JP2004161781A - Oral vaccine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare an oral vaccine effectively producing an antibody after oral administration and obtaining infection protective effect. <P>SOLUTION: This vaccine for oral administration comprises an antigen and a lipid, wherein the lipid contains a glycolipid combined with mannose. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention relates to an oral vaccine that efficiently produces antibodies after oral administration. The oral vaccine of the present invention, after oral administration to a living body, efficiently produces an antibody against the retained antigen, and provides a protective effect against infection.

  Most of the current vaccines are injections that are injected subcutaneously, but side effects that occur infrequently and pain during administration are clinical problems. Although various administration methods have been studied to improve this, oral vaccines are the most desired dosage form for early development because of safety and convenient administration methods.

  Oral vaccines have been studied using buffers, enteric-coated tablets, microparticles, nanoparticles, adjuvant substances, etc., but have not been successful yet (Non-Patent Documents 1 and 2). ). In these reviews, applications to oral vaccines using liposomes with orthodox membrane composition are also introduced, but although the antibody titer slightly increased, it was far from practical use. Have been.

  In any case, it can be said that there was no means for efficiently producing antibodies after oral administration in the prior art.

  It is known that liposomes whose membranes are modified with phosphatidylserine are injected intravenously (Non-Patent Document 3), and that liposomes whose membranes are modified with a mannose derivative or a mannan derivative accumulate in liver and lung. (Non-Patent Document 4, Non-Patent Document 5).

International Journal of Pharmaceutics, 75, 1 (1991) Journal of Clinical Pharmacy and Therapeutics, 16, 309 (1991) Cancer Research, 40,4460 (1980) Biochimica et Biophysica Acta 632,562 (1980) Pathophysiology 6, 771 (1985)

  An object of the present invention is to provide an oral vaccine capable of efficiently producing an antibody against a retained microbial antigen or an antigen such as an attenuated microorganism after oral administration to a living body.

  The present invention relates to a vaccine for oral administration which is a complex of an antigen and a lipid, and this complex can increase the antibody titer even by oral administration by containing a glycolipid binding mannose as a lipid. it can.

  The oral vaccine of the present invention is capable of efficiently producing an antibody against the retained antigen after oral administration to a living body. Therefore, an oral vaccine of a microbial antigen or an attenuated microbe that has not been absorbed orally and could not produce an antibody in the past has been made possible.

  The complex can be classified into a case where the protein or glycoprotein as an antigen and the lipid are irregularly arranged by hydrophobic bonds and / or hydrogen bonds, and a case where the complex is a lipid membrane structure.

  The lipid membrane structure of the present invention should be interpreted in a broad sense, and may have a lipid and a lipid forming a membrane or an oil component and a water component may form a membrane as a boundary, The particles having a membrane structure in which the polar groups of the amphiphilic lipid are arranged toward the aqueous phase side of the interface can also be referred to as liposomes, emulsions or water-soluble micelles. The present invention has been made based on the finding that the above problem can be solved by the fact that the complex contains glycolipids that bind mannose as a lipid and / or phospholipids that are phosphatidylserine.

  The vaccine of the present invention may optionally further comprise an adjuvant substance.

  The antigens used in vaccines are well understood by researchers in this field. For example, antigens of microorganisms or attenuated microorganisms include influenza virus A, influenza virus B, influenza virus C, and 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 type T cell leukemia) virus, coxsackie virus, enterovirus, sudden rash virus (HHV-6), measles virus, rubella virus, mumps (mumps) virus, viruses such as polio virus, Japanese encephalitis virus, rabies virus, caries streptococci, Cholera Protein antigens of microorganisms such as Haemophilus influenzae, Streptococcus pneumoniae, B. pertussis, diphtheria, tetanus, rickettsiae such as chlamydia, and protozoa such as malaria pathogens, or attenuation that weakens the virulence of these microorganisms themselves Microorganisms and the like can be mentioned. In the case of an influenza virus, examples of the protein antigens of microorganisms include hemagglutinin (HA) and neuraminidase (NA) alone or as a mixture that is held by the oral vaccine of the present invention.

