KR20160069204A - immune inducing agent including Human Cathelicidin LL-37, oral vaccine including of the same, and immune inducing system using of the same - Google Patents

Abstract

The present invention relates to an immune activation inducer including human cathelicidin LL-37 which is capable of activating mucosal immune system by activating follicular dendritic cell (FDC). The present invention further relates to an oral vaccine containing the same, and to a method for inducing immune activation using the same. Used in the immune activation inducer of the present invention is human cathelicidin LL-37 peptide or a recombinant protein containing the LL-37 peptide, leading to the activation of follicular dendritic cell and further inducing activation of an acquired immune system. The immune activation inducer of the present invention can be used in an oral vaccine, allergy treatment, and mucosal disease treatment since the inducer activates the acquired immune system. In addition, the use of the immune activation inducing method enables effective induction of immune activation for the target object.

Description

[0001] The present invention relates to an immunological activity inducer comprising human cathelicidin LL-37, an oral vaccine containing the same, and a method for inducing immunological activity using the agent, including human cathelicidin LL-37, oral vaccine including the same, and immune inducing system using the same}

The present invention relates to a pancreatic dendritic cell activator, an immunological activity inducer, an oral vaccine containing the same, and a method for inducing immunological activity using the same, which comprises human cathelicidin LL-37 for activating mucosal immunity.

The inner surface of the respiratory, digestive, urinary, and genital parts of the body is covered by mucous membranes and is always exposed to a large number of antigens and pathogens coming from the outside. Therefore, the mucosa does not only serve as a transit pathway, but also acts as a primary and immunological defense system through the mucosal immune system. The mucosal immune system maintains its immune system through the close relationship between congenital and acquired immunity. Among them, Peyer's patch (PP: PP) acts as a site for mucosal immunity inducing IgA production, a typical immunoglobulin of the mucosa. When PP induces mucosal immunity, secretory IgA (S-IgA) antibody is produced at the mucosal effect site, and the S-IgA antibody thus generated has a protective effect on the antigen and pathogen It will play a defense role.

On the other hand, as an antimicrobial peptide, it is named on the basis of the N-terminal cathelin domain, which plays an important role in the innate immunity stage by destroying the membrane of the bacterium and, through interaction with receptors on the immune response, Is a multifunctional molecule that participates as a modulator in the cell. Among them, human cathelicidin LL-37 regulates macrophages, dendritic cells, B cells and T cells.

Although this antimicrobial peptide LL-37 has been suggested as a potential modulator of the mucosal immune system, the technology for use as an immune activity inducer has been lacking, and in particular, as an immunity enhancer used alone, .

[Related patents]

1. Korean Patent Application No. 10-2010-7001436

2. Korean Patent Application No. 10-2012-0135114

The present invention uses a recombinant protein comprising human catecholysidine LL-37 peptide or LL-37 peptide, which activates the congenital immune system and activates the acquired immune system by activating pancreatic dendritic cells, Inducing agent, an oral vaccine containing the same, an allergy therapeutic agent, and a therapeutic agent for mucosal diseases.

The present invention relates to a method for activating a follicular dendritic cell (FDC) using a recombinant protein comprising a human cathelicidin LL-37 peptide or an LL-37 peptide to promote the production of a germinal center (GC) And to provide a method for inducing immunological activity.

In one aspect,

An immunostimulatory activity inducer comprising a recombinant protein comprising a human catechiricidin LL-37 peptide or an LL-37 peptide, wherein the inducer has an immunological activity It concerns the inducer.

In another aspect,

A human recombinant protein comprising a human cathelicidin LL-37 peptide or a human cathelicidin LL-37 peptide, wherein said human cathelicidin LL-37 peptide activates pancreatic dendritic cell activation It deals with the issue.

In yet another aspect,

Preparing a recombinant protein comprising a human catechiricidin LL-37 peptide or an LL-37 peptide; Introducing the recombinant protein comprising the human citalyclidine LL-37 peptide or the LL-37 peptide into the immunoreactivity inducing substance; And activating the innate immunity and / or acquired immunity of the immunostimulatory activity-inducing subject.

The vesicle dendritic cell activators and immunostimulatory agents of the present invention can activate the acquired immune system by activating the pancreatic dendritic cells (FDC) by using recombinant proteins including human celecidic LL-37 peptide or LL-37 peptide .

The pancreatic dendritic cell activator and immunostimulatory agent of the present invention can activate the congenital immune system by using a recombinant protein comprising human catecholizidine LL-37 peptide or LL-37 peptide.

The vesicle dendritic cell activator and immunostimulating agent of the present invention can be used for oral vaccine, allergy treatment agent, mucosal disease treatment agent and the like by activating the congenital and acquired immune system. In particular, the present invention can be applied to the development of a recombinant oral vaccine by applying a recombinant protein containing LL-37 peptide to various other antigens. These recombinant oral vaccines are economically advantageous because they can be produced in large quantities using E. coli and plant systems, and can be administered without professional medical personnel through edible methods. Oral vaccines can be applied to both livestock and humans.

