JP2002504521A - Mucosal particulate conjugate vaccine - Google Patents

Abstract

  (57) [Summary] Kind Code: A1 Abstract: Disclosed are mucosal, especially oral, microparticle conjugate vaccines against specific pathogenic microorganisms, especially intracellular pathogenic microorganisms. The immunizing component of this type of vaccine is probably through a linker to biodegradable microparticles (particularly starch microparticles), such as Mycobacterium tuberculosis or Enteritidis (cross-linked starch microparticles (eg, polyacryl starch microparticles)). (Utilized)), consisting of recovery-generation tests from specific pathogenic microorganisms. In addition, methods of inducing protective immunity against specific pathogenic microorganisms in mammals and the use of recovery-generation tests are likely to leverage linkers for biodegradable microparticles for the production of mucosal microparticle conjugate vaccines It is described as being derived from a particular pathogenic microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a microparticle conjugate vaccine. And mucous membranes (eg, oral examination, administration to mammals, including humans). The vaccine is directed against certain pathogenic microorganisms, such as Mycobacterium tuberculosis or Enteritidis, especially those in cells. The present invention also provides a method for inducing protective immunity against such microorganisms, and the use of recovery-generation tests derived from such microorganisms to utilize biodegradable microparticles for the production of vaccines. About.

BACKGROUND [0002] Usually, vaccines are today formulated for parenteral administration. Use only a few vaccine arcs verbally, and thus for characteristic purposes. Therefore, the oral cholera vaccine aims to produce antibodies against the cholera toxin B-subunit CTB. It causes diarrhea in infected people by disrupting the salt and water balance on the bowel. The antibody is supposed to suppress toxin binding through the CTB unit to epithelial disc seed receptors (GMT receptors). Further, for the efficacy discussed, some vaccines containing reduced poliovirus are approved for use in some countries. However, carrier systems for oral use with sequestration tests have not yet been approved for humans.

[0003] There are some obvious effects of having an oral vaccine. They are, like nurses,
It is easier to use than the parenteral one, so that administration does not require professional personnel, and oral administration avoids the stresses caused by injection, especially in children. In addition to that, the production of oral products is easier and less expensive than parenteral products that do not produce seed for a. The potentially improved effect of oral vaccination over one parenteral drug in neonates is, however, more significant. There, the immune system intestinal area of the mucosa develops quickly in other parts of the meat mass. There, the parenteral vaccine is active. Also, for the elderly, the mucosal reaction is probably due to oral vaccination,
Would be better.

[0004] An important feature of the immune response is memory function, which is characteristic B cells, differentiated and mediated by proliferation to determine whether they are attributable to characteristic antigenic structures. And a healthy working set of memory cells would want to give a vaccinated person lifelong recovery. The test is then discriminated experimentally by the so-called booster effect obtained after irradiation. Furthermore, recovery against invading microorganisms is also provided by a cellular response, which can be detected by a so-called delayed-type hypersensitivity reaction. It is usually performed in the mouse's ears and extermination. These immune response arcs are often seen after parenteral vaccination. Oral vaccination usually gave a mucosal response. Then, it was detected by the production of a localized antibody of subtype immunoglobulin A (sigA). However, what is being done is against pathogenic microorganisms that confer memory functions and cellular responses in addition to the desired mucosal, preferably oral, mucosal immunoglobulin A production of the vaccine. Furthermore, as cellular immunity appears to be the most important defense against host bacterial pathogens, an efficient vaccine against this type of pathogen must stimulate a T cell immune response.

[0005] In addition, some experimental and epidemiological indications suggest that the cellular immune response is of primary importance among Th1-types, especially for resisting viral and parasitic infections. The Th1 response is also thought to better mimic the response seen after natural infection and reduce the risk of development after allergy.

[0006] A few vaccination studies have been performed on particulate antigens using the parenteral immunization pathway. Vordermeier et al. Showed a 38kDa protein test (DL-lactide co-glycolide) from Mycobacterium tuberculosis entrapped in granular adjuvant polyester fiber, particles were humoral Th1-test properties and cellular immune response Triggered. And it did not, however, protect against intravenous challenge with Mycobacterium tuberculosis (Vordermeier et al .. 1995).

Previous experimental vaccination studies with protective antigens from Mycobacterium tuberculosis (ie, secreted proteins) against tuberculosis have shown somewhat different parenteral adjuvants (eg, Freund's incomplete adjuvant) (FIA), dimethyldeoctadesilamonium chloride (DDA), polyester fiber (DL-lactide co-glycolide) particles, liposomes, aluminum hydroxide and RIBI adjuvant) were successfully carried out (Pal, and Horwitz (1992) Andersen, 1994a, Roberts et al. 1995, Vordermeier et al. (1995) Lindblad et al., 1997, and Sinha et al. (1997)).

[0008] Until recently, alum sediment (eg, aluminum hydroxide) was the only adjuvant approved in the United States and in Sweden for human use. In a recent study by L. indblad et al. (1997), the use of aluminum hydroxide with an experimental Vaccine secreted test from M. tuberculosis was queried. It elicited a Th2 response. And indeed, it is M. tuberculosis (Lindblad et al. 199
The following countermeasures with 7) increased the susceptibility of poultry and animals. This result mark that available adjuvants use today for humans must be replaced with new safe adjuvants for future cell-free vaccines against endothelial pathogens such as Mycobacterium tuberculosis.

