamic acid (gamma-PGA) as an efficient mucosubstantial system for influenza vaccines, Lt; RTI ID = 0.0 > vaccine < / RTI >

The poly-gamma -glutamic acid (y-PGA) -based vaccine zbuntu system not only acts as mucosal adhesion delivery vehicle for sM2HA2, but also acts as a hydrophobic immunostimulatory 3-O-desacyl-4'-monophosphoryl lipid A ) ≪ / RTI > and < RTI ID = 0.0 > QS21. ≪ / RTI > The intranasal co-administration of the sM2HA2 and the combined azturate gamma-PGA / MPLA / QS21 (CA-PMQ) can induce a mucosal, systemic, cell-mediated immune response with a high degree of protection have. The sM2HA2 / CA-PMQ immunization can prevent disease symptoms and provide complete protection against lethal infection by various influenza subtypes (H5N1, H1N1, H5N2, H7N3 and H9N2) for at least 6 months Can be given.

Thus, the present application shows that mucosal administration of sM2HA2 in combination with CA-PMQ can be a powerful strategy for a wide range of cross-protective influenza vaccines and that CA-PMQ as a mucosal avant-bundle can be used against an effective mucosal vaccine do.

Figure 1a shows the effect of various combinations of recombinant protein antigens (sM2HA2) and mucoadhesive polymer (gamma-PGA) -based combination aztamiv (CA-PMQ, gamma-PGA / MPLA / QS21) (A) The chemical and molecular structure of γ-PGA, MPLA, QS21, and sM2HA2.
Figure 1b shows a CA-PMQ-based vaccination for anti-viral mucosal and systemic immunity. [Gamma] -PGA may serve as a mucoadhesive delivery vehicle for the sM2HA2 vaccine.
Figures 2A-2E illustrate the characterization and the avant-bunt characteristic of the CA-PMQ in one embodiment of the present invention.
Figure 3 is a graph depicting the results of a single intraperitoneal injection in a mouse vaccinated with 1: OVA alone (20 μg), 2: OVA + γ-PGA / MPLA / QS21 (PMQ), 3: OVA + CT (D) IL-4 and (E) IFN-y cytokines in (A) serum IgG, (B) nasal wash IgA and (C) serum IgG isotype.
Figure 4 illustrates the enhancement of the immune response to sM2 and HA2 when CA-PMQ is delivered with the recombinant sM2HA2 vaccine in one embodiment of the invention.
Figure 5 shows that, in one embodiment of the present invention, the CA-PMQ-succumbed sM2HA2 vaccine confer improved cross-protection against various influenza subtypes.
Figure 6 shows that CA-PMQ-succumbed sM2HA2 induces long-lasting immune responses and protective efficacy against lethal infections by various influenza subtypes in one embodiment of the invention.
Figure 7, for the screening of ¹²³ I- labeled OVA (OVA ⁽¹²³ I)) having a high labeling efficiency according to one embodiment of the present size-determined using a rule PD-10 desalting column, ¹²³ I activity ( Bar) and OVA elution amount (yellow portion of 4-2).
Figure 8 shows that CA-PMQ induces IgG sub-classes specific for sM2 and HA2 in one embodiment of the invention.
Figure 9 shows the results of a preliminary experiment to determine the proper dose and efficacy of sM2HA2 protein against influenza virus infection in one embodiment of the present invention.

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains.

It should be understood, however, that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout this specification, when a member is "on " another member, it includes not only when the member is in contact with the other member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise. The terms "about "," substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) "or" step "used to the extent that it is used throughout the specification does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Throughout the present specification, the microorganism used as the transformed host cell can be any microorganism which is not toxic or attenuated when applied to a living organism, such as Gram-negative bacteria Escherichia coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacterium bovis, And lactic acid bacteria such as Bacillus, Lactobacillus, Lactococcus, Staphylococcus, Listeria monocytogenes, Streptococcus and the like which can be used as the Gram-Yan bacteria can be used. . The lactic acid bacteria may include Lactobacillus genus, Streptococcus genus, and Bifidobacterium genus. Typically, the genus Bacillus is Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus permentum, Lactobacillus delbrueus Lactobacillus jones, Lactobacillus family, Lactobacillus bulgaricus and Lactobacillus casei; Strap Tortoise Cocker Strap Tortoise Cocker Spaniel Moss; The genus of Bifidobacterium is Bifidobacterium, Bifidobacterium bifidum, Bifidobacterium, Bifidobacterium, Bifidobacterium, Bifidobacterium, Bifidobacterium, Bifidobacterium, And the like can be used, and more preferably, Lactobacillus lactobacillus casein can be used, but the present invention is not limited thereto.

Throughout this specification, the term "vector" means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. As the plasmid is the most commonly used form of the current vector, the term "plasmid" and "vector" are sometimes used interchangeably throughout the specification. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Although there is no appropriate truncated enzyme cleavage site, the synthetic oligonucleotide adapters according to conventional methods can easily ligate the vector and foreign DNA using a linker. After ligation, the vector should be transformed into the appropriate host cell. Transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook, et al., Supra. Alternatively, electroporation (Neumann, et al., EMBO J., 1: 841, 1982) can also be used to transform such cells, but is not limited thereto.

As a vector used for cell surface expression of an antigen according to the present invention, a vector known in the art can be used.

On the other hand, in order to increase the expression level of a transfected gene in a host cell, the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in a selected expression host. Preferably, the expression control sequence and the gene are contained within a recombinant vector containing a bacterial selection marker and a replication origin.

Throughout the specification, the term " transformation " means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration.

Throughout this specification, the term "vaccine" means an agent used to stimulate the immune system using biological organisms for the purpose of prevention against disease. Immunization refers to a process for efficiently removing an antigen by stimulating the production of antibodies, stimulation of T-lymphocytes, or other immune cells (such as macrophages) in an organism. An overview of the detailed immunology above is readily understood by those of ordinary skill in the art (Barrett, J. T., Textbook of Immunology, 1983). The subject to be transfected with the transformed microbial vaccine expressing the target protein as an antigen may be a mammal, more preferably a human.

Throughout this specification, the term " mucoadhesiveness " may be intended to refer to the property that the substance is attached to the mucosa through interaction of the hydroxyl group of the protein per mucosal layer of the epithelial cell with hydrogen bond or the like, But may not be limited.

Throughout this specification, one of ordinary skill in the art can make appropriate choices among a variety of vectors, expression control sequences, and hosts without undue experimentation, without departing from the scope of the present invention. For example, in selecting a vector, the host should be considered because the vector must be replicated within it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered.

One aspect of the disclosure includes a mucoadhesive adhesive budent composition and an antigen, wherein the mucoadhesive adhesive composition comprises a complex of an anionic polymer, a toll-like receptor agonist, and a saponin Wherein said antigen comprises influenza A virus comprising an sM2 antigen protein having the amino acid sequence of SEQ ID NO: 1 and a protein having an amino acid residue 15-137 sequence in the HA2 antigen protein of A / EM / Korea / W149 / 06 (H5N1) There is provided a therapeutic vaccine composition.

In one embodiment of the invention, the influenza A therapeutic vaccine composition is cross-protected against an influenza A subtype selected from the group consisting of subtypes H5N1, H1N1, H5N2, H7N3 and H9N2 of the influenza A .

In one embodiment of the present invention, the sM2 protein is synthesized by modifying the M2 protein conserved sequence of H5N1, H5N2, H1N1, H7N3, and H9N2 influenza viruses, and is a cone containing only the extracellular and cytoplasmic domain portions from which the transmembrane domain has been removed. It can be expressed as a census sequence (SEQ ID NO: 1).

In one embodiment of the invention, the HA2 protein is a protein having an amino acid residue 15-137 sequence in the HA2 antigen protein of A / EM / Korea / W149 / 06 (H5N1) virus.

In one embodiment of the present invention, the sM2HA2 protein comprises the amino acid sequence of the sM2 antigen protein having the amino acid sequence of SEQ ID NO: 1 and the amino acid residue 15-137 of the HA2 antigen protein of the A / EM / Korea / W149 / 06 (H5N1) Lt; / RTI >

In one embodiment of this application, the anionic polymer is a biocompatible polymer containing a carboxyl group and / or a hydroxy group and may be selected from the group consisting of polygamma glutamic acid, hyaluronic acid, cellulose, polyacrylic acid, polysaccharides, derivatives thereof, ≪ / RTI > and the like.

