CA2226392A1 - Intranasal vaccination against gastrointestinal disease - Google Patents

Abstract

The invention features intranasal immunization methods for inducing immune responses in distal mucosal sites, e.g., the gastrointestinal or genitourinary tracts. The methods of the invention may be used to induce protective and/or therapeutic immune responses against pathogens (e.g., bacteria of the genus Clostridium, e.g., C. difficile) which infect these distal sites. Also included in the invention are vaccination methods in which combinations of mucosal (e.g., oral or intranasal) and parenteral (e.g., subcutaneous or intraperitoneal) routes of administration are used.

Description

CA 02226392 l998-0l-06 W 097/02835 PCT~US96/10987 INTRANASAL VACCINATION AGAINST GASTROINTESTINAL DISEASE
Backqround of the Invention This invention relates to intrAn~ vaccination ~ 5 methods for preventing and/or treating gastrointestinal disease.
Clostridium difficile is a gram-positive, spore-forming, toxigenic bacterium that causes antibiotic-associated diarrhea which can progress into severe and 10 sometimes fatal colitis. Upon disruption of the normal intestinal flora by, e.g., antibiotic or anti-neoplastic therapy, C. difficile may become established in the colon where it produces two high molecular weight toxins, Toxin A and Toxin B. Both of these polypeptides are 15 cytotoxins, but Toxin B is greater than 1000-fold more potent than Toxin A. Toxin A is also an enterotoxin, as it causes ac~ lation of fluid in ligated ~ni ~1 intestinal loops.

Summary of the Invention We have shown that intranasal, and combined mucosal ( e . g ., oral or intranasal) and systemic ( e . g ., subcutaneous or intraperitoneal), vaccination regimens, even in the absence of an adjuvant, are effective in inducing mucosal ; ~ responses at distal mucosal sites (e.g., the gastrointestinal and/or genitourinary tracts).
Vaccination of hamsters with C. difficile toxins A or B
(or toxoids) using either of these methods gives rise to protection of these ~n; ~l ~ from subsequent C. difficile challenge.
Accordingly, the invention features a method of inducing a distal mucosal ; c response (i.e., a mucosal immune response outside of the upper respiratory tract, e . g ., in the gastrointestinal and/or genitourinary tracts) to a gastrointestinal or genitourinary tract W 097/02835 PCT/US96tlO987 pathogen in a ~ ~1. In this method, a non-replicatable polypeptide antigen which is dissolved in a pharmaceutically acceptable diluent, and which is capable of inducing the distal ; ? response to the pathogen, 5 is a~ ;n;~tered intranasally to the mammal.
The invention also features a method of inducing a distal mucosal immune response to a pathogen in a involving: (1) a~~ ;n;~tering an antigen capable of inducing the distal ; c response to a mucosal surface 10 of the ~m~l, and (2) parenterally a~m; n; .ctering the antigen to the ~~ ~1. Any order of combined mucosal and parenteral a~;n;~tration is included in the invention.
For example, mucosal (e.g., intranasal, oral, ocular, gastric, rectal, vaginal, gastrointestinal, or urinary 15 tract) administration may precede parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) ~ ;n;~tration, or parenteral ~1~ ; n; ~tration may precede mucosal a~ ;n;.ctration. As an example, three weekly doses may be ~ ;n;~tered mucosally (e.g., intr~n~lly) and, on the fourth week, combined mucosal (e.g., intr~n~ ) and parenteral (e.g., intraperitoneal) a~ ;n;~tration may carried out.
Pathogens to which mucosal ; c responses may be ; n~lC~ in the methods of the invention, and from which 25 the antigens (e.g., non-replicatable polypeptide antigens) may be derived, include, but are not limited to gastrointestinal pathogens such as ~elicobacters (e.g., H. pylori, H. felis, and ~. hei7 -nii), Campylobacters (e.g., C. jeiuni), pathogens which cause diarrhea and 30 colitis (e.g., Clostridia (e.g., C. difficile, C. novyi, and C. sordellii), enterotoxigenic E. coli, Shigella, Vibrio cholerae, and Salmonella typhi), and genitourinary tract pathogens (e.g., human immunodeficiency virus, herpes simplex viruses, papilloma viruses, Treponema 35 pallidum, Chlamydia, and Neisseria gonorrhoeae).

W O 97/02835 PCT~US96/10987 Specific examples of antigens (e.g., non-replicatable polypeptide antigens) that may be used in the methods of the invention include, but are not limited to, bacterial toxins. For example, toxins from ~ 5 Clostridia (e.g., C. difficile, C. novyi, and C.
sordellii), such as C. difficile Toxin A and/or B Toxoid, C. novyi ~-toxin (Bette et al., Toxicon 29(7):877-887, 1991), C. sordellii lethal toxin (Bette et al., supra), and immunogenic fragments and derivatives thereof, may be 10 used. The antigens used in the methods of the invention may be obtained by standard methods known in the art, e.g., purification from a culture of the pathogen from which it is derived, recombinant DNA methods, and chemical synthetic methods.
The invention may employ Clostridium (e.g., C. difficile) toxoids as vaccine antigens. A toxoid is a toxin (or mixture of toxins, e.g., C. difficile Toxin A
and Toxin B) that has been treated so as to decrease the toxic properties of the toxin(s), but to retain 2 0 antigenicity. Toxoids included in the invention are made using stAn~A~d methods including, but not limited to, chemical (e.g., formaldehyde or glutaraldehyde) treatment, protease cleavage, and recombinant methods (e.g., by ~k;ng fragments or mutations (e.g., point 25 mutations) of the toxin(s)).
The method of the invention may be carried out in order to prevent or decrease the c-hAnc~ of a LuLule infection by a pathogen (i.e., to induce a protective ; c response) and/or to treat an ongoing infection (i.e., to induce a therapeutic ; ~ response). In the case of intestinal pathogens, for example, the method of the invention may be used to treat a ~ ~1 that is at risk of developing, but does not have, diarrhea caused by the pathogen (e.g., C. difficile), or a mammal that has 35 diarrhea caused by the pathogen. MA ~ which may be CA 02226392 l998-0l-06 W O 97/02835 PCTrUS96/10987 treated according to the method of the invention include, e . g ., humans, cows, horses, pigs, dogs, cats, sheep, and goats.
An advantage of the methods of the invention is 5 that, for at least some antigens (e .g., C. difficile toxins and toxoids), mucosal ad]uvants are not required for induction of an immune response (e.g., a protective immune response).
Other features and advantages of the invention 10 will be apparent from the following detailed description of the preferred embodiments thereof, and from the claims.

