JPH11510793A - Intranasal vaccination for gastrointestinal disease - Google Patents

Abstract

  (57) [Summary] The invention features intranasal immunization to induce an immune response in a distal mucosal site, for example, the gastrointestinal tract or genitourinary tract. The methods of the present invention can be used to induce a prophylactic and / or therapeutic immune response against pathogens that infect these peripheral sites (eg, Clostridium bacteria, eg, C. difficile). The invention also includes vaccination methods using a combination of a mucosal (eg, oral or nasal) route of administration and a parenteral (eg, subcutaneous or intraperitoneal) route of administration.

Description

DETAILED DESCRIPTION OF THE INVENTION                     Intranasal vaccination for gastrointestinal disease                                Background of the Invention   The present invention relates to an intranasal vaccination method for the prevention and / or treatment of gastrointestinal diseases. About.   Clostridium Difficile can be severe and sometimes Gram-positive, causing antibiotic-related diarrhea that can lead to fatal colitis It is a sporulating, toxin-producing bacterium. For example antibiotic or anti-neoplastic treatment Disruption of the normal intestinal flora by difficile now colonizes the colon Toxin A and toxin B, producing two high molecular weight toxins. these Both polypeptides are cytotoxins, but toxin B is 1000-fold more than toxin A More toxic. Toxin A also causes accumulation in the intestinal loop fluid of ligated animals. It is enteric toxin.                                Summary of the Invention   We consider intranasal and mucosal (eg, oral or nasal) and systemic (Eg, subcutaneously or intraperitoneally). Even in the absence of adjuvant, distal mucosal sites (eg, gastrointestinal And / or genitourinary tract) Was. C. using any of these methods. difficile toxin A or B (or Vaccination of hamsters with C. xoid. d These animals are protected from ifficile inoculation.   Accordingly, the present invention provides a method for treating gastrointestinal or urogenital tract pathogens in mammals. A distal mucosal immune response (ie, a mucosal immune response outside the upper respiratory tract, such as , Gastrointestinal tract and / or genitourinary tract). This method Distant immune response to pathogens A non-replicatable polypeptide antigen capable of inducing The product is administered intranasally.   The present invention also elicits a distal mucosal immune response to a pathogen in a mammal (1) Immunity distal to the mucosal surface of a mammal Yes Administering an antigen capable of inducing a response to the mammal, and (2) Parenteral administration of the drug substance. Any order combination of mucosal and parenteral administration The combination is also included in the present invention. For example, mucosal (eg, intranasal, mouth, eye, stomach, Intestinal, vaginal, gastrointestinal, or ureteral administration is first given orally (eg, intravenously, subcutaneously, intraperitoneally). Or intramuscular), or parenteral administration prior to mucosal administration. May be performed. By way of example, three doses per week may be administered mucosally (eg, nasal Intravenously) and at week 4 mucosa (eg, intranasally) and parenterally (eg, abdomen). Administration in combination with (cavity) administration may be performed.   An antigen (e.g., capable of replicating) that induces a mucosal immune response in the methods of the invention. Pathogens that originate from non-polypeptide antigens include Helicoba gastrointestinal (eg, H. pylori, H. felis, and H. heilmanii) Pathogens, Campylobacter (eg, C. jejuni), diarrhea And pathogens that cause colitis (eg, Clostridia (eg. For example, C. difficile, C.I. novyi, C.I. sordellii), enterotoxigenic E. coli, Shigella, Cholera, and Salmonella typhi) and genitourinary tract pathogens (eg, human immunodeficiency disease) Virus, herpes simplex virus, papilloma virus, syphilis treponema, Lamidia, and Neisseria gonorrhoeae).   Antigens that can be used in the methods of the invention (eg, Particular examples of (tide antigens) include, but are not limited to, bacterial toxins. Was For example, C. difficile toxin A and / or B toxoid; novyi's α-g Xin (Bette et al., Toxicon 29 (7): 877-887, 1991), C.I. sordellii Clostridium (clo) such as the lethal toxin (Bette et al., Supra) stridia) (eg, C. difficile, C. novyi, and C. sodellii). Xins and immunogenic fragments and derivatives having them can be used. Antigens used in the methods of the invention can be prepared by standard methods known in the art, for example, Purification of pathogens from cultures, DNA recombination methods and chemical synthesis methods Is obtained.   The present invention relates to the production of Clostridium (eg, C. difficile). Xenoids may be used as antigens in vaccines. Toxoid is a toxin Yes Toxins (or toxins) that have been treated to reduce toxic properties but retain antigenicity Mixtures of the toxins, e.g. difficile toxin A and toxin B) You. The toxoids included in the present invention are made using standard methods and are chemically (Eg, formaldehyde or glutaraldehyde) treatment, protease Fragmentation and recombinant methods (eg, toxin fragments or mutants (eg, Mutants), but is not limited thereto.   The method of the present invention is used to prevent or reduce the chance of future infection by a pathogen. (Ie, to induce a prophylactic immune response), and / or a sense of progress Performed to treat the stain (ie, to induce a therapeutic immune response) You. In the case of a pathogen in the intestine, for example, the method of the invention may involve the pathogen (eg, C. d. ifficile), but may still develop diarrhea Treat mammals that have not died or have diarrhea caused by a pathogen Can be used to Mammals that can be treated according to the methods of the present invention include, for example, Examples include humans, cows, horses, pigs, dogs, cats, sheep, and goats.   An advantage of the method of the present invention is that at least some antigens (eg, C. difficile Mucosal adjuvants respond to immune responses (e.g., toxins and toxoids) Not required for the induction of a protective immune response).   Other features and advantages of the invention will be set forth in the following detailed description of preferred embodiments of the invention. It will be apparent from the detailed description and the appended claims.                                Detailed description   First, the drawings will be described.Drawing   FIG. 1 shows C.I. in hamsters immunized with difficile antigen , C. 4 is a graph showing the level of prevention against disease caused by difficile. Clinda Level of protection from systemic (lethal) and intestinal (diarrheal) diseases after administration of mycin (See Table 1 for a description of the immune pathway).   