The immunological activity inducing method using the recombinant protein comprising the human catechiricidin LL-37 peptide or the LL-37 peptide of the present invention can be used to induce an effective immunological activity on the immunoactivity inducing subject.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing an improvement in the expression level of cytokines in the Fir plate by treatment of recombinant proteins including LL-37 or LL-37.
FIG. 2 is a graph showing the proliferation of proinflammatory cytokine secreting cells in the Peyer's patches, excluding B cells and T cells, by LL-37 treatment.
FIG. 3 is a graph showing the increase of Th17 cell polarity by LL-37 treated antigen presenting cells.
4 is a graph showing the increase of IL-6 secreting CD11c ⁺ cells in the fire plate on the second day after oral administration of EGFP-LL-37.
FIG. 5 is a graph showing the increase in cytokine secretion of the fire plate on the 14th day after oral administration of EGFP-LL-37. FIG.
FIG. 6 is an image showing the recruitment of CD11c ⁺ CD70 ⁺ cells into a fire plate by oral administration of EGFP-LL-37. FIG.
FIG. 7 shows the results of flow cytometry showing the increase of Fir-CD11c ⁺ CD40 ⁺ cells by oral administration of EGFP-LL-37.
Figure 8 is an image showing the characterization of a Firman tissue sample by binding of antibodies reactive with FDC-M1, FPR-2, and GL-7.
FIG. 9 shows results of flow cytometry showing the expression of VCAM-1, TLR2, and FPR-2 in B220-CD-FDC-M1 cells.
10 is an image showing calcium influx in FDC-M1 cells.
11 is a graph showing the calcium inflow in FDC-M1 cells monitored for 200 seconds.
Figure 12 is an RT ² Profiler PCR analysis image of FDC stimulated with LL-37 peptide.
FIG. 13 is an image showing the characterization of a sample of a firefly tissue sample by binding of antibodies reactive with IgA, GL7, and B220 at 7 days after oral administration of EGFP-LL-37.
FIG. 14 is an image showing the characterization of a sample of a firefly tissue sample by binding of antibodies reactive with IgA, GL7, and B220 at 7 days after oral administration of EGFP.
FIG. 15 shows the results of flow cytometry showing the increase of B200 ⁺ GL7 ⁺ Fas ⁺ PP cells after oral administration of PBS, EGFP, and EGFP-LL-37.
FIG. 16 is an image showing the characterization of a sample of a firefly tissue sample by binding of antibodies reactive with IgA, GL7, and B220 at 14 days after oral administration of EGFP-LL-37. FIG.
FIG. 17 is an image showing the characterization of a sample of a firefly tissue sample by binding of antibodies reactive with IgA, GL7 and B220 at 14 days after oral administration of EGFP-LL-37. FIG.
FIG. 18 shows the results of flow cytometry showing the increase of B200 ⁺ GL7 ⁺ Fas ⁺ MLN cells after oral administration of PBS, EGFP, and EGFP-LL-37.
FIG. 19 is a graph showing the increase in total SIgA level after oral administration of EGFP-LL-37. FIG.
20 is a graph showing an increase in antigen-specific SIgA levels after oral administration of EGFP-LL-37.
21 is a graph showing the increase in the number of antigen-specific IgA-secreting cells in the fire plate and the lamina propria after oral administration of EGFP-LL-37.

The present invention uses a recombinant protein comprising a human cathelicidin LL-37 peptide or an LL-37 peptide activating the acquired immune system by activating the congenital immune system and activating the pancreatic dendritic cells of the mucosal immune system to form a pancreatic dendritic cell activator, An immunological activity inducing agent, an oral vaccine containing the same, an allergy therapeutic agent, and a therapeutic agent for mucosal diseases. The inducing agent is characterized by enhancing the immunity when administered alone or enhancing the immune response when administered in combination with an antigen.

In one aspect, the present invention provides an immunological activity inducing agent comprising a recombinant protein comprising a human cathelicidin LL-37 peptide or an LL-37 peptide, wherein the inducing agent enhances the immune response or enhances the immune response when administered in combination with an antigen ≪ / RTI >

Congenital immunity is a cell or mechanism that protects a host from infection in a nonspecific way, indicating an immune system that does not remember a particular pathogen and responds instantaneously. Unlike acquired immunity, immunity does not last long and treats pathogens in a comprehensive way. Acquired immunity refers to immunity obtained by infecting a disease or vaccination, and is typically exemplified by immunization by an antibody. The immunoactivity inducer of the present invention can activate both the innate immune system and the acquired immune system. The immunoactivity inducing agent may be used for an allergic treatment agent, a mucosal disease therapeutic agent, and an adjuvant for a vaccine, using the inherent immunological activity characteristics and the acquired immune system activity characteristics of the immunological activity inducer.

The mucosal immunity of the present invention is a term well understood in the art and includes the production of secretory IgA and / or mucosal CTL (cytotoxic T lymphocyte) in mucosal tissues such as gastrointestinal tissue, vaginal tissue, : Cytotoxic T cells) refers to an immune response characterized by stimulation of the response. The immune activity inducer of the present invention activates the mucosal immune system. In particular, the immune activity directing agent of the present invention, which comprises a recombinant protein comprising human catechiricidin LL-37 peptide or human cathelicidin LL-37, is a follicular dendritic cell of firefly playing an important role in the mucosal immune system cell: FDC) to activate the germinal center (GC) and activate mucosal immunity.

