JP2005274366A - Screening method for vaccine target substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening method for a substance capable of making transmission of a mucosa vaccine efficient, and the mucosa vaccine using the substance. <P>SOLUTION: The purpose of the present invention is attained by the screening method for the substance for promoting conveyance or transmission of an antigen to an M cell, using the M cell (cilium M cell) of a cilium epithelial cell layer, for example, the screening method for the substance for promoting the conveyance or the transmission of the antigen to the M cell including a process for bringing the antigen and/or a testing compound into contact with the cilium M cell, a vaccine target substance identified by the screening method, and the vaccine or the like using the substance as a target. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for screening a substance that promotes the transport or delivery of an antigen to M cells (hereinafter also referred to as “chorionic M cells”) present in the chorionic epithelial cell layer, a vaccine target substance identified by the screening, The present invention relates to a mucosal vaccine using the substance.

  The immune system exists as a defense system against the entry of foreign objects from the outside world. Immune systems can be broadly classified into (1) systemic and peripheral immune systems centering on thymus and bone marrow, and (2) mucosal immune systems.

  The mucous membrane is the interface with the external environment that covers the oral cavity, nasal cavity, respiratory organs, digestive organs, and genitals. The mucous membrane is in contact with various foreign substances through food intake, breathing, and the like, and is a major route for invading the host body for pathogenic microorganisms, for example. Therefore, the immune defense mechanism in the mucous membrane is important as a life barrier.

  In order to induce antigen-specific immunity in mucosal immunity, antigens are induced in mucosal immunity-inducing tissue such as gut associated lymphoreticular tissue (hereinafter abbreviated as GALT) present on the mucosal surface of each organ. Need to be captured. In the epithelial cell layer of the mucosal immunity-inducing tissue, there are M cells specialized for the antigen uptake function. Antigen-specific immunity induction is initiated only when the antigen taken up by the M cell is delivered to antigen-presenting cells such as macrophages and dendritic cells present in the lower layer of the M cell (for example, Yuki Y, Kiyono H: New generation of mucosal adjuvants for the induction of protective immunity. See Rev Med Virol 13. 293-310. 2003: Non-patent document 1).

  Vaccines are used to activate the immune system. The administration form by injection employed in many current vaccines is effective for inducing an immune response via the systemic immune system, but hardly induces an immune response via the mucosal immune system. In order to induce an immune response in this mucosal immune system, it is necessary to take an oral or nasal administration form. Furthermore, it has been known that the immune response is induced simultaneously in the systemic immune system by administering the vaccine orally or nasally.

The current development of mucosal vaccines, such as oral vaccines, efficiently transports vaccine antigens to M cells, which are specialized cells for antigen uptake existing in mucosal immunity-inducing tissues such as Peyer's patches, which is a representative of GALT. It is done in consideration of delivery.
Yuki Y, Kiyono H: New generation of mucosal adjuvants for the induction of protective immunity. Rev Med Virol 13. 293-310. 2003

  However, M cells, which are antigen uptake ports, are scattered in the epithelial cell layer and less than 0.1% of all epithelial cells, and no specific marker that can be used for detection is known. Under such circumstances, it is desired to screen for molecules that enable the delivery and delivery of vaccine antigens by utilizing the functions of M cells, or to develop new mucosal vaccines targeting the obtained molecules.

  As a result of repeated studies on M cells involved in mucosal immunity, the present inventors surprisingly found that M cells that were thought to exist only in mucosal immunity-inducing tissues such as Peyer's patches for the first time were intestinal absorption. The present invention was completed by discovering that it also exists in the villi epithelial cell layer composed of epithelial cells. Specifically, the present invention provides a method for screening a vaccine target substance as shown below, a vaccine target substance identified by the screening, a mucosal vaccine using the substance, and the like.

(1) A method for screening for a substance that promotes transport or delivery of an antigen to an M cell using M cells present in the chorionic epithelial cell layer.
(2) The screening method according to (1), which comprises a step of bringing a test compound into contact with the M cell.
(3) The method according to (2) above, comprising the step of observing M cells after contacting the test compound with an electron microscope and / or a confocal image analysis system, using the test compound labeled as the test compound. Screening method.