FIG. 2 shows that hamsters were administered three doses of vaccine by the indicated route. C. in serum obtained from against difficile toxin A, toxin B, and whole cell antigen FIG. 10 is a graph showing the mean (+ SE) antibody titer as determined by ELISA. You. (See Table 1 for a description of the immune pathway). Ham after three doses of vaccine Serum from the star was assayed for specific IgG. The titer is greater than 0.3 It was defined as the highest dilution with luminosity. Each bar represents the mean (+ SE) of five animals Show.   FIG. 3 shows hamsters receiving three doses of vaccine by the indicated route. 4 is a graph showing the biological activity of the serum obtained from. Serum was used in IMR-90 cells. B. inhibition of itotoxin A or cytotoxin B activity; difficile cells The test was performed on a collection; the titer was defined as the highest dilution of biological activity. Each bar represents the mean (+ SE) of five animals. (See Table 1 for a description of the immune pathway ).   FIG. 4 shows the long-term antibody response in hamsters immunized with i.n.i.p. and sc. This is a graph. Before administering clindamycin (i.n.i.p.-I and s.c.-I) And response at 140 days after administration of clindamycin (i.n.i.p.-II and s.c.-II) A comparison is shown. Serum against toxin A, toxin B, and whole cell antigens The test is performed by ELISA and the titer is the highest dilution with an absorbance greater than 0.3. Each bar represents the mean (+ SE) of 5 animals.   FIG. 5 shows the long-term antibody response in hamsters immunized with i.n.i.p. and sc. It is a graph shown. Before administering clindamycin (i.n.i.p.-I and s.c.-I ) And 140 days after administration of clindamycin (i.n.i.p.-II and s.c.-II) A comparison is shown. Serum inhibits cytotoxin in IMR-90 cells and And C. Tested for aggregation of difficile cells; titer is biologically active This was the highest dilution of serum. Each bar represents the mean (+ SE) of five animals.   6A-6B show the results of intranasal immunization with toxoid in the presence or absence of CT. Anti-toxin A (Figure 6A) in mouse serum, feces, saliva, and vaginal secretions FIG. 6 is a graph showing the anti-toxin B (FIG. 6B) IgA response.   7A-7C show serum anti-toxin B following intranasal immunization of mice with toxoid. Cytotoxicity (Fig. 7A), serum anti-toxin A after intranasal immunization with toxoid Cytotoxicity (Fig. 7B) and salivary and vaginal fractions after intranasal immunization with toxoid FIG. 7C is a graph showing the cytotoxicity of anti-toxin A in the urine (FIG. 7C).   FIG. 8 shows rat sera from mice immunized intranasally with toxoid. FIG. 4 is a graph showing the level of passive protection of ligated small intestinal loops from toxin A. .   FIG. 9 shows toxoids after lethal administration with toxin A or toxin B. It is a graph which shows the survival rate of the intranasally immunized mouse.   Figure 10 shows intranasal immunization with toxoid followed by ligated small intestinal loops in mice. 4 is a graph showing the level of intestinal toxicity of toxin A.   11A-11B show toxin A features following immunization with GST-ARU by the indicated route. FIG. 12 is a graph showing differential systemic (FIG. 11A) and mucosal (FIG. 11B) IgA responses. ( (See Table 5 for a description of the immune pathway)   12A-12B show toxin A cytotoxicity of serum collected 40 days after immunization with GST-ARU. It is a graph which shows an inhibition level. (See Table 5 for a description of the immune pathway)   FIGS. 13A-13B are immune sera from mice immunized with GST-ARU, Is a graph showing the level of passively inhibiting the intestinal toxicity of toxin A in tomato . (See Table 5 for a description of the immune pathway)   Figure 14 shows lethal toxin after immunization with recombinant toxin A repeat (ARU). 5 is a graph showing the survival rate from administration of A (see Table 5 for a description of the immune pathway). ).   FIG. 15 shows toxin A in the intestinal loop of ligated mice after immunization with GST-ARU 5 is a graph showing the level of protection from intestinal toxicity in rats (see Table 5 for a description of immune pathways). See).Intranasal and mucosal-systemic combinations to induce mucosal immune responses at distal sites Combined vaccination method   We provide a method of administration intranasally or a combination of mucosal and systemic administration Elicited a mucosal immune response in the gastrointestinal and genitourinary tracts.   The method of the present invention may be used for Helicobacter (eg, H. pylori, H. pylori). felis, and H. heilmanii), Campylobacter (for example, , C. jejuni), including but not limited to gastrointestinal pathogens, Pathogens (eg Clostridia), enterotoxin (Primary Escherichia coli, Shigella, Vibrio cholerae, and Salmonella typhi); or genitourinary tract pathogens ( For example, human immunodeficiency virus, herpes simplex virus, papilloma virus , Treponema pallidum, Chlamydia, and Neisseria gonorrhoeae) Can be used to induce a therapeutic immune response. Prevention and prevention using the method of the present invention. And / or appropriate to the pathogen causing the condition desired to be treated Vaccine antigens (eg, polypeptide antigens) are readily selected by those skilled in the art. . The method of the present invention may comprise C as a specific example of a vaccine antigen used in the method of the present invention. . refer to antigens derived from difficile (eg, toxins or toxoids) However, it is explained as follows.C. as vaccine Use of difficile toxins and toxoids   C. which may be used in the methods and compositions of the present invention. difficile toxin polypeptide Can be prepared using several standard methods. For example, toxin (Eg, toxin A and / or toxin B) difficile culture filtrate (Eg, Kim et al., Infection and Immunity 55: 2984-2). 992, 1987; and see Example I below).   