A peyer's patch (PP) refers to a lymphoid tissue that collects in a plane on the mucosal layer of the mammalian small intestine. The Firefly plays an important role in immunity related to the defense of the body, which is called the superficial immunity. On the other hand, pancreatic dendritic cells (FDCs) are cells found in the lymph nodes, and are cells with membrane-like protrusions existing in a specific compartment called the plexus of activated B cells. The follicular dendritic cells are antigen presenting cells that capture antigen by complexing with antibodies or complement products and present these antigens on the surface to allow B lymphocytes to recognize the antigen. When the pancreatic dendritic cells are activated in the main stimulation site of the mucosal immune system, secretory IgA (secretory IgA: SIgA) is secreted from the affected area of the mucosal immune system to form mucosal immunity. Therefore, the immunostimulatory agent of the present invention comprising the recombinant protein comprising human catechiricidin LL-37 peptide or human cathelicidin LL-37 induces an effective pelvic center structure by activating pancreatic dendritic cells, Can be activated.

Mucosal vaccines can induce mucosal immune responses that produce antigen-specific secretory IgA on the mucosal surface as well as systemic immune reactions that produce antigen-specific serum IgG, as opposed to the systemic immune response alone There is an advantage. Therefore, the immune activity inducer of the present invention, which activates the mucosal immune system and can be used as a mucosal vaccine, is capable of simultaneously activating not only mucosal immunity but also whole body immunity. Using the above characteristics, the present immunostimulatory agent can be used as a vaccine and vaccine inducer for all infectious diseases, and more preferably, it can be used more effectively for mucosal immune diseases.

When the immunostimulatory agent of the present invention is used for mucosal immune diseases, the antigen can be used without any particular limitation as long as it is derived from pathogenic microorganisms such as viruses, bacteria and fungi that can be defended in the mucosal immune system. That is, the antigen can be used without particular limitation as long as it is derived from a virus, a bacterium, a fungus or the like derived from a disease which is infected through the mucosa. For example, the antigen may be derived from the group consisting of dengue virus, cholera, food poisoning, brucellosis.

The immunoactivity inducer may improve the immunity of an object when administered alone to an object desired to be immunized. That is, it is administered without co-administered antigen to enhance the immune system for the treatment of patients with chronic infectious diseases, particularly those with impaired immune system. Strengthening the immune system in this way may be useful as a preventive measure to limit the risk of hospital infection and post-operative infection. For example, the immunoactivity inducing agent can be used for an allergy treatment agent, a mucosal therapeutic agent, and the like.

 In addition, the immunoactivity inducer may be used as an adjuvant for effectively enhancing the immune response of the subject, rather than administering the antigen alone. For example, the immunological activity inducing agent may be used as an adjuvant for an oral vaccine.

The immunological activity inducing agent may be present in an aqueous formulation, a solid formulation, or an emulsion formulation. Since the immunoactivity inducing agent is intended to be administered orally or orally, it can be used without particular limitation as long as it is an oral administration or oral administration to an immunity-inducing subject. In addition, the immunoactivity inducing agent may be present as a low-rate sustained-release preparation that lowers the rate of the inducing agent.

In another aspect, the invention features a recombinant protein comprising human catecholysidine LL-37 peptide or human catechirizidine LL-37 peptide, wherein said human catechiricidin LL-37 peptide is characterized by activating pancreatic dendritic cells Lt; RTI ID = 0.0 > dendritic < / RTI > cell activator. With respect to the above phagocytic dendritic cell activator, the contents described in relation to the immunological activity inducing agent can be equally applied.

In another aspect, the invention provides a method of producing a recombinant protein comprising the steps of: preparing a recombinant protein comprising human catecholysidine LL-37 peptide or LL-37 peptide; Introducing the recombinant protein comprising the human citalyclidine LL-37 peptide or the LL-37 peptide into the immunoreactivity inducing substance; And activating the innate immunity and / or acquired immunity of the immunostimulatory activity-inducing subject.

The step of preparing the human cathelicidin LL-37 peptide can be carried out by any method known in the art for producing the peptide without any particular limitation.

The step of preparing a recombinant protein comprising the human catechiricidin LL-37 peptide peptide can be produced by a method known in the art for producing a recombinant protein. For example, expression may be expressed in prokaryotes, eukaryotes, yeast cells, plant cells, and mammalian cells by inserting the LL-37 gene into any expression vector. A vector refers to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell, which may include a nucleic acid sequence that enables it to replicate in a host cell, such as the origin of replication. Representative vectors may include vectors for expression in E. coli and Salmonella, or viral vectors. In addition to the LL-37 nucleic acid molecule in the production of the recombinant protein comprising the human cathelicidin LL-37, the nucleic acid molecule encoding one or more pathogen antigens may be expressed together in a vector.

The injection can be used without any particular limitation as long as it can activate the mucosal immune system. For example, the infusion may be performed by any one method selected from the group consisting of nasal administration, oral administration, and oral administration.

The injection may be carried out at a constant number of times and at a constant unit dose for a certain period of time. For example, the injection may be carried out at 14 to 30 days and at least one to three times and at two unit doses.