Detailed DescriPtion The drawings are first described.
15 Drawings Fig. 1 is a graph showing the levels of protection against C. difficile disease in hamsters ; ;zed with C. difficile antigens by the indicated routes. The levels of protection from systemic (death) and intestinal (diarrhea) disease a~ter cl;n~A ~cin challenge are shown.
(See Table 1 for a description of the ;~lln;~ation routes.) Fig. 2 is a graph showing the mean (+SE) antibody titers to C. difficile Toxin A, Toxin B, and whole cell 25 antigens in sera from hamsters after 3 doses of vaccine atl~; n; ~tered by the routes indicated, as determined by ELISA. (See Table 1 for a description of the routes of ; ;~ation.) Sera from hamsters after 3 doses of vaccine were assayed for specific IgG; the titer was 30 defined as the ~; dilution with an absorbance of >0.3. Each bar represents the mean (~SE) of five An; ~1 ~
Fig. 3 is a graph showing the biological activity of sera from hamsters ~t- ;n;~tered 3 doses of vaccine by W O 97/02835 PCTrUS96/10987 the indicated routes. Sera were tested for inhibition of cytotoxin A or cytotoxin B activity in IMR-90 cells, and for agglutination of C. difficile cells; titers were defined as the ~ 1 dilution with biological activity.
~ 5 Each bar represents the mean (+SE) of five An; ~ (See Table 1 for a description of the routes of ; ln; zation.) Fig. 4 is a graph showing the long term antibody response in i.n.i.p. and s.c. ; ln; zed hamsters.
Comparisons of the responses before clindamycin challenge (i.n.i.p.-I and s.c.-I) and 140 days after cl;n~ ycin challenge (i.n.i.p.-II and s.c.-II) are shown. Sera were tested by ELISA against Toxin A, Toxin B, and whole cell antigens, and the titers were expressed as the ~
dilution with absorbance >0.3; each bar represents the 15 mean (+SE) of five An;~
Fig. 5 is a graph showing the long term antibody response in i.n.i.p. and s.c. immunized hamsters.
C~ ~-~isons of the responses before clindamycin challenge (i.n.i.p.-I and s.c.-I) and 140 days after clindamycin 20 challenge (i.n.i.p.-II and s.c.-II) are shown. Sera was tested for inhibition of cytotoxins in IMR-go cells and for agglutination of C . difficile cells; the titer was the -~; =l dilution of serum with biological activity.
Each bar represents the mean (+SE) of five animals.
Figs. 6A-6B are graphs showing the anti-Toxin A
(Fig. 6A) and anti-Toxin B (Fig. 6B) IgA responses in serum, feces, saliva, and vaginal secretions of mice after intrAnA~ n;zation with toxoid, in the presence or absence of CT.
Figs. 7A-7C are graphs showing the serum anti-Toxin B cytotoxicity after intrAn~l immunization of mice with toxoid (Fig. 7A), the serum anti-Toxin A
cytotoxicity after intr~nA~s~l ; n; ~ation with toxoid ~ (Fig. 7B), and the salivary and vaginal secretion anti-CA 02226392 l998-0l-06 W O 97/02835 PCT~US96/10987 Toxin A cytotoxicity after intranasal immunization with toxoid (Fig. 7C).
Fig. 8 is a graph showing the level of passive protection of ligated small intestinal loops of rats from 5 Toxin A using sera from mice ; ;zed intranasally with toxoid.
Fig. 9 is a graph showing the percent survival of mice intranasally ; ;zed with toxoid after lethal challenge with Toxin A or Toxin B.
Fig. 10 is a graph showing the level of Toxin A
enterotoxicity in ligated intestinal loops of mice after intranasal immunization of toxoid.
Figs. llA-llB are graphs showing the Toxin A-specific systemic (Fig. llA) and mucosal (Fig. llB) IgA
15 responses after immunization with GST-ARU by the indicated routes. (See Table 5 for a description of the routes of immunization.) Figs. 12A-12B are graphs showing the levels of Toxin A cytotoxicity inhibition of sera taken 40 days 20 after ; ;zation with GST-ARU. (See Table 5 for a description of the routes of ; ;zation.) Figs. 13A-13B are graphs showing the levels of passive inhibition of Toxin A enterotoxicity in rat intestinal loops with ; ? sera from GST-ARU ; ;zed 25 mice. (See Table 5 for a description of the routes of ; ;~ation.) Fig. 14 is a graph showing the percent survival from lethal Toxin A challenge after ; ;~ation with recombinant Toxin A repeats (ARU). (See Table 5 for a 30 description of the routes of immunization.) Fig. 15 is a graph showing the levels of protection from enterotoxicity of Toxin A in ligated mouse intestinal loops after ; ;zation with GST-ARU.
(See Table 5 for a description of the routes of 35 ; ;~ation.) W O 97/02835 PCT~US96/10987 Intranasal and Combined Mucosal-Systemic Vaccination Methods for Inducinq Mucosal Immune Responses at Distal Sites ~ We have shown that intr~n~s~l, or combined mucosal 5 and systemic, administration regimens give rise to mucosal immune responses in the gastrointestinal and genitourinary tracts.
The methods of the invention may be used to induce protective and/or therapeutic ; c responses to 10 gastrointestinal pathogens including, but not limited to, Helicobacters (e.g., H. pylori, H. felis, and H.
hei71 ~nii) Campylobacters (e.g., C. jejuni), and pathogens which cause diarrhea and colitis, e.g., Clostridia, enterotoxigenic E. coli, Shigella, Vibrio 15 cholerae, and Salmonella typhi; or genitourinary tract pathogens (e.g., human immunodeficiency virus, herpes simplex viruses, papilloma viruses, Treponema pallidum, Chlamydia, and Neisseria gonorrhoeae). Appropriate vaccine antigens (e.g., polypeptide antigens), 20 corresponding to the pathogen which causes the condition desired to be prevented and/or treated using the method of the invention, are readily selected by one skilled in the art. The methods of the invention are described, as follows, referring to antigens from c. difficile (e.g., 25 toxins or toxoids) as specific examples of vaccine antigens which may be used in the methods of the invention.