In addition, C.I. The difficile toxin polypeptide can be prepared using standard DNA recombination techniques. Can also be produced (eg, Ausubel et al., Eds., Current Protocols  in Molecular Biology, John Wiley & Sons, Inc., 1994). These methods The appropriate expression including all or part of the nucleic acid fragment encoding the toxin The vector is transformed into a suitable host cell. (C. salt of difficile toxin A The base sequence and the deduced amino acid sequence, and the nucleic acid sequence of toxin B, respectively, Dove et al., Infection and Immunity 58: 480-488, 1990, and Baroque. See Barroso et al., Nucleic Acids Research 18: 4004, 1990. ) Wide variety Any of the expression systems can be used to produce a recombinant toxin. example The toxin polypeptide can be a prokaryotic host (eg, E. coli) or a eukaryotic cell. Hosts (eg, yeast cells (eg, baker's yeast), mammalian cells (eg, COS1, NIH3T3, or JEG3 cells), or arthropod cells (eg, Spodoptera Giperda (Spodoptera frugiperda) (SF9) cells. this Such cells can be obtained from a number of different sources known to those of skill in the art, for example, American Type Cu lture Collection (ATCC), Rockland, MD (eg, Ausberg ( Ausubel ) Et al., Supra). Transfection / transformation methods and expression vectors used The choice of the reactor is described, for example, in Ausubel et al., Supra. As such, depends on the host system chosen. Expression vectors (for example, plasmids or Or viral vectors) are described, for example, in "Cloning Vectors: Laboratory Manual. (Cloning Vectors: A Laboratory Manual) (Pouwels et al., 1985) Supplement, 1987; Ausubel et al., Supra). " Can be selected from those that have been   C. difficile toxin polypeptides, especially for short fragments, can also be chemically synthesized, e.g. For example, see Solid Phase Peptide Synthesis, 1984, Second Edition, Stewart and Young ed., Pierce Chemical Co., Rockf ord, methods as described in IL, and standard in vitro translation methods. Can also be produced.   C. difficile toxoids can also be used in the methods and compositions of the present invention. . Toxoids are those that have lost or reduced the toxicity of the toxin but maintain antigenicity. Toxin that has been treated to Toxoid uses standard methods For example, chemical (eg, glutaraldehyde or formaldehyde) treatment (Eg, Libby et al., Infection and Immunity 36: 822-829, 1982). Can be prepared by Toxoid can also be used as described above. Mutating the gene encoding syn and expressing the mutant gene in an expression system And can be prepared. Mutable toxin A and / or toxin Region B includes conserved cysteine residues, nucleic acid binding regions, internal hydrophobic And / or a carboxy-terminal repeat sequence domain. Book C. that can be used in the invention A specific example of such a mutation in the difficile toxin is See, for example, "Barroso et al., Microbial Pathogenesis 16: 297-303, 199. 4 ".   Other methods of producing toxoids that can be used in the present invention are essential for toxicity. Includes chemical modifications of amino acids that are not related to antigenicity. For example, including SH Specific amino acid, lysine, tyrosine, tryptophan, or histidine residues Chemically modifying reagents may be used in the present invention and are known in the art (eg, See, for example, Cohen et al., Ann. Rev. Biochem. 37: 683-695, 1968). Sa Furthermore, using an azide-bonded substrate analog such as UDP glucose, Thus, a standard photoaffinity labeling method that is covalently bound to the active site of the toxin , Can be used to produce toxoids.   Original full length C. In addition to difficile toxins, polypeptide fragments of toxins, or Or toxins containing mutations (such as toxoids or not) Is a polypeptide fragment of toxin), if the antigenicity is maintained, Can be used. C. Examples of difficile toxin fragments include, for example, "price ( Price) et al., Current Microbology 16: 55-60, 1987; Lyerly et al. Current Microbiology 21: 29-32, 1990; and Frey et al., Infection. and Immunity 60: 2488-2492, 1992 ". C. difficile toxin Genes encoding toxin fragments containing and / or mutations can be prepared using standard methods. (Eg, Ausubel et al., Supra). Included in the present invention The fragments, derivatives, and toxoids can be prepared using standard methods in the art. For example, induction of mucosal immune response (see below), induction of prophylactic immunity (see below) Or screen for antigenicity by measuring induction of a therapeutic immune response. Can be performed.   Although not required, an adjuvant may be administered with the vaccine in the method of the invention. You may. Any of the many adjuvants known to those skilled in the art can be used . For example, cholera toxin (CT), heat-denaturable enterotoxin (LT) from Escherichia coli Or a fragment or derivative thereof having adjuvant activity for mucosal administration Can be used for Horse mackerel like RIBI (ImmunoChem, Hamilton, MT) Luvant or aluminum hydroxide can be used for parenteral administration.   For example, an adjuvant (eg, CT, LT, or the like having adjuvant activity). Fragment or derivative). difficile toxin (or a fragment or A fusion protein containing the derivative is also included in the present invention and can be prepared by standard methods (eg, Au (See Ausubel et al., Supra). Furthermore, the present invention Cutin can be covalently conjugated or cross-linked to an adjuvant. Aju on antigen Methods for covalently conjugating or chemically cross-linking bunts are described in, for example, Cr yz) et al., Vaccine 13: 67-71, 1994; Liang et al. Immunology 141: 14 95-1501, 1988; and Czerkinsky et al., Infection and Im. munity 57: 1072-1077, 1989 ".   Vaccine composition (with or without adjuvant) as described above The substance is administered intranasally according to the method of the present invention. The mode of administration combination is also used. For example, an initial dose of water may be applied to mucosal (eg, intranasal or oral) surfaces. Kuching is administered and booster immunization is administered parenterally (eg, intraperitoneally or subcutaneously). ) Can be administered; this combination gives surprisingly good results. For example Parenteral booster immunization may be performed one week after the initial mucosal administration.   The amount of vaccine administered will depend on the particular vaccine, as determined by those skilled in the art. Whether the antigen, adjuvant is administered together with the vaccine antigen, the method and frequency of administration And the desired effect (eg, prevention and / or treatment). Typically The vaccine antigen of the present invention is administered in an amount ranging, for example, from 1 μg to 100 mg. . When an adjuvant is administered with the vaccine, for example, in the range of 1 ng to 1 mg. Enclosed quantities can be used. Administration is as needed, as determined by one of skill in the art. Repeated. For example, after the first dose, three boosters at one week intervals It can be performed. The vaccine can be any pharmaceutically acceptable carrier or diluent (Eg, water, saline, (eg, phosphate buffered saline), or bicarbonate solution (eg, For example, 0.2 M NaHCO_Three)). Carrier or dilution used in the present invention The product will be selected based on the method and route of administration, and standard pharmaceutical practice. You. Appropriate pharmacy, as well as the pharmaceutical need for use in pharmaceutical formulations Typical carriers and dilutions are standard reference texts in the art and USP / NF. In Remington's Pharmaceutical Sciences It is listed.   