  These antigens can be obtained from microorganisms by methods such as processing and purification, but can also be obtained by synthesis, and can also be produced by genetic engineering.

  Further, the antigen that the oral vaccine of the present invention can hold varies depending on the type of lipid membrane structure. For example, what can be held by the liposome is not particularly limited, and examples thereof include water-soluble or fat-soluble microorganism antigens and attenuated microorganisms. In the case of an emulsion, a lipid-soluble antigen can be used, and in the case of a micelle, a water-insoluble antigen can be used.

  As the phosphatidylserine according to the present invention, soybean, phosphatidylserine derived from natural products such as egg yolk, hydrogenated phosphatidylserine obtained by hydrogenating them, or soybean, obtained by semisynthesis from phosphatidylcholine derived from natural products such as egg yolk. Phosphatidylserine, hydrogenated phosphatidylserine obtained by hydrogenating them, semi-synthesized or fully synthesized dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, distearoylphosphatidylserine, and the like, and preferably hydrogenated phosphatidylserine, dipalmitoyl Phosphatidylserine and distearoylphosphatidylserine.

  The glycolipids to which mannose is bound according to the present invention include phosphatidylethanolamine derivatives having mannose residues (Biocheimica et Biophysica Acta, 632 562 (1980)) and cholesterol derivatives (Proc. Natl. Acad. Sci. USA, 77, 4430 (1980)), dimannosyl diglyceride (Biochemical and Biophysical Research Communications, 110, 140 (1983)), mannobiose fatty acid esters and amides (Japanese Unexamined Patent Publication (Kokai) No. 1-104088), phosphatidyl mannose, and the like. And derivatives in which cholesterol is partially substituted (Pathophysiology, 6,771 (1985)). More specific compounds include 4-0- (6-O-eicosanoyl-β-D-mannopyranosyl) -D-mannopyranose and N- (2-N′-cholesterylcarboxymethyl) carbamylmethylmannan. be able to.

  These phospholipids (phosphatidylserine) and glycolipids as the membrane components may be added alone or as a mixture to a lipid membrane structure mainly composed of other membrane components such as phosphatidylcholines (lecithin) and sphingomyelin, These alone may form a lipid membrane structure.

  In other words, phosphatidylserines alone can form liposomes and emulsions, and glycolipids (mannobiose fatty acid esters and amides) that bind mannose alone can form emulsions and micelles. It is. Therefore, in the present invention, the addition ratio of phosphatidylserine and mannose-bound glycolipid used as a membrane component in the present lipid membrane structure is not limited at all in the upper limit. The lower limit is preferably 1% or more in terms of mole fraction based on all lipid membrane components, and it is desirable to add 5 to 50%.

  Other membrane components include phosphatidylcholines (lecithin), for example, phosphatidylcholine, hydrogenated phosphatidylcholine obtained by hydrogenating phosphatidylcholine derived from natural products such as soybean and egg yolk, semi-synthetic or fully synthetic dimyristoyl phosphatidylcholine, semi-synthetic or fully synthetic dicholine. Examples include lipids such as palmitoyl phosphatidylcholine and semi-synthetic or fully synthetic distearoyl phosphatidylcholine, and sphingomyelin.

  In addition, charged lipids can be added as other lipids to achieve physical stabilization of the liposome. Examples of charged lipids can include dialkyl phosphates, diacylphosphatidic acids, stearylamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine or ionic surfactants (acidic or basic).

  Further, sterols such as cholesterol may be added in order to achieve physical and biological stability of the liposome.

In the present invention, if necessary, an adjuvant substance can be further added. Examples of the adjuvant substance include, for example, (N-acetylmuramoyl) -L-alanyl-D-isoglutamine (muramyl dipeptide, MDP) which is the least effective adjuvant substance on the cell wall of Mycobacterium tuberculosis and derivatives thereof, for example, 6-O- (2-tetradecylhexadecanoyl) -N-acetylmuramoyl) -L-alanyl-D-isoglutamine (B30-MDP), 6-O- (2-tetradecylhexadecanoyl) -N -Acetylmuramoyl) -L-alanyl-D-glutamamide (B30-MDP amide), N ² -[(N-acetylmuramoyl) -L-alanyl-D-isoglutaminyl] -N ⁶ -stearoyl-L -Lysine (MDP-Lys [L18]) and the like. In the case of the water-soluble muramyl dipeptide, a liposome can be used as the lipid membrane structure, and the lipid membrane structure may be held in the internal aqueous phase of the liposome. The amount of muramyl dipeptide to be added is not limited at all, and can be added up to the saturation solubility in an aqueous solvent. In the case of lipophilic muramyl dipeptide derivatives, liposomes, emulsions, and micelles can be used as the lipid membrane structure. The addition ratio to the total lipid membrane component is not limited at all, but it is preferably 0.1% or more in terms of molar fraction, and it is preferable to add 1 to 30%.