[0009] The novel adjuvants were the last approved drop consisting of synthetic drugs, spherical viruses with hemagglutinin, and neuraminidase from influenza viruses, which did not cause hepatitis A-virus. Adjuvants are claimed to have fewer side effects than conventional aluminum adjuvants. (Gluck R. l995).

[0010] Biodegradable microparticles, such as starch particles linked in a crossover (particularly starch particles) have been disclosed in the prior art. Polyacrylic starch microspheres utilized as protective antigens for use in the experimental part of the present description of the invention have been previously disclosed as a parenteral adjuvant for test deliveries (Degling and Stjarnkvist (1995)). Was. The particles are weak macrophage activators without triggering an immune response (Artursson et al. (1985)).

[0011] The lack of a general vaccination system for oral use is due to the challenges associated with testing proteins or carbohydrate acquired sequestration of cells of the immune system and administration of their uptake by intestinal epithelium and transport. First of all, the test must be protected proteolytic degradation during transit by gastrointestinal fluff to immunocompetent areas of the intestine. At least, it is crucial that the relevant epitopes of the test are stored, possibly to be taken up by Peyer's patch M-cells, and then transported to the antigen-presenting cells of the patch. Thus, vaccines must be formulated until the test is wound up by immunocompetent bacteria in such a way that the test epitope is protected.

DESCRIPTION OF THE INVENTION The present invention defines-unexpectedly, recovery of mammalian gastrointestinal tract tests, as shown in mice by conjugation of the recovery-generation test, requires that biodegradable microparticles (
(Eg starch carriers). And it is porous. Tests are obviously not available inside the pores for enzymes and they are not able to spill out of the pores due to covalent bonds. In addition, the ability to transport only carriers of a small size and having a superficial structure in or near the submicron region that the M-cells and / or other uptake cells of the intestine winds up, The current understanding. Unexpectedly, the mucosal particulate conjugate vaccines of the present invention can be partially downgraded to this size and structure, which is taken up by M-cells and is then optimal for producing an immune response It is. And it protects against related microbial opposition.

[0013] In addition, as detected by the delayed type hypersensitivity test, the present invention provides an improved test within a barren systemic immunoglobulin M / utilizing particulate vaccine that unexpectedly triggers this type of cellular response. The mucosal sigA response as well as the immunoglobulin G response, which gives a recovery against microbial challenge, even when the stability is taken into account.

More precisely, the present invention relates to a specific pathogenic microorganism (as an immunizing component,
For biodegradable microparticles, which consist of T cells activating the amount of recovery-production tests derived from said micro-organisms, which are probably exploited through a linker). The biodegradable microparticle is preferably a starch particle (eg, a crosslinked starch particle). In a preferred embodiment of the present invention, the crosslinked starch particles are polyacrylic starch microparticles. In another preferred embodiment of the invention, the mucosal vaccine is an oral vaccine.
Pathogenic microorganisms are, for example, intracellular pathogenic microorganisms. And in a preferred embodiment of the present invention it is selected from the group consisting of Mycobacterium tuberculosis and Enteritidis.

[0015] Pathogenic microorganisms within a particular cell include, for example, a wide variety of different microorganisms, viruses such as mycobacterial species, Salmonella species, Shigella species, Leishmania species, rotavirus, herpes species, Vassinia virus and influenza. From viruses, Meningococcus, B. pertussis, Streptococcus species, toxinogenic Escherichia coli, Helicobacter pylorus, Campiobacter jejuni, Toxoplasma gondii, schistosome species, Listeria monocytogenes, Trypanozoma cruzi and other species (Chlamydia species) HIV species that can be selected, etc. The recovery-production test from a particular microorganism may be an intracellular test, a cell wall antigen or a secreted test.

[0016] Another aspect of the invention is directed to a method of inducing protective immunity to certain pathogenic microorganisms in mammals, including humans. And, especially for biodegradable microparticles as an immunizing component, activating the amount of recovery-generation tests derived from said micro-organisms, which are likely to be exploited through a linker, in the Th1-type, before T cells The method comprises the administration of mucosa to the mammal.

In a preferred embodiment of the invention, the administration of the mucosa is oral and the recovery-production test from said microorganism is secreted proteins from M. tuberculosis or Enteritidis. Yet another embodiment of the present invention relates to the use of a recovery-generation test derived from a specific pathogenic microorganism, possibly utilizing a linker for biodegradable microparticles for the production of a mucosal microparticle conjugate vaccine against said specific pathogen. Aim for direction.

[0018] The mucosal vaccine of a preferred embodiment of this aspect of the invention is an oral vaccine, the test is derived from Mycobacterium tuberculosis or Enteritidis, and the biodegradable microparticles include polyacrylic starch microparticles Starch particles (eg, starch particles linked in a cross). In most preferred embodiments of the invention, the recovery-production test is the secreted proteins from Harlingen strain Mycobacterium tuberculosis (TB).

With respect to an individual's mammal, as well as the immunological and general state of aging and species of the vaccinated individual, the T cells activating the amount of the conjugate of the present invention may be expressed in the test body. And dependent on several factors, such as chemical and biological traits. Recommended doses are given by the manufacturer based on clinical trials. Not only can the conjugates of the present invention activate T cells and especially Th1-cells (even though the amount of conjugate of the vaccine is calculated for T cell activation to ensure immune memory) It should also be understood that secretory IgA and systemic immunoglobulin M / immunoglobulin G responses can be induced.