The following examples are intended to illustrate, without limiting, the method of the present invention. Things. Modifications of the conditions and parameters described below, which will be apparent to those skilled in the art. Variations are included within the scope of the present invention.                                  Example   Two model systems, mouse and hamster Used to evaluate the law. Hamsters have human-like antibiotic-related diarrhea Hamster model is C. Vaccination against difficile disease Was used to directly evaluate the prophylactic effect of Hamster C. difficile Infection causes severe hemorrhagic cecumitis, the colon observed in human disease states Reminds of fire. Further, C.I. One time clindamycin in combination with difficile Oral or systemic doses in hamsters can cause severe diarrhea , Eventually leading to animal death.   Using a variety of assays, the hamster model was also Can be used to monitor the immune response induced by the method. For example, immune Of serum and mucosal samples from hamsters subjected to cysteine block cytotoxicity in vitro Can be used to measure In addition, the ligated bowel of the immunized hamster The loop evaluates the inhibition of the enterotoxic activity of toxin A induced by vaccination Used to Besides, C. in hamsters. difficile colonization is faeces It can be monitored by culture, or toxin A or And / or presence of toxin B is determined by ELISA and / or cytotoxicity analysis Can be done.   The mouse model is characterized by the immune response induced by the vaccination method of the present invention. There are advantages in assessing In particular, monoclonal antibodies that recognize mouse IgA Commercially available, thus facilitating evaluation of mucosal immune response in mice . In contrast, such reagents are useful for assessing hamster mucosal immune responses. Not available. A further advantage of the mouse model is that it provides a sample of the mucosal surface of the mouse Methods have been developed that can describe mucosal responses to various immunization methods You.   Once the immunogenicity of the vaccine candidate has been established, for example, by ELISA analysis, Mouse serum samples were tested for the characteristics of antibodies likely to be associated with an effective vaccine Can be used for For example, serum from immunized mice contains (1) toxin A and And / or the ability of toxin B to block cytotoxicity in vitro, or (2) Toxin A was isolated using a ligated intestinal loop from a mouse or rat to which toxin A had been administered. Analyzes for the ability to block intestinal toxicity can be made. Immunized mice also G To determine protection from death or secretion buildup due to intestinal toxicity of xin A, It may be administered orally or in its ligated intestinal loop. Finally immunized Mice are given a toxin systemically at doses known to be lethal. sell.Example IC. Immunization of hamsters with a vaccine composition containing difficile toxin   The following method analyzes the efficacy of the immunization method of the present invention in a hamster model system. Used to C. Preparation of difficile toxoid vaccine   C. A culture filtrate of difficile was prepared and used by Libby et al. (Infection and I mmunity 36: 822-829, 1982). Concise If you write in C. difficileVPI strain 10463 (ATCC Deposit No. 43255) in a dialysis flask Cultured for 3 days, centrifuged and sterile filtered. 1 ml for 100 ml culture filtrate Of formaldehyde was added and the mixture was incubated at 37 ° C. for 1 hour. Culture filtrates have a toxin A concentration of about 50 μg / ml as determined by ELISA (Lyerly et al., Infection and Immunity 47: 349-352, 1985) ), Toxin B as determined by cell culture cytotoxicity assay. Then 10⁶Cytotoxicity titer (Ehrich et al., Infection and I mmunity 28: 1041-1043, 1980). Toxoid is a 500 ml cell concentrator (Amicon, B everly, MA) by ultrafiltration through a 30 kD membrane, 3 volumes of phosphate buffered saline (PBS), pH 7.4. Toxoid is concentrated 10-fold and destroyed by the filter Bacteria were stored at 4 ° C. until use. Toxin size (3 for toxin A) 08 kD and 269 kD for toxin B). It was speculated that there was no significant loss of the synprotein, and each inactivated toxin Was estimated to be a concentration of 500 μg / ml in a 10-fold solution. Toxoid material is IRM- Has any detectable cytotoxic activity against 90 cells (ATCC accession number CCL186). I didn't. Preparation of whole cell vaccine   C. difficileVPI strain 10463 (ATCC Deposit No. 43255) is a strain that minimizes sporulation. Proteose peptone-yeast extract culture at 37 ° C for 36 hours under aerobic conditions Earth (PPY; Holbrook et al., J. Appl. Bacteriol. 42: 259-273, 1977. ). The culture was centrifuged and the pelleted cells were washed three times with PBS. . After the last wash, the pelleted cells contain 1% (volume: volume) formaldehyde. And incubated at 4 ° C. for 24 hours. Excess formua The aldehyde was removed by washing three times with PBS, and C.I. di The suspension of fficile cells was stored at 4 ° C. Ten⁹C. difficile colony forming unit (CFU) (cell suspension with OD 1.0 at 550 mn) Even when the cells were cultured under anaerobic conditions at 37 ° C. for 36 hours, no proliferation occurred. animal   Six to eight week old female Sicilian hamsters (Mesocrisetus aura) at the time of immunization Tuss (Mesocricetusu auratus), Charles River, Kingston, NY Used in the experiment. Animals were caged in groups of 5 for immunization periods, after which C. Individuals were caged while inoculating difficile. Immunization method   Seven different immunization methods were analyzed (Table 1). For intranasal (i.n.) immunity , 5 μg of each toxoid (inactivated toxin A and 10 μl in 10 μl of 10 times toxoid) 5 μg of cholera toxin (Calbiochem, La Jol) la, CA). This 15 μl antigen adjuvant mixture Use a micropipette to divide the nose into the nostril of the hamster and halve the dose in each nostril. The administration was performed so that each administration was performed. For intragastric (i.g.) immunity, each toxoid 100 μg is mixed with 10 μg cholera toxin, made up to 1 ml volume with PBS, and by gastric tube Was administered. For intraperitoneal (i.p.) and subcutaneous (s.c.) immunizations, 5 μg each of toxoid Was mixed with 0.3 ml of RIBI adjuvant (RIBI, ImmumoChem, Hamilton, MT) . For rectal (r.) Immunization, 50 μg of each toxoid in 100 μl of toxoid is equivalent to 10 μg of toxoid. It was mixed with 1 μl solution containing relatoxin. 5 for rectal administration of whole cells (w.c.r.)  