In one embodiment of the invention, the anionic polymer enables the poorly soluble immunostimulatory substance to be easily and reproducibly dispersed in an aqueous solution without a surfactant. The anionic polymer serves as a lyophilization stabilizer so that the mucoadhesive adhesive lotion composition can be stably prepared in powder form, but it is not limited thereto. The anionic polymer can interact with the antigen of the vaccine through electrostatic attraction or lipophilic linkage.

In one embodiment of the invention, the polygamma glutamic acid (gamma -PGA) is naturally synthesized by microorganism species (Vasili) and is a high-anion used in a variety of applications (e.g., food, cosmetics, and pharmaceuticals) And exhibits excellent biocompatibility and non-cytotoxicity. The γ-PGA may serve as a mucosal adhesion delivery vehicle for recombinant protein antigens and may also bind a hydrophobic immunostimulatory compound such as 3-O-desacyl-4'-monophosphoryl lipid A (MPLA) and QS21 It can provide a simple but powerful strategy.

In one embodiment, the toll-like receptor agonist is selected from the group consisting of MPLA (monophosphoryl lipid A), imiquimod, recipe mod, flagellin, CpG, poly (I : C), and combinations thereof.

In one embodiment herein, the MPLA is a detoxified derivative of a lipopolysaccharide (LPS) derived from the Re595 strain of Salmonella minnesota , which acts through TLR4, a member of the toll-like receptor (TLR) family . MPLA-stimulated dendritic cells acting as antigen-presenting cells (APCs) express increased levels of co-stimulatory molecules and secrete cytokines that are important for activation and differentiation of B- and T-cells. However, MPLA is a poorly soluble compound that is difficult to disperse in aqueous solution. Accordingly, various approaches have been used to increase solubility, including formulation into emulsions, formulation into aqueous dispersions containing small amounts of surfactant or helper lipids, and inclusion in liposomes.

In one embodiment of the invention, the saponin may be selected from the group consisting of QS21, Quil A, QS7, QS17, beta-eskin, digitonin, and combinations thereof.

In one embodiment of the invention, the mucoadhesive ejbant composition may be in powder form.

In one embodiment herein, the anionic polymer, the toll-like receptor agonist, and the saponin have a weight ratio of about 1: about 0.001 to about 1: about 0.01 to about 10 , For example, about 1: about 0.01: about 0.01, or about 1: about 1: about 1.

In one embodiment herein, the mucoadhesive ejbant composition may be used by dissolving it in an aqueous solution, but may not be limited thereto.

In one embodiment of the present invention, the solvent dissolving the anionic polymer, the tol-like receptor agonist and the saponin is selected from the group consisting of dimethylsulfoxide (DMSO), ethanol, aceton, dimethylformamide (DMF ), Chloroform (chloroform), and combinations thereof.

In one embodiment of the invention, the mucosal adhesion enhancer composition and the antigen may be included in a weight ratio of about 1: about 0.1 to about 10, but may not be limited thereto.

In one embodiment of the invention, the virus is type A influenza virus.

In one embodiment of the invention, the vaccine is preferably capable of ingestion for oral use or food, and is preferably for nasal administration, but is not limited thereto.

In one embodiment of the invention, the vaccine is preferably for human and animal prophylaxis, more preferably wild birds such as chickens, turkeys, geese, ducks, pheasants, quail, doves, ostriches, poultry, Cat, and the like, but the present invention is not limited thereto.

1 schematically illustrates a method of constructing a vaccine composition by combining MPLA / QS21 / antigen using polygamma glutamic acid as an anionic polymer in one embodiment of the present invention. The γ-PGA provides a simple and robust strategy for binding of hydrophobic MPLA, QS21 and protein antigens. sM2HA2 protein and the consensus sequence sM2 over the extracellular and cytoplasmic domain residues except for the transmembrane domain, wherein sM2 is the fusion peptide HA2 derived from A / EM / Korea / W149 / 06 (H5N1) in the pRSET A vector (Residues 15-137). The purified sM2HA2 protein from the prokaryotic expression system is detected by SDS-PAGE. Kumasi Brilliant Blue staining (left panel) shows that the protein has a molecular weight of 25 kDa. Furthermore, the protein was confirmed by Western blot analysis using anti-M2 (middle panel) and anti-HA2 antibody (right panel): lane 1: whole cell lysate of E. coli containing pRSET A vector without sM2HA2 gene ; Lane 2: Purified sM2HA2 protein.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by these Examples.

[Example]

Ethics Declaration

All experiments with animals were carried out in accordance with the Korean NIH Guidelines for the Management and Use of Laboratory Animals. All mouse experimental procedures were approved by the animal experiment committee, protocol number BSL-ABSL-13-010 of BioLeaders Corporation, Daejeon, Korea, for animal care and use. BioReaders' IACUC is registered in the Republic of Korea's Animal and Plant Quarantine Agency (QIA). We did our best to minimize the pain of the animals.

< Production of? -PGA / MPLA / QS21 complex>

γ-PGA (Mw, 5 kDa) was prepared from BioReaders Corporation (Daejeon, Korea). MPL (Avanti Polar Lipids Inc), QS21 (Desert King Chile Inc.) and OVA (Boryung Pharmaceutical Co.). MPLA (1 mg, 0.57 mmol) and QS21 (1 mg, 0.50 mmol) were dissolved in a mixed solvent of DMSO and 20% THF, respectively. The two solvents were mixed with γ-PGA (13.3 mg) and dissolved in 10 mL of distilled water with vigorous stirring at room temperature for 5 minutes.

After this reaction, the solution was incubated with shaking for 24 hours at 25 &lt; 0 &gt; C. The solution was lyophilized and re-dissolved in 10 mL of distilled water. The solution was dialyzed against cellulose membrane tubes (MWCO 12-14 kDa) in deionized water for 3 days.

Thereafter, the solution was lyophilized to obtain the final product (? -PGA / MPLA / QS21 complex) as a powder.

<Characterization of γ-PGA / MPLA / QS21 complex>

The γ-PGA / MPLA complex (γ-PGA: MPLA (1: 5 (w / w)) was dissolved in DMSO-d ₆ : D ₂ O (8: PGA: QS-21 (1: 5 (w / w)) was also added to a solution of DMSO-d ₆ : D ₂ O (8: 2 (v / v)).

The molecular weight and molar radius of the γ-PGA conjugate sample were measured by size-exclusion chromatography (SEC) equipped with a refractive index (RI) and a multi-angle laser light scattering (MALLS) detector. Each retention time of the SEC chromatogram was measured with a static laser light-scattering detector. The scattering intensity is related to the concentration at each retention time, as measured by a separate concentration-sensitive RI detector. The system was eluted at a flow rate of 0.8 mL / min when a viscosity detector was attached. Three consecutively connected HPLC-SEC columns (TSKgel GMPWXL, TOSHO) were used.

This characterization was carried out at 40 &lt; 0 &gt; C in a column oven. The γ-PGA and γ-PGA complexes (γ-PGA / MPLA / QS21 and γ-PGA / MPLA / QS21 + OVA) were dissolved in 0.1 M NaNO ₃ as a mobile phase in SEC analysis, 220 nm; Millex-GV), and injected (200 μl). Light scattering was measured with a DAWN Heleos II MALLS detector. A Viscotek VE3580 RI detector was used as the RI detector. Excitation and fluorescence emission spectra of the γ-PGA / MPLA / QS21 + OVA resuspended in PBS were obtained using a fluorescence spectrophotometer (FS-2, Scinco, Korea). The anti-freeze activities of γ-PGA were measured using DSC (Differential Scanning Calorimetry), and the calculated values are shown in Table 1 below.

[Table 1]

Table 1 shows the calculated activity of γ-PGA in γ-PGA / MPLA / QS21 after DSC measurement.

<Storage stability of PGA / MPLA / QS21 and a very bunt effect>

In order to test the storage stability and the avant-bulb effect of the γ-PGA / MPLA / QS21 (CA-PMQ) complex, the OVA antigen-specific IgG response in the serum was stored for 4 months in a shelf- After storage (22-29 &lt; 0 &gt; C). The titers of the antibodies were determined using ELISA (enzyme-linked immunosorbent assay) on serum samples from each mouse (n = 5).