Use of C. difficile toxins and toxoids as vaccines C. difficile toxin polypeptides which may be used 30 in the methods and compositions of the invention can be prepared using any of several st~n~d methods. For example, the toxins (e.g., Toxin A and/or Toxin B) can be purified from C. dif~icile culture filtrates (see, e.g., CA 02226392 l998-0l-06 W O 97/0283S PCT~US96/10987 Kim et al., Infection and Immunity 55:2984-2992, 1987;
and see Example I, below).
C. difficile toxin polypeptides can also be produced using standard recombinant DNA methods (see, 5 e.g., Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1994). In these methods, a suitable host cell is transformed with an appropriate expression vector containing all or part of a toxin-encoding nucleic acid fragment (see Dove et al., Infection and Immunity 58:480-488, 1990, and Barroso et al., Nucleic Acids Research 18:4004, 1990, for the nucleotide and deduced amino acid sequences of C. Difficile Toxin A, and the nucleotide sequence of Toxin B, respectively). Any of a variety of expression 15 systems can be used to produce the recombinant toxins.
For example, the toxin polypeptides can be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., yeast cells (e.g., Saccharomyces cerevisiae), - -l;an cells (e.g., COSl, NIH3T3, or JEG3 cells), or 20 arthropod cells (e.g., Spodoptera frugiperda (SF9) cells)). Such cells are available from a number of different sources known to those skilled in the art, e.g., the American Type Culture Collection, Rockville, MD
(also see, e.g., Ausubel et al., supra). The 25 transfection/transformation method used, and the choice of expression vector, will depend on the host system selected, as is described by, e.g., Ausubel et al., supra. Expression vectors (e.g., plasmid or viral vectors) can be chosen from, e.g., those described in 30 Cloning Vectors: A Laboratory MAn77A7 (Pouwels et al., 1985, Supp. 1987; also see, e.g., Ausubel et al., supra).
C. difficile toxin polypeptides, particularly short ~ragments, can also be proAIlc~ by chemical synthesis, e.g., by the method described in Solid Phase 35 Peptide Synthesis, 1984, 2nd ed., Stewart and Young, W O 97/02835 PCT~US96/10987 Eds., Pierce Chemical Co., Rockford, IL, and by standard in vitro translation methods.
Toxoids of C. di f f icil e toxins can also be used in the methods of the invention. A toxoid is a toxin that 5 has been treated so that the toxicity of the toxin is eliminated or reduced, but the antigenicity is maintained. Toxoids may be prepared using stAn~d methods, for example, by chemical (e.g., glutaraldehyde or formaldehyde) treatment (see, e.g., Libby et al., 10 Infection and Immunity 36:822-829, 1982). Toxoids may also be prepared by making mutations in the genes encoding the toxins and expressing the mutated genes in an expression system, as is described above. Regions in Toxin A and/or Toxin B that can be mutated include, e.g., 15 the conserved cysteine residues, the nucleotide binding region, the internal hydrophobic region, and/or the carboxyl-teL ;n~l repeat regions. Specific examples of such mutations in C. di f f icil e toxins which can be used in the invention are described by, e.g., Barroso et al ., 20 Microbial Pathogenesis 16:297-303, 1994.
Other methods of producing toxoids that can be used in the invention include chemical modification of amino acids which are critical for toxicity, but are not related to antigenicity. For example, reagents which 25 specifically modify SH-cont~;n;ng amino acids, lysine, tyrosine, tryptophan, or histidine residues are known in the art (see, e.g., Cohen et al ., Ann. Rev. Biochem.
37:683-695, 1968). In addition, azido-linked substrate analogs, such as UDP-glucose, which can be covalently 30 linked to toxin active sites by ultraviolet irradiation, can be used to produce toxoids.
~ In addition to native, full length, C. difficile toxins, polypeptide fragments of toxins, or toxins (or polypeptide fragments of to~;n~) cont~;n;ng mutations (which may or may not be toxoids) can be used in the W O 97/02835 PCT~US96/10987 invention, provided that antigenicity is retained. For examples of fragments o~ C. di~icile toxins, see, e.g., Price et al ., Current Microbiology 16:55-60, 1987; Lyerly et al., Current Microbiology 21:29-32, 1990; and Frey et 5 al ., Infection and T ;ty 60:2488-2492, 1992. Genes encoding fragments of C. difficile toxins, and/or toxins cont~;n;ng mutations, are made using st~n~d methods (see, e.g., Ausubel et al., supra). Fragments, derivatives, and toxoids included in the invention can be 10 screened for antigenicity using standard methods in the art, e.g., by measuring induction of a mucosal immune response (see below), induction of protective ; ;ty (see below), or induction of a therapeutic immune response.
Although not required, adjuvants may be a~;n;~tered with the vaccines in the methods of the invention. Any of a number of adjuvants that are known to one skilled in the art may be used. For example, a cholera toxin (CT), the heat-labile enterotoxin of 20 Escherichia coli (LT), or fragments or derivatives thereof having adjuvant activity, can be used for mucosal ~;n;~tration. An adjuvant such as RIBI (ImmunoChem, Hamilton, NT) or aluminum hydroxide can be used for parenteral a~;n;~tration.
Fusion proteins cont~;n;ng a C. difficile toxin (or a fragment or derivative thereof) ~used to, e.g., an adjuvant (e.g., CT, LT, or a fragment or derivative thereof having adjuvant activity), are also included in the invention, and can be prepared using st~n~d methods (see, e.g., Ausubel et al., supra). In addition, the vaccines of the invention can be covalently coupled or cross-linked to adjuvants. Methods for covalently coupling and chemically cross-linking adjuvants to antigens are described by, e.g., Cryz et al., Vaccine 35 13:67-71, 1994; Liang et al., J. Immunology 141:1495-W O 97/02835 PCT~US96/10987 ~501, 1988; and Czerkinsky et al., Infection and Immunity 57:1072-1077, 1989.
- As is mentioned above, vaccine compositions (with or without adjuvants) are a~;n;ctered intranasally ~ 5 according to the methods of the invention. Combined modes of al~ ;n;stration may also be used, e.g., the first dose of the vaccine can be a~;n;-ctered to a mucosal (e.g., intranasal or oral) surface, and booster immunizations can be administered parenterally (e.g., 10 intraperitoneally or subcutaneously); this combination gives unexpectedly good results. For example, a parenteral booster ;~lln;zation may be given one week after the first, mucosal a~;n;ctration.
The amount of vaccine a~ ; n; -ctered depends on the 15 particular vaccine antigen, whether an adjuvant is co-a~; n; ctered with the vaccine antigen, the mode and frequency of a~ ; n; ctration, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art. In general, the vaccine 20 antigens of the invention are a~ ; n; ctered in amounts ranging between, e.g., 1 ~g and 100 mg. If adjuvants are ~- ;n;stered with the vaccines, amounts ranging between, e.g., 1 ng and 1 mg can be used. A~' ;n;~:tration is repeated as n~c~ccA~y, as can be determined by one 25 skilled in the art. For example, a priming dose can be followed by 3 booster doses at weekly intervals.
Vaccines may be administered in any pharmaceutically acceptable carrier or diluent (e.g., water, a saline solution (e.g., phosphate-buffered saline), or a 30 bicarbonate solution (e.g., 0.2 M NaHC03)). The carriers and diluents used in the invention are selected on the ~ basis of the mode and route of ~l ;n;stration, and st~n~d pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as 35 pharmaceutical necessities for their use in CA 02226392 l998-0l-06 W 097/02835 PCTrUS96/10987 pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a stAn~A~d reference text in this field, and in the USP/NF.
The following examples are meant to illustrate, 5 but not to limit, the methods of the invention.
Modifications of the conditions and parameters set forth below that are apparent to one skilled in the art are included in the invention.

EXAMPLES
Two model systems, the mouse and the hamster, were used to evaluate the vaccination methods of the invention. Because hamsters are susceptible to antibiotic-associated diarrhea which is similar to that of humans, the hamster model was used to directly 15 evaluate the protective efficacy of vaccination against C. difficile disease. C. difficile infection of hamsters causes severe hemorrhagic cecitis, which is r~ ; n i ~cent of the colitis observed in the human disease state. In addition, oral or systemic A~- ;n; ~tration to a hamster of 20 a single dose of clindamycin, in combination with C.
difficile, results in severe diarrhea, which ultimately leads to death of the An; ~ l ~
Using a variety of assays, the hamster model may also be used to monitor the ; e response ;n~llc~ by 25 the vaccination methods of the invention. For example, serum and mucosal samples from immunized hamsters can be used to measure inhibition of in vitro cytotoxicity. In addition, ligated intestinal loops of immunized hamsters can be used to evaluate the inhibition of the enterotoxic 30 activity of Toxin A induced by vaccination. Further, colonization of hamsters with C. di f f icil e can be monitored by fecal culture, or the presence of Toxin A
and/or Toxin B in hamster feces can be determined by ELISA and/or cytotoxicity analysis.