  The vaccine of the present invention is characterized in that it protects against infection by raising the antibody titer by oral administration, but the dose is 50 μg to 5 mg, usually 200 μg to 2 mg, per influenza vaccine per adult. It should be administered once, twice weekly, twice twice weekly, twice twice weekly, or twice monthly. It can also be applied to Furthermore, administration may be performed three times, four times, five times, or six times at the same interval. It is usually a single dose or 2-4 doses at monthly intervals. In the case of the hepatitis B vaccine, the dose is 20 μg to 5 mg, and usually 50 μg to 2 mg, per adult. The usage can be considered in the same manner as for an injectable vaccine. With respect to vaccines against other antigens, the amount may be the same as that of a vaccine for injection in the same amount to 20-fold, and administration may be appropriately performed according to these. Of course, the number may be appropriately increased or decreased according to the immune responsiveness, age, etc. of the administered individual.

  Since phosphatidylcholine, phosphatidylserine, cholesterol, and the like used in the present invention are biological components, they are considered to have low toxicity. YMRustum et al. Have tested the acute toxicity of liposomes prepared with phosphatidylcholine, phosphatidylserine and cholesterol from egg yolk, with a 50% lethal toxicity of 5.5 g / kg or more (Cancer Research 39, 1390-1395 (1979)). reference).

Next, a method for producing the vaccine of the present invention will be described.
a) A method for producing a liposome-type vaccine An antigen of a microorganism or an attenuated microorganism itself, a membrane component substance such as phosphatidylserine (lecithin), sphingomyelin, and a glycolipid binding phosphatidylserine and / or mannose. In some cases, an aqueous dispersion of liposomes is prepared using an adjuvant substance according to a known method (eg, Annual Review of Biophysics and Bioengeneering, 9, 467 (1980)). Such liposomes may contain sterols such as cholesterol, charged lipids such as dialkyl phosphate, diacylphosphatidic acid, stearylamine and antioxidants such as α-tocopherol as stabilizers.
b) Production method of emulsion type vaccine Antigen of microorganism or attenuated microorganism itself, amphipathic substance such as lecithin, polyoxyethylene sorbitan fatty acid ester (Tween), fat such as soybean oil, phosphatidylserine and / or mannose An emulsion is prepared according to a known emulsion preparation method using a glycolipid binding to and optionally an adjuvant substance.
c) Method for producing micelle-type vaccine Antigen of microorganisms or attenuated microorganisms themselves and micelle-forming surfactants such as polyoxyethylene sorbitan fatty acid ester (Tween), fatty acid sodium, polyoxyethylene hydrogenated castor oil, etc. Is added to the aqueous solvent at a concentration of), a glycolipid binding phosphatidylserine and / or mannose, and optionally an adjuvant substance, to prepare an aqueous dispersion of micelles according to a known micelle preparation method.