First, a 96-well immunoadsorption plate (Snow, Denmark) was sensitized with 100 μL of each antigen (1 μg mL ^-1 ) overnight at 4 ° C. After blocking the plate (2% BSA in PBST, 1 hour at 37 ° C) to prevent nonspecific binding, 100 μl of a 2-fold serial diluted sample diluted in blocking buffer was added to the plate (37 ° C For 1 hour), HRP-conjugated goat anti-mouse IgG antibody (Southern Biotech) diluted 1: 4000 was added. After incubation at room temperature for 1 hour, 100 μL of peroxidase substrate tetramethylbenzidine (TMB; BD Pharmingen) was added to each well. The reaction was terminated by the addition of 2M H ₂ SO ₄ . Absorbance was recorded with an ELISA reader (Molecular Devices, Sunnyvale CA) at a wavelength of 450 nm. The endpoint reversal was determined using an optical density (OD) cutoff value of 0.2.

< in vivo  SPECT / CT Imaging>

OVA was radiolabeled with ¹²³ I using an iodine tube method. Briefly, Na ¹²³ I was added to an iodine tube (Pierce, Rockford, Ill., USA) and stirred gently for 5 minutes. To this solution was added 0.4 mg of OVA dissolved in 1 mL of PBS (phosphate buffered saline, pH 7.4) and allowed to react with Na ¹²³ I at room temperature for 1 hour. The radiolabeled OVA ( ¹²³ I) was purified using a PD-10 column (Pierce) and the product was condensed using a centrifuge (cut-off: 10 kDa, Millipore, Billerica, Mass., USA) 7).

The OVA ( ¹²³ I) was then mixed with PBS (control) or the CA-PMQ was dissolved in PBS and allowed to react for 30 minutes prior to use. The mass ratio of OVA ( ¹²³ I): CA-PMQ was 1: 5. Approximately 40 [mu] l of sample was intranasally administered to BALB / c mice (6 weeks old, approximately 20 g). Animal SPECT and CT were performed in Inbion PET / CT / SPECT (Siemens, Knoxville, TN, USA) of Korea Basic Science Research Center (Cheongwon, Korea). After 6 hours and 12 hours of administration, the mice were scanned

&Lt; Preparation of recombinant sM2HA2 fusion protein >

Consensus sM2 across the extracellular and cytoplasmic residues except the transmembrane domain was obtained from the analysis of the H5N1, H1N1 and H9N2 subtype sequences in the database.

Previous studies have shown that the extracellular domain is recognized as an epitope for the induction of monoclonal antibodies that are well-conserved among influenza virus subtypes (amino acids) and can protect against influenza virus infection .

In order to ensure optimal homology with the other subtypes used in the examples herein, the residues at positions 14 (EG) and 18 (RK) of H5N1 were modified to account for the conserved epitope (amino acid) Respectively. The consensus template was amplified by polymerase chain reaction (PCR) using a specific primer containing restriction enzyme sites (SecI and Eco RI) at the ends. The gene was then cloned into pGEM-T Easy vector using the TA cloning method (Promega, USA). The gene fragment was then cut and inserted in-frame into the multi-cloning site of the pRSET A vector (Invitrogen, USA) using the corresponding site to form the plasmid pRSET A-sM2 .

Similarly, the HA2 gene (residue 15-137) from A / EM / Korea / W149 / 06 (H5N1) was modified by inserting Eco RI and Hind III sites at the 5 'and 3' end sites, respectively, Gt; pRSET-A-sM2HA2 &lt; / RTI &gt; for the expression of the prokaryotic nuclei of pRSET-A-sM2HA2. The plasmid construct was confirmed by restriction enzyme cleavage and proved by sequencing (Solgent, Daejeon, Korea).

E. coli BL21 (DE3) cells were transformed with the pRSET-A-sM2HA2 plasmid using the heat shock method. Thereafter, the colonies were treated with 100 μg / mL of ampicillin (96.9% purity according to high performance liquid chromatography (HLPC); Calbiochem, USA) and 35 mg / mL chloramphenicol (according to high performance liquid chromatography (HLPC) 97 % Purity; Calbiochem) was seeded in 5 mL of LB broth and re-grown with stirring at 37 &lt; 0 &gt; C.

The cells were then incubated overnight in a large volume LB medium at 37 DEG C with stirring at 200 rpm. When the incubation reached an optical density (OD ₆₀₀ ) of 0.6, 2 mM isopropyl -? - D-thiogalactopyranoside (99% purity; Bio-Basic, Ontario, Canada) For an additional 12 hours to induce the expression of the target protein. The cultures were obtained by centrifugation at 6,000 x g for 20 minutes at 4 ° C. The cell pellet was then incubated with 20 Ml of a solution containing 20 mM Tris-HCl, 0.5 M NaCl, 10% glycerol and protease inhibitor (1 mM phenylmethylsulfonyl fluoride, Sigma-Aldrich, &Lt; / RTI &gt; in cold buffer. Bacterial lysis was performed by ultrasonication at 25% amplitude for 3 minutes at intervals of 2-second pulse and 1-second stop, followed by centrifugation (12,000 x g, 20 minutes) at 4 ° C.

The supernatant was then removed and the remaining pellets remained as inclusion bodies (IBs). The inclusion bodies were re-suspended in denaturing buffer (20 mM Tris-HCl, 0.5 M NaCl, 10% glycerol and 6 M urea), sonicated for 1 minute and centrifuged immediately before. The supernatant suspension was removed and the residual pellet was re-suspended in denaturing buffer II (20 mM Tris-HCl, 0.5 M NaCl, 8 M urea, pH 8.0) and stirred overnight at 4 ° C. The pellet was sonicated and immediately centrifuged as before.

The supernatant was then separated from the inclusion bodies into rescued proteins and purified by His-tag affinity chromatography (Qiagen, USA). The purified protein was then dialyzed at 4 ° C and in PBS using a permeable cellulose membrane (molecular cut-off: 12-14 kDa, Spectrum Research, East Damascus, Auckland, USA).

The target protein was identified by SDS-PAGE and its concentration was determined using Bradford assay (Bio-Rad, Hercules, Calif., USA). Western blot analysis using a mouse anti-histidine antibody (1: 500, Invitrogen, Carlsbad, CA, USA) and rabbit anti-M2 and anti-HA2 antibody (1: 1000) was performed for immunodetection. The rabbit anti-M2 and anti-HA2 antibodies used in this experiment were obtained by intramuscular inoculation of KLH-conjugated M2 and HA2 peptides in rabbits twice weekly. The purified protein was used as a vaccine antigen.

<Animal Grouping and Vaccination>

Specific pathogen free (SPF) BALB / c mice (n = 380) were purchased from Samtako (Seoul, Korea). The mice were stored in SPF environment and obtained the approval of IACUC (Daejeon, Korea). The mice were separated into five experimental sets, each containing five groups. Of the 5 sets, 3 sets had 14 mice per group and 2 sets had 17 mice per group. Three groups of mice in each set were intranasally vaccinated with 20 [mu] l of sM2HA2 (2 [mu] g) alone or with CA-PMQ or CT azantes at 1-week intervals. As a control group, two of each set were similarly treated with PBS or CA-PMQ alone.

All mice were anesthetized with ether prior to inoculation and the present invention was performed in a biosafety level (BSL) -2 laboratory facility. Blood samples were obtained in retro-orbital reticulation at day -1 (pre-experiment), 7 (first), 21 (second), 35 (third), and 180 The serum was separated by centrifugation and stored at -20 ° C for later analysis. After 1 and 24 weeks of the last vaccination, 3 mice from each group were randomly sacrificed to obtain samples from the lungs and intestines and stored at 70 [deg.] C for mucosal IgA study. To analyze the CTL response, spleens were aseptically obtained from arbitrary 3 mice in one of the sets on day 35.

< Specific antibodies to sM2 and HA2 >

Serum IgG values, including isotype (IgG1 and IgG2a), and mucosal IgA (lung and bowel) were determined using an ELISA using sM2 and HA2 proteins as coating antigens as described above. The coating antigens used in the examples herein were obtained by E. coli expression of pRSET A-sM2 and pRSET A-HA2 plasmids and purified by His-tag-affinity chromatography (quiagen, USA) as described above . 96-well ELISA plates (Snow, Rochelle, Dermat) were coated with 300 ng / well sM2 or HA2 protein overnight at 4 占 폚 and in coating buffer (pH 9.6). After washing with PBS containing 0.05% Tween 20 (PBST), the plate was blocked with 300 μL of 10% skim milk.