(4) The screening method according to (1), which comprises a step of contacting the M cell with an antigen and a test compound.
(5) The method according to (4), including the step of observing M cells after contacting the antigen and the test compound with an electron microscope and / or a confocal image analysis system using the labeled antigen as the antigen. Screening method.
(6) The screening method according to (5), wherein the observation by the electron microscope and / or the confocal image analysis system is based on an immunohistological technique.

(7) A method for screening a substance that promotes transport or delivery of an antigen to these M cells by using M cells present in the villi epithelial cell layer and M cells in the mucosal immunity-induced tissue epithelial cell layer.
(8) The screening method according to the above (7), which comprises a step of bringing an antigen and / or a test compound into contact with each of M cells present in the chorionic epithelial cell layer and M cells in the mucosal immunity-induced tissue epithelial cell layer.

(9) A substance that facilitates transport or delivery of an antigen to M cells, identified by the method of any one of claims 1 to 8.
(10) An adjuvant comprising the substance according to (9).
(11) A mucosal vaccine comprising the adjuvant according to (10).
(12) A mucosal vaccine targeting the substance according to (9).
(13) A method for evaluating the delivery effect of a mucosal vaccine by using M cells present in the chorionic epithelial cell layer and M cells in the mucosal immunity-induced tissue epithelial cell layer.

  The present invention is not limited to the conventionally known mucosal immunity-inducing tissue epithelial cell layer M cells such as Peyer's patch (hereinafter also referred to as “Peier's patch M cells”), but also the chorionic epithelial cell layer composed of absorptive epithelial cells. Of Molecules (Villus M Cells) in the Molecules, Discovery of Molecules that Enables the Delivery and Delivery of Vaccine Antigens Specific to the Latter or Both M Cells and Development of New Mucosal Vaccines Targeting It Provides a means to Furthermore, it provides a means for observing whether the existing transmucosal antigen administration methods are transported and delivered to the latter or the former as well as both. Furthermore, elucidation of a new route of infection via villus M cells provides a means to develop new prevention methods.

  In addition, the fact that many serious infections such as influenza, SARS, AIDS, dysentery, and cholera are caused by mucosa-covered tissues such as the nasal cavity, oral cavity, upper bronchus, intestinal tract, and genitourinary mucosa. It is clear that elucidation of the invasion mechanism and its defense mechanism is the most effective in establishing infection prevention methods. Conventional injection vaccine administration methods effectively induce systemic immunity, and Peyer's patch M cell targeted oral vaccines effectively induce mucosal immune responses. The present invention relates to identification / separation of M cells present in the intestinal villi absorbing epithelial cell layer, which is the key to developing a mucosal vaccine targeting newly discovered villous M cells, and antigen uptake / pathogenic microorganism invasion analysis. Yes, it is useful for identifying new vaccine delivery molecules and evaluating existing mucosal vaccines.

  According to the first aspect of the present invention, there is provided a method of screening for a substance that promotes the transport or delivery of an antigen to the M cell using the villi M cell. Specifically, an antigen and / or a test compound is brought into contact with the villus M cell to test whether or not the test compound is taken into the M cell and to what extent it is taken up.

  The M cells used here are newly found villous M cells, and are not particularly limited, but are collected from the small intestine, large intestine, ileum, etc. of mammals such as mice, rats, and rabbits. In the above screening experiment, it may be used as M cells present in tissue pieces such as small intestine, large intestine, ileum, etc., and in some cases, cultured cells can be used. M cell culture can be performed based on a known technique (reference: Kerneis, S. et al .: Science 277, 949-52 (1997).

  When the above screening experiment is performed efficiently, for example, a tissue piece having M cells or a culture solution is dispensed into a 96-well plate, and an antigen and / or a test compound is added thereto, and a certain time, for example, 5 Incubation is performed at ˜120 minutes, preferably 10-30 minutes, at a constant temperature, for example 30-40 ° C., preferably 35-37 ° C. This reaction is preferably carried out in a medium suitable for cells, tissues, etc., for example, RPMI, MEM, etc., or near neutral isotonic buffer such as PBS.