X 10⁸Cells were 50 μg of each toxoid in 100 μl of toxoid, plus 10 μg of cholera It was mixed with a 1 μl solution containing toxin. r. and w.c.r. The feed is made by inserting a disposable 20 x 1 1/2 feeding needle 3 cm into the rectum. Was administered. Isofluorene lightly hemp for immunization of i.n., i.g., i.p., and s.c. Drunk Animals were immunized. r. and w.c.r.immunizations were anesthetized with pentobarbital Made on animals. In the control intranasal group (c.i.n.), 5 μg of cholera ibis Shin received. The control subcutaneous group (c.s.c.) received 0.3 ml of RIBI adjuvant subcutaneously. I did. Groups of five animals were used for all immunization methods. In all groups, Vaccine (or adjuvant control) on days 0, 7, 14, and 28 ) Received a total of four doses.   Blood samples (200-400 μl) are anesthetized with isofluorene to assess the immune response Obtained from the posterior orbital sinus of the lower hamster on days 0, 2, 4, 7, and 36. blood The solution was left overnight at 4 ° C. until an aggregate was formed, and serum was obtained by centrifugation. blood Only clear antibodies were evaluated; secreted IgA due to lack of suitable anti-hamster IgA reagent Was not measured. C. Fecal samples survive after difficile inoculation Every other day to assess the extent of colonization and the presence of toxins. Mixed with 2 volumes of PPY medium (see below) C. difficile inoculation   On day 38 (10 days after the fourth immunization) of all hamsters, 0.5 mg Dandamycin is administered orally, followed by removal of free toxin 3 hours later. Washed with PPY medium for 10^FiveViable CFU of CFU. difficile 10463 strain (ATCC Deposit No. 43 255) Oral stomach inoculation was performed. After inoculation, the hamsters will have diarrhea and disease Observed daily. 0 diarrhea: no diarrhea; 1+, loose feces but no wet tail 2+, perianal and tail areas wet; and 3+, tail, feet, and lower abdomen Wet parts (animals with such findings are usually round and crouched and do not No). Evaluation of tissue destruction   A sick hamster was euthanized. Euthanized hamsters and chestnuts Derived from survival from all immunization methods collected 8 days after danmycin administration Cecal samples were fixed in 10% neutral buffer formalin. Fixed in formalin Tissues are embedded in paraffin, sliced at 5 μM, hematoxylin and Osin was stained and examined by light microscopy. The criteria for the histological stage are: 0, Minimal infiltration of lymphocytes, plasma cells, and eosinophils; 1+, lymphocytes, plasma cells Mild infiltration of vesicles, neutrophils and eosinophils, as well as intestinal lymphoid tissue proliferation Mild congestion of mucosa, with or without; 2+, moderate mixed inflammatory cells Moderate infiltration of the lamina propria with or without infiltration of globular cells Blood and edema, necrosis or vacuolation of individual surface cells, and crypt enlargement; 3+, severe inflammation of the mucous membranes, congestion, edema and bleeding, necrosis of surface cells, or petals Degeneration with cancer or ulcer. Evaluation of infection   The feces obtained after clindamycin administration were C.I. The presence of difficile was checked. Cycloserine (125 μg / ml) and serum diluted 10-fold in PPY medium Inoculated in selective medium containing fuoxitin (8 μg / ml) and incubated for 48 hours under anaerobic conditions After incubation, counting was performed. The presence of toxin A in feces Explained by the manufacturer using a Toxin A kit (Tech Lab, Blacksburg, VA) It has been decided to be. After 15 minutes with the substrate, 0.D. was read at 450 nm. The concentration of toxin was estimated from a standard curve of toxin A facilitated on each plate. Was. This estimate is based on Softmax software (Molecular Devices, Sunnyvale, CA) Made with For quantification of toxin B, the fecal suspension is centrifuged and filtered. IMR-90 fibroblast cultures on a 10-fold diluted sample Were tested as described below. ELISA for antibodies to toxin A and toxin B   Microtiter plates (Corning, New York, NY) are carbonate-bicarbonate buffered Coated with 100 ng / well of purified toxin A or B in buffer, pH 9.3 And incubated at 4 ° C. overnight. Wash the plate and add phosphate buffered saline (PB S), blocked with 2.5% nonfat dry milk (NFDM) in pH 7.4. Serum sample At a 2x dilution ranging from 500x to 64,000x, and incubate the plate at 37 ° C for 1 hour. Incubated. Anti-hamster IgG conjugated with alkaline phosphatase (100 0x, Southern Biotech, Birmingham, AL) and incubate at 37 ° C for 1 hour. Washing was performed prior to the addition of the p-nitrophenyl phosphate substrate. The positive control is Included in each plate; wells were diluted by two-fold dilutions ranging from 100 to 0.8 ng / ml. Coat with Syn A or Toxin B and react with specific goat anti-toxin (TechLab) And then reacted with alkaline phosphatase-conjugated anti-goat IgG. Negative control Is coated with purified toxin and contains alkaline phosphatase-conjugated anti-hamster IgG. Reacted. At 405 nm, 0.D. is read, and the titer is 0.D. It was defined as the reciprocal of the maximum dilution of the charge. ELISA for antibodies to whole cell antigens   Plates were prepared with 100 μl of 0.D. adjusted to 0.2 at 550 nm formalin killed C.I. di Coat with fficile suspension, then incubate overnight at 150 rpm on a swirl shaker. I did it. Cells were fixed on plates by incubating at 70 ° C. for 2 hours. . After washing, add a serum sample at a 2-fold dilution ranging from 100-fold to 12,800-fold, at 37 ° C. Incubated for 1 hour. Anti-hamster IgG and substrate were added as above . The positive control was mouse C. from 500 to 64,000 times. Use difficile whole cell antisera It was included in the plate. (Anti-serum was prepared using standard methods, VPI strain 10463 Produced against). Wells coated with whole cells serve as negative controls It was directly reacted with rephosphatase-conjugated anti-hamster IgG. Inhibition of cytotoxicity   IMR-90 fibroblasts were cultured in D-MEM medium containing 10% fetal bovine serum (Gibco, Grand Island , NY) in 96-well plates until confluent. Cells The minimum dose of Toxin A or Toxin B required to be 100% spherical is 1 cytotoxic Sex unit (CTU₁₀₀). 