Unlabelled supernatant from homogenized tissue for detection of 1: 200 diluted mouse serum in PBS containing 2% skim milk for detection of IgG, IgG1, IgG2a, or local IgA (lung and intestine) Were added to designated wells and cultured at 37 DEG C for 2 hours.

Thereafter, horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, IgG2a or IgA (Sigma-Aldrich, St. Louis, USA) diluted 1: 3,000 was added. After 2 hours of incubation at 37 ° C, 100 μL of substrate solution containing tetramethylbenzidine (TMB, Millipore, Bedford, Mass.) Was added to the plate and the reaction was allowed to proceed for 10 minutes. After completion of the reaction by adding 2N H ₂ SO ₄ , the optical density at 450 nm was measured using an ELISA automatic reader (Molecular Devices).

<T-cell ELISPOT specific for sM2 and HA2>

Interferon (IFN) -γ and interleukin (IL) -4 ELISPOT assays in spleen cells were performed using mouse IFN-? And IL-4 ELISPOT kits (BD Biosciences, USA) as described above.

Briefly, BD ELISPOT 96-well plates were coated with anti-mouse IFN- [gamma] and IL-4 capture antibodies (5 [mu] g / mL) in PBS overnight at 4 [deg.] C. After removing the antibody, the plate was blocked with a solution containing complete RPMI 1640 medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, Calif., USA) and incubated at room temperature for 2 hours.

Zygotic cells from vaccinated mice were aseptically isolated and incubated with either sM2 or HA2 protein (1 ug / well), medium alone (negative control), avant alone or 5 ug / mL phytohemagglutinin (positive control, bit halogen, Ted Carlsbad, CA, USA was added) ⁵ × 10 5 cells / well in medium containing. After 24 hours of incubation at 37 ° C under 5% CO ₂ , the plates were treated sequentially with biotin-conjugated anti-mouse IFN-γ and IL-4 antibody, streptavidin-horseradish peroxidase, and substrate solution .

Finally, spots were counted using an ImmunoScan Entry analyzer (Cellular Technologies, Shaker Heights, USA).

<Virus and Attack Inoculation Test>

In order to investigate and compare the protective efficacy of various influenza viruses against the highly fluxed sM2HA2 vaccine with CA-PMQ, mice were treated with the pathogens A / EM / Korea / W149 / 06 (H5N1), A / Puerto Rico / 8 / Infected with the A / Aquatic bird / Korea / W81 / 2005 (H5N2), A / Aquatic bird / Korea / W44 / 2005 (H7N3) or A / Chicken / Korea / 116/2004 (H9N2) influenza subtypes. All the viruses used in the examples of this application were received from Dr. Choi, Young-Gi (Medical School and Medical Research Center, National University of Chungbuk, Cheongju, Korea).

The mice were anesthetized and one week after the last vaccination, they were intranasally infected with a 10-fold mouse-adapted virus of MLD ₅₀ contained in 20 μL of PBS. Mice were observed for 13 days and time points were fixed to measure weight loss and survival. Six mice in each group were sacrificed at 3 and 5 dpi to check for viral titer in the lungs and 5 mice in each group were left for use in the survival test. Survival was determined based on cut-off of 25% weight loss, which is the time of death or euthanasia of the animal. All efforts to minimize pain were made and all mice surviving after the final observation were humanely euthanized by inhalation of CO ₂ for 5 minutes.

<Virus Titer and Histopathological Analysis of the Lung>

A 50% tissue culture infectious dose (TCID ₅₀ ) assay was performed to determine the viral titer of the lungs (33) as described above. Briefly, lung tissue was homogenized in PBS containing antimicrobial and antifungal agents (Gibco, Grand Island, NY, USA) and centrifuged at 12,000 x g to remove cellular floats. All samples were immediately serially diluted 10-fold and incubated with confluent MDCK cells for 1 hour at 37 ° C and a humid atmosphere. An intermediate layer medium containing L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) trypsin (Thermofisher Scientific, Rockford, USA) was added to the infected cells and incubated for 72 hours.

The virus titre was determined based on the hemagglutinin (HA) test after observing the cytopathic effect (CPE), calculated by the Reed and Muench method, and the Log ₁₀ TCID ₅₀ / lung tissue Respectively. In terms of histopathology, lung tissue was obtained from an ether coma-anesthetized mouse at 5 dpi. The tissue was immediately fixed in 10% neutral buffered formalin, embedded in paraffin wax, flaked, placed on a slide, and stained with eosin dye. Histopathologic changes were observed with an optical microscope as described above.

<Statistical Analysis>

Data are presented as means ± SD (SD) and represent at least three independent experiments. The differences between the groups were analyzed using ANOVA and the averages were compared by Student T-test. Values of <0.05 and <0.01 were considered significant and very significant, respectively. Comparisons of survival were performed in a log-rank test using GraphPad Prism 6.0.

Preparation of < sM2HA2 Recombinant Protein and [gamma] -PGA / MPLA / QS21 &

1 is a graph showing the effect of the recombinant fusion protein sM2HA2 and the mucoadhesive combination succulent gamma-PGA / MPLA / QS21 (CA-PMQ (SEQ ID NO: 2)) containing the Stach domain of consensus matrix proteins- &Lt; / RTI &gt; of the vaccine system.

Figure 1A shows a simple and robust strategy for simultaneous binding of recombinant proteins using hydrophobic MPLA, QS21, and anionic gamma-PGA in this example. The vaccine system is prepared by the formation of hydrogen-binding and hydrophobic interaction-induced complexes between γ-PGA and MPLA / QS21 and the formation of ionic and hydrophobic interaction-induced complexes between γ-PGA and protein antigens (Fig. 1A, upper and middle panels). To improve the range of protection against influenza A virus, well-conserved sM2 and HA2 fusion constructs were obtained (Fig. 1A, bottom panel). The recombinant protein (sM2HA2) was refolded from the inclusion bodies of E. coli, purified by His-tagged affinity chromatography, and dialyzed using a permeable cellulose membrane. The purified protein was confirmed by SDS-PAGE and Western blotting using rabbit anti-M2 and anti-HA2 antibody. Through coomassie staining and immunoblotting, the protein was observed at a molecular weight of 25 kDa as predicted (Fig. 1A, bottom panel).

The presence of a carboxyl group in the? -PGA can provide a strong interaction moiety with the mucosal layer. These interactions are thought to be the result of hydrogen bonding between γ-PGA and proton-acceptor in the mucous glycoprotein. Thus, CA-PMQ can act as a transmucosal delivery vehicle for sM2HA2 and can induce strong mucosal and systemic anti-viral immunity (FIG. 1B). When γ-PGA is mixed with MPLA and QS21, water-insoluble MPLA and amphipathic QS21 can be easily dispersed in an aqueous solution. Due to the electrostatic repulsion from ionized carboxy groups at pH 5.5-7.4, the uncoiled expanded state of? -PGA facilitates hydrogen bonding and secondary interaction with MPLA and QS21.

The results of the FTIR-ATR spectroscopy in the present example are shown in FIG. 2A. Specifically, the FTIR spectra of γ-PGA, MPLA, QS21, γ-PGA / MPLA and γ-PGA / QS21 are shown. Encircled in the figure is the protonated carboxylic acid (Protonation) and increased hydrogen bonding of reducing ^{γ-PGA (2,509.11 cm -1)} - it shows the following chemical shifts (3,435.31 cm ^1).

As shown in Figure 2a, it supports hydrogen-binding interactions with MPLA and QS21 of gamma-PGA. The interaction with MPLA and QS21 in the γ-PGA was analyzed characteristics, the two parts were noteworthy (for example, hydrogen, and carboxylic acid salts of carboxylic acids in combination peak, 3,416.31 cm ^-1 at 2,509.11 cm ^-1). 3416.31 ^cm-1, the hydrogen representing the point-bonding molecules with MPLA and QS21 in the γ-PGA-increased to cross hydrogen bond.