WO 97/0283S PCTrUS96/10987 Features of the mouse model are advantageous in evaluating the immune responses induced by the ~ vaccination methods of the invention. Specifically, monoclonal antibodies which recognize mouse IgA are - 5 commercially available, and thus facilitate evaluation of the mouse mucosal immune response. In contrast, such reagents are not available for evaluating the hamster mucosal immune response. An additional advantage of the mouse model is that methods for sampling mouse mucosal 10 surfaces have been developed which allow mucosal responses to various ; ;zation regimens to be mapped.

once the immunogenicity of a vaccine candidate is establ ;~::h~ by, e.g., ELISA analysis, mouse serum samples can be used to investigate properties of the antibodies 15 which are likely to be associated with effective vaccines. For example, serum from immunized mice can be analyzed for its ability (1) to inhibit in vitro cytotoxicity of Toxin A and/or Toxin B, or (2) to inhibit the enterotoxicity of Toxin A using ligated intestinal 20 loops of mice or rats challenged with Toxin A. T ln; zed mice may also be challenged orally, or in their ligated intestinal loops, to determine protection from death or fluid ac~~ tion due to Toxin A enterotoxicity.
Finally, ; ln;zed mice may be challenged with toxins 25 systemically with doses known to be lethal.

ExamPle I. T -; zation of Hamsters with Vaccine Compositions Containinq C. difficile Toxins The following methods were used to analyze the efficacy of the immunization methods of the invention in 30 the hamster model system.
Preparation of a C. difficile Toxoid Vaccine C. difficile culture filtrate was prepared and inactivated as described by Libby, et al. (Infection and CA 02226392 l998-0l-06 W 097/02835 PCT~US96/10987 T ln;ty 36:822-829, 1982). Briefly, C. difficile VPI
strain 10463 (ATCC accession number 43Z55) was grown for 3 days in dialysis flasks, centrifuged, and filter sterilized. One ml of formaldehyde was added to 100 ml 5 of the culture filtrate, and the mixture was incllh~ted at 37~C for 1 hour. The culture filtrate had a concentration of approximately 50 ~g/ml of Toxin A, as determined by ELISA (Lyerly, et al., Infection and T - ; ty 47:349-352, 1985), and a cytotoxic titer of 106 10 for Toxin B, as determined by a cell culture cytotoxicity assay (Ehrich, et al ., Infection and Immunity 28:1041-1043, 1980). The toxoid was washed with 3 volumes of phosphate buffered saline (PBS), pH 7.4, by ultrafiltration through a 30 kD membrane in a 500 ml cell concentrator (Amicon, Beverly, MA). The toxoid was concentrated 10-fold, filter-sterilized, and stored at 4~C until used. Based on the sizes of the toxins ( 308 kD
for toxin A and 269 kD for toxin B), no significant loss of toxin protein during the ~onc~ntration step was assumed, and a concentration of 500 ~g/ml of each inactivated toxin in the lOx solution was estimated. The toxoid material was devoid of any detectable cytotoxic activity against IRM-90 cells (ATCC accession number CCL
186).
25 Proparation of a Whole Cell Vaccine C. difficile VPI strain 10463 (ATCC accession ll~ h~ 43255) was grown in proteose peptone--yeast extract media (PPY; Holbrook, et al. J. Appl. Bacteriol. 42:259--273, 1977) at 37~C for 36 hours under anaerobic conditions to ;n; ;~e spore formation. The cultures were centrifuged and the pelleted cells were washed 3 times with PBS. After the final wash, the pelleted cells were resuspended in PBS cont~;n;n~ 1% (vol:vol) formaldehyde and ;ncllh~ted at 4~C for 24 hours. ~Yc~
formaldehyde was ~ t- ~ ed by 3 washes with PBS, and the CA 02226392 l998-0l-06 W O 97/02835 PCTrUS96/10987 formalinized C. difficile cell suspension was stored at 4~C. Inoculation of the equivalent of 109 C. difficile colony-forming units (CFU) (a cell suspension with an O.D. of 1.0 at 550 nm) into PPY media yielded no growth 5 after 36 hours of culture at 37~C under anaerobic conditions.
Animals Female Syrian hamsters (Mesocricetus auratus, Charles River, Kingston, NY), 6-8 weeks old at the time 10 of immunization, were used in all of the experiments.
The ~3n; :~ls were caged in groups of 5 during the immunization period, and then caged individually during C . di f f icil e challenge.
Immunization Regimons Seven different ; ;zation regimens were analyzed (Table 1). For intranasal (i.n.) ; n;zation, 5 ~g of each toxoid (inactivated Toxin A and inactivated Toxin B), in 10 ~1 of the lOx toxoid, were mixed with a 5 ~1 solution contA;n;ng 5 ~g of cholera toxin (Calbiochem, 20 La Jolla, CA). The 15 ~1 antigen-adjuvant mixture was a~; n; ~tered into the external nares of the hamsters with a micropipettor, with half of the dose al ; n; ~tered to each nostril. For intragastric (i.g.) ; ;~ation, 100 ~g of each toxoid were mixed with 10 ~g of cholera toxin, 25 adjusted to a volume of 1 ml with PBS, and al~ ;n;~tered by gavage. For intraperitoneal (i.p.) and subcutaneous (s.c.) immunizations, 5 ~g of each toxoid were mixed with 0.3 ml of RIBI adjuvant (RIBI, ImmunoChem, Hamilton, NT).
For rectal (r.) ; ln;~ation, 50 ~g of each toxoid, in 30 100 ~1 of toxoid, were mixed with a 1 ~1 solution cont~in;ng 10 ~g of cholera toxin. For rectal al ;n;~tration of whole cells (w.c.r.), 5 x 108 cells were mixed with 50 ~g of each toxoid, in 100 ~1 of - toxoid, plus a 1 ~1 solution cont~;n;ng 10 ~g cholera 35 toxin. For both r. and w.c.r. groups, the sample was WO 97/02835 PCTrUS96/10987 applied with a disposable 20 x 1~ feeding needle inserted 3 cm into the rectum. The i.n., i.g., i.p., and s.c.
i ;~ations were performed in An;~1~ lightly anaesthetized with isofluorane. The r. and w.c.r.
5 immunizations were done in pentobarbital anaesthetized An; ~ A control intranasal group (c.i.n.) received 5 ~g of cholera toxin intranasally. A control subcutaneous group (c.s.c.) received 0.3 ml of RIBI adjuvant subcutaneously. Groups of 5 An; ~l ~ were used for all 10 immunization regimens. All groups received a total of 4 doses of the vaccine (or adjuvant control) on days 0, 7, 14, and 28 of the experiment.