  As a vaccine antigen, an antigen derived from a microorganism, an attenuated microorganism, or a purified antigen can be used, but it is desirable to label the antigen so that the subsequent microscopic observation process is facilitated. As a labeling agent used for labeling, for example, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin, GFP (green fluorescent protein) and the like are used.

  Here, the test compound refers to a candidate substance that promotes the transport / delivery of vaccine antigens to M cells. For example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plants It is selected from extracts, animal tissue extracts and the like. These may also be novel compounds or known compounds. Label the test compound as necessary. When labeling both the antigen and the test compound, it is preferable to use another labeling substance so that the two can be distinguished.

  After contacting the M cell with the antigen and / or test compound as described above, the extent to which the antigen and / or test compound has been taken up by the M cell is confirmed using an appropriate technique. For example, a molecule that, after incubation, analyzes a labeled sample using a scanning or transmission electron microscope, a confocal image analysis system, etc., and allows M cells to carry and deliver a vaccine antigen. Select candidates for. Usually, such analysis is performed based on immunohistological techniques known to those skilled in the art.

  In this way, a molecule having a high ability to deliver an antigen to villi M cells can be screened. In the above example, only the villi M cells are shown. However, in addition to the villi M cells, mucosal immunity-derived tissue-derived M cells are used, so that the vaccine is specific for either M cell or both. Molecules with high antigen delivery capability can be screened.

  In another aspect of the present invention, a substance that facilitates the transport or delivery of an antigen to M cells identified by the above screening method can be provided. Such substances can be used as adjuvants for mucosal vaccines.

Moreover, according to another aspect of the present invention, a mucosal vaccine comprising such an adjuvant can be provided.
Among antigens used in vaccines, for example, as microbial antigens or attenuated microorganisms, influenza virus type A, influenza virus type B, influenza virus type C, rotavirus, cytomegalovirus, RS virus, adenovirus, AIDS virus (HIV) ), Hepatitis C virus, hepatitis A virus, hepatitis B virus, varicella zoster virus, herpes simplex virus types 1 and 2, ATL (adult T cell leukemia) virus, coxsackie virus, enterovirus, idiopathic rash virus ( HHV-6), measles virus, rubella virus, mumps virus, poliovirus, Japanese encephalitis virus, rabies virus, rotavirus, noo Oak virus, foot-and-mouth disease virus, infectious gastroenteritis virus, smallpox virus Lus, Tengu virus, Yellow fever virus, West Nile virus, SARS (Coronavirus), Ebola hemorrhagic fever virus (Filovirus), Marburg virus (Firovirus), Lassa fever virus, Hantavirus, Nipah virus, etc. Cocci, periodontopathic bacteria, cholera, influenza, pneumococci, pertussis, diphtheria, tetanus, botulinum, anthrax, pathogenic Escherichia coli, plague, wild boar, Escherichia coli O157, Lyme disease Borrelia, Bacteria containing pathogenic bacteria such as Salmonella, MRSA (Staphylococcus aureus), VRE (Enterococcus), Mycobacterium tuberculosis, Shigella, Salmonella typhi, Salmonella paratyphi, Legionella, Brucella (Wavy fever), Chlamydia, Q Rickettsia such as heat rickettsia, amoeba dysentery, malaria pathogen Protein antigens such as protozoa, or can be given an attenuated microorganism which weakened pathogenic of these microorganisms themselves. In addition, as a protein antigen of microorganisms, in the case of influenza virus, hemagglutinin (HA) or neuraminidase (NA) alone or a mixture can be exemplified as what is retained in the oral vaccine of the present invention. These antigens can be obtained by methods such as processing and purification from microorganisms, but can also be obtained by synthesis, and can also be produced by genetic engineering.

  Further, the antigen that can be held by the oral vaccine shown in the present invention varies depending on the type of lipid membrane structure. For example, there are no particular restrictions on what the liposomes can hold, and examples include water-soluble or fat-soluble microbial antigens or attenuated microorganisms. In the case of an emulsion, a fat-soluble antigen can be mentioned, and in the case of a micelle, a poorly water-soluble antigen can be retained.