The above carboxylic acid peak (at 2,509.11 cm ^-1 ) also decreased after complex formulation as the carboxylic acid groups of? -PGA were used to form hydrogen bonds with MPLA and QS21. The hydrophobic main chain of? -PGA can also interact by hydrophobic interaction with the hydrophobic alkyl chains of MPLA and QS21.

To investigate antigen-specific immune responses, OVA (ovalbumin), a protein antigen model, can also be coupled to γ-PGA. The efficient dispersion of OVA through ionic and hydrophobic interactions in the γ-PGA matrix was studied using fluorescence spectrophotometry. 2B shows the excitation and emission spectrum of the γ-PGA / OVA (FITC) complex according to this embodiment. As shown in FIG. 2B, when OVA (FITC) is mixed with γ-PGA, OVA FITC) were both increased. Furthermore, the emission peak of OVA (FITC) showed a blue-shift whereas the excitation band of OVA (FITC) became red-shifted, indicating that γ-PGA acts as a dispersant and has a hydrophobic interaction with OVA (FITC) (FITC), which is the main cause of OVA.

) 결과를 나타내었다. 상기 도 2c 에 나타낸 GPC-MALLS 데이터와 같이, γ-PGA 를 MPLA, QS21 및 OVA 와 혼합한 후에, 기울기 (몰 반경을 몰 질량으로 나눈 것) 가 감소하였음을 나타내었으며, 이는 γ-PGA, MPLA, QS21, 및 OVA 의 복합체가 형성되었음을 제시하는 것이다. MPLA 및 QS21 과의 결합 후에, CA-PMQ 복합체는, 백신 아주번트의 서로 다른 형태의 추후의 제형화 및 투여 전에 다양한 바이러스성 항원의 조합을 위한 중간 물질로 사용될 수 있는 파우더 형태로 보관될 수 있다 (입자-기반 또는 하이드로겔-타입 백신 아주번트). 동결건조된 CA-PMQ 복합체는 저장 안정성 측면에서 냉장(냉동) 보관을 요하는 액체 제형인 전통적인 아주번트에 비하여 이점을 갖는다. On the other hand, FIG. 2C shows the moles of γ-PGA (black square), γ-PGA / MPLA / QS21 complex (blue triangle) and γ-PGA / MPLA / QS21-OVA complex GPC-MALLS analysis of moles radius to mass (

) Results. As shown in the GPC-MALLS data shown in FIG. 2C, it was shown that the slope (the molar radius divided by the molar mass) decreased after mixing the γ-PGA with MPLA, QS21 and OVA, , &Lt; / RTI &gt; QS21, and OVA were formed. After conjugation with MPLA and QS21, the CA-PMQ complex can be stored in powder form that can be used as an intermediate for the combination of various viral antigens prior to further formulation and administration of different forms of vaccine embryo (Particle-based or hydrogel-type vaccine). The lyophilized CA-PMQ composites have advantages over traditional aztans, which are liquid formulations that require refrigeration (frozen) storage in terms of storage stability.

Furthermore, in the lyophilization process of the CA-PMQ complex,? -PGA can act as an antidandruff agent for MPLA and QS21. During sample preparation by lyophilization, the freezing and thawing process frequently damages the biologically active material. Γ-PGA produced in Vasily is expected to have high immobilization activity due to its high acidic amino acid composition. The immobilized activity of γ-PGA was measured by DSC and the results are shown in Table 1. The immobilized activity of γ-PGA was significant (Table 1).

The immunostimulatory activity of the lyophilized CA-PMQ complexes in this example was checked at 4 months after manufacture and the results are shown in FIG. 2d: Immediately after OVA alone (1), very burnt to CA-PMQ Of OVA-specific antibody titers tested in OVA (2) and 4 months after formulation (3). As shown in FIG. 2D, the antibody titer values were almost the same. The results of these experiments suggest that the? -PGA-based materials can be used for binding hydrophobic immunostimulatory materials by a simple and robust manufacturing strategy.

The present example also examined mucoadhesive properties of a combined vaccine system consisting of OVA (as an antigen model) and CA-PMQ, and the residence time of the intranasal OVA antigen determined using SPECT / CT is shown in Figure 2e : SPECT images at 6 hours and 12 hours after nasal administration of [ ¹²³ I] -labeled OVA (OVA ( ¹²³ I)) in the presence and absence of CA-PMQ (red dot circle: nasal cavity, yellow arrow: ). To investigate the mucoadhesive properties of γ-PGA, OVA was added to CA-PMQ and the mixture was intranasally administered. In order to keep track of in vivo (in vivo) of the administered dose condition, was labeled OVA as iodine ⁽¹²³ I), was carried out micro SPECT / CT (single positron emission computed tomography / computed tomography) imaging. Specifically, approximately 500 μL of each 4.5 mL fraction was obtained and then condensed. The reaction was performed simultaneously in two batches and a total of 1 mL of OVA ( ¹²³ I) was produced.

As shown in FIG. 2e, by performing micro SPECT / CT system imaging, we observed a strong SPECT signal of OVA ( ¹²³ I) administered with CA-PMQ. The signal remained strong after 12 hours. In contrast, the signal when OVA ( ¹²³ I) was administered alone sharply decreased within 6 hours by the mucociliary clearance mechanism.

These experimental results suggest that γ-PGA increases the residence time of OVA antigen co-transferred into the mucosal layer and acts as a controlled-release reservoir for nasal epithelial cells. Through the present example, it can be assumed that gamma -PGA can interact with the mucin molecule through physical chain entanglement and form a hydrogen bond with the sugar residue of the oligosaccharide chain. Since the γ-PGA is in an uncoiled state at the pH of the nasal secretion (pH 6.0-6.5), the electrostatic repulsive force generated from the ionized functional groups is such that γ-PGA is involved in the mucus glycoprotein and mechanical chain entanglement and 2 Thereby facilitating vehicle interactions.

To determine the inducing effect of CA-PMQ on the immune response, OVA protein as a protein antigen model was administered via the intranasal route with CA-PMQ, and antibody titers were determined using an ELISA assay. The level of OVA-specific serum IgG induced by the CA-PMQ / OVA group was significantly higher than that induced by the OVA alone group (FIG. 3A). Similarly, high levels of OVA-specific IgA titers were detected at the inoculation site (FIG. 3B). In addition, OVA-specific IgG1 and IgG2a levels were also detected, and both IgG1 and IgG2a were significantly increased in OVA-inoculated group with CA-PMQ or CT compared to OVA alone (FIG. 3C).

Cellular immune responses were analyzed through the generation of (D) IL-4 and (E) IFN-y cytokines in splenocytes from vaccinated mice. Spleen cells of vaccinated mice (n = 5) with OVA peptide (SIINFEKL, 5 μg mL ^-1 ) were stimulated and determined using ELISPOT analysis. Compared with OVA alone immunized mice or controls, mice vaccinated with CA-PMQ showed more IL-4 and IFN-y-secretory cells in the spleen stimulated with OVA protein (Figures 3D and 3E) . These results indicate that the CA-PMQ system can function as an effective multinucleate capable of strongly inducing strong mucosal immunity as well as lean body and cellular immune response.

All data were obtained three times and expressed as mean ± SD. Asterisks show significant differences between OVA / PMQ, OVA / CT and controls (** P <0.01; * P <0.05).

- sM2HA2  CA- PMQ  With mucosa Vaccinated  Thing sM2 - &lt; / RTI &gt; and HA2-specific immune response:

To determine the ability of CA-PMQ to induce an immune response, groups of mice were intranasally immunized with 2 [mu] g of sM2HA2 alone or a combination of CA-PMQ. The components of the CA-PMQ are actually generally recognized as safe (GRAS), and their safety profile has already been measured. Following intra-mouse administration of CA-PMQ, weight loss and respiratory distress were not observed. To compare the effects of well-known mucosal very cholera toxin (CT) and CA-PMQ, mouse groups were immunized in a similar manner using CT and sM2HA2 together.