The present invention will be described with reference to Examples and Test Examples using bovine serum albumin (BSA), influenza virus antigen and hepatitis B virus antigen as a model of a protein antigen of a microorganism, but the present invention is not limited thereto. Absent.
In the examples, without being encapsulated in the lipid membrane structure, there may be a case where the antigen protein is present free from the lipid membrane structure or a complex of another form exists, but if necessary, It may be used after being removed by an operation such as ultracentrifugation or gel filtration, or may be used without being removed.
Liposomal oral vaccine holding bovine serum albumin (BSA) a) Preparation of liposome encapsulating BSA The membrane composition ratio is distearoyl phosphatidylcholine (DSPC): hydrogenated phosphatidylserine (H-PS): cholesterol (Chol) = 1: 1: To 160 mg of the lipid consisting of 2 (molar ratio), 20 ml of a 9: 1 mixture of chloroform and methanol was added and dissolved, and the solvent was distilled off while heating at 37 ° C. using a rotary evaporator. It was further dried under vacuum for 2 hours to prepare a lipid film. To this was added 4 ml of each concentration of BSA dissolved in physiological saline (see Table 1), and the mixture was stirred with a vortex mixer at 50 ° C. for 10 minutes to prepare multilamellar liposomes (MLV, multilamellar vesicle). Subsequently, centrifugation (35,000 × g, 4 ° C., 20 minutes) was performed, the supernatant was removed, and the precipitate was resuspended by adding 4 ml of physiological saline. This centrifugation operation was repeated three times to remove free BSA. The liposome suspension thus prepared was dialyzed against 3 liters of physiological saline for 12 hours using a 0.1 μm polycarbonate membrane filter to remove liposomes having a particle diameter of 0.1 μm or less. In Table 1, the BSA-encapsulated liposome-type oral vaccine shown on the right was used.

Final liposome suspension: DSPC / H-PS / Chol = 54.4 / 51.2 / 54.4 (mg)

Since 0.5 ml is administered once per mouse, the lipid dose is 10 μmol and the BSA dose is 50 μg (Example 1) to 400 μg (Example 4).

b) Immunity test The prepared BSA-encapsulated liposome suspension or an aqueous solution of BSA alone as a control (BSA dose was matched with that of the liposome group) was used according to the schedule shown in Table 2, and BALB / c mice ( (Male, 7 weeks old) orally 0.1 ml per dose (6 animals per group). To measure antibody titers in blood and saliva, blood was collected by orbital blood sampling, and saliva was anesthetized by intraperitoneally administering 0.1 ml of 12 mg / ml pentobarbital to mice, and 0.2 mg / ml pilocarpine was administered. After intraperitoneal administration, a Pasteur pipette was inserted into the mouth and collected.

c) Measurement of antibody titer IgG, IgA in serum and IgA in saliva were measured using the ELISA method shown below.
[ELISA method]
50 μl of 100 μg / ml BSA dissolved in carbonate buffer (pH 9.4) was added to each well of the microplate, and the mixture was allowed to stand at 4 ° C. overnight to immobilize BSA as an antigen on the microplate. Wash with phosphate buffer (PBST) containing 100 μl / well three times to remove excess BSA. Next, after blocking with 3% skim milk and washing three times with PBST to remove excess skim milk, 50 μl / well of a serum sampled from a mouse diluted twice with PBST was added thereto at 25 ° C. for 2 hours. Incubate. The serum was removed by washing three times with 100 μl / well of PBST, and 50 μl of anti-mouse antibody IgG or IgA conjugate horseradish peroxidase at a concentration of 500 ng / ml was added to each well and incubated at 25 ° C. for 2 hours. I do. After washing three times with 100 μl / well of PBST to remove excess anti-mouse antibody, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) containing 0.015% hydrogen peroxide (500 ng / well) ml) and incubated at 37 ° C. for 20 minutes, and the absorbance at 405 nm is measured with a microplate reader. Antibody titers (Antibody titer), in absorbance serum 0.2, in saliva and 2n maximum dilution ratio exceeds 0.1, expressed in log ₂ 2 ^n.
d) Test Results The serum IgG and IgA antibody titers and the salivary IgA antibody titers when tested according to the schedule shown in Table 2 are shown in Tables 3-1 to 4 and 4-1 to 3.
In the case of continuous administration for 5 days in Test Example 1 (Tables 3-1 to 4), in the BSA aqueous solution administration group of the control example, the serum IgG antibody titer slightly increased, but the oral vaccine administration of the example was performed. The increase in the antibody titer was obviously larger in the group. Regarding the IgA antibody titer in the serum, no change was observed even when the dose was increased in the BSA aqueous solution group, but a positive increase in the antibody titer could be recognized in the oral vaccine administration group.