  The vaccine shown in the present invention is characterized by raising the antibody titer by oral administration, and the dose is, for example, 10 to 1000 μg per adult. Do this once, or twice at one week intervals, twice at two week intervals, twice at three week intervals, or twice at one month intervals It can also be applied to. Furthermore, it can be administered three times, four times, five times, and six times at similar intervals. Usually it is a single dose or 2 to 4 doses at one month intervals. The usage can be considered in the same manner as an injectable vaccine. With respect to vaccines against other antigens, the same amount or 20 times the amount of vaccine for injection may be used, and the usage may be appropriately administered according to these. Moreover, it may be appropriately increased or decreased according to the immune responsiveness or age of the individual to be administered.

The vaccine shown by this invention can be manufactured as follows, for example.
a) Method for producing liposome-type vaccine In the case of microbial antigens or attenuated microorganisms themselves, membrane component substances such as phosphatidylserines (lecithin), sphingomyelin, and glycolipids that bind phosphatidylserine and / or mannose In some cases, further using an adjuvant substance, an aqueous dispersion of liposome is prepared according to a known method (for example, Annual Review of Biophysics and Bioengeneering, 9, 467 (1980)). Such liposomes may contain sterols such as cholesterol, charged lipids such as dialkyl phosphate, diacyl phosphatidic acid, stearylamine and antioxidants such as α-tocopherol as stabilizers.

b) Production method of emulsion type vaccine Microorganism antigen or attenuated microorganism itself, amphiphile such as lecithin, polyoxyethylene sorbitan fatty acid ester (Tween), fat and oil such as soybean oil, phosphatidylserine and / or mannose An emulsion is prepared according to a known emulsion preparation method, using a glycolipid bound to, and optionally an adjuvant substance.

c) Production method of micelle vaccine Micelle forming surfactant such as microbial antigen or attenuated microorganism itself, polyoxyethylene sorbitan fatty acid ester (Tween), fatty acid sodium, polyoxyethylene hydrogenated castor oil, etc. And an aqueous solution of phosphatidylserine and / or mannose, and optionally an adjuvant substance, to prepare an aqueous micelle dispersion according to a known micelle preparation method.

  The vaccine as described above may be used after adding a known adjuvant in some cases. As long as the adjuvant for mucosal immune vaccine used in the present invention is a protein antigen used as a mucosal immune vaccine, it can be used together with any protein antigen as a mucosal adjuvant. Examples of protein antigens include cell membrane proteins of Haemophilus influenzae and outer membrane proteins of Streptococcus mutans. Further, an adjuvant can be used together with a protein antigen or the like usually used for pertussis vaccine, measles vaccine, rubella vaccine, mycoplasma vaccine and the like. In addition, the adjuvant may be a carbohydrate, lipid, virus, bacterium itself, and the like.

  The adjuvant used in the present invention is usually administered by nasal administration or oral administration together with a protein antigen used as a vaccine for transmucosal administration. It is particularly preferable to administer by oral administration. The adjuvant used in the present invention is administered together with the protein antigen, usually in liquid or powder form, by dropping, spraying or spraying into the nasal cavity or oral cavity. Examples of the dosage form of the adjuvant administered in this manner include liquids, suspensions, powders and the like. Examples of the liquid agent include those dissolved in purified water, buffer solution and the like together with protein antigen. Examples of the suspending agent include those suspended in purified water, buffer solution and the like together with methyl cellulose, hydroxymethyl cellulose, polyvinyl pyrrolidone, gelatin, casein and the like. Examples of the powder include those mixed well with methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose and the like. In these preparations, absorption promoters, surfactants, preservatives, stabilizers, moisture-proofing agents, moisturizing agents, solubilizing agents and the like that are usually used can be added as necessary.

  The adjuvant used in the present invention varies depending on the subject to be administered, administration method, administration form, etc., but is usually administered in the range of 100 μg to 1000 μg, preferably 100 μg to 500 μg per adult, at the same time as the protein antigen. . Production of antibodies specific to protein antigens used as vaccines in both saliva, nasal cavity, digestive tract, respiratory organs, genital secretions and serum by administering mucosal immune vaccine together with mucosal immune vaccine Is significantly enhanced. Therefore, the adjuvant used in the present invention is effective as an adjuvant for various mucosal immune vaccines.