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~ 0 1~ 0 W 097/02835 PCTrUS96/10987 To evaluate the immune responses, samples (200-400 ~1) of blood were obt~;ne~ on days 0, 2, 4, 7, and 36 from the retro-orbital sinus of the hamsters under isofluorane anesthesia. The blood was left to clot 5 overnight at 4~C, and the serum was obtained by centrifugation. Only serum antibodies were evaluated;
secretory IgA was not measured because of a lack of a suitable anti-hamster IgA reagent. After C. di f f icil e challenge, a sample of feces was obtained every other day 10 from the surviving animals and mixed with 2 volumes of ppy ~~; A for evaluation of the degree of colonization and presence of toxins (see below).
C. di~icile Challenge All hamsters were challenged on day 38 (10 days 15 after the 4th ; n;zation) with 0.5 mg of clindamycin a~;n;~tered orogastrically, followed 3 hours later by an orogastric inoculation of 105 CFU of viable C. dif~icile 10463 strain (ATCC accession 11l h~ 43255), which were washed with PPY media, in order to ~l;~;nAte free tox;n~.
20 After challenge, the hamsters were observed daily for diarrhea and illness. The severity of the diarrhea was scored as: 0, no diarrhea; 1+, loose feces, but no wet tail; 2+, peri-anal and tail region wet; and 3+, tail, paws, and lower abdomen wet (An; ~lc with this appearance 25 were usually hunched and inactive).
Ev~luation of Tis3ue Damage Severely ill hamsters were euthanized. Samples of cecum from the euthAn;~ed hamsters, and from the survivors from every ; ;7ation regimen, taken 8 days 30 after cl;n~A y~in challenge, were fixed in 10% neutral buffered formalin. FOL -1 ;n--fixed tissues were embedded in paraffin, sectioned at 5 ~M, s~;ne~ with hematoxylin and eosin, and ~A ; ned by light microscopy. Histologic grading criteria were: 0, minimal infiltration of 35 lymphocytes, plasma cells, and eosinophils; 1+, mild CA 02226392 l998-0l-06 infiltration of lymphocytes, plasma cells, neutrophils, and eosinophils, plus mild congestion of the mucosa, with or without hyperplasia of gut associated lymphoid tissue;
2+, moderate infiltration of mixed inflammatory cells, - 5 moderate congestion and edema of the lamina propria, with or without goblet cell hyperplasia, individual surface cell necrosis or vacuolization, and crypt dilatation; and 3+, severe inflammation, congestion, edema, and hemorrhage in the mucosa, surface cell necrosis, or 10 degeneration with erosions or ulcers.
Evaluation of Infections Feces obtained after clindamycin challenge were studied for the presence of C. difficile. Ten-fold dilutions in PPY media were inoculated onto selective 15 media cont~;ning cycloserine (125 ~g/ml) and cefoxitin (8 ~g/ml), and colonies were counted after 48 hours of incubation under anaerobic conditions. The presence of Toxin A in feces was determined using a Toxin A kit ~T~hT~h, Blacksburg, VA), as described by the 20 manufacturer. After 15 minutes with substrate, the O.D.
was read at 450 nm, and the concentration of toxin was estimated from a st~n~d curve of Toxin A prepared in each plate. The estimations were carried out using Softmax software (Molecular Devices, Sunnyvale, CA). For 25 quantification of Toxin B, fecal suspensions were centrifuged and filter-sterilized, and ten-fold dilutions of the samples were tested for cytopathic effects on IMR-90 fibroblast cell cultures, as is described below.
E~ISA for Ant;~o~;es to Toxin A and Toxin B
Microtiter plates (Corning, New York, NY) were coated with 100 ng/well of purified Toxin A or Toxin B in carbonate-bicarbonate buffer, pH 9.3, and incubated overnight at 4~C. The plates were washed and blocked with 2.5% non-fat dry milk (NFDM) in phosphate buffered 35 saline solution (PBS), pH 7.4. Serum samples were added CA 02226392 l998-0l-06 W 097/02835 PCT~US96/10987 at two-fold dilutions ranging ~rom 1:500 to 1:64,000, and the plates were incubated for 1 hour at 37~C. Anti-hamster IgG (1:1000, Southern Biotech, Birmingham, AL) conjugated with alkaline phosphatase, was added, 5 incubated for 1 hour at 37~C, and washed prior to addition of a p-nitrophenyl phosphate substrate. A
positive control was included in each plate; wells were coated with Toxin A or Toxin B in two-fold dilutions ranging from 100 to 0.8 ng/ml, and reacted with specific 10 goat anti-toxin (T~-hT~h), followed by an anti-goat IgG
alkaline phosphatase conjugate. Negative controls were wells coated with purified toxin and reacted with an anti-hamster IgG alkaline phosphatase conjugate. The O.D. was read at 405 nm, and the titer was defined as the 15 reciprocal of the highest dilution of sample giving an O.D. 2 0.3-~rT~ for Ant;~o~;e~ to Whole Cell Antigen Plates were coated with 100 ~1 of a formalin-killed C. difficile suspension adjusted to an O.D. of 0.2 at 550 nm, and then ;ncllh~ted overnight in an orbital shaker at 150 rpm. The cells were fixed to the plates by ;n~ tion at 70~C for 2 hours. After washing, serum samples were added at two-fold dilutions ranging from 1:100 to 1:12,800 and incubated for 1 hour at 37~C.
25 Anti-hamster IgG and substrate were added as is described above. A positive control was included in each plate using mouse C. difficile whole cell antiserum at 1:500 to 1:64,000. (The antiserum was produced against VPI strain 10463, using st~n~d methods). The wells coated with 30 whole cells were reacted directly with the anti-hamster IgG Al~ ;ne phosphatase conjugate as negative controls.
Inhibition o~ Cytotoxicity IMR-90 fibroblast cells were grown to confluence in 96-well plates in D-MEM media (Gibco, Grand Island, 35 NY) cont~;n;ng 10% fetal calf serum. The ;n; ~1 dose of CA 02226392 l998-0l-06 W 097/02835 PCTfUS96/10987 - 21 -~
Toxin A or Toxin B needed to cause 100% rounding of the cells was defined as 1 cytotoxic unit (CTUloo). For - Toxin A, 6.3 ng/ml, and for Toxin B, 125 pg/ml, were defined as 1 CTUloo. Two-fold dilutions of the hamster - 5 serum samples, ranging from 1:100 to 12,800, were mixed with 4CTUloo of either toxin, incubated for 1 hour at 37~C, and the mixture was then added to the cells. Goat anti-Toxin A and goat anti-Toxin B served as positive controls. Cells were observed after 24 hours, and the 10 proportion of round cells was determined. The titers of the samples were defined as the reciprocal of the highest dilution of sera inhibiting 250% cell rounding.
Agglutination Twenty-five ~l samples of hamster serum were diluted ranging from 1:25 to 1:3,200. The dilutions were prepared in 96-well U-bottom microplates (Falcon, Oxnard, CA). The formalin-killed C. difficile suspension was adjusted to an O.D. of 1.O at 550 nm, and 25 ,ul of the suspension were added to the serum dilutions. Mouse anti-C. di f f icil e whole cell anti-serum served as a positive control, and PBS was used as a negative control.
The plates were incubated overnight at 4~C, and the agglutination was then scored. Endpoint titers were defined as the reciprocal of the highest dilution of serum causing agglutination.
Western Blot Analysi~
C. difficile VPI strain 10463 (ATCC accession number 43255), and the strains isolated from hamsters after cl;n~lA ycin challenge were grown in 5 ml of PPY
media at 37~C under anaerobic conditions for 36 hours.
The cultures were centrifuged and the pellets were washed three times with PBS. The pellets were resuspended in 250 ,ul of 3% SDS in PBS, and the lysates were fractionated by electrophoresis in a 12% preparative SDS-polyacrylamide gel (Bio--Rad, Hercules, CA) at 200 volts W O 97/02835 PCT~US96/10987 for 1 hour. Proteins were transferred from the gel to nitrocellulose at 150 volts for 1. 2 hours in a Bethesda Research Laboratories Mini-V 8-10 chamber (Life Technologies, Grand Island, NY). The membranes were 5 blocked with 5~ non-fat dry milk in PBS for 1 hour, washed, and mounted in a multiscreen apparatus (BioRad, Hercules, CA). A 1:200 dilution of each hamster serum sample was then added and incubated for 1 hour, and the reaction was developed with NBT/BCIP (Gibco, 10 Gaithersburg, MD). Mouse anti-C. difficile 10463 whole cell serum served as a positive control. To type C.
di~ficile strains isolated from feces, SDS-lysates from isolates were fractionated by electrophoresis, transferred to nitrocellulose, and reacted with whole 15 cell mouse antiserum, as is described above.
Statistical Analysis The ; - responses to the different C. difficile antigens was studied for possible significant correlation with the outcome of the hamsters after clindamycin challenge using the Kruskal--Wallis test (Quick-STATISTICA
software, StatSoft, Tulsa, OK).