The results of Test Example 2 in the case of administration 5 times a week (Tables 4-1 to 3) were as follows. That is, in the BSA aqueous solution oral administration group of the control example, the serum IgG antibody titer showed only a slight increasing tendency as in Test Example 1, and no difference based on the administration method was observed. On the other hand, in the liposome oral administration group of Example, the administration schedule of Test Example 2 showed a tendency to further increase the antibody titer, and the antibody titer was clearly higher than that of the control BSA aqueous solution group. Was. Regarding the serum IgA antibody titer, no change was observed in the BSA aqueous solution group even when the dose was increased, as in Test Example 1. However, a steady increase in the antibody titer was observed in the liposome-administered group. Was completed. The salivary IgA antibody titer was clearly higher in the liposome administration group than in the BSA aqueous solution administration group.

From the above, it has been clarified that the vaccine of the present invention produced using bovine serum albumin (BSA) as a model antigen can efficiently produce an antibody in a living body by oral administration.

a) Preparation of influenza HA antigen-encapsulated liposome-type oral vaccine

A 9: 1 mixture of chloroform and methanol is added to 160 mg of lipid having a membrane composition ratio of distearoylphosphatidylcholine (DSPC): hydrogenated phosphatidylserine (H-PS): cholesterol (Chol) = 1: 1: 2 (molar ratio). 20 ml is added and dissolved, and the solvent is distilled off while heating at 37 ° C. using a rotary evaporator. Further drying is performed under vacuum for 2 hours to prepare a lipid film. To this was added 4 ml of each concentration of influenza HA antigen (see Table 5) dispersed in phosphate buffered saline (PBS, pH 7.4), and the mixture was stirred with a vortex mixer for 10 minutes to obtain multilamellar liposomes (MLV, multilamellar vesicle). Finally, these are diluted 3.5-fold with PBS to obtain an influenza HA antigen-encapsulated liposome-type oral vaccine shown in Table 5, right.

Final liposome suspension: DSPC / H-PS / Chol = 54.4 / 51.2 / 54.4 (mg)

Since 0.5 ml is administered once per mouse, the lipid dose is 10 μmol and the HA antigen dose is 10 μg (Example 5) to 80 μg (Example 8).

b) Immunity test The prepared influenza HA antigen-containing liposome suspension or a PBS solution of HA antigen alone as a control (having the same amount of HA antigen as that of the liposome group) was prepared according to the schedule shown in Table 6 using a gastric tube with BALB. / C mice (male, orally administered 0.5 ml per dose at 7 weeks of age (6 mice per group). To measure antibody titers in blood and saliva, blood was collected by orbital blood sampling and saliva was collected. A mouse is anesthetized by intraperitoneally administering 0.1 mg of pentobarbital at 12 mg / l, and intraperitoneally administering 0.2 mg / ml of pilocarpine, followed by inserting a Pasteur pipette into the mouth and collecting.

HA antigen dose in Test Example 3: 10, 20, 40, 80 μg / time / mouse

c) Measurement of antibody titer The measurement of IgG, IgA and secretory IgA antibody titers in serum was performed in the same manner as in the case of the BSA-encapsulated liposome vaccine, and it was confirmed that the increase in the antibody titer of the HA antigen-encapsulated liposome was excellent. Can be

a) Preparation of liposome-type oral vaccine encapsulating influenza virus antigen or hepatitis B virus antigen

To 19.3 mg of distearoyl phosphatidylcholine (DSPC), 19.3 mg of cholesterol (Chol) and 18.2 mg of hydrogenated soybean phosphatidylserine (HSPS), add 50 ml of a chloroform: methanol mixture (9: 1 by volume), dissolve, and use a rotary evaporator. The solvent was distilled off while heating to 50 ° C. Then, drying was performed under vacuum for 2 hours to prepare a lipid film. To this, 2 ml of an antigen (see Table 7) suspended in phosphate buffered saline (PBS) was added, and the mixture was stirred with a vortex mixer at 50 ° C. for 10 minutes. The mixture was heated and filtered twice at a temperature of 50 ° C. twice with a polycarbonate membrane filter having a pore size of 0.6 μm to prepare multilamellar liposomes.