  According to still another aspect of the present invention, a method for evaluating the delivery effect of a mucosal vaccine can be provided by using villi M cells and M cells of a mucosal immunity-induced tissue epithelial cell layer. According to this method, an existing mucosal vaccine can be evaluated.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. First, materials and general methods used in the present invention will be described.

[Mouse to use]
BALB / c mice and C57BL / 6 mice are purchased from Nippon Claire Co., Ltd. (Tokyo). Mice treated with LTβR-Ig fusion protein and mice knocked out of both TNF and LT-α (TNF / LT-α ^{− / −} ; 129 × C57BL / 6) were prepared by Eugster et al., Int. Immunol. 8 , 23 -36 (1996) and Rennert et al., J. Exp. Med. 184 , 1999-2006 (1996). LT-α ^{− / −} (C57BL / 6) mice are obtained from Jackson Laboratory (Bar Harbor, Maine). Id2 ^{− / −} (129 / Sv) mice are generated by the method described by Yokota et al., Nature 397 , 702-706 (1999).

[M cell staining method]
The standard lectin staining method described in Gebert et al., Am. J. Pathol. 154 , 1573-1582 (1999) is used to detect murine M cells. The small intestine from which the mucus was removed from BALB / c mice or C57BL / 6 mice with or without Peyer's patches (PP) or without Peyer's patches was fixed with 4% paraformaldehyde for 1 hour and washed. And then blocked with 10% FBS dissolved in PBS containing 0.1% glycine. Lectin labeling experiments were performed at a concentration of 20 μg / ml using TRITC-coupled gorse agglutinin (UEA-1-TRITC, Vector, Burlingame, Calif.) And wheat embryo agglutinin conjugated with FITC (WGA-FITC). 2 hours. After washing with PBS, samples are stored in Tris buffer containing 30% glycerol and 0.1% sodium nitride. Samples are observed with a Bio-Rad MRC-600 confocal imaging system (Bio-Rad, Hercules, CA). Alkaline phosphatase activity and Alcian blue staining are performed on the entire fixed small intestine by the method described by Debard et al., Gastenterology 120 , 1173-1182 (2001).

[Observation with electron microscope]
For analysis by scanning electron microscope (SEM), mucus was first removed from a small intestine section, which was dissolved in PBS containing 100 mM HEPES at room temperature, 1% in 2% glutaraldehyde and 2% paraformaldehyde. Time fixed. After washing with PBS, the sample is treated with 1% osmium tetroxide at room temperature for 1 hour and dehydrated using a graded alcohol solution. The dehydrated sample is critical point dried with CO ₂ and sputter coated, and then observed using a Hitachi S-700 scanning electron microscope (Hitachi). Tissue samples are fixed in 2% formaldehyde and 0.05% glutaraldehyde dissolved in cacodylate buffer (pH 7.3) for 4 hours for analysis by transmission electron microscopy (TEM). After blocking for 4 hours with PBS containing 1% L-lysine, the samples were UEA-1-biotin (25 μg / ml), UEA-1-TRITC (12.5 μg / ml) and WGA-FITC (20 μg / ml). Stain with After staining, a sample containing villus M cells is isolated while observing with a confocal image analysis system comprising a confocal microscope. Next, the obtained tissue is labeled with PBS containing a goat anti-biotin antibody bound with a 20 nm gold colloid, and observed using a Hitachi H-7100 transmission electron microscope.