6.3 ng / ml for toxin A 125 pg / ml at 1 CTU₁₀₀Defined. Range from 100x to 12,800x Two-fold dilutions of hamster serum samples from 4 to 4 CTUs of either toxin₁₀₀Mixed with , 37 ° C for 1 hour and the mixture was then added to the cells. Goat anti-toxic A and goat anti-toxin B served as positive controls. Cells are observed after 24 hours The ratio of round cells was determined. The titer of the sample should prevent more than 50% of the cells from rolling. It was defined as the reciprocal of the highest dilution of harmful serum. Aggregation   A 25 μl sample of hamster serum was diluted from 25-fold to 3,200-fold. Dilution Prepared in 96-well U-bottom microplates (Falcon, Oxnard, CA). Holma C. killed by phosphorus Adjust the difficile suspension so that 0.D. is 1.0 at 550 mn, 2 5 μl of the suspension was added to the serum dilution. Mouse anti-C. difficile whole cell antiserum positive And PBS was used as a negative control. Plates are incubated overnight at 4 ° C. Bait and aggregation were subsequently evaluated. The end point of the titer is determined by the serum It was defined as the reciprocal of the large dilution. Western blot analysis   C. difficile VPI strain 10463 (ATCC accession number 43255), and clindamycin Throw After the administration, the strain isolated from the hamster was subjected to anaerobic conditions at 37 ° C for 36 hours in 5 ml of PPY medium. Growth. The culture was centrifuged and the pellet was washed three times with PBS. Pellet the PBS Suspend in 250 μl of 3% SDS in lysate and lysate is 12% preparative SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) by electrophoresis at 200 volts for 1 hour. . Bethesda Research Laboratories Mini-V8-10 Chamber (Life Technologies Proteins from the gel at 150 volts for 1.2 hours using Grand Island, NY). Transferred to Lurose membrane. The membrane was blocked with 5% non-fat dry milk in PBS for 1 hour, Washed and mounted in multi-screen instrument (BioRad, Hercules, CA). 200 times dilution Of each hamster serum sample was then added and incubated for 1 hour. Images were taken with CIP (Gibco, Gaithersburg, MD). Mouse anti-anti-C. difficile10463 all Cell serum was served as a positive control. C. isolated from feces Classify difficile strains Therefore, as described above, the SDS lysate from the isolate is electrophoretically The cells were transferred to a nitrocellulose membrane and reacted with mouse antiserum of all cells.result Results after clindamycin administration   Ten days after the last immunization of hamsters, clindamycin and C.I. Administer difficile did.   All sham-immunized, intranasal control (c.i.n.) and subcutaneous control (c.s.c) animals For all animals, within 48 hours of dosing, animals died, mostly severe (3+) diarrhea Was accompanied. Acute diffuse necrotic hemorrhagic cecumitis (grade 3+) found by pathological examination Was done. The crypt epithelium was proliferative and the enlarged crypt was full of neutrophils. Lymphocytes, plasma cells and neutrophils infiltrated the lamina propria. Died of inoculation The vaccinated hamsters have also died within the first 48 hours. Most have grade 3+ diarrhea and grade 3+ cecumitis based on histopathology I was Animals that survived treatment did not develop diarrhea or had 1+ to 3+ He had a degree of diarrhea. The degree of diarrhea is the degree of cecumitis on pathological examination And was correlated. Animals with 3+ diarrhea are subacute grade 2-2.5 + diffuse Had mucopurulent cecumitis. Neutrophils, lymphocytes, and plasma cells are Multifocal crypt abscesses were also noted, invading the lamina propria. Had 2+ diarrhea animal Developed subacute to chronic grade 1.5-2 + moderate cecumitis. Mild Animals with diarrhea (1+) have mild lymphocytic cecumitis and are gray Was 1.0-1.5 +. Grade 1+ mild lymphocytic cecumitis causes diarrhea Not even hamsters were evident.   The results of clindamycin administration for all vaccine groups are shown in FIG. Toxoid vaccine by intragastric (i.g.) or rectal (r.) Route or rectum Immunization with mucosal immunization by whole cell toxoid vaccine (w.c.r.) The animals had minimal protection against death and all had diarrhea. For death, r. And w.c.r immunization methods protect only 20% of hamsters On the other hand, the i.g. method protected 40% of hamsters. Immunized parenterally (i.p. And s.c.) animals were completely protected against diarrhea. Similar results in the nasal cavity In animals immunized with (i.n.). i.n.Immunized animals receive toxoid intraperitoneally When receiving booster doses (i.n.i.p.), 10 for death and diarrhea 0% defense was done. C. in feces. Difficile and toxin presence   A stool sample obtained after administration of clindamycin was treated with C.I. difficile, toxic A and toxin B were analyzed. Similar patterns all survive Was observed in animals. Two days after administration, C.I. difficile is about 10 in feces⁹CFU / ml reached Colonization was slightly reduced, at least in spite of the presence of diarrhea. About 10 for 9 days⁸CFU / ml remained. In contrast, toxin A and toxin B is constantly decreasing and by day 9 despite constant colonization, Toxins have almost disappeared in feces. All isolated C.I. difficile strain Western blot was performed using whole cell antiserum against VPI 10463 strain (ATCC Deposit No. 43255). Classified by blot analysis. Simply from different animals and different immunization groups The isolated strains were analyzed and all were found to be identical to each other, Suggests that only one strain has colonized all hamsters. I However, this strain differs from the VPI10463 strain used for administration. Was. Immune response   C. Serum antibodies to the difficile antigen were obtained from hamsters from all experimental groups. Measured. Immune response to toxin A after early immunization Was tested by ELISA. Two days, four days, and And 7 days, animals vaccinated by the i.n.i.p., r., And w.c.r. No specific IgG was detected. Parenterally immunized animals (i.p. and s.c. ), No response was evident after 2 and 4 days, but a slight A rise in body was observed. In contrast, measured after the last vaccine dose (day 36). Antibody responses showed seroconversion in all groups. First time The lack of an initial (pre-existing) antibody response to the tin dose indicates that the animal No infection and no sensitization to toxin A are doing.   C. To study the systemic antigen response to the difficile antigen, three protocols Were used: 1) recognition of immobilized antigen by ELISA; 2) cell culture assays. And 3) bacterial aggregation. Toxin A, toxin B, and all Vesicles were used as antigen. Against toxin A, toxin B, and whole cell antigens Antibody responses were present in all groups as determined by ELISA (Fig. 2). Hamsters immunized by the i.n.i.p, i.n., i.g. r. and w.c.r.) and sc. And high response to toxin B. Antibody levels against whole cell antigens And a similar pattern to that observed with toxins.   