The step of activating the innate immunity and / or acquired immunity of the immunoactivation-inducing subject may include promoting the production of the embryo center by inducing the activity of the follicular dendritic cell (FDC) of the immunoactivity inducing subject.

Human cathelicidin LL-37 is able to activate the innate immune system of the immune activity inducing subject. For example, human cathelicidin LL-37 is able to induce the differentiation of Th17 cells and to express the cytokines IL-6, IL-23 and IL-1. In addition, human cathelicidin LL-37 can secrete IL-6, a highly cytokine, in CD11c ⁺ cells, the most representative cells mediating acquired immunity among innate immune cells.

Human cathelicidin LL-37 can also activate the acquired immune system. More specifically, it is as follows. Human cathelicidin LL-37 activates pancreatic dendritic cells. Due to the activity of the pancreatic dendritic cells, germinal B cells collect to form the follicle, and the embryo center is formed. In particular, pancreatic dendritic cells highly express CXCL13 mRNA transcripts, which are very important for embryogenesis by stimulation of LL-37.

The immunological activity induction method may be performed in an ex vivo manner. For example, the immunological activity induction method can be carried out by using mucosal immunological tissue or the like obtained through tissue culture as an immunological activity inducing subject.

In addition, the immunological activity induction method may be performed in vivo. For example, the immunological activity inducing subject may be an animal other than a human. Preferably, the animal is a mammal in which a firefly, which is a mucosal immunological tissue, is present.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  : LL -37- Production of complex recombinant antigens

Oligomers complementary to the LL-37 sequence were heated in boiling water (10 mM Tris HCl (pH 7.5), 100 mM NaCl, and 1 mM EDTA) for 2 minutes in boiling water to make LL-37 conjugated with EGFP or ED, Followed by cooling and annealing. The complementary oligomer sequence of LL-37 is 5CCG ACC CGC TGC TGG GTG ATT TCT TCC GGA AAT CTA AAG AGA AGT TTG GCA AAG AGT TTA AAA GAA TTG TCC AGA GAA TCA AGG ATT TTT TGC GGA ATC TTG TAC CCA GGA CAG AGT CCT AGG -3 and 5TCT AGG ACT CTG TCC TGG GTA CAA GAT TCC GCA AAA AAT CCT TGA TTC TCT GGA CAA TTC TTT TAA ACT CTT TGC CAA TCT TCT CTT TAG ATT TCC GGA AGA AAT CAC CCA GCA GCG GGT CG-3 '.

The EGFP gene was amplified by PCR from pEGFP-1 (Clontech, Mountain View, Calif.). For the preparation of EGFP containing LL-37 or not containing LL-37, the following primers were used: forward primer, 5T GA GCT C AT GGT GAG CAA GGG CGA GGA G-3 Sac site), reverse primer, 5TCC GTA CAG CTC GTC GAT-3 or 5TCC CTT GTA CAG CTC GTC GAT-3

ED was amplified from C6 / 36 cells infected with DENV-2 and the following primer set was used to prepare ED containing LL-37 or not containing LL-37: forward primer, 5 GGA TCC ATG TCA TAC TCT ATG TGT-3 (underlined letters represent BamHI site), reverse primer, 5 GGA TCC TTT CTT GAA CCA CTT-3 or 5 GAA TTC CTA TTT CTT GAA CCA CTT-3 (underlined letters represent BamHI site or EcoR site). The amplified gene product was cloned into a pCold vector (TaKaRa Bio Inc., Shiga, Japan) containing double-stranded LL-37 oligomer. Recombinant proteins were expressed using a BL21 (DE3) expression host and purified according to manufacturer's recommendations using Bio-Scale Mini Profinity IMAC Cartifidge in BioLogic Low-Pressure Chromatography systems (Bio-Rad Laboratories, Inc., CA, USA). Sample lysates containing His-tagged proteins, balanced by native lysis buffer, were loaded into the cartridge at 2 ml / min, washed, and eluted. Purified proteins were exchanged in PBS via dialysis and analyzed by SDS-PAGE. Endotoxin levels were measured by endotoxin assay kit (GencScript USA Inc., NJ, USA).

Experiment 1: Cell separation and flow cytometry

PP (fire plate) was collected from the small intestine of rats. PP intracellular lymphocytes (IEL) and epithelial cells were prepared by stirring in 1 mM EDTA for 30 min at 37 ° C. PP cells and epithelial cells except IEL were collected after digesting with collagenase D (0.5 mg / ml) and DNase (100 g / ml) for 60 min. The IEL and epithelial cells were removed using 0.5 mM EDTA to obtain lamina propria cells. Small intestinal tissue without PP was cut into small pieces and stained with collagenase D (0.5 mg / ml) and DNase (100 g / ml) Min. ≪ / RTI > Spleen lymphocytes (SPL) and mesenteric lymph node lymphocytes (MLN) are prepared in the same digestion state. CD3 ^- B220 ^- cells in PPL were prepared using Dynabeads Biotin Binder (Invitrogen). CD11c ⁺ cells or CD4 ⁺ cells in PP cells were prepared with CDllc microbeads or CD4 ⁺ T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec lnc.).