The particle size of the obtained liposome-type oral vaccine was measured by a quasi-elastic light scattering method (DLS-700, dynamic light scattering meter, manufactured by Otsuka Electronics Co., Ltd.). Table 8 shows the results. Liposomes prepared in the absence of antigen protein served as control.

  The forms of these oral vaccines were stained by a negative staining method and observed by a transmission electron microscope. It was confirmed that both Examples and Controls were in the form of liposomes.

As phosphatidylserine, an oral vaccine using hydrogenated egg yolk phosphatidylserine, dimyristoylphosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, distearoylphosphatidylserine or bovine brain-derived phosphatidylserine can be similarly prepared.

(Example 12)

4-0- (6-O-eicosanoyl-β-D-mannopyranosyl) -D-mannopyranose 6.4 mg, distearoylphosphatidylcholine 32.0 mg, cholesterol 19.3 mg, dicetyl phosphate 5.5 mg, A / PR / 8 as antigen Multilamellar liposomes were prepared in the same manner as in Example 9 using 2 ml of the influenza virus-inactivated antigen (2 mg / ml) derived from (H1N1).
(Example 13)

Preparation of oral vaccine containing mannan derivative in membrane component

In the same manner as in Example 9, 32.0 mg of distearoyl phosphatidylcholine, 19.3 mg of cholesterol and 5.5 mg of dicetyl phosphate were used as antigens and 2 ml of an influenza virus inactivated antigen derived from A / PR / 8 (H1N1) (2 mg / ml). Layered liposomes were prepared.

  10.0 mg of N- (2-N′-cholesterylcarboxyaminomethyl) carboxymethylmannan was dissolved in 10 ml of PBS, added to the above liposome suspension, and stirred for 2 hours while maintaining the liquid temperature at 20 ° C. A liposome-type oral vaccine containing a mannan derivative was obtained.

  The particle size of the oral vaccine obtained in Examples 12 and 13 was measured in the same manner as described above. The weight average particle diameter ± standard deviation (nm) was 443.7 ± 208 (nm) in Example 12 and 450.7 ± 211 (nm) in Example 13. In addition, transmission electron microscopy was performed in the same manner to confirm that the liposome was in the form.

As a mannose derivative, an oral vaccine using a phosphatidylethanolamine derivative having a mannose residue, a cholesterol derivative having a mannose residue, dimannosyl diglyceride, or phosphatidyl mannose can be similarly prepared.

(Example 14)

Preparation of oral vaccine containing mannose derivative or mannan derivative and phosphatidylserine as membrane components

Multilamellar liposomes were produced in the same manner as in Example 9 using 20.0 mg of hydrogenated soybean phosphatidylserine, 20.0 mg of distearoylphosphatidylcholine and 19.3 mg of cholesterol.

  Dissolve 10.0 mg of N- (2-N′-cholesterylcarboxymethyl) carbamylmethylmannan in 10 ml of PBS, add this to the above liposome suspension, and keep the solution at 20 ° C. for 2 hours. The mixture was stirred to obtain an oral vaccine.

The weight average particle size ± standard deviation of the obtained oral vaccine was 476.9 ± 206 (nm). In addition, it was confirmed by transmission electron microscope observation that the liposome was in the form.

(Example 15)

a) Preparation of oral vaccine with adjuvant substance

(6-O- (2-tetradecylhexadecanoyl) -N-acetylmuramoyl) -L-alanyl-D-glutamine 10mg, hydrogenated soy phosphatidylserine 20.0mg, distearoyl phosphatidylcholine 20.0mg and cholesterol 19.3mg The same treatment as in Example 9 was performed to prepare a multilamellar liposome. The weight average particle size ± standard deviation was 461.2 ± 210 (nm). In addition, it was confirmed by transmission electron microscope observation that the liposome was in the form.