Antibodies to whole cell antigen were recovered from hamsters after clindamycin administration. Western blot analysis using whole cell lysates from selected C. difficile strains , Was further characterized. Animals were immunized with 70 kD protein and 200  For proteins with a size of kD or more (this is considered a toxin) Antibody was expressed. Animals immunized by the i.n.i.p. route have the strongest immune response showed that. A variety of other proteins are available via the i.n.i.p., i.p., and s.c. Recognized by serum from immunized animals, but mucosally (r. And w.c.r. groups) These proteins are not apparent in sera from immunized animals, or It was not very clear. Purification of hamster serum in another assay Is Immunoblots were performed in parallel on the toxins and whole cell lysates. This In these assays, low-molecular-weight proteins that reacted with hamster serum were converted to toxins. Not a fragment.   Serum antibodies with biological function may be different from those obtained by ELISA. The turn was shown (FIG. 3). Antibodies that inhibit cytotoxicity by toxin A Detected in animals. Hamsters immunized by the i.n.i.p. and i.p. High anti-toxin A activity (mean + SE 22,000 ± 4,900 and 18,000 ± 2,000, respectively) ), While animals immunized mucosally (i.n., i.g., r., And w.c.r.) Lower activity (580 ± 280, 280 ± 146, 1720 ± 560, and 2760 ± 290, respectively) Was. High anti-toxin B responses were obtained in all groups except animals rectally immunized. Was. Aggregated antibodies can be administered parenterally (i.p. and s.c.) or combined viscosities. Membrane-Detected only in animals that received the toxoid vaccine by parenteral route (i.n.i.p) Was issued. Implications of immune response and defense   Animals immunized with i.n.i.p are fully protected against death and diarrhea, Provides the highest serum immune response when considered by both LISA and biological activity (FIGS. 2 and 3). Complete protection against death is due to parenteral vaccine injection Or provided by all immunization designs, including only intranasal immunity. Contrast In addition, animals rectally immunized have a protective ratio and a serum antibody response, especially toxin B. Neutralizing antibody was the lowest (FIG. 3). However, with the i.n. Despite the resulting high protection (Figure 1), similar antibody responses were observed in the i.n. and i.g. groups. As explained by the answer (FIGS. 2 and 3), the immunological correlation is universal. Was not.   Animals from all groups were analyzed together to define immunological correlations. The results of the administration were compared to the immune response (Table 2). Average anti- Body levels are higher than those that resulted in death, except for whole-cell ELISA. The remaining animals were significantly higher. Hamsters with severe diarrhea (3+) Is significantly lower in all assays when compared to animals without diarrhea. It was a clear immune response (Table 3). Have mild to moderate diarrhea (1+ and 2+) C The antibody response in Muster, except for the aggregated antibody response in animals with 1+ diarrhea, There was no significant difference from animals without diarrhea (p <.05). a Kruskal-Wallis test a p-value as compared to the group that does not cause diarrhea, Kruskal-Wallis test Long-term defense   4 surviving animals from the i.n.i.p group and 4 animals from the sc group It was kept for 140 days after the administration of syn. On day 140, blood and fecal samples were collected and Received clindamycin again. Of the four animals in each group, three (75%) Survived the second dose. In the i.n.i.p. group, 2 out of 4 animals (50%), s.c. In the group, 0/4 (0%) were protected against diarrhea. Immune response before re-dose Was compared to the response obtained before the first dose. ELISA analysis shows that all cell antigens Toxin A and toxin B antibodies were significantly lower It did not decrease (FIG. 4). Before re-dosing when biological activities are compared Showed a marked decrease in anticytotoxin activity and C. difficile aggregation. (FIG. 5).Example II C. Immunization of mice with a vaccine composition containing difficile toxin   The following method analyzes the efficacy of the immunization method of the present invention in a mouse model system Used for ELISA   The ELISA method used in the following experiments is described above. Briefly, Toxin-specific immune responses can be achieved in 96-well plates with toxin A or toxin B. Coat and block wells with skim milk in PBS-Tween, Add sample and use commercially available anti-mouse alkaline phosphatase (AP) binding reagent The detection was carried out by detecting at. Plate is Sigma 104 Alkaline Phosphor Develop with Phatase (AP) substrate (Sigma Chemical Company, St. Louis, MO) Was. Data from these assays are shown as absorbance at 405 nm (hereinafter reference). Cytotoxicity   Both toxin A and toxin B are cytotoxic to various cell lines Bring. This toxicity is evident by rounding fibroblasts (IMR-90) . IMR-90 cells are sensitive to 10-100 pg of toxin A and 0.1-1.0 pg of toxin B is there. The dose of toxin used for cytotoxicity inhibition experiments was This corresponds to eight times the amount required to round 50% of the R-90 monolayer culture. Serum or Dilute secretions appropriately and mix with either toxin A or toxin B for 1 hour at 37 ° C For a while. The toxin is then transferred to a confluent well in a 96-well cell culture plate. And incubated overnight. Data round 50% of monolayer culture Is shown as the highest dilution that prevents Vaccine preparation   Toxoid and recombinant toxin A (GST-ARU) as described above Was prepared. Enteric toxicity   Toxin A intestinal toxicity was assessed using ligated intestinal loops administered with toxin A did. Antibodies that inhibit enterotoxicity are preincubated with serum or secretions containing the antibodies. It was determined by administering the toxin A to the loop. Using rats They were fasted prior to use, anesthetized, and intestinal sections were cut. Each loop is complete Contains blood vessels, no feces. Pretreatment with immunized serum, and In contrast, toxin A (1-10 μg) was administered to the lumen of each loop. 4 o'clock After a short time, the loop was removed and the contents weighed. Mouse and hams A similar loop is used to determine the effectiveness of immunity against intestinal toxicity. It can be administered directly. In mouse loop assays, loops treated with PBS Volume compared to loops treated with toxin A. Data is ligated loop c Shown as the weight of the contents per m. Systemic administration   Mice receive LD of each toxin administered intraperitoneally₅₀Was administered ten times. The data shown in the figure (see below) are the percentage of animals that survived the treatment. To hamster 2 mg clindamycin and 1 x 10⁷Vegetatively grown C. difficil e organism was administered.result Intranasal immunization of mice with toxoid   Five female Swiss Webster mice (Taconic Farms, Germantown, NY) with 5 μg CT as a mucosal adjuvant. Toxoid (15 μg of each toxin) by intranasal route, with or without so Immunization was performed once a week. The immunization design is summarized