B220 ^- PP cells were incubated with CD45R microbeads with an autoMACS ^™ Separator (Miltenyi Biotec Inc.) to increase the amount.

B200 ^- PPL was stained with allophycocyanin-Cy7-conjugated anti-CD3 and FDC-M1 was then stained with biotin-conjugated anti-rat IgG2c, allophycocyanin-conjugated streptavidin and 7-aminoactinomycine D (7-AAD) (BD Biosciences). Dead cells were removed and B220 ^- CD3 ^- FDC-M1 ⁺ cells were sorted using FACSAria (BD Biosciences).

Classified FDC-M1 ⁺ cells stained with anti-TLR2 (Abcam), anti -VCAM-1 (BD Biosciences), or anti-FPR-2 (Santa Cruz Biotechnology) staining was followed by FITC-conjugated secondary antibody (Abcam) in Respectively. For the analysis of secretory cells FPR-2, PP cells were stained with anti-FPR-2 and then stained with FITC conjugated secondary antibody, allophycocyanin-Cy7 conjugated anti-CD3 and anti-CD45R, allophycocyanin conjugated CD11b, anti-FDC M1 antibody , Followed by biotin conjugated anti-rat IgG2c and PE-CF594 conjugated streptavidin, and 7-AAD. IEL and epithelial cells were stained with anti-FPR-2, stained with FITC conjugated secondary antibody, biotin conjugated anti-GP2 (MBL, Woburn, MA) and stained with allophycocyanin conjugated streptavidin, PE-Cy7 conjugated CD45, 7-ADD Respectively. For characterization of PP cells, PP cells were stained with Alexa Flour 630 conjugated CDl1c (Biolegend), FITC-conjugated CD40, and 7-AAD. To analyze GC B cells, PP cells were stained with allophycocyanin-Cy7 conjugated B220, PE conjugated CD95 (Fas), FITC conjugated GL-7 (Biolegend) and 7-AAD. All cells were monitored by flow cytometer and data were analyzed with FlowJo software (Trec Star Inc., Asland, OR).

Experiment 2: Cytokine ( cytokine ) analysis

PP cells prepared from BALB / c were stimulated with the designated protein for 3 hours on the third day after in vitro stimulation and the medium was collected. PP CD4 ⁺ T cells from immunized mice were incubated with the antigen protein for 17 hours and the medium was collected. Th1 / Th2 / Th17 cytokine kit (BD Biosciences) was used according to the protocol of the manufacturer. The results were analyzed by FCAP Array software.

PP CD3 ^- B220 ^- cells prepared from BALB / c were stimulated with pLL - 37 (10M) for 3 hours and the cells were cultured in an ELISPOT plate coated with purified antibody against the designated cytokine, respectively. , LL-37 The PP CD3 stimulation in order to analyze the biological activity of the cytokine ^- B220 ^- from the ELISPOT plate coated with cells into a pre-purified IL-17 antibodies for 3 days were cultured with CD4 ⁺ T cells. CD11c ⁺ cells purified from immunized rat PP were cultured for 24 hours on ELISPOT coated with purified anti-IL-6 antibody. PP lymphocytes and spleen lymphocytes were prepared from PP cells as Percoll gradients. Splenocytes were obtained from immunized mice and cultured with antigen EGFP for 17 hours on ELISPOT plates coated with purified antibodies against the designated cytokines, respectively. After removal of the cells from the plate, biotin-conjugated antibodies to the designated cytokines were bound, followed by HRP-conjugated avidin, and spots were developed with a 3-amini-9-ethyl carbazole substrate solution.

Experiment 3: Antigen-specific IgG Wow IgA of level  decision

Antigen (50 g) was orally administered to five female female BALB / c mice per group for two weeks once per week without anesthesia, followed by monitoring of feces for four weeks. After 4 weeks of sampling with the same antigen (100 g) that did not contain LL-37, it was boosted into the abdominal cavity in mice immunized on days 10 or 14. Serum and excreta were prepared on the third day. Diluted serum or excrement was added to the pre-coated ELISA plate with purified anti-mouse IgA or the same antigen (100 ng). After removing the sample, anti-mouse IgA conjugated with HRP was added and each well was developed with TMB substrate solution or 3-amini-9-ethyl carbazole substrate solution.

Experiment 4: Plaque  Decrease neutralization test method ( Plaque Reduction Neutralization Test : PRNT )

Neutralizing antibody levels for DENV-2 infection in C6 / 36 cells were measured by PRNT using a 96-well ELlSPOT plate. The diluted sera were heat-inactivated at 56 for 30 minutes. Subsequently, samples were incubated with 40 plaques forming DENV-2 units in each well for 37 to 60 minutes, and then adsorbed onto a C6 / 36 cell monolayer for 28 to 90 minutes, washed with PBS, and resuspended in 0.75% methylcellulose Overwrapped. The virus was reacted with anti-dengue virus antibody, stained with alkaline phosphate-conjugated anti-mouse IgG, and spot development was observed with BCIP / NBT substrate.