A possible linker between the two components of the conjugation of the invention is used to facilitate the conjugation reaction or enhance the test presentation. The linker may be an amino acid residue such as K or a di-, tri- or polypeptide amino acid sequence. The mucosal microparticle conjugate vaccines of the invention can be presented in different pharmaceutical formulations, according to the actual intended route of administration (conjugate and solubility of the species and stability of the test or test).

[0021] It may be possible to do so by reducing the depletion of the particulate carrier by enzymes and / or may be acidic, to ensure the efficacy of the vaccine formulation, and to ensure the efficacy of the vaccine process in the stomach and / or in the small intestine, By improving vaccine uptake by antigen presenting cells by modifying the formulation of the vaccine in different ways. In this way (for example)-the degree of cross-linking of the microparticles can be easily controlled by the degree of derivatization of the starch used in the production of the microparticles, since higher cross-linking yields more resistant particles Or-the size of the microparticles is controlled during production by emulsion variability prior to the polymerization of the induced starch in the acrylic group, so that the larger particles give a more stable product Vaccine microparticles are materials that protect the stomach (eg, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate or acrylic acid) for the vaccine to be released after delivery by the stomach and upper small intestine Salt polymer (Eudragit trademark)
)) Can be dispensed in hard gelatin capsules covered by, or-particles are protected during transport by the stomach and upper small intestine, and later freed from The vaccine microparticles can be individually covered, for example, by coacervation with a shellac or cellulose acetate phthalate-phase separation or porosity centrifugal process, for example from protecting the stomach, or- The vaccine microparticles can be suspended in an alkaline buffer such as sodium bicarbonate, neutralizing the acidic pH of the small intestine and / or the vaccine microparticles can be bulking agents (eg, lactose, microcrystals) Disintegrants, such as fibrous cellulose, can be used for uptake of vaccine microparticles by antigen presenting cells In the small intestine, it can be compressed into tablets with a lubricant such as magnesium stearate in a manner that disintegrates slowly; or-the vaccine microparticles are bulking agents (eg lactose, microcrystalline (A disintegrant such as fibrin, a lubricant such as magnesium stearate). And it is then covered by a gastroprotective material such as cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate or acrylate polymer (Eudragit®), or -Can be covered by formable material. And it protects the vaccine by delivery through the stomach and upper small intestine. The invention is illustrated in further detail using descriptions of experiments and results. However, experiments must not be considered so as to limit the scope of the claimed invention.

Description of the Experiment Experiment 1 Murine mice were immunized with polyacryl starch microspheres having covalently attached extracellular proteins from Mycobacterium tuberculosis (Harlingen strain) to investigate the potential of conjugation as an oral vaccine. Was done. The humoral and cellular immune response was investigated, and the recovery after the challenge was determined.

Materials Maltodextrin was a gift from Dr. Lars Svensson (Stadex, Malmo, Sweden). The glycidyl acrylate is from Fluka (Buchs, Switzerland) and is available from Polysciences (PA, USA), N, N, N ', N'-tetramethylethylenediamine (TEMED) and 4-nitrophenyl phosphate. Disodium salt from Merck (Darmstadt, Germany), Biorad protein assay kit and horseradish peroxidase with goat anti-mouse immunoglobulin G to utilize Biorad from CA (USA), Freund incomplete Auxiliary substance is Difco Lab
oratories (MI (USA)) had an effect, BCG vaccine had Stat
ens Serum Institute (Copenhagen, Denmark), alkaline phosphate-utilized human anti-mouse immune IgG / IgM is from Biosource (CA (USA)), carbonyldimidazole, bovine serum albumin (grade V), phenylmethyl Goat anti-mouse immunoglobulin A and 4-chloro-I-naphthol utilizing sulfonyl fluoride, trypsin inhibitor, alkaline phosphate were purchased from Sigma (St. Louis, MO, US
A)

Purification of extracellular proteins from M. tuberculosis Mycobacterium tuberculosis (Harlingen strain) was produced by Proskaue at Smittskyddsinstitutet in Stockholm.
One, two or three weeks of r-Beck medium (corresponding to protein solutions w1, w and w3) were grown. The three (w1, w2 and w3) protein solutions were handled separately during the purification process. Bacteria were removed by centrifugation at 30 minutes of supernatant filtered and 5000 rpm for culture. And the two continuous ones are filtered by a centralized -YM10 filter (Amicon, MA, USA), filtering about 50 micrometres. Ammonium sulfate (final concentration 4.24M) was added to the concentrate during stirring. After centrifugation at 8000 rpm for 30 minutes, the sediment was dissolved in phosphate buffered saline (pH 7.0). Proteins (solutions w1, w2 and w3) were extensively dialyzed in Spectra / Por®), cleaved against buffer with 0.25 M boric acid and 0.15 M NaCl 35
Dialysis membrane with a molecular weight of 00 (antimicrobial spectrum, CA, USA) pH 8.5. Protein enrichment was determined for Coomassie Blue according to Bradford (Bradford (1976)). Bovine serum albumin was used as a standard. Proteins are stored-80w C until further use.