[In situ antigen uptake]
As the Salmonella typhimurium PhoPc strain transformed with the plasmid pKKGFP, the one provided by Dr. F. Niedergang is used. Furthermore, Yersinia pseudotuberculosis, E. coli invasin and E. coli expressing GFP are prepared by known methods. Mice are anesthetized by intraperitoneal injection of 2 mg ketamine (Sigma, St. Louis, MO) per mouse. Approximately 10 cm long pieces of the small intestine of TNF / LT-α ^{− / −} mice and wild type mice are ligated at both ends with surgical sutures. Bacteria expressing 5 × 10 ⁸ GFP are suspended in 1.0 ml, inoculated into loops and incubated in situ. Ten minutes later, Peyer's patches and fragments without intestinal tracts are removed and washed thoroughly with RPMI medium containing chilled PBS and 100 μg / ml gentamicin. Intestinal epithelial cells are isolated from Peyer's patches and intestinal fragments by the method described in Yamamoto et al., J. Immunol. 160 , 2188-2196 (1998). The isolated cells are fixed with 4% paraformaldehyde, washed with 10% FBS in PBS, and labeled with UEA-1-TRITC. The percentage of double positive intestinal epithelial cells is analyzed with a FACS Caliber flow cytometer (Becton Dickinson, San Jose, Calif.). Using the selected mice, the entire small intestine fragment is processed as described above with a confocal microscope. In order to remove weakly adherent and / or extracellular bacteria, vigorous washing with RPMI 1640 medium containing chilled PBS and gentamicin is performed with chorionic epithelial tissue and epithelial cells containing M cells after bacterial infection. In the separation process. Gentamicin is selected as an antibiotic because it has a lethal effect on Salmonella.

[Immunity]
The recombinant S. typhimurium BRD 847 strain used for immunization experiments is derived from the plasmid pTETnir15 under the control of the nirB promoter induced under anaerobic conditions from the non-toxic immunogenic 50-kDa tetanus toxoid. AroA aroC double mutant (rSalmonella-ToxC) expressing ToxC fragment. As a control, rSalmonella that does not express ToxC is used. Recombinant Salmonella is suspended in PBS at a concentration of 2.5 × 10 ¹⁰ per ml. This suspension of Salmonella is orally administered by 0.2 ml per mouse from a tube. The antibody titer in the serum is measured by the enzyme immunoassay described in Yamamoto et al., J. Immunol. 164 , 5184-5191 (2000).

[Data analysis]
Data are expressed as mean ± standard deviation and evaluated by Mann-Whitney U test. A P value of less than 0.05 is assumed to be substantially significant.

Identification of UEA-1 cells in intestinal villous epithelial tissue
M cells exist in the intestinal tract related lymphoid tissue (GALT), one of mucosal immunity-inducing tissues, for example, the dome epithelial cell layer (or FAE) of Peyer's patch, and have been considered to grow only there. However, using confocal image analysis of the whole intestine of a mouse stained with agglutinin agglutinin (UEA-1) bound to TRITC and wheat embryo agglutinin (WGA) labeled with FITC, as shown in FIG. In addition to the FAE region (jm), UEA-1 ⁺ WGA ⁻ cells (chorionic M cells) were found in the chorionic epithelial tissue (ai). In FIG. 1, a portion observed as white or gray (actually green) at af, j and k is a portion stained with WGA, and a portion indicated by an arrow (actually red) is UEA-1. It is the part dyed by. UEA-1 has specificity for carbohydrate structures containing α- (1-2) -fucose, but selectively binds to the entire plasma membrane of Peyer's patch M cells and not WGA ⁺ columnar epithelial cells. . In order to further confirm the specificity of UEA-1 staining, a blocking experiment using 50 mM soluble fucose was performed. Pre-incubation of UEA-1 with soluble fucose for 1 hour clearly blocked UEA-1 staining with FACS, data not shown, but immunohistochemical analysis further showed the specificity of the UEA-1 staining method . In addition, as shown in FIG. 1, two types of villi UEA-1 ⁺ WGA ⁻ cells (villi M cells), that is, a dense type (a and b) and a diffusion type (d and e) are the same cell population. Distinguish based on density.

Growth of UEA-1 ⁺ villus M cell populations in various mice without Peyer's patches We tested whether villus M cells could grow in Peyer's patch (or GALT) -deficient mice. As the mice, mice treated with LTβR-Ig in the uterus, LTα ^{− / −} mice, TNF / LTα ^{− / −} mice, and Id2 ^{− / −} mice were used. Also in FIG. 2, the white portion (actually green) is the portion stained with WGA, and the portion observed in a darker color (actually red) is the portion stained with UEA-1. . This dark portion indicates the location of M cells. As shown in FIG. 2, M cells showing characteristic UEA-1 ⁺ WGA ⁻ staining were found in the tip region of the intestinal villi of all Peyer's patch-deficient mice. This indicates that there is a growth path of M cells independent of FAE. Furthermore, although data are not shown, it was shown that M cells were also present in TNF / LTα ^{− / −} mice lacking the newly described isolated lymph node (ILF) in addition to Peyer's patches. Furthermore, the data is supported. To determine the distribution and number of villus M cells in wild type mice and mice without GALT, the density of villus M cells in the entire small intestine was examined using a confocal image analysis system. Wild-type mice were found to have about 40 to 50 density villous M cell groups per whole small intestine, and TNF / LTα ^{− / −} mice were also found to have about 50 to 60 density villous M cell groups. The similarity of both values suggests that the growth of villi M cells is not completely dependent on GALT and FAE.