BALB / c mice were immunized with sM2HA2 alone, sM2HA2, CA-PMQ alone, or PBS with CA-PMQ or CT ejbant at 0, 2 and 4 weeks. Serum samples were collected at -1 (pre), 7 (first), 21 (second) and 35 (third) days. Serum IgG, lung and intestinal IgA were detected by ELISA and absorbance at 450 nm was measured. For the measurement of sM2- or HA2-specific systemic and local antibodies, ELISA plates were coated with purified sM2 or HA2 protein (3 [mu] g / mL). (A) Serum IgG antibodies specific to sM2 (B) Specific mucosal IgA in the lungs (left panel) and intima (right panel). (C) serum IgG antibodies specific for HA2 and (D) HA2-specific mucosal IgA in the lungs (left panel) and intestines (right panel). Spleen cells were obtained 10 days after the last immunization. Cells were re-stimulated in vitro with sM2 or HA2 protein and cytokine-forming cell points were determined by analysis. IFN-y and IL-4 locus-forming cells were determined per 5 x 10 ⁵ splenocytes.

 Serum IgG responses specific for sM2 (Fig. 4A) and HA2 (Fig. 4C) were induced to a much higher level in the sM2HA2 group with CA-PMQ aztunate than in the sM2HA2 alone group. Controls vaccinated with PBS and CA-PMQ alone failed to induce higher levels of sM2- or HA2-specific immune responses than those seen in the CA-PMQ and sM2HA2 without CT groups, -PMQ or CT. &Lt; / RTI &gt; Similarly, high IgA titers specific for sM2 and HA2 were detected in both inoculated and non-inoculated sites.

As shown in Figures 4B and D (left panel), mice immunized with sM2HA2 with CA-PMQ or CT azantes were significantly more immunocompetent than mice immunized with sM2HA2 alone or with the control, IgA. Interestingly, vaccination with sM2HA2 / CA-PMQ or sM2HA2 / CT also induced high levels of sM2- and HA2-specific IgA in the gut (Figs. 4B and 4D, right panel).

In addition, sM2- and HA2-specific IgG1 and IgG2a levels were also measured. Specifically, BALB / c mice were intranasally immunized with sM2HA2 alone, sM2HA2, CA-PMQ alone, or PBS with CA-PMQ or CT eubtane at 0, 2 and 4 weeks. Serum samples were collected at -1 (pre-experiment), 7 (first), 21 (second) and 35 (third) days. For the measurement of sM2- or HA2-specific IgG1 and IgG2a antibodies, ELISA plates were coated with purified sM2 or HA2 protein (3 μg / mL). Absorbance of the antibody was measured by absorbance at 450 nm using an indirect ELISA.

Figure 8A shows the results of IgG1 (left panel) and IgG2a (right panel) antibodies specific for sM2, and Figure 8B shows the results of IgG1 (left panel) and IgG2a (right panel) antibodies specific for HA2.

As shown in Fig. 8, both IgG1 and IgG2a increased to a very high level when compared to sM2HA2 alone and the control group, in the group in which sM2HA2 was inoculated with CA-PMQ or CT after immunization enhancement (Figs. 8A and 8B ). IgG1 was more prominent than IgG2a.

Taken together, these results indicate that vaccination with the sM2HA2 / CA-PMQ vaccine resulted in more effective induction of sM2- and HA2-specific IgG and IgA responses than either sM2HA2 alone or CA-PMQ , Which is similar to or somewhat improved by powerful mucosal avant-bleated CT. Additionally, since the sM2HA2 / CA-PMQ induces significant levels of IgG1 and IgG2a in the mouse and increases the production of IgG2a and IgG1 antibodies, respectively, into Th1 and Th2 cells, the newly introduced vaccine approach Suggesting that it is possible to effectively induce cell lysing and cell-mediated immunity.

Bar indications represent the ± standard deviation of the group (n = 5). The data represent three independent experiments. The asterisk indicates significant differences between the sM2HA2 / CA-PMQ, sM2HA2 / CT and control groups (** P <0.01; * P <0.05).

- CA- PMQ  The sM2HA2  Vaccine-induced sM2 - &lt; / RTI &gt; and HA2-specific T-cell response:

T-cell responses are important in the regulation of an effective immune response and are known to contribute to a wide range of cross-protective immunity. To investigate this protective immune mechanism, the induction of intracellular IFN-y and IL-4-secreting cells was detected by ELISPOT one week after the final boosting. The spleen cells producing IFN-y and IL-4 were stimulated with HA2 protein (Fig. 4E) and sM2 (Fig. 4F). Mice vaccinated with CA-PMQ showed a very high number of IFN-y-secreting cell populations in the spleen stimulated with sM2 and HA2 proteins as compared to the mice immunized with sM2HA2 alone or the control (Fig. 4E and Fig. 4E) 4F, left panel).

In addition, a significantly increased number of IL-4-secreting cells were detected in the spleen of mice inoculated with sM2HA2 with CA-PMQ compared to the mice vaccinated with sM2HA2 alone or the control (Fig. 4E and 4F, Right panel). However, sM2HA2 recombinant protein was also detected in the spleen after being stimulated with the sM2 and HA2 proteins, without any mutant, while levels of IFN- [gamma] - secretase were detected in the spleen, whereas in the control only sole background levels of cytokine- gamma and IL-4-secreting cells (Fig. 4E and 4F), which can induce cellular immunity as the fusion protein sM2HA2 itself,

The CA-PMQ indicates that this immunity can be greatly enhanced. Importantly, mice vaccinated with sM2HA2 with CT showed similar levels of IFN-y and IL-4-secreting cells in the spleen after stimulation with the sM2 and HA2 proteins. These results provide evidence that intranasal immunization with sM2HA2 in combination with CA-PMQ enhances the sM2- and HA2-specific T-cell immune responses to levels comparable to the use of CT known as strong mucosal avant-bundle.

Bar indications represent the ± standard deviation of the group (n = 5). The data represent three independent experiments. The asterisk indicates significant differences between the sM2HA2 / CA-PMQ, sM2HA2 / CT and control groups (** P <0.01; * P <0.05).

- CA- PMQ - Very bunched sM2HA2  The vaccine ensures protection and improves lung characteristics against lethal infections by various influenza subtypes:

To evaluate sM2HA2-induced immunoreactivity against lethal infections by influenza virus, vaccinated mice were challenged with 10 LD50 mouse-adapted H5N1, H1N1, H5N2, H7N3 or H9N2 influenza A subtypes Respectively. Protection efficacy was assessed as survival rate and weight loss and observed daily (dpi) for 13 days post-infection.

Specifically, BALB / c mice were immunized with sM2HA2 alone, sM2HA2, CA-PMQ alone or with PBS with CA-PMQ or CT esbunt, at 0, 2 and 4 weeks. Mice were intranasally infected with 10 LD ₅₀ mouse-adapted influenza virus subtypes 2 weeks after the last boost. Virus titers in lung tissue were determined by TCID ₅₀ in MDCK cell lines at 3 and 5 dpi. At 5 dpi, the lungs were randomly extracted from one set of each group and fixed and stained with eosin prior to observation under an optical microscope.

Figure 5A shows the body weight (upper panel), survival (intermediate panel, log-rank test, P = 0.00063) after infection with A / EM / Korea / W149 / 06 (H5N1) 5B shows the change in body weight (upper panel), survival (middle panel, logarithmic panel) after infection with A / Puerto Rico / 8/34 (H1N1) in this example, 5C shows the change in body weight after infection with A / Aquatic bird / Korea / W81 / 2005 (H5N2) in the present example (upper panel, ), Survival (middle panel, log-rank test, P = 0.00063) and lung virus titer (lower panel), and FIG. 5D shows the change in A / Aquatic bird / Korea / W44 / 2005 ), Survival (middle panel, log-rank test, P = 0.00063) and lung virus titers (lower panel) (Middle panel, log-rank test, P = 0.0001) after infecting with A / Chicken / Korea / 116/2004 (H9N2) in this example, 0.00063) and lung virus titers (lower panel). As shown in Figure 5F, in contrast to the lungs of sM2HA2 or vaccinated mice as a control, the lungs of mice vaccinated with sM2HA2 / CA-PMQ showed clear lung vacuoles free of inflammatory cell infiltration.