[0025] Polyacrylated starch microparticles (as mentioned earlier (Artursson et al., 1984 and Laakso et al. (1986)), the fine particles are formed by polymerization of the acrylated starch in the emulsion.
Prepared). Briefly, 500 mg of acrylated starch was dissolved in 5 ml of 0.2 M sodium phosphate buffer, pH 7.5, 1 mM EDTA. Ammonium peroxide sulfate (200.mu.l) was added to give a final concentration of 0.8M in the aqueous phase. And it was then homogenized in 300 ml of toluene: chloroform (4: 1). TEMED was used to initiate the polymerization.
The microparticle composition is characterized by D-TC nomenclature (characterized by Hjerten 1962 and Edman et al. (1980)) and the amount of TEME. D represents acrylated starch (g / 100 ml); T is the total concentration of acryl groups expressed as acrylamide equivalents (g / 100 ml); and C is any additional Crosslinking agents (eg, bisacrylamide;% w / w) are also relative amounts. The microparticles used in this study had a D-TC value of 10-0.5-0, and 100 μl of TEMED was added.

Conjugation of Extracellular Protein (TB) from Mycobacterium tuberculosis to Microparticles Extracellular proteins (TB) w1 and w2 + w3 are Bethell coupled to microparticles using the CD-I-method Others (Bethell et al. (1981)). Microparticles (5 mg / ml)
At room temperature, a dry dose-modifying factor for 1 h CD-I (50 mg / ml) was activated. After several washes with a centrifuge with a dose modifier to remove unreacted CDI, the particles (50 mg) were suspended in 10 ml of binding buffer (0.15 M N
Amount of mg w1 or w2 + w3. containing 0.250 M boric acid with aCl (pH 8.5). The mixture was rotated end over end at 4-6 ° C. for 48 hours. The TB-microparticles were then flowed through PBS, filtered through a 10 micrometer filter and stored at 4-6 ° C. The amount of w1 and the linked w2 + w3 were determined by amino acid analysis after acidic hydrolysis of the microparticles.

[0027] Particle size determination The TB-particles are dried and scanned with a scanning electron microscope (SEM) at 5000 magnification (Jeol T330).
Photo was taken at The particle size determined from scanning electron micrographs is less than 2 micrometer. In previous studies, 98% of the particles were less than 2.5 micrometers in diameter and were determined by the Coulter Counter (Degling and Stj-rnkvist (1995)).

Immunization BA.LB/c A murine strain of ABom (Bomholtgard, Ry, Denmark). And female (old 8
-10 weeks) was used. Mice (5-6 / group) were immunized orally by gastric intubation (4 hours on 3 consecutive days) with TB-particles containing w1 and w2 + w3 proteins . The murine group also has TB-particles containing w1 and w2-I-w3 proteins or 0.1 ml, the corresponding amount of physiological saline solution, having soluble w1 and w2 + w3. It was im. As one positive control, the group of mice was injected ip with w1 + w2 + w3 in Freund's Incomplete Adjuvant (FIA). 0.1m as other positive control mice were immunized
The sc with l diluted the BCG vaccine (with saline). Soluble w1 + w2 + w
When a low dose of 3 was controlled, the carrier protein BSA was controlled to minimize protein adsorption on 0.1% co-glassware. Table 1-1 (Immunization Schedule) appears for detailed information.

Blood sample collection and preparation (0, 7, 15, 34, 42, 49, 57 and 65 with heparinized capillaries subway from the electron orbital plex, blood collected samples). The tubes are centrifuged and collected and frozen serum-20 ° C until further use.

Stool Collection and Extraction Stool (4-6) from each mouse is collected for 5 consecutive days after immunization into Ellerman tubes, dried and frozen. Dry weight was determined and 50 mM EDTA
A solution containing 5% milk powder, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mg soybean trypsin inhibitor / ml, phosphate buffered saline (PBS-A) was added (20%). μl / mg stool). The solids are crushed and for 15 minutes
Separated by centrifugation at 13000 rpm / min and aupernatants frozen at -20oC until further use.

Determination of anti-TB IgG and IgM and sIgA with ELISA An equal mixture of -w1, w2 and w3 proteins in the protein solution was obtained with 0.05% NaN3
(18 μg / ml) (pH 9.6) diluted with 0.05 M sodium bicarbonate buffer with
Then, Nunc Immunoplate Maxisorb F96 plate is covered (100 μl / container)
And then incubated in the wet room until morning at 4 ° C. The plate was shaken dry and 1% OVA in 1 mM PBS-A, pH 7.4 was added (200 μl / container), and then the plate was removed with unclear fasteners at room temperature. Incubate for 2h in a moist chamber to avoid. 5 washes with 0.05% Tween 20 in saline with Titertek microplate flow lab in washer 120), serum / stool samples were added to the plate in a two-fold dilution system, 2 Washed as before before incubating for time and plate. Alkaline phosphatase-utilized secondary antibody (human anti-mouse immunoglobulin G and immunoglobulin M or goat anti-mouse immunoglobulin A) 1:10
00/1: 250 of PBS-A with 0.2% Tween 20 (PBS-T) was diluted (100 μl / vessel) and the plates were incubated for 2.5 hours. After washing (substrate), 4-nitrophenyl phosphate (1 mg / ml, MgCl3 and 0.02, 0.5 mM% NaN3 (pH 9.
8) In a 10% diethanolamine buffer with) added and absorbance Mu
Measured after 10 minutes (12 minimal for immunoglobulin A) at 405 nm with an ltiscan MCC / 34O microtiter plate spectrophotometer (Labsystem). The co-sponsored negative serum was used on each plate (
Immunoglobulin G / immunoglobulin M measurement). The average of the absorbance values was calculated very often from the beginning (1:20 dilution); means = 0.130 (sd = 0.045 n = 19.) Means means + 3
If above Xsd (and therefore above 0.265), the sample was considered positive. Positive samples (serum from mice immunized for 100.mu.g w1w2w3 of Frewad's incomplete supplement) were also added to each plate and treated in the same as standards. There were other samples. The titer is
-Log2 given (dilution x 10).