RES~LTS
outcome After Clindamycin Challenge Hamsters were challenged with cl ;n~ ycin and 25 C. difficile 10 days after the last ; ;zation. All sham-; ;~ed, control intranasal (c.i.n.), and control subcutaneous (c.s.c.) ~n; ~1~ died within 48 hours of challenge, most with severe (3+) diarrhea. Acute, diffuse necrohemorrhagic typhlitis (grade 3+) was found 30 on pathologic ~;n~tion. Crypt epithelium was hyperplastic and dilated crypts were filled with neutrophils. Lymphocytes, plasma cells, and neutrophils infiltrated the lamina propria. Vaccinated hamsters that suc~;, h-~-l to challenge also died within the first 48 CA 02226392 l998-0l-06 W O 97/02835 PCTrUS96/10987 hours, and most had grade 3+ diarrhea and grade 3+
typhlitis on histopathologic ~ ;n~tion. Animals that - survived challenge either had no diarrhea or had diarrhea ranging in severity from 1+ to 3+. The severity of 5 diarrhea correlated with the severity of typhlitis on pathologic e~A~;nation. Animals with 3+ diarrhea had subacute, diffuse mucopurulent typhlitis grades 2-2.5+.
Neutrophils, lymphocytes, and plasma cells infiltrated the lamina propria, and multifocal crypt abscesses also 10 were noted. Those ~n;~ with 2+ diarrhea had subacute to chronic, moderate typhlitis grade 1.5-2+. ~ni ~1~
with mild diarrhea (l+) had a mild lymphocytic typhlitis, grades 1.0-1.5+. Mild lymphocytic typhlitis grade 1+ was also evident in hamsters without diarrhea.
The outcome of the cli n~- ycin challenge in all vaccine groups is shown in Fig. 1. Mucosally ; ln; 7ed ~n; ~1~ that received toxoid vaccine by the intragastric (i.g.) or rectal (r.) routes, or whole cell-toxoid vaccine by the rectal route (w.c.r.), were ~;n; ~lly 20 protected against death, and all had diarrhea. The r.
and w.c.r. ;~lln;zation regimens protected only 20% o~
hamsters, while the i.g. regimen protected 40% of hamsters against death. Parenterally ; ;zed (i.p. and s.C. ) ~n; ~1 ~ were fully protected against diarrhea. A
25 similar outcome was observed in ~n; ~1~ intranasally immunized (i.n.). When i.n. immunized ~n; ~1 S received a booster dose of toxoid intraperitoneally (group i.n.i.p.), 100% protection was achieved against both death and diarrhea.
30 Pr~ne~ of C. dif~icile and Toxins in Feces Fecal samples obtained after clindamycin challenge were analyzed for the presence of C. difficile, Toxin A, and Toxin B. A similar pattern was observed in all surviving ~n;~l ~. Two days after challenge, C.
35 difficile reached approximately 109 CFU/ml in feces;

CA 02226392 l998-0l-06 thereafter, colonization decreased slightly and remained at about 108 CFU/ml for at least 9 days, regardless of the presence of diarrhea. In contrast, the levels of Toxin A and Toxin B steadily decreased, and by day 9, 5 almost no toxins were found in feces, in spite of continuous colonization. All isolated C. difficile strains were typed by Western blot analysis using whole cell antiserum against the VPI 10463 strain (ATCC
accession number 43255). Strains isolated from different 10 animals and from different ; ln; zation groups were analyzed, and were all found to be similar to one another, suggesting that only one strain was colonizing all hamsters. However, this strain was different from VPI 10463, which is the strain used for challenge.
I une Responses Serum antibodies against C. difficile antigens were measured in hamsters from all experimental groups.
Immune responses to Toxin A after the priming ; ln;~ation were studied by ELISA in some of the groups.
20 No specific IgG was detected in An;~lc vaccinated by the i.n.i.p., r., and w.c.r. routes on days 2, 4, and 7 after the initial vaccine dose. In the parenterally ;-~lln; zed An; -lc (i.p. and s.c.), no response was evident after days 2 and 4, but at day 7, a slight rise in antibody titer was observed. In contrast, the antibody responses measured after the last vaccine dose (day 36) demonstrated sero-conversion in all groups. The absence of early (anamnestic) antibody responses to the first vaccine dose shows that the ~n; ~l c were immunologically naive and had not previously been primed with Toxin A.
Three approaches were used to study the systemic antibody responses to C. di~icile antigens: 1) recognition of immobilized antigens by ELISA; 2) inhibition of cytotoxicity in a cell culture assay; and 3) agglutination of bacteria. Toxin A, Toxin B, and CA 02226392 l998-0l-06 W O 97/02835 PCTrUS96/10987 whole cells were used as antigens. Antibody responses to Toxin A, Toxin B, and whole cell antigen were present in all groups, as determined by ELISA (Fig. 2). Hamsters immunized by the i.n.i.p., i.n., i.g., and i.p. regimens 5 had higher responses against Toxin A and Toxin B, than those immunized by the rectal (r. and w.c.r.) and s.c.
routes. Antibody levels against whole cell antigens showed a pattern similar to that observed with toxins.
Antibodies to whole cell antigens were further 10 characterized by Western blot analysis with whole cell lysates from the C. difficile strain isolated from the hamsters after cl; n~ ycin challenge. Animals in all immunization regimens developed antibodies to a 70 kD
protein and to proteins with sizes of 2200 kD (which are 15 likely to be the toxins); ~n; ~ n; zed by the i.n.i.p. route had the strongest immune responses. A
variety of other proteins were recognized by sera from ~n; ~l~; ;zed by the i.n.i.p., i.p., and s.c. routes, but these proteins were not apparent, or were less 20 prominent, in sera from animals ; -n; zed mucosally (r.
and w.c.r. groups). In other assays, hamster serum was immunoblotted in parallel against purified toxins and whole cell lysates. These assays demonstrated that the lower molecular weight proteins which reacted with the 25 hamster sera were not toxin fragments.
Serum antibodies with biological functions showed a different pattern from that obtained by ELISA (Fig. 3).
Antibodies inhibiting cytotoxicity by Toxin A were elicited in all An;~ Hamsters ; ;zed by the 30 i.n.i.p. and i.p. routes developed the highest anti-toxin A activity (mean +SE 22,000+4,900 and 18,000+2,000, respectively), whereas mucosally ;~lln;zed ~n;~l ~ (i.n., i.g., r. and w.c.r.) had lower activities (580+280, 280+146, 1720+560, and 2760+290, respectively). High 35 anti-Toxin B responses were obtained in all groups, CA 02226392 l998-0l-06 W O 97/02835 PCTrUS96/10987 except in rectally ; ;zed An;~lc. Agglutinating antibodies were elicited only in An; ~ls that received toxoid vaccine parenterally (i.p. and s.c.), or by a combined mucosal-parenteral route (i.n.i.p.).
5 Correlation of the I7n7nune Re~ponse and Protection The i.n.i.p. immunized An; ~lc were fully protected against death and diarrhea and had the highest serum immune responses when both ELISA and biological activity were considered (Figs. 2 and 3). Complete 10 protection against death was provided by all ; 7n; ~ation schemes that included parenteral injection of the vaccine or intranasal ; ;zation alone. In contrast, rectally ; -n; ~ed An; ~ls had the lowest protection ratios and serum antibody responses, particularly neutralizing 15 antibody against Toxin B (Fig. 3). Immunological correlations were not consistent, however, as illustrated by the similar antibody responses in i.n. and i.g. groups (Figs. 2 and 3), despite the greater protection afforded by the i.n. vaccine (Fig. 1).
To define the immunologic correlations, animals from all groups were analyzed together, and the outcome of the challenges was compared with the immune responses (Table 2). Mean antibody levels in all tests, except the whole cell ELISA, were significantly higher in survivor 25 2~n; -lc than in ;~n; -l.c with a lethal outcome. Hamsters with severe diarrhea (3+) had significantly lower serum ; c responses by all assays, as compared to those An; - l ~c without diarrhea (Table 3). Antibody responses in hamsters with mild-moderate diarrhea (1+ and 2+) did 30 not differ significantly from those without diarrhea, except for the agglutinating antibody responses in those with l+ diarrhea (p<. 05).