As adjuvant substances, (6-O- (2-tetradecylhexadecanoyl) -N-acetylmuramoyl) -L-alanyl-D-glutamamide and N ^2- [N-acetylmuramoyl] -L-alanyl-D Oral vaccines can be prepared in a similar manner using [-isoglutaminyl] -N ⁶ -stearoyl-L-lysine.

b) Immunity test The liposome suspension containing inactivated influenza virus particles (inactivated whole virus particles, fixed with 0.05% formalin) obtained in Example 9 was subjected to BALB / cCr using a gastric tube for mice according to the schedule shown in Table 9. Slc mice (female, 7 weeks old) were orally administered in an amount of 0.2 ml each time (5 mice per group). Three weeks after the start of immunization (21 or 22 days), serum and intestinal lavage fluid were collected from each group of mice, and IgG and IgA antibody titers specific to the administered antigen were measured using ELISA.

The following procedure and method were used for the preparation of serum and intestinal washings.
Under ether anesthesia, the axillary vein of the mouse was cut and blood was collected. The obtained blood was centrifuged at 3000 rpm for 10 minutes to separate serum, and then subjected to antibody measurement. After blood collection, the same mouse was laparotomized, the intestine was removed, and the intestine was cut at the junction of the ileum and cecum. From the stomach and duodenum junction, 5 ml of a sampling solution (ELISAmate: Diluent / Bloking Solution from KPL) was slowly injected into a syringe with a 25-needle (for 10 ml), and the intestinal lavage fluid coming out of the terminal part of the ileum was received in a petri dish. After the washing solution was centrifuged at 2000 rpm for 10 minutes, the supernatant was subjected to antibody measurement. Each sample was stored at -20 ° C until the time of measurement.

c) Measurement of antibody titer IgG and IgA antibody titers in serum and intestinal lavage fluid specific to the administered antigen were measured using an ELISA measurement kit (ELISAmate) of KPL (Kirlegaard & Perry Lab. Inc.). This was performed according to the method described in the protocol.

As a result, the production of IgA and IgG specific to the antigen in the serum and the production of IgA in the intestinal washings were confirmed.

[ELISA method]
50 μl of 2.5 μg / ml antigen dissolved in the coating buffer is added to each well of a 96-well microplate, and the mixture is allowed to stand at room temperature for 1 hour to fix the antigen to the microplate. After discarding the solution in each well and thoroughly removing the solution by thoroughly flicking the plate on a paper towel, add 100 μl of the blocking solution to each well, and allow to stand at room temperature for 5 minutes. After removing the well solution, 50 μl each of a two-fold serial dilution of serum and intestinal lavage fluid prepared from the mouse is added to each well, and the mixture is allowed to stand at room temperature for 1 hour. After removing the well solution, repeat washing 4 times with 200 to 300 μl of washing solution. After removing the well, 50 μl of a 200-fold diluted solution of a peroxidase-labeled anti-mouse IgG (γ) antibody or a peroxidase-labeled anti-mouse IgA (α) antibody is added to each well, and the mixture is left to stand at room temperature for 1 hour. The well solution is removed, and the well is washed four times with 200 to 300 μl of a washing solution, and then the well is filled with the washing solution and allowed to stand at room temperature for another 5 minutes. After removing the well solution, 50 μl of the peroxidase substrate solution is added to each well, and allowed to stand at room temperature for 20 minutes. Then, 50 μl of a peroxidase reaction stop solution is added to each well to stop the reaction, followed by stirring with a plate mixer. The absorbance at 405 nm of each well is measured by ELISA READER (Multiskan PLUS: Titertek). The antibody titer is determined by calculating the absorbance at each dilution of a standard sample (sample having a known antibody titer) measured simultaneously with the logit-log conversion formula shown in Equation 1, and the vertical axis represents logit Y and the horizontal axis represents the antibody titer. The logarithmic values are plotted, a standard line is drawn, and the antibody titer of each sample is calculated based on the standard line.

Claims (2)

A liposome containing an antigen and a lipid, which contains at least one of an influenza virus inactivating antigen, an influenza virus HA antigen and a hepatitis B virus antigen as an antigen, and a glycolipid having mannose bound as a lipid. A vaccine for oral administration characterized by containing. A liposome containing an antigen and a lipid, comprising at least one of an influenza virus inactivating antigen, an influenza virus HA antigen or a hepatitis B virus antigen as an antigen, and a glycolipid having mannose bound as a lipid. A vaccine for oral administration, comprising: increasing an IgG antibody titer and an IgA antibody titer.