Experiment 5: Fluorescent antibody assay

PP and MLN immunized mice were prepared and frozen. After the sample was acetone fixed, the Fc receptor was blocked using purified standard IgG. To monitor congenital cells, the PP slices containing FAE were stained with Alexa Flour 647 conjugated CD11c, PE conjugated CD70, anti-FPR-2, then stained with FITC conjugated secondary antibody, and stained with DAPI. In order to examine the GC B cell follicles, the specimens were stained with anti-GL-7 antibody and stained with FITC-conjugated anti-rat IgM (Bethyl Laboratories, Inc., Montgomery, TX), anti-mouse IgA or IgG1 antibody stained with rhodamine-conjugated anti-goat IgG (Abcam) and biotin-conjugated B220 antibody (eBiosciencc) and then stained with Alexa Fluor 450-conjugated streptavidin (eBioscience). In order to confirm the expression pattern of FPR-2 in PP, the PP samples obtained from BALB / c mice were stained with anti-FDC-M1, stained with FITC conjugated secondary antibody, anti-FPR-2 and Alexa Flour 700 conjugated secondary (Molecular probe) and anti-GL-7, followed by Alexa Flour 594 conjugated anti-rat IgM (Molecular probe) and then stained with DAPI. CX3CR1 ^{+ / GFP} The PP samples obtained from rats were stained with anti-M cell antibody (NKM 16-2-4), stained with Alexa 680 conjugated secondary antibody, anti-FPR-2, stained with PE conjugated secondary antibody and stained with DAPI. The sorted FDC-M1 ⁺ cells were cultured for 3 days in a 2% gelatin coated dish containing TNF- (5 ng / ml) and anti-LTR (1 g / ml). After treatment with biotin conjugated LL-37 (10M) for 15 min, cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100. The cells were stained with anti-FPR-2 antibody, stained with FITC conjugated secondary antibody and PE conjugated streptavidin, and stained with DAPI. The samples were analyzed by confocal laser scanning microscopy (CLSM) (LSM 510 META; Carl Zeiss, Thomwood, NY).

Experiment 6: Calcium Of the calcium flux  Measure

The separated and cultured FDC-M1 ⁺ cells for 3 days were treated with FPR-2 antagonist peptide WRWWWW (10M) for 1 hour and then incubated with Fluo-4, AM according to the recommendations of the manufacturer of Fluo-4 calcium imaging kit . Cells were loaded with Fluo-4A, AM and 37 for 30 min, then loaded at room temperature for 30 min, washed, and then prepared in live cell imaging solution containing 2 mM glucose. Cells were placed in CLSM, stimulated with pLL-37 for 200 s, and then measured for calcium flux at 506 nm fluorescence emission. Data were analyzed with LSM 501 software.

Experiment 7: RT ² Profiler PCR array

mouse cytokines & chemokines (PAMM-150ZA) RT ² Profiler PCR Array Kit PR2 SYBRGreen ROX ^TM processed using the ABI 7500 system with qPCR Master Mix and analyzed according to the manufacturer's recommendations. Classified FDC-M1 ⁺ cells were not treated, or treated with pLL-37 (10M) for 15 min, RNA was prepared in the RNeasy Plus Micro Kit (Qiagen) complementary DNA are RT ^2, including RT ² PreAMP pathway primer mix PreAMP cDNA synthesis kit (mouse cytokines & chemokines).

2x RT ² SYBR Green Mastermix and PCR mix containing cDNA were distributed on an RT ² Profiler PCR array plate and the threshold cycle (CT) for each well was calculated using real-time cycler software. The relative amount of cDNA was calculated using the Ct method using three normalization genes, heat shock protein 90, and actin in the PCR Array Data Analysis Web portal (www.sabiosciences.com/pcrarraydataanalysis.php). Experimental results were obtained in three independent experiments, and all genes were measured three times.

p -value were calculated based on the FDC-M1 ⁺ cells with LL-37-treated ^{FDC-M1 + Student's t-} test of the fold exchange value for each gene in the cell. The p-values are shown below 0.001. Heat map data were analyzed using MeV software.

Statistical analysis

Statistical analysis was performed using SigmaPlot (Systat Software, Chicago, IL). The results were expressed as mean standard deviation. Unpaired Student's t-test was used to compare groups, and p-values less than 0.05 were considered significant.