Subsequent Type Hypersensitivity (DTH) test (cells tested by DTH that developed an arbitrated and resolved immune response to TB performed on day 52 (ie, one week after the third immunization) In order to assess if it was done). Mus musculus, a tuberculosis protein mixture of saline wl-w3 (1 mg
/ mI) was given an intradermal injection (10 .mu.l) of the left ear with the left ear. Control 10.m
As ul, saline was injected in the correct ear. After 48 and 72 h, the ear thickness was measured against the test and 24 before the dial thickness gauge (Mitsutoyo Scandinavian Peninsula,
Upplands Vasby, Sweden). The DTH reaction was calculated (A1-B1 / A
0) * l00, where A1 = the ear ear thickness that is challenged for the test at time t, the increase from the ear thickness at ear time 0 is the challenge B1 for saline at time t =
And, prior to the challenge (Degling and Stjarnkvist (1995)), the ear thickness of the ear increases from A0 = time 0 for the test challenged.

Experimental infection of mice. Immunized mice and control mice will have 5 × 10 6 CPU M. tuberculosis (Harlingen strain) iv via tail vein on day 106 (18 days after last immunization). I was challenged. The weight of the mouse was determined and Infectio
15 days after n.

Determination of protective immunity At killed 121 infected mice (15 days after infection) the day there were mice and the spleen and lungs were aseptically removed. The CFU of M. tuberculosis was determined by homogenizing each tissue of the tissue homogenate in PBS and 10 folds over time prior to culturing dilutions on duplicate plates of 7H10 agar. Colony forming units were counted after 3 weeks of incubation at 370C.

SDS-polyacrylamide gel electrophoresis and immunoblotting, T of an equal mixture of fractions w1, w2, w3 and w1w2w3
He proteins were isolated from PhastSystem (r) (Pharmacia (Uppsala.) Swede
n) Gel electrophoresis instrument using 10-15% SDS PhastGel (r) (Pharmacia Biotechnology, Uppsala, Sweden). Gel is both silver stained and Coomas
sie dirty with blue. The separated proteins can be obtained from nitrocellulose membrane (Pharmacia Biotech, Uppsala).
(Sweden, Sweden) and incubated for 2 h at RT with a solution containing 5% milk powder in PBS-T on a shaker. After washing with PBS-T, in 0.5% OVA PBS-T with serum from groups 1-6 (1:20, diluted), the six membranes were incubated for 20 h at RT on a shaker. . After washing with PBS-T, a secondary antibody (goat anti-mouse immunoglobulin G utilizing horseradish peroxidase, 1: 20,000 in 0.5% of PBS-T
The membrane was incubated for 2 h at 37 ° C on a shaker. Substrate (4-chloro-l-naphtol (10 mg dissolved in 3.3 ml MeOH and 20 mM Tris (3
500 mM NaCI buffer with 0.mu.l H202 (37%) was added to 16.7 ml))) PBS
Added after washing with -T. The reaction was stopped after 20 ml with distilled water. Statistics-Unpaired t-test was a pefformed comparison of two independent sapmles. Differences were considered significant if p <0.05.

Results Conjugation of Tuberculosis Proteins to Polyacrylic Starch Microparticles-The first of the w1 proteins to be subdivided is the From, 5.63.mu.g protein per mg, and the microparticles are coupled (23% protein conjugate yield). The microparticles (6.4% protein yield) were coupled per mg from the next conjugate with a .mu.g w1 protein of 1.38 in the supernatant. Additional conjugation of protein fraction w1 was performed and 3.93.mu.
g protein was ligated (corresponding to a protein yield of 15.9%). From the conjugate with fraction w2 + w3 to 4.16.mu.g protein / mg, the microparticles were coupled (corresponding to a protein yield of 55%) to give 0.89 mg of protein w2 + w3 in the supernatant. Based on the following conjugate, the yield of the bound protein was 10.1% per fine particle). Analysis of extracellular M. tuberculosis proteins by SDS-polyacrylamide gel electrophoresis and immunoblotting

The three protein fractions (ie w1), w2 and w3. Were analyzed by SDS-polyacrylamide gel electrophoresis to determine the protein size of the mixture used in the immunization experiments . Several bands of kDa in the region 14.4-30 and 43-94 kDa were observed by SDS-polyacrylamide gel electrophoresis analysis (not at all).
12 bands). There were no differences between the w1, w2 and w3 protein fractions. (Not presented result).