CA 02226392 l998-0l-06 W 097/02835 PCTrUs96/l0987 Table 2. Correlation betwQen i ~une response against C. dif~icile antigens and protection against death Antibody titer according to:
deathsurvival (n=ll)(n=23) T --%~ay Total ~ean + SEMean i SEp valuea studi~d (n=34) Toxin A ELISA 5210i9602750il3606390ill80 .0290 5 Toxin B ELISA 8610il7603800i2840lO910i2050 .0063 Whole-cell ELISA 6560il8908363+56105704+980 .1382 Anti-cytotoxin A 7370il6201470i39010195i2170 .0001 Anti-cytotoxin B 8690il9001790il2409330i2610 .0197 C. dif~icile 130+25 25ilO 180+31 .0032 agglutination aKruskal-Wallis test Table 3. Correlation between i ;une response against C. di~icil e antigens and severity of diarrhe~

no diarrhea diarrhea, 3+
(n=10) (n=9) I., inno~s-y Mean + SE Mean + SE p valuea toxin A ELISA9400+19701333+160 .0003 toxin B ELISA17,800+3430870+230 .0001 whole cell ELISA 9000+1660 1820+310 .0001 anti-cytotoxin A15840+36903220+610 .0016 anti-cytotoxin B11540+4770670+440 .005 C. difficile 265+49 55+28 .0098 agglutination ap values when compared with the no diarrhea group, Kruskal-Wallis test.

W O 97/02835 PCTrUS96/10987 Long Tel~n Protection Four surviving ~ni ~l~ from the i.n.i.p. group and four from the s.c. group were held for a period of 140 days after clindamycin challenge. On day 140, samples of 5 blood and feces were taken, and the ~n;~l ~ were re- -challenged with cl;n~ ycin. Three of four ~n;~l~ (75%) in each group survived re-challenge. Two of four ~n;~
(50~) in the i.n.i.p. group and 0/4 (0~) in the s.c.
group were protected against diarrhea. The ; lne 10 responses before re-challenge were compared with the responses obtained before the first challenge. In the ELISA analysis, Toxin A and Toxin B antibodies were not reduced, although levels against whole cell antigens were markedly decreased (Fig. 4). When the biological 15 activities were compared, marked decreases in anti-cytotoxin activity and in C. difficile agglutination were observed prior to re-challenge (Fig. 5).

Example II. Immunization of Mice with Vaccine Compositions Containinq C. di f f icil e Toxins The following methods were used to analyze the efficacy of the i ization methods of the invention in the mouse model system.
ELISA
The ELISA methods used in the following 25 experiments are described above. Briefly, toxin-specific immune responses were detected by coating 96-well plates with Toxin A or Toxin B, blocking the wells with skim milk in PBS-tween, addition of samples from the mice, and detection with a ~- ~cial anti-mouse alkaline 30 phosphatase (AP) conjugated reagent. The plates were developed with Sigma 104 alkaline phosphatase (AP) substrate (Sigma Chemical Company, St. Louis, MO). Data from these assays are represented as the absorbance at 405 nm (see below).

CA 02226392 l998-0l-06 W 097102835 PCT~US96/10987 Cytotoxicity Both Toxin A and Toxin B mediate cytotoxicity against a variety of cell lines. The toxicity is manifested by the rounding of fibroblast cells (IMR-90).
5 IMR-90 cells are sensitive to 10-100 pg of Toxin A and 0.1-1.0 pg of Toxin B. The dosage of toxins used in cytotoxicity inhibition experiments corresponds to 8x the amount required to cause rounding of 50~ of a confluent monolayer of IMR-90 cells. Serum or secretions were 10 diluted appropriately and mixed with either Toxin A or Toxin B for 1 hour at 37~C. The toxins were then added to confluent wells of a 96-well cell culture plate and incubated overnight. Plates were read using a phase contrast microscope. Data are presented as the highest 15 dilution that protects 50~ of the monolayer from rounding.
~accino Preparation Toxoid and recombinant Toxin A (GST-ARU) were prepared as is described above.
20 Enterotoxicity Toxin A enterotoxicity was assessed using ligated intestinal loops challenged with Toxin A. Antibodies inhibiting enterotoxicity were measured by challenging loops with Toxin A pre-incubated with sera or secretions 25 cont~;n;ng antibodies. Rats were fasted prior to use, anesthetized, and sections of intestine were tied off.
Each loop contains intact blood vessels and is free of feces. Toxin A (1-10 ~g) was administered to the lumen of each loop, with and without pre-treatment with ; c 30 sera. After 4 hours, the loops were removed and the contents weighed. Mouse and hamster loops can be directly challenged in a similar fashion to determine the efficacy of ; ;zation against enterotoxicity. In the mouse loop assay, the volume in PBS treated loops is W O 97/02835 PCT~US96/10987 compared to Toxin A treated loops. The data is presented as the mg of contents per cm of ligated loop.
8y~temic Challenge Mice were challenged with lOx the LD50 of each 5 toxin al~ ; n; ~tered intraperitoneally. The data shown in the figure (see below) is the % animals surviving the challenge. Hamsters were challenged with 2 mg cl;n~ ycin and 1 x 107 vegetative C. difficile orgAn;~ ~