result

1. LL-37 produces an immunostimulatory microenvironment in the induction compartment of the mucosal immune system.

Although LL-37 was found on the mucosal surface, little was known about the modulatory activity of LL-37 in mucosal compartments. In order to understand the modulatory effects of LL-37 in the mucosal induction compartment, the expression pattern of cytokines after pLL-37 treatment was observed (Fig. 1), since effective immune response induction was determined by specific cytokines and chemokines. For this purpose, cells of Peyer's patch were isolated and then peptide LL-37 (pLL-37). After stimulation with scrambled peptide GL-37 (pGL-37), recombinant protein EGFP (EGFP) and recombinant protein EGFP-LL-37 (-LL-37) containing c-terminal LL-37, secretion of cytokines Respectively. PP cells express IL-4, IL-6 and IL-17 very well in the test tube compared to control scrambled GL-37 peptide (pGL-37) 3 days after treatment with pLL-37. To further determine whether this immune response was caused by antigen participation, recombinant EGFP or LL-37 conjugated EGFP was treated. As a result, the expression of IL-6 and IL-17 was further improved when LL-37-conjugated EGFP was treated with EGFP alone. Therefore, LL-37 itself induces immunomodulatory effects in PP. It was then assumed that the regulation of cytokine expression by LL-37 is closely related to the activity of PP congenital immune cells. Our idea is supported by an increase in the number of IL-6, IL-23, IL-1 secreting cells, excluding B cells and T cells stimulated with pLL-37 (FIG. 2). In addition, PP-innate cells stimulated by pLL-37 can induce polarization of Th17 cells from enhanced PP CD4 ⁺ T cells by adding IL-6 and TGF- (FIG. 3). These results suggest that DCs between them are a major population of responses to LL-37 because PP congenital cells are activated by LL-37 and the development of effective T cells requires stimulation of mature DCs. Therefore, the cytokine expression pattern of CD11c ⁺ PP cells obtained from mice administered orally with EGFP or EGFP-LL-37 was analyzed. Although we did not find expression of IL-23, IL-1 in CD11c ⁺ PP cells, we found that PP cells regulated by EGFP-LL-37 via oral administration had more IL-6 secretion CD11c ⁺ Cell number (Fig. 4). In addition, oral administration of EGFP-LL-37 induced an increase in Th17 number in EGFP treatment compared to EGFP alone (FIG. 5). Based on these results, the APC-modulating effect of LL-37 links PP congenital and adaptive immune responses.

2. In vivo stimulated PP APCs with LL-37 conjugated reporter proteins induce GC B cell development.

To confirm PP APC-modulating effect by LL-37 in the body, we investigated whether PP CD11c ⁺ expresses CD70. CD70 is transiently expressed in activated DC, B cells, and T cells, and is closely related to the survival and proliferation of activated B cells and T cells. The expression of FPR-2, one of the host receptors interacting with LL-37, was observed to examine chemotaxis activity (FIG. 6). Immature CD70 ^- CD11 ⁺ cells and FPR - 2 - expressing cells are clustered in PP FAE (follicle - associated epithelium) in only steady state with PBS or EGFP alone. However, oral administration of EGFP-LL-37 increased the accumulation of CD70 ⁺ or CD11c ⁺ cells in PP FAE. To further investigate the activity of PP APCs on oral administration of EGFP-LL-37, the degree of expression of CD40 in PP cells was monitored. CD40 is associated with T cell dependent immunoglobulin class switching, memory B cell development, and GC formation. As expected, the oral administration of EGFP-LL-37 resulted in an increase in the expression of CD40 in PP cells, and a 5-fold increase in the expression of CD40 in PP CD11c ⁺ cells, especially when administered with EGFP alone ). Therefore, it is believed that the PP congenital immune system was activated by stimulation of LL-37.

3. FPR-2 expressing DCs corresponding to pLL-37

To produce neutralizing antibodies with high affinity for pathogens, somatic cells, mutations and affinity maturation are required in GC, and these reactions depend on the activity of FDCs. Based on the above results, it is assumed that LL-37 is involved in the activity of FDC through interaction with one of the host receptors responsive to LL-37, FPR-2. Observation of the distribution of FPR-2 expressing congenital cells in PPs by flow cytometry revealed that cells expressing FPR-2 contained FDCs defined by B220 ^- CD3 ^- CD11b ^- FDC-M1 ⁺ cells (Fig. 8). To further understand the role of LL-37 in FDC-M1 ⁺ , B200 ^- CD ^- FDC-M1 ⁺ cells between B220 with reduced PP cells were isolated and identified by expression of VCAM-1 or TLR2. Then, the expression of FPR-2 was reaffirmed in these cells (Fig. 9). Isolated FDC-M1 ⁺ cells were cultured for 3 days with TNF- and anti-LTR. In order to test the biological activity by interaction between FPR-2 and LL-37, calcium influx was monitored for 200 seconds in FDC-M1 cells loaded in Fura-4, AM. As shown in FIGS. 10 and 11, treatment of pLL-37 induces calcium influx in FDC-M1, but this response is blocked in FDC-M1 in which the FPR-2 antagonist peptide WRWWWW has been pretreated. In addition, the LL-37 stimulation of FDC-M1 cells enhanced the expression of chemokines such as Tgfb2, IL-4, and Cxcl13 and the expression of cytokines, And is highly correlated with the development of cell-dependent IgA ⁺ cells (Fig. 12). In addition, the activity of FDC by LL-37 is supported by enhanced development of B cell follicles and GC B cells in PP after oral administration of EGFP-LL-37 (FIGS. 13 and 14). Seven days after the in vivo stimulation of EGFP-LL-37, we found that B-cell follicles were well formed in PPs rather than EGFP alone and were well-recognized by IgA ⁺ isotype-switching expressing GL-7. Although the PP GC is well developed in chronic mucosal environment, the increase of GC B cells in the PP from EGFP-LL-37 orally administered rats was significantly increased ( p <0.05) in B200 ⁺ GL7 ⁺ Fas ⁺ (Fig. 15). We can also confirm the formation of GC B cell follicles in the mesenteric lymph node (MLN). Although the larger B cell follicles can be identified in the MLN of mice administered EGFP-LL-37 in vitro compared to EGFP alone (Figures 16 and 17), the number of B220 ⁺ GL7 ⁺ Fas ⁺ PP cells is significantly different (Fig. 18). Therefore, the activity of FDC by LL-37 is important for linking PP congenital immunity with acquired immune system.