Late Type Hypersensitivity (DTH) As shown in Table 1-2, TB-microparticles after -24, 48 and 72 hours had an increase in ear thickness of the orally immunized group. And, however, the increase was not quite high for other groups. The DTH-response etiology of basal immunized with TB-microparticles was significantly higher than the control group after 24 h. After 48 and 72 hours, DTH-r
esponse is both control and base DTH-response is saline free TB-
Increased due to being much stronger than immunizing im with antigen. After 72 hours, the DTH-response of this group was also significantly higher than the response of the BCG group, and the response of the group immunized for the Freunds incomplete adjuvant TB-antigen was comparable.
Two mice in the control group showed a 40-50% increase in vehicle thickness, and three mice did not respond at all. This explains the standard error (SD) with a high average within this group after 48-72 h.

Humoral Immune Response (im shows that Freund's incomplete adjuvant and basic TB-proteins immunized group have immunized im with physiological saline frec TB-proteins) (The group is immunized for TB-microparticles).
The reaction was also significantly higher for the control and BCG groups. Orally immunized groups for TB-microparticles did not elicit humoral (immunoglobulin G and immunoglobulin M) responses. Mucosomal (Table 1-3)

(Signature A) Immune response (sigA immune response after the third immunization of the group and im the results of which are given verbally microparticles, two days after Freund's test for incomplete Freund's adjuvant) Preliminary indicating that immunization was performed). The reaction of the BCG group was below. However, further studies must be performed to confirm these results.

Recovery Experiments The recovery level is determined by two parameters following infection, weight loss during infection and Mt of the lungs.
Determined by uberculosis CFU. As shown in Tables 1-4, control mice and vaccinated groups lose weight during infection. M. in the lungs after infection.
Tuberculosis CFU is presented in Tables 1-5. Protective immunity has been demonstrated in birds immunized orally for TBmicroparticles. Viable M. tuberculosis reduction of lung at least 10-100 unimm
The effect was similar to that seen after immunization with the BCG vaccine in the folds compared to unified controls. The recovery after intramuscular immunization with TB-microparticles (Table 1-5) was somewhat less responsive after oral administration of TB-microparticles' reduced viable M. tuberculosis in the lungs Had at least 10 folds. As shown in Table 1-5, protective immunity was achieved with TB-anti
No gen or intraperitoneally FIA and TB-antigen were shown in intramuscularly immunized poultry.

[0042]

[Table 1]

[0043]

[Table 2]

[0044]

[Table 3]

[0045]

[Table 4]

[0046]

[Table 5]

Experiment 2 Extracellular proteins were isolated from S. Enteritidis linked to polyacryl starch microparticles and by covalent binding. Immunogenicity of induced recovery against conjugation and challenge with live bacteria following oral administration to mice was followed.

Materials and Methods Materials In addition to them, items were specified in Experiment 1. Bacto-tryptone and Bactoyeast-extract were from Sigma (MO (USA)) to Difco (MI (USA)), alkaline phosphatase-utilized goat anti-mouse immunoglobulin A and murine immunoglobulin A-kappa, and RPMI 1640, H
EPES and glutamine were from Life Technologies LTD (Paisley, Scotland).

Purification of extracellular protein from S. Enteritidis. S. Enteritidis wild type is Luria-Bertani 2 ml (LB) broth (1% Bacto-tryptone / 0.
(5% Bacto-yeast-extract / 1% sodium chloride) and shaking at 37 ° C, 200 rpm / min overnight. The next day, the culture was diluted in 500 ml LB and grown under the same conditions until OD = 1. After centrifugation (1,500 × g for 60 minutes at 4 ° C.), the bacterial pellet was resuspended in RPMI 1640 with 20 mM HEPES and 4 mM glutamine. The mixture was centrifuged toward 1500Xg (200 rpm / min) at 4 oC. The culture supernatant was used for the Stirred Cell
Off the Ultrafilter (Amicon, MA (USA)), filter through a 0.22 micrometer Millipore Express Filter by filtering 000 cuts of YM 10,000, concentrate and concentrate in conjugate buffer (0.15 M NaCI (pH 8.5) with 0.250 M boric acid). Protein concentration was determined for Coomassie Blue according to Bradford (Bradford (1976)) and for a ready prepared reagent from Bio-rad. Then, bovine serum albumin was used as a standard.

Preparation of Polyacrylic Starch Microparticles, Conjugation of Salmonella Tests and Characterization of Antigen Particle Combination Salmonella Tests of Polyacrylic Starch-containing microparticles were prepared, Ex
Characterized as described in periment 1.

Immunization Procedure From its own breeding of the Balb / c strain, mice were divided into 5 groups (4 mice / group). The In which each of the first groups attacked was an immunized ip with 10.5 .mu.g protein in 0.1 ml Freund's adjuvant. (In the challenge experiment, 6-12 mice were included in each group.) The second group was awarded an im injection with 10.5 .mu.g protein that utilized 1 mg microparticles. . For a 31.5 .mu.g protein that utilizes 3 mg microparticics divided into doses given for three consecutive days, the third group of mice was orally immunounized by gastric incubation. Group 4 was an untreated control group and Group 5 was a hyperimmunized group. And it was awarded 50 .mu.g protein in 0.1 ml Freund's adjuvant (30 .mu.g proteins as booster dose). Booster challenge was given after 21 days.

Collection and Handling of Blood and Stool Samples The sampling procedures and handling used for blood and stool collection are described in Ex.
presented in periment 1.

Assessment of Immune Response Analysis of whole body IgG response as well as mucosal immunoglobulin A response is conventional.
Performed by ELISA technology. And it is described in Experiment I. As presented in Experiment 1, the cellular response was delayed-type hypersensitivity test (DTH-
rest).