Experiments were performed to evaluate bacterial adhesion by villus M cells and the ability of internalized villus M cells to take up pathogenic microorganisms. RSalmonella typhimurium (rSalmonella-GFP), Yersinia pseudotuberculosis (yersinia-GFP), E. coli expressing yersinia invasin (E. coli-invasin-GFP) and wild-type E. coli-GFP Was inoculated. After 10 minutes of in situ incubation for each bacterium, immunohistochemical analysis was performed and shown in FIG. 3A a and b (wild type mice) and e and f (TNF / LTα ^{− / −} mice). Thus, it was revealed that rSalmonella-GFP is present in UEA-1 ⁺ cells (chorionic M cells) of the chorionic epithelium. Furthermore, as shown in FIG. 3A c and d (wild-type mice) and g and h (TNF / LTα ^{− / −} mice), Yersinia-GFP also specifically expressed villi UEA-1 ⁺ cells (chorionic M cells). ). As shown in i and j of FIG. 3A, immunohistological analysis using frozen sections revealed that rSalmonella-GFP was localized in the apical membrane region of villi UEA-1 ⁺ cells (chorionic M cells). . In order to show the bacterial uptake ability of villi UEA-1 ⁺ cells (chorionic M cells), ileal loop infection experiments using rSalmonella-GFP were performed, and the localization of the bacteria was analyzed using a confocal microscope. The results are shown in FIG. 3B. As shown to a, b, and c of FIG. 3B, it became clear that rSalmonella-GFP was localized in the intracellular area | region. Furthermore, as shown in d of FIG. 3B, rSalmonella-GFP was found in the intracellular region of villi UEA-1 ⁺ cells (chorionic M cells) prepared by cytospin.

Intestinal epithelial cells were further separated from chorionic epithelial tissue and Peyer's patch, and TRITC-UEA1 was reverse-stained for ileal loop infection experiments and flow cytometry analysis of microorganisms expressing GFP. As shown in FIG. 3C, the fraction of UEA-1 ⁺ cells (chorionic M cells) is more frequently expressed than that of UEA-1 ⁻ cells isolated from chorionic epithelial tissue, rSalmonella-GFP, Yersinia-GFP, or E. coli − Cells containing invasin-GFP appeared. Furthermore, similar patterns were obtained with UEA-1 ⁺ cells (FAE-M cells) and UEA-1 ⁻ cells isolated from the dome region of Peyer's patches. In addition, as shown in FIG. 3C, many cells containing rSalmonella-GFP, Yersinia-GFP, or E. coli-invasin-GFP were isolated from chorionic epithelial tissue of TNF / LTα ^{− / −} mice lacking GALT. Recovered from UEA-1 ⁺ cells but not UEA-1 ⁻ cells. These results indicate that villus UEA-1 ⁺ cells (villus M cells) have the ability to take up several different bacteria from the luminal side that are known to be taken up by FAE-M cells. . Furthermore, the delivery effect of an antigen or a vaccine can be easily evaluated by using the above method.

Analysis of Villous M Cells by Scanning and Transmission Electron Microscopy From scanning electron microscopy (SEM) analysis, villous M cells are prominent features observed in Peyer's patch M cells, ie as shown in FIGS. It was found to have a depressed surface with short irregular microvilli. Transmission electron microscope (TEM) analysis shown in FIGS. 4f and g also showed that UEA-1 cells bound with gold particles bound to villi M cells. Furthermore, as shown in FIG. 4h, infiltrating mononuclear cells were observed in the pockets of villi M cells. SEM also showed that bacteria bind to the cell membrane of villi M cells in the small intestine of TNF / LTα ^{− / −} mice without FAE after exposure of Salmonella to the intestine, as shown in FIGS. 4i to l. . These results suggest that newly identified villous M cells form another gateway for antigen uptake and / or an entrance from the luminal side of intestinal villous epithelial tissue. These results are the first evidence that M cells grow and localize not only in Peyer's patch FAEs, but also in chorionic epithelium.