4. In vivo stimulation of PP of LL-37-conjugated reporter protein leads to the generation of T-cell dependent high affinity IgA.

In order to examine whether the PP-induced immune response activated by the body stimulation of EGFP-LL-37 acts as an amplifying agent in acquired immunity, the antigen-specific mucosal immune response and induction of systemic immune response were confirmed. When measuring the extent of SIgA in fecal extracts, mice immunized with EGFP with EGFP-LL-37 or CT showed more total SIgA compared to mice immunized with control PBS or EGFP (Fig. 19). The induction of EGFP-specific IgA in mice immunized with EGFP-LL-37 rapidly reached and persisted for 4 weeks, and these values are comparable to those of mice administered with EGFP and CT (FIG. 20). To further study the biological activity of antigen-specific acquired immunity induced in LL-37, we investigated the possibility of neutralizing antibody development because the development of neutralizing antibodies requires APC activity and T-cell dependent GC response . We therefore applied LL-37 in an oral vaccine model using the pathogenic antigen envelope domain (ED) of dengue virus serotype 2 (DENV-2). In fact, DENV infection occurs systemically, but the immune response to DENV is required in the mucosal compartment PP because of the resident site after infection. Serum prepared from rats to which ED-LL-37 was orally administered and systemically boosted with ED showed the highest level of neutralizing antibody against DENV-2, which is also comparable to the serum of rats immunized with ED with CT (Fig. 21).

In the mucosal milieu, homeostasis between microorganisms and the host immune system is closely linked to the production of SIgA and induction of tolerance. In particular, the occurrence of SIgA, which has a high affinity for microorganisms, antigens and allergen-inducing antigens, is closely related to the activities of innate immunity and acquired immunity. However, little is known about the linkage mechanism of the two immune responses. LL-37 is not only constitutively present in mucosal compartments, but also has immunoregulatory roles such as pre-inflammatory and anti-inflammatory functions and chemotactic properties by formyl peptide receptor 2 (FPR-2) , We propose LL-37 as a mucosal linker that produces T cell dependent SIgA in a peyer'patch. In the present invention, the collection of CD11c ⁺ cells secreting IL-6 or expressing CD40 well, the increase of B200 ⁺ GL7 ⁺ Fas ⁺ papillary B cells, and the oral administration of reporter protein conjugated with LL- Provides evidence of the role of LL-37 modulating activity in PP by enhancing total IgA and antigen-specific secretory IgA levels after administration. These results suggest that PP follicular dendritic cells (FDCs) express FPR-2, that the LL-37-induced calcium flux in PP FDC is blocked by the FPR-2 antagonist WRWWWW peptide, and that LL- The activity of PP FDCs has been shown to improve Tgfb2, IL-4, and Cxcl13 transcript levels associated with IgA isotype switching of follicular B cells in the germinal center. Overall, the present invention suggests the role of LL-37 as an immunological linker in PP by improving the innate immune system and forming acquired immunity.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments.

Claims (13)

Human cathelicidin LL-37 peptide
Or a recombinant protein comprising a human catechiricidin LL-37 peptide, wherein the inducing agent enhances an immune response when the immunogenicity is enhanced or when it is co-administered with an antigen. The inducer of immunological activity according to claim 1, wherein the human cathelicidin LL-37 activates pancreatic dendritic cells (FDC). 2. The inducer of immunological activity according to claim 1, wherein the immunological activity inducing agent activates mucosal immunity. 2. The immunological activity inducer according to claim 1, wherein the antigen is derived from the group consisting of dengue virus, cholera virus, food poisoning bacteria, and brucellosis pathogens. Human cathelicidin LL-37 peptide
Or a recombinant protein comprising a human cathelicidin LL-37 peptide, wherein said human cathelicidin LL-37 peptide activates pancreatic dendritic cells. An oral vaccine comprising the immunological activity inducing agent according to any one of claims 1 to 4. A therapeutic agent for allergy comprising the immunostimulatory agent according to any one of claims 1 to 4. A therapeutic agent for mucosal diseases comprising the immunological activity inducing agent according to any one of claims 1 to 4. Preparing a recombinant protein comprising a human catechiricidin LL-37 peptide or an LL-37 peptide;
Introducing the recombinant protein comprising the human citalyclidine LL-37 peptide or the LL-37 peptide into the immunoreactivity inducing substance; And
Activating the innate immunity and / or acquired immunity of the immunoactivity inducing subject
Immune activity induction method. 10. The method of claim 9, wherein the step of activating the innate immunity and / or acquired immunity of the immunoactivity inducing entity comprises the step of inducing the activity of the follicular dendritic cell (FDC) of the immunoactivity inducing subject to produce a germinal center &Lt; / RTI &gt; 10. The method according to claim 9, wherein the immunoactivity inducing substance is an animal other than human. 12. The method of claim 11, wherein the animal is a mammal. [Claim 11] The method according to claim 9, wherein the infusion is performed by any one method selected from the group consisting of nasal administration, oral administration, and oral administration.

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