Challenge of immunized mice The challenge with S. Enteritidis (3 X104-CFU / Mice) was performed 6 weeks after booster challenge. The mice were crushed seven days after the challenge. Liver and spleen homogenates were cultured on LB-agar plates overnight, and the number of CFU was counted.

Results Test-micraparticic Conjugate Characterization The starch microparticles utilized contained 10 mg Salmonella test per mg. All particle preparations used contained less than 33 mm and more than 90% of the particles having a diameter.

Humoral Immune Response When the particles are controlled im (Table 2I), the Salmonella proteins linked to the polyacryl starch microparticles are immunoglobulin G / The immunoglobulin M response was comparable to, but less than, that of the immunized group for Freund's adjuvant proteins. Similarly, stool species immunoglobulin A reactions show peaks on Salmonella proteins linked to polyacryl starch microparticles and on days 27 and 28, and verbs on groups immunized for Freunds adjuvant proteins. The igA response attributable to the base immunized species was lower, although comparable in the groups immunized in (Table 2-2).

Celluler inmmune response A relatively high, sustained increase in ear thickness was noted for microparticles in groups that were orally immnunized. The response was comparable to that of positively immunized (Table 2-3) immobilized less than that obtained for positive control (of Freund's adjuvant).

Competition of immunized Mus musculus The results from the challenge of immunized Mus musculus with living Salmonella bacteria are shown in Tables 2-4 and 2-5. The reduction of CFU was shown for groups immunized orally for test-bound microparticles (only microparticles with or with soluble antigen) compared to the control group. Maximum recovery indicated to the group to be immunized for test or utilized to starch microparticles. This was also seen when studying average weight loss. And it is a control group, a 4.0% reduction for the orally immunized groups for soluble antigens, a 3.6% reduction for the orally immunized groups for microparticles with soluble antigen and test-coverage. For bound microparticles (not presented in any table), it showed a 10.3% reduction for a 1.8% reduction for orally immunized groups. The results of this study, which secreted a test derived from Salmonella for polyacryl microparlticles, could be administered as an oral vaccine capable of eliciting a local secretory source and a systemic immune response.

In addition, strong species of immunoglobulin A with significant observed inter-individual variability were observed in this study. A good recovery to the challenge was also shown indirectly by following weight loss after the challenge. For the test-utilized microparticles, the orally treated groups were significantly less than the weight (1.8%) compared to the control group (not treated at all). After the competition, he released 10.3% of his weight.

Table 2-1 Table of sera after immunization with S. enteitidis test of different formulations-one S
pecific liquid reaction. Titers are given as mean +/- SEM. (N = 4). Method of administration Day 0 Titer Day 35 Oral immunization 0.0 0.0 Test preparation Test utilized 5.5 + 1- 0.3 microparaicles Im immunization Test utilized 9.0 + 1- 0.4 Particle Ip immunization Soluble antigen in 0.0 7.0 + / -. 0.0 Freund's Adjuvant

Table 2-2 Stool species mucosal response (immunoglobulin A) after immunization with S. enteritidis test of different formulations. Values are given as means +/- SEM. (N = 4). Administration method Immunoglobulin A (ng / mg stool) Antigen preparation Day 26 Day 27 Day 28 Test used for oral immunization 1.1 + t. 0.5 2.1 + 1- 0.55 2.25 + / .. 0.8 Test used for fine particle Im immunization 0.4 + 1- 0,2 0.9 +/- 0.35 0.6 + 1-0.2 Particulate Soluble antigen in Ip immunization 1.3 + 1- 0.55 1.4 + 1- 0.52.4 +/- 0.75 Freund's adjuvant

Table 2-3 Cellular immune response (as test on delayed type hypersensitivity) After immunization with S. enteritidis test of different formulations. The results are given as an average% increase in the thickness of the challenged ear (+/- S.E.M). (N = 2-4) Method of administration% Increased hours after challenge test formulation 24 48 72 Oral immunization Tests utilized 41 +/- 10 67 +/- 3 99 +/- 15 Microparticle Im immunization Tests utilized 107 +/- 13 138 +/- 24 179 +/- 12 Particulate Ip Immunization soluble antigen in 190 +/- 22 209 +/- 25 214 +/- 21 Freund's adjuvant Non-immunized 10+ /-1 23 +/- 4 38 +/- 2 house mouse (control)

Table 2-4 Colonies in which the murine In liver formed a immunized unit. ~ (CFU) for the S. entertidis test after challenge with 3 X104 CFU. The mice were challenged 6 weeks after booster challenge and crushed 7 days after challenge. Livers were homogenized and total CFU was counted after overnight incubation on LB-agar. Results are presented as geometric means and ranges; n is given in parentheses. Method of Administration The CFU of the more viable Antigen formulation is average Range Ingestion Tests utilized 1.8 x 103 (6) 2-3.12 x 106 particles Oral Solubleantigens 0.69 x 103 (6) 1-1.5 x 104 Particles
4.5 x 103 (6) 1-5.0 x 104 non-immunized mice of soluble antigen 2.6
x 106 (12) 1.1x103-1.9X107 (control)