Induction of antigen-specific immune response in PP-deficient mice As shown in FIG. 3, chorionic M cells are effective antigen uptake ports for bacterial antigens. To be induced through When a GALT-deficient mouse lacking the TNF / LT-α gene is orally immunized with a recombinant Salmonella BRD 847 strain expressing the 50-kDa ToxC fragment of the tetanus toxoid vaccine antigen, an antibody against tetanus toxoid (TT) specific serum IgG As shown in FIG. 5, the titer was comparable between the serum of TNF / LTα mice and wild-type mice immunized orally. As expected, the antibody titer of TT-specific serum IgG could not be detected when wild-type and TNF / LTα mice were orally immunized with recombinant Salmonella that does not express Tox-C. These results suggest that villus M cells are an important antigen uptake port for inducing an antigen-specific immune response to an antigen in the gastrointestinal environment.

  This patent relates to the identification and isolation of M cells present in the intestinal villi absorbing epithelial cell layer, which is the key to the development of mucosal vaccines targeting newly discovered villous M cells, and antigen uptake and pathogenic microorganism invasion analysis. Yes, it is useful for identifying new vaccine delivery molecules and evaluating existing mucosal vaccines.

Confocal of FAE epithelial tissue of Peyer's patch dome in normal BALB / c mice and UEA-1 ⁺ M cells (Peier patch M cells) present therein and UEA-1 ⁺ M cells (chorionic M cells) in chorionic epithelium It is an observation figure by a microscope. It is a figure which shows the presence of the villus M cell in various mice without a Peyer's patch. (A) It is a figure which shows the result of the immunohistological analysis of the antigen uptake by UEA-1 ⁺ villus M cell. (B) It is a figure which shows the localization of Salmonella which expresses GFP in the intracellular region of UEA-1 ⁺ villus M cell. (C) Antigen uptake by UEA-1 ⁺ villus M cells. It is a figure which shows the observation result by a scanning electron microscope and a transmission electron microscope of a villus M cell and Peyer's patch M cell. It is a figure which shows the induction | guidance | derivation of an antigen-specific immune response in a Peyer's patch defect mouse.

Claims (13)

  A method for screening a substance that promotes transport or delivery of an antigen to an M cell, using the M cell present in a villi epithelial cell layer.   The screening method according to claim 1, comprising a step of bringing the test compound into contact with the M cell.   3. The screening method according to claim 2, comprising a step of observing M cells after contacting the test compound with an electron microscope and / or a confocal image analysis system, using the test compound labeled as the test compound.   The screening method according to claim 1, comprising a step of contacting the M cell with an antigen and a test compound.   The screening method according to claim 4, comprising a step of using an antigen labeled as the antigen and observing M cells after contacting the antigen and the test compound with an electron microscope and / or a confocal image analysis system. .   6. The screening method according to claim 5, wherein the observation by the electron microscope and / or the confocal image analysis system is based on an immunohistological technique.   A method for screening a substance that promotes transport or delivery of an antigen to these M cells by using M cells present in the villi epithelial cell layer and M cells in the mucosal immunity-induced tissue epithelial cell layer.   8. The screening method according to claim 7, comprising a step of bringing an antigen and / or a test compound into contact with each of the M cells present in the chorionic epithelial cell layer and the M cells in the mucosal immunity-induced tissue epithelial cell layer.   A substance that facilitates transport or delivery of an antigen to M cells, identified by the method of any one of claims 1-8.   An adjuvant comprising the substance according to claim 9.   A mucosal vaccine comprising the adjuvant according to claim 10.   A mucosal vaccine targeting the substance of claim 9. A method for evaluating the delivery effect of a mucosal vaccine by using M cells present in the villi epithelial cell layer and M cells in the mucosal immunity-induced tissue epithelial cell layer.

Images