As expected, mice in the non-immunized group in the control group did not survive the lethal influenza infection. Significant weight loss was observed in mice vaccinated with sM2HA2 only without CA-PMQ or CT (Fig. 5A-5E, top panel) and 0-40% of individuals survived against various lethal infections of various subtypes 5A-5E, middle panel). In contrast, mice administered sM2HA2 with CA-PMQ survived 100% against the lethal infection of H1N1, H5N1, H5N2 and H9N2 (FIG. 5A-5C and 5E, middle panel) 80% survived (Figure 5D, middle panel). mice vaccinated with sM2HA2 / CT survived 100% against the lethal infections of the H1N1, H7N3 and H9N2 influenza subtypes (Figures 5B, 5D and 5E, middle panel), and the lethality of the H5N1 and H5N2 influenza subtypes (Figure 5A, middle panel) and 80% (Figure 5C, middle panel), respectively, against infection. mice immunized with sM2HA2 / CT showed a similar level of protection (60-100%) to mice immunized with sM2HA2 / CA-PMQ, whereas in the case of sM2HA2 / CT-vaccinated mice significant weight loss (Figures 5A-5E, top panel), &lt; RTI ID = 0.0 &gt;

This indicates that protection against lethal infection is not effective in the CT-very bunted group. These results are consistent with previous studies demonstrating that intranasal vaccination with 3M2eC + CT could improve mortality but induce antibodies that can not be improved to morbidity. In contrast, mice administered with sM2HA2 with CA-PMQ exhibited minor signs of disease and only showed a temporary and slight decrease in body weight.

To further better understand the protective efficacy observed with the sM2HA2 / CA-PMQ vaccine, the viral titer of the lungs of the inoculated mice was measured at 3 and 5 dpi to evaluate the viral clearance. The sM2HA2 / CA-PMQ-immunized mice exhibited reduced lung viral titers at day 3 when challenged with H5N2, H7N3 and H9N2 viruses, and completely cleared the infection at day 5 (Fig. 5C-5E, bottom panel).

Similarly, in the case of the lethal infection of H5N1 and H1N1, mice immunized with CA-PMQ-very burnt sM2HA2 showed a significant decrease in lung viral titer at 5 dpi compared to sM2HA2 alone immunized mice or control mice (Figs. 5A and 5B, bottom panel). In addition, the virus clearance of the lungs of sM2HA2 / CT-immunized mice was observed to be associated with survival outcomes for infection with various influenza subtypes.

Histopathological examination was performed to clarify the association with the clearance of the virus in the lung. Representative samples of the lungs of mice challenged with the H5N2 virus were obtained and examined using an optical microscope. Mice immunized with sM2HA2 / CA-PMQ showed reduced or no pulmonary inflammation, while apparent signs of severe pulmonary inflammation were observed in the lungs of mice or controls immunized with sM2HA2 (Fig. 5F).

Taken together, these results demonstrate that sM2HA2 in combination with the CA-PMQ conserved fusion protein significantly improves the protective efficacy against various influenza subtypes, and that the sM2- and HA2-specific It can be seen that the immune response contributes to the efficient control of viral replication.

Error bars represent the mean ± SD of body weight. The asterisk indicates a significant difference in body weight between the groups (n = 5). The data represent three independent experiments. In pulmonary viral titer, an asterisk indicates a significant difference between sM2HA2 / CA-PMQ, sM2HA2 / CT and the control group at 5 dpi (** P <0.01; * P <0.05).

- CA- PMQ - Very bunched sM2HA2  Long term Persistent  Immune response and protection were induced:

Immune responses and the duration of protection are important criteria for effective vaccines. Thus, the persistence of immunity induced by sM2HA2 / CA-PMQ was examined through the detection of serum IgG and mucosal IgA by ELISA 6 months after immunization.

Specifically, mouse sets were intranasally vaccinated with PBS, CA-PMQ, sM2HA2, sM2HA2 / CA-PMQ or sM2HA2 / CT according to schedule. Serum was obtained 0, 35, and 180 days after the last vaccination. Lung and bowel samples were obtained on 180 day tea. To confirm long-lasting antibody levels of long-lasting IgG, IgG1, IgG2a and IgA, ELISA was performed three times using sM2 or HA2 protein as a coating antigen. Six months after the final vaccination, the mice were inoculated with lethal doses of A / Puerto Rico / 8/34 (H1N1) virus (10 MLD ₅₀ ). FIG. 5A shows IgG, IgG1 and IgG2a antibody levels specific to sM2 (left) and HA2 (right) in this example, and FIG. 5B shows antibody levels specific for lung (left) and intestinal (Left) and survival (right) percent change after infecting with the H1N1 virus in the present example, and FIG. 5C shows the IgA antibody level Lt; / RTI &gt;

In mice immunized with sM2HA2 / CA-PMQ, similar levels of sM2 and HA2-specific serum IgG as well as lung and intestinal IgA were detected after 6 months of boosting (FIGS. 6A and 6B). Similarly, in order to determine whether the T-helper cell subtype also acts after 6 months of the final vaccination with sM2HA2 / CA-PMQ, IgG subtypes such as IgG1 associated with Th2 and IgG2a associated with Th1 were measured by ELISA Respectively. Interestingly, mice vaccinated with sM2HA2 / CA-PMQ showed significant levels of IgG1 and IgG2a specific for both sM2 and HA2 after vaccination, and IgG1 and IgG2a levels were detected at approximately the same level 6A).

Taken together, these results indicate that sM2HA2 / CA-PMQ induces both fluid and cell-mediated immune responses lasting up to at least 6 months after the final vaccination. To determine the duration of protection, immunized mice were challenged with influenza A / Puerto Rico / 8/34 (H1N1) heterosubtype. Weight change and survival were observed up to 13 dpi. The control mice exhibited > 30% weight loss and died 9 days after infection (Fig. 6C, left panel).

In contrast, the sM2HA2 / CA-PMQ-vaccinated mice exhibited an unexpectedly constant percentage weight loss, but the body weight recovered to 13 dpi and the mice survived 100% against the infection, (Figure 6C, right panel). Furthermore, mice immunized with sM2HA2 alone showed a rapid weight loss and were 100% mice and succumbed to 11 dpi, indicating that the CA-PMQ contributes to long-lasting immunity.

Taken together, these results indicate that the CA-PMQ not only improves the immune response and protective efficacy of the recombinant sM2HA2 vaccine against various influenza subtypes, but also assures long-term persistence of immunity.

The bars indicate mean ± standard deviation. The asterisk indicates a significant difference between sM2HA2 / CA-PMQ, sM2HA2 / CT and the control group. (** P <0.01; * P < 0.05).

Current influenza vaccines based on various surface antigens are not adequate to provide effective protection against the onset of unforeseen pandemic influenza. Over the past several decades, various approaches have been attempted to develop new vaccines that can provide for ease of manufacture and administration as well as extensive cross-protection. In particular, studies were conducted to induce cross-protection using M2e and HA2. Given the above studies, it has been hypothesized that the embodiments of the present invention allow a fusion vaccine based on sM2 and HA2 (sM2HA2) to cause significant cross-protection. Thus, the examples herein constituted the fusion plasmids of the two promising targets, and the fusion plasmids were expressed in a highly efficient microbial expression system (E. coli) and tested for extensive cross-protection immunity. The selection of the appropriate route and azuret to stimulate the functional immune response is critical to the success of the development of the subunit vaccine.

Previous studies have shown that the nasal mucosal pathway is superior to inducing cross-protective immunity in mice than systemic administration. Other studies have focused on the combination of chemical and genetic conjugates (carrier molecules or viral particles) or recombinant or live influenza vaccines and DNA to improve cross-protection. Unfortunately, these vaccines did not exhibit perfect protection, as vaccinated animals exhibited significant weight loss despite being administered with very bunt.

In the present example, a new combination adjuvant CA-PMQ rule was first manufactured and administered to improve cross-protection. More importantly, vaccination with only 2 [mu] g of two well-conserved fusion proteins (sM2 and HA2) with CA-PMQ through the examples herein resulted in a relatively high dose of attack of the lethal influenza A subtype It has proven to give broad protection against inoculation.

Preliminary experiments were performed to determine the dose and efficacy of sM2HA2 on influenza virus infection. Specifically, the mice were intranasally immunized intranasally with varying doses (2, 5 and 10 [mu] g) of sM2HA2 protein at 0, 2 and 4 weeks, and after 2 weeks of final boosting 10 LD ₅₀ mouse- (H1N1) subtype of influenza A / Puerto Rico / 8/34. (A) initial body weight and (B)% survival after infection with the H1N1 virus.