Table 2-5 After challenge with 3 x 104 CFU., Mice spleen colony forming units (CFU) of mice immunized for testing from S. enteritidls mice after booster challenge The challenged 6 weeks, after the challenge, killed 7 days. The liver was homogenized and the total CFU after LB-agar overnight incubation
Counted. Results are presented as geometric means and ranges; n is given in parentheses. Method of administration CFU average of spleen Antigen range Average range Internal test Tests used
3.22 x l03 (6) 3 -2.48 x 106 microparticles with oral soluble antigen 1.43 x l03 (6) 1-5.70 x 104 microparticles Internal 6.04 x l03 (6) 1-3.30 x 105 with soluble antigen 2.32 x 106 (12) 2.3x105-1.5x107 non-immunized mice

References Andersen, P., a. Effective vaccination of mice against M. tuberculosis infection with a soluble mixture of secreted Mycobacterium proteins. Infect. Immun. (62 (1 994a) 2536-2 544). Artursson. P., Edman, P., Laakso, T. and Sjoholm I. Characterization of polyacryl starch granules as carrier for proteins and drugs. J. Pharmaceutical Sciences (73 (1984) 1507-1513). Artursson, P., Edman, P., and Autologous Proteins Modified for Biodegradable Sjoholm (I.) Microsphere Immune Responses and Entrapped in Polyacrylic Starch Microparticles. i Pharmacol. Erp.Ther (234 (1985) 2
55-259). Investigation of the activation of various insoluble polysaccmides with Bethell, GS, Ayers, JS, Hahn, MTW, and Hancock, w. 1,1 -carbonyldiimidazole and of active matrix properties. J. Chromatography. 219 (
1981) 361-372. Bradford, MM, Fast A and Sensitive Anal for the weighing of microgram quantities of protein utilizing the principles of protein-dye biridning. Biochem. (72 (1976) 248-254). Degling, L. and Stjlrnkvist (P.). Biodegradable microsphere XVIII: polya with human serum albumin to be used
Adjuvant effect of cryl starch microparticles. Vaccine..13 (1995) 629-636. Edman, P., Elcman, B., and Sjoholm. Immobilization of proteins of biodegradable polyacryldextran. Rnicrospheres i
Pharrn. Sound. 69 (1980) 838-842. Giluck R. Liposornal presentation of tests for human use. Vaccine design: Subunits and adjuvants approach. Michael F. Powell and Target
Edited by .1. Newman. High pressure Press New york 1995. pp 325-345. S
Molecular sieve chromatography on Hjerten, S polyacrylamide gels
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Drip): Factor affecting distribution and microparricle of polyacrylic starch
I'm a P / warm. Science (75 (1986)) 962-967. Irrunu against Lindblad (EB) Elhay MJ (Silva) R., Appelberg, R. and Andersen (P.) adjuvant tuberculosis tuberculosis subunit vaccine.
Infectious immunity of ne-reaction 65, (] 997) 623.629. Pal, P. G. and Horwicz. Im with kind
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-4792). Roberts, AD, Sonnenberg, MGOrdway DJ Furney, SK, Brennan, PJ
., Belisle, J .; Protective immunity traits generated by vaccination of mice with T. and Orme IM purified culture filter Ad'ycobaciaraum protein tests: uberculosis. Immunology 85 (1995) 502-508. Sinha, RK, Verma, 1. And Khuller G K. Mycobac, erium tuberculosis H37 Ra
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──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF Term (reference) 4C076 AA31 BB01 CC06 CC41 EE38 4C085 AA03 BA07 BA09 BA24 CC07 CC33 EE01

Claims (10)

[Claims] 1. Particular pathogeziic microorganisms (as imn1unizin-as components, for biodegradable microparticles, consist of T cells activating the amount of recovery-generation tests derived from said microorganisms, which are probably exploited through a linker. V) mucosal microparticle conjugate vaccine. 2. A vaccine according to claim 1. Biodegradable rnicroparticles are starch particles, including crosslinked-linkeci starch particles. 3. The vaccine according to claim 2, wherein the crosslinked starch particles are polyacrylic starch microparticles. 4. A vaccine according to any one of claims 1-3, wherein m
A ucosal vaccine is an oral vaccine. 5. A vaccine-4 according to any one of the preceding claims, wherein the pathogenic microorganism is an intraceliuilar pathogenic microorganism. 6. The vaccine according to claim 5, wherein the intracellular pathogenic microorganism is selected from the group consisting of Mycobacterlum tuberculosis and Salmonella enieritidis. 7. A method for inducing protective immunity against specific pathogenic microorganisms in mammals, including humans, including biodegradable microp as an immunizing component.
amlicles. consisting of mucosal administration to the mammal of T cells activating the amount of a recovery-generation test derived from the microorganism, which is probably exploited through a linker. 8. The method according to claim 7, wherein the administration of the mucosa, the mucosal administration, and the secreted proteins which are oral and recovery-production tests derived from the microorganism, are from Mycobacterium tuberculosis or Enteritidis. 9. The use of a recovery-generation test derived from a particular pathogenic microorganism, possibly utilizing a linker for biodegradable microparticles for the production of a mucosal microparticle conjugate vaccine against said specific pathogen. 10. Use according to claim 7, wherein the mucosal vaccine is an oral vaccine, said test is derived from M. tuberculosis or Enteritidis, and biodegradable micropaziicles linked to a cross Stiff particles are starch particles, including barren polyacryl starch microparticles.