10 RESULTg Intr~n~ Immunization of Mice with Toxoid Groups of 5 female Swiss Webster mice (Taconic Farms, Germantown, NY) were i n; zed weekly by the intranasal route with toxoid (15 ~g of each toxin), with 15 or without 5 ~g CT as a mucosal adjuvant. The ; ;zation scheme is summarized in Table 4. Serum, saliva, feces, and vaginal secretions were obtained after im ln; ~ation. Specific IgA and IgG antibodies can be detected in serum, and specific IgA antibodies against 20 both Toxin A and Toxin B can be detected in saliva, feces and on mucosal surfaces against both Toxin A and Toxin B
after ; ln;7ation (Figs. 6A and 6B). This response was apparent regardless of the a~r; n; ~tration of CT along with the toxoid. Serum from ; n; 7ed ~n;~l c also 25 inhibited the cytotoxicity of both Toxin A and Toxin B
(Figs. 7A-7C). Immune sera was used to passively protect rat loops from the enterotoxic effects of Toxin A (Fig.
8) . ~n; -1 c were challenged with 10 LD50 of Toxin A
followed by 10 LD50 of Toxin B one week later. All 30 ; ;zed ~n; -1~ survived this challenge, while controls did not (Fig. 9). Finally, ligated intestinal loops from ; ;7ed ~n; ~lc were challenged with Toxin A, directly demonstrating the induction of ant;ho~;es~ which were probably mucosal IgA antibodies, capable of inhibiting W O 97/02835 PCT~US96/10987 fluid accumulation and, presumably, diarrhea (Fig. 10).
These data demonstrate the C. difficile vaccine elicits a strong protective mucosal immune response when a~ ; n; ~tered to a mucosal surface, and that does not 5 require a mucosal adjuvant.
Table 4. Mucosal i une respons~ a~ter intr~n~Q-l ~ nuniz~tion of mice with C. dif~icile toxoid, ~/-cholera toxin ~CT); Immunizations were carried out on days O, 7, 21, 35, 49, and 59. Challenge with toxin A/B
10 took plnce on day 70. Samples (serum, feces, saliva, and vaginal secretiOn8) were taken on day . (CT = cholera toxin; PBS = phosphate-buffered saline) Antigen Dose (~g) Adjuvant # Animals Toxoid 15 5 ~g CT 5 Toxoid 15 none 5 PBS O 5 ~g CT 5 Example III. Immunization of Mice with Vaccine Compositions Containinq GST-ARU fusion proteins The COOH-terminal region of C. di f f icil e Toxin A
20 contains a series of repeating amino acid units which are thought to be involved in binding of the toxin to carbohydrate residues on target cells (see, e.g., Lyerly et al., Current Microbiology 21:29-32, 1990; Frey et al., Infection and T ; ty 60:2488--2492,1992; and references 25 cited therein). A fusion protein consisting of the carboxyl - ter ; n~ 1 region of C. difficile Toxin A fused to glutathione S-transferase (GST) was constructed, as follows. Using st~n~rd methods, a Sau3A fragment cont~;n;ng nucleotides which encode the 794 carboxyl-30 terminal amino acids of Toxin A was isolated (see, e . g .,Dove et al ., su pra , for the sequence of the Toxin A
gene). The sticky ends of the fragment were filled in, ~ and the blunt-ended fragment was ligated into SmaI -digested pGEX3X. Clones containing a plasmid encoding 35 GST-ARU were grown in E. coli, and the GST-ARU fusion protein was purified on a glutathione-agarose affinity W O 97/02835 PCTrUS96/10987 column, eluted from the column by free glutathione, and dialyzed to remove the glutathione, using s~n~d methods.
Groups (n=5) of female Swiss Webster mice were i ;zed with Toxin A fusion protein (GST-ARU) by the intragastric (IG; 100 ~g), intranasal (IN; 50 ~g), or intraperitoneal (IP; 25 ~g) routes, with or without CT
(5 ~g) as a mucosal adjuvant, in four weekly doses (Table 5). After the final dose, samples were obtained for ; c analysis. All routes induced good serum immune responses against Toxin A. Intranasal (IN) a~; n; ctration resulted in mucosal IgA responses, even without CT. T ; zation by the IG route was also effective, but seemed to be enhanced by the presence of a 15 mucosal adjuvant. T ; zation by the IP route induced good systemic and fecal responses, but not in salivary or vaginal samples (Figs. llA-llB). Serum antibodies did not significantly inhibit the in vitro cytotoxicity of Toxin A, but were able to passively protect rat loops 20 from enterotoxicity, suggesting that the carboxyl-terminal binding domain is only required for in vivo toxicity (Figs. 12A-12B and 13A-13B). An;r~l.c ;mrlln; ~ed by the IN and IP routes were protected from lethal challenge, but the IG ; ;zed ~n;mAlc were not (Fig. 14). Survivors of the systemic challenge also demonstrated the presence of enterotoxin neutralizing ant; hoA; es in ligated intestinal loops (Fig. 15). IN
; ;~ation resulted in circulating antibodies that protected An; ~lc from the lethal effects of Toxin A, as 30 well as the enterotoxic effects of Toxin A on the intestinal mucosa, even without CT. Some protection was observed by all routes tested, but the IN route of a~~ ; n; ~tration appeared to be more effective in eliciting a mucosal response.

W O 97/02835 PCTrUS96/10987 Table 5. Mucosal immune response after immunization of mice with recombinant C. di~icile toxin A COOH terminus ~GST-ARU). ization~ were carried out on days O, 7, ~ 14, and 21. Challenge with toxin A took place on day 35.
5 Bamples (serum, feces, saliva, and vaginal secretions) wero taken on day 28. (CT = x ~g cholera toxin; IG =
- intragastric; IN = intr~n~ ; IP = intraperitoneal; PBS
= pho-~phate-buffered saline) Antigen Dose (~g) Adjuvant Route #
10 Animals GST - ARU100 none IG 5 GST-ARU 5 0 none IN 5 GST-ARU 25 none IP 5 Other embodiments are within the following claims.

What is claimed is:

Claims (15)

1. A composition comprising non-replicatable polypeptide antigen which is dissolved in a pharmaceutically acceptable diluent and which is capable of inducing a distal mucosal immune response to a gastrointestinal or genitourinary tract pathogen in a mammal, when administered intranasally.

2. The composition of claim 1, wherein said distal mucosal immune response is in the gastrointestinal tract.
. .

3. The composition of claim 1, wherein said distal mucosal immune response is in the genitourinary tract.

4. The composition of claim 1, wherein said pathogen causes diarrhea.

5. The composition of claim 4, wherein said pathogen is from the genus Clostridium.

6. The composition of claim 5, wherein said pathogen is Clostridium difficile.

7. The composition of claim 1, wherein said antigen comprises a toxin of said pathogen, or an immunogenic fragment or derivative thereof.

8. The composition of claim 7, wherein said antigen comprises Clostridium difficile Toxin A, or an immunogenic fragment or derivative thereof.

9. The composition of claim 7, wherein said antigen comprises Clostridium difficile Toxin B, or an immunogenic fragment or derivative thereof.

10. The composition of claim 8, wherein said antigen comprises Clostridium difficile Toxin A and Clostridium difficile Toxin B.

11. The composition of claim 1, wherein said antigen comprises a toxoid.

12. The composition of claim 11, wherein said toxoid is a Clostridium difficile toxoid.

13. A method of inducing a distal mucosal immune response to a pathogen in a mammal, said method comprising the steps of:
a. administering an antigen capable of inducing said immune response to a mucosal surface of said and b. parenterally administering said antigen to said mammal.

14. The method of claim 13, wherein said mammal is at risk of developing, but does not have, diarrhea caused by said pathogen.

15. The method of claim 14, wherein said antigen is a C. difficile toxoid.