As shown in FIG. 9, when sM2HA2 was administered 3 times (2 μg / dose) through the intranasal route without very bunting, 40% survival and rapid weight loss were observed against the lethal infection of H1N1. Based on the above results, the level of sM2HA2-specific antibody was enhanced when CA-PMQ-very burnt sM2HA2 was administered. Because the vaccine was delivered through the mucosal route, significant levels of mucosal IgA were observed in both the lungs and intestines. Furthermore, significant levels of serum IgG, especially IgG1 and IgG2a, were also induced by the CA-PMQ-very burnt sM2HA2 vaccine. Generally, Th1 cells induce IgG1 antibodies in mice whereas Th1 cells enhance IgG2a antibody production. Although both IgG subtypes have potent neutralizing abilities, IgG2a is known to interact effectively with complement and Fc receptors and can contribute to virus clearance.

Thus, a reduction in the viral titer of sM2HA2 / CA-PMQ-vaccinated mouse lung following a lethal infection indicates the induction of sM2- and HA2-specific IgG1 or IgG2a in the virus clearance. The cellular immune response plays an important role in vaccination. The Th1-type (IFN-y) and Th2-type (IL-4) cytokine responses were observed by ELISPOT in the examples herein and the response by stimulation of both the sM2 and HA2 proteins in the highly burnt sM2HA2 immunized mouse 0.0 &gt; IFN-y, &lt; / RTI &gt; but not in sM2HA2 alone or in control mice. IL-4 in mice immunized with CA-PMQ-very burnt sM2HA2 was also observed at a much higher level than unimmunized mice. These results suggest that the mucosal vaccine system of CAM-PMQ-succumbed sM2HA2 induces a potent cellular immune response and that mice receive a very different influenza subtype from group 1 (H1, H5, H9) and group 2 - to protect against.

In addition, the present example demonstrated that CA-PMQ-succumbed recombinant sM2HA2 induced long-lasting immunity even after 6 months of the final vaccination and provided protection against lethal infection by various influenza subtypes (Fig. 6). This is important if you are a successful vaccine. Immunity A very bun is a huge heterozygote, and can be divided into two groups: immune stimulants and delivery systems. The second type is to enhance the immune response by overcoming mucosal obstacles and increasing the uptake of antigen, while the first category of aztans stimulates the immune system by interacting with specific receptors.

In the pharmaceutical industry, new formulation of the same material has a very large impact on effectiveness and performance and is thus recognized as a new substance. The use of multi-bun formulations and the ease of production are also essential in improving bioavailability. Embodiments herein provide a simple and robust formulation strategy for the combination of mucoadhesive polymer-based (e.g., gamma-PGA) antigen delivery systems and poorly soluble immunostimulatory compounds (e. G., MPLA and QS21) to provide. In this regard, the novel combination of the delivery system of Zambant and the immunostimulatory compound based on the &lt; RTI ID = 0.0 &gt; y-PGA / MPLA / QS21 &lt; / RTI &gt; complex is a powerful candidate group for inducing a potent immune response.

The nasal passages are a promising alternative form of needle-free administration due to their physiological and immunological characteristics. Indeed, the World Health Organization (WHO) and leading organizations in the world's health, such as the Centers for Disease Control and Prevention, have advocated the need to invest research efforts in developing advanced delivery technologies that can simplify immunization schedules. Achieving this goal can improve the range of vaccination against a number of infectious diseases, especially in developing countries. Although the mucus vaccine delivery strategy is evaluated as a powerful method of needle-free vaccine delivery, the mucociliary clearance mechanism has limited its ability to inhibit the delivery of the antigen in the immune system.

Accordingly, there is a growing need for a novel approach to a mucosal vaccine delivery system that can improve the passage of antigens, such as intestinal and nasal mucosa, to biological barriers. Challenges for effective mucosal vaccination involve the generation of effective mucosal immunity and the difficulty of a safe and effective mucosal delivery and delivery system. The CA-PMQ system used in the examples herein is composed of natural ingredients that promote both sedimentation and cellular immunity. The γ-PGA, which is naturally synthesized by a microbial species (basil), can serve as a mucoadhesive delivery vehicle for recombinant protein antigens and can also provide a simple and robust strategy for binding hydrophobic MPLA and QS21 .

In the present example, it is suggested that the presence of non-ionized carboxy group in? -PGA is critical for strong interaction with mucus. This interaction is the result of hydrogen bonding between the γ-PGA and the proton-acceptor in the mucous glycoprotein. The mucoadhesive polymer can interact with the mucin molecule with physical chain entanglement and subsequently hydrogen bonds with sugar moieties on the oligosaccharide chain to form an enhanced gel network and thus the polymer is adherent for extended periods of time It is possible to reach the conclusion that it keeps it as it is. Since the polymer at the pH of the medium (6.2) is in an uncoiled and unexpanded state due to the electrostatic repulsion generated from the ionized functional groups, the polymer can more easily facilitate physical chain entanglement and secondary interaction with mucus glycoprotein have. Since a large number of carboxyl groups are present in γ-PGA, the polymer has a macromolecular morphology, which is more advantageous for increasing the accessibility of its hydrogen-bonding group when compared to other polymers.

To date, most researchers involved in the development of vaccines have focused on the discovery and production of antigens specific for target diseases or pathogens. They also tested the antigenicity of an antigen when combined with only a few of the very bunts that could utilize a single immunostimulant. In order to produce optimal prophylactic or therapeutic immunity, the intensity and persistence of the induced immunity had to be controlled according to the type of disease.

However, the development of effective vaccines with limited knowledge or ability to formulate complex avant-systems including immunostimulants and delivery vehicles has been limited. The CA-PMQ eubuntus, disclosed in the Examples herein, is formulated in a combination of aztans and is capable of inducing a potent, long-lasting humoral and cellular immune response. From this viewpoint, the CA-PMQ can be an excellent candidate for researchers who are engaged in the development of vaccines against cancer diseases as well as cancer immunotherapies.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the foregoing detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are to be interpreted as being included in the scope of the present invention.

<110> Research & Business Foundation SUNGKYUNKWAN UNIVERSITY          The Industry & Academic Cooperation in Chungnam National University (IAC) <120> MUCOADHESIVE POLYMER ADJUVANT-BASED INFLUENZA VACCINE <130> DP20160351KR <150> KR 15/0102465 <151> 2015-07-20 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 50 <212> PRT <213> Influenzae Virus <400> 1 Met Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu   1 5 10 15 Cys Lys Cys Ser Asp Ser Ser Asp Pro Asp Arg Leu Phe Phe Lys Cys              20 25 30 Ile Tyr Arg Arg Leu Lys Tyr Gly Leu Lys Arg Gly Pro Ser Thr Glu          35 40 45 Gly Val      50

Claims (6)

Mucoadhesive &lt; RTI ID = 0.0 &gt; adherent &lt; / RTI &gt; composition and antigen,
The mucoadhesive adhesion enhancer composition comprises a complex of an anionic polymer, a toll-like receptor agonist, and a saponin,
Wherein the antigen is a protein having an amino acid sequence of SEQ ID NO: 1 and a protein having an amino acid residue 15-137 sequence in the HA2 antigen protein of A / EM / Korea / W149 / 06 (H5N1)
Vaccine composition for treating influenza A
The method according to claim 1,
Can induce cross-protection against the influenza A subtype selected from the group consisting of the subtypes of influenza A, H5N1, H1N1, H5N2, H7N3 and H9N2.
Vaccine composition for treating influenza A
The method according to claim 1,
Wherein the anionic polymer is selected from the group consisting of polygamma glutamic acid, hyaluronic acid, cellulose, polyacrylic acid, polysaccharides, derivatives thereof, and combinations thereof.
Vaccine composition for treating influenza A
The method according to claim 1,
The toll-like receptor agonist may be selected from the group consisting of MPLA (monophosphoryl lipid A), imiquimod, recyquimod, flagellin, CpG, poly (I: C) &Lt; / RTI &gt; and combinations thereof.
Vaccine composition for treating influenza A
The method according to claim 1,
Wherein said saponin is selected from the group consisting of QS21, Quil A, QS7, QS17, beta-eskin, digitonin, and combinations thereof.
Vaccine composition for treating influenza A
The method according to claim 1,
Wherein the mucoadhesive adhesive is a powder.
Vaccine composition for treating influenza A

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