in Table 4. Serum, saliva, feces , And vaginal secretions were obtained after immunization. Detect specific IgA and IgG antibodies in serum Specific IgA antibodies to both toxin A and toxin B, On mucosal surfaces against both saliva, feces and post-immunization toxin A and toxin B It can be detected (FIGS. 6A and 6B). This response was based on whether CT was administered with toxoid Regardless, it is clear. Serum from immunized animals also contains toxin A and And toxin B both inhibited cytotoxicity (FIGS. 7A-7C). Immune serum, Toxic It was used to passively protect the rat loop from the enterotoxic effect of A. Movement For objects, LD₅₀10 times the dose of toxin A, followed by LD one week later₅₀10 times The amount of toxin A was administered. All immunized animals survived this treatment while the control Did not survive (FIG. 9). Finally, the ligated intestinal loop from the immunized animal Administration of toxin A showed direct antibody induction, but this was And possibly with mucosal IgA antibodies that can inhibit diarrhea (FIG. 10) These data indicate that C.D. diffi Cile vaccine elicits strong protective mucosal immune response and requires mucosal adjuvant And not. Example III Immunization of Mice with a Vaccine Composition Containing a GST-ARU Fusion Protein   C. The COOH-terminal region of difficile toxin A binds to carbohydrate residues in target cells Series of repeating amino acids that are thought to be involved in binding to Includes unit. (See, eg, Lyerly et al., Current Microbio logy 21 :: 29-32, 1990; Frey et al., Infection and Immunity 60: 2488. ~ 2 492, 1992; and references cited therein). Glutathione S-G C. fusion to transferrinase (GST) Carboxy end of difficile toxin A A fusion protein containing the terminal region was constructed as follows. Using standard methods, Including the nucleotides encoding the 794 carboxy terminal amino acids of toxin A The Sau3A fragment was recovered (for the sequence of the toxin A gene, Dove) et al., Supra). The protruding ends of this fragment were filled in and blunt ends The piece was ligated into pGEX3X digested with SmaI. GST-ARU using standard methods Clone containing the plasmid encoding GST-ARU fusion protein Quality is purified on a glutathione-agarose affinity column and free glutathione Eluted from the column with Tathione and dialyzed to remove glutathione .   Intragastric (IG; 100 μg), intranasal (IN; 50 μg), or intraperitoneally (IP; 25 μg) , One week with or without CT (5 μg) as mucosal adjuvant By giving four doses to the female Swiss Webster mouse (Sw iss Webster mice) (n = 5) immunized with a toxin A fusion protein (GST-ARU) Was done. Samples were obtained for immunological analysis after the last dose. Toki on all routes A good serum immune response against Syn A was induced. Intranasal (IN) administration without CT Both elicited mucosal IgA responses. Although immunization by the IG pathway is also effective , Appeared to be enhanced by the presence of a mucosal adjuvant. Exemption by IP route Plagues induce good systemic and fecal responses, but are not induced in saliva or vaginal samples. Did not. (FIGS. 11A-11B). Serum antibodies have toxin A cytotoxicity in vitro Did not inhibit the rat loop passively from intestinal toxicity. Suggests that carboxy-terminal binding domain is required only for in vivo toxicity (FIGS. 12A-12B and 13A-13B). Animals immunized by the IN and IP routes Protected from lethal dosing, but not IG immunized animals (FIG. 14). Also Survival of systemic administration also results in the presence of neutralizing antibodies to enterotoxin in the ligated intestinal loop As shown (FIG. 15). IN immunity is an enterotoxin of toxin A in the intestinal mucosa, even without CT Circulating antibodies that protect animals from the lethal effects of toxin A as well as their effects cause. Some protection was observed by all routes tested, The administration of the IN route appeared to be more effective in eliciting a mucosal response.   Other embodiments are within the following claims.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/00 615 A61K 31/00 615 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG) ), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BB , BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR , KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, UZ, VN (72) Inventor Tres-Lopez Francisco Mexico Dee. F. 01740 Mexico City Colonia San Clemente Ja Calandas 107-204 (72) Inventor Zang Senksi, Cambridge, Massachusetts, United States, Cambridge Linde Avenue # 8 362 (72) Inventor Ray Wende, Cambridge, Massachusetts, Cambridge, England # 8 362 (72) Inventor Liary David M. Radford MacArthur Avenue, Virginia, United States of America 204 (72) Inventor Moncliff James S. U.S.A.Cristian Jansburg, Walter Drive, Virginia 695

Claims (1)

[Claims] 1. When dissolved in pharmaceutically acceptable diluents and administered intranasally to mammals Induces a mucosal immune response in the gastrointestinal tract or urogenital tract in A composition comprising a capable, non-replicatable polypeptide antigen. 2. 2. The composition of claim 1, wherein the distal mucosal immune response occurs in the gastrointestinal tract. 3. 2. The composition of claim 1, wherein the distal mucosal immune response occurs in the genitourinary tract. 4. 2. The composition of claim 1, wherein the pathogen causes diarrhea. 5. 5. The pathogen is from the genus Clostridium. A composition as described. 6. The pathogen is Clostridium difficile The composition of claim 5, wherein 7. The antigen is a pathogen toxin, or an immunogenic fragment or derivative thereof The composition of claim 1 comprising a body. 8. If the antigen is a Clostridium difficile 8. The method according to claim 7, which comprises toxin A or an immunogenic fragment or derivative thereof. Composition. 9. If the antigen is a Clostridium difficile 9. The method according to claim 7, which comprises toxin B, or an immunogenic fragment or derivative thereof. Composition. Ten. If the antigen is a Clostridium difficile Toxin A and Clostridium difficile 9. The composition of claim 8, wherein the composition comprises toxin B. 11． The composition of claim 1, wherein the antigen comprises a toxoid. 12． Toxoid is Clostridium difficile The composition according to claim 11, which is a toxoid. 13. a. Administer an antigen capable of inducing an immune response on the mucosal surface of a mammal Stages and b. Parenterally administering the antigen to the mammal. A method for inducing a distal mucosal immune response to a pathogen in a mammal. 14. Mammals may develop diarrhea caused by pathogens 14. The method of claim 13, wherein the method does not cause diarrhea. 15． The antigen is C. 15. The method of claim 14, which is a difficile toxoid.