WO1999002188A1

WO1999002188A1 - Hen egg yolk antibodies to clostridium difficile antigens and use in therapy for pseudomembranous colitis

Info

Publication number: WO1999002188A1
Application number: PCT/US1998/014401
Authority: WO
Inventors: J. Glenn Songer; Vitaliano A. Cama; Charles R. Sterling
Original assignee: The Arizona Board Of Regents On Behalf Of The University Of Arizona
Priority date: 1997-07-11
Filing date: 1998-07-10
Publication date: 1999-01-21
Also published as: AU8395498A

Abstract

Egg yolks and egg yolk fractions containing avian antibodies against Clostridium difficile, and methods of providing passive immunization for the prevention or treatment of pseudomembranous colitis or diarrhea by means of enteral administration of anti-Clostridium difficile yolk antibodies harvested from the eggs of hyperimmunized avian hens.

Description

HEN EGG YOLK ANTIBODIES TO CLOSTRIDIUM

DIFFICILE ANTIGENS AND USE IN THERAPY FOR

PSEUDOMEMBRANOUS COLITIS

FIELD OF THE INVENTION

The present invention relates to the passive transfer of immunity; it relates particularly to the passive transfer of immunity by egg yolk antibodies to the antigens of Clostridium difficile produced in hyperimmunized hens. BACKGROUND OF THE INVENTION

Passive immunization is carried out by transferring immune elements, usually IgG, from an immunized animal to a non-immune recipient. Passive immunization is an invaluable therapeutic strategy for providing immune protection to a mammal which cannot develop itself an effective autologous immune response to a threatened or existing infection. Examples of such patients are newborns that are not yet immunocompetent, as well as infants or adults that are immunocompromised by another disease, such as HIV infection, as a result of therapy in connection with organ transplants, or as a consequence of malnutrition.

Passive immunization may also be employed in therapies for subjects having intact immune systems, including patients suffering from an infectious disease that cannot be controlled by medication or an autologous immune response. In such applications, the administration of effective amounts of antibodies that can mitigate the symptoms of infection facilitate a more rapid recovery than would otherwise be possible. Parenteral passive immune transfer, typically by injection, risks sensitizing the recipient. To avoid this problem, hyperimmune bovine colostrum (HBC) from lactating immunized animals has been given orally. An example of this approach is presented in U. S. Patent No. 4,324,782 to Beck who discloses that dental caries is prevented in rats by administering bovine milk containing Streptococcus mutans antibodies to young animals by oral ingestion or direct application to the teeth.

Hyperimmune bovine colostrum therapy, however, implies a continuous supply of high-titer HBC. Supply can be disrupted by the breeding cycle of cows and the brief period of colostrum production during lactation. Assuring HBC product uniformity and establishing and monitoring sanitary methods of collection and storage is expensive. Yolken, R. (New Engl J Med 312:605-610 (1985)) also has found that the processing of immune bovine milk products to make them suitable for newborn feeding markedly reduces the concentration of antibodies.

The use of egg yolks of hyperimmunized hens (HEY antibody) for passive immunity transfer overcomes the limitations of HBC therapy by providing a continuous source of uniform immunizing agent which can be produced, collected and stored in substantial volume. Hen egg yolk antibodies are particularly resistant to digestive degradation. As a result, they can be introduced into the digestive system, for example as food, to neutralize infectious organisrηs in the stomach and small intestine.

The manufacture and collection of hen egg antibodies is well understood (U.S. Patent Nos. 4,387,272 and 4,550,019 to Poison; and Losch, U., J Veterinary Medicine

B 33:609-619 (1986)). Hen egg yolk antibodies thus produced have been used in a number of applications for passive transfer of immunity. U.S. Patent No. 4,748,018 to Stolle, et al. discloses a method of passive immunization against bacterial infection comprising a preliminary development of tolerance to HEY by repeated oral ingestion of egg yolk, followed by parenteral injection of HEY antibody to a selected bacterial antigen. U.S. Patent No. 5,080,895 to Tokoro discloses prevention of E. coli diarrhea in newborn piglets by oral administration of anti-bacterial hen egg yolk antibodies. Hamada, S., Infection and Immunity 59(11):4161-4167 (1991); and Otake, S., J. Dental Research 70(3):162-166 (1991) reproduced the results of Beck in protecting rats against dental caries by means of passive immunization with orally administered hen egg yolk antibodies against S. mutans. Bartz, C. et al., J Infect Disease 142(3):439-441 (1980) prevented murine rotaviral infection in mice by the oral administration of the water-soluble fraction of the eggs of immunized hens; Yokoyama, H, et al. Infection and Immunity 60(3):998-1007 (1992) succeeded in passively protecting neonatal piglets from fatal enterotoxigenic E. coli infection by oral administration of a crude yolk immunoglobulin fraction from the eggs of immunized hens. On the basis of animal studies, Yolken, R. et al., Pediatrics 81(2):291-295 (1988); and J Clin Immunol 10(6):80S-87S (1990), proposed the oral administration of antiviral HEY immunoglobulin for the prevention and treatment of enteric infections, including rotaviral infection in humans. Methods and formulations for the oral administration of immune globulin are known (U.S. Patent No. 4,477,432 to Hardie).

Sterling and Cama discovered that the yolk of eggs from hens immunized against Cryptosporidium parvum was an effective anti-Cryptosporidiosis agent when administered orally, and it also has the advantages of being an attractive, well- packaged and appetizing therapeutic substance to ingest, having the taste of real food.

(Cama, V. and C. Sterling (1991) J Protozool 18(6); U.S. Patent No. 5,753,228, which is incorporated herein by reference).

Clostridium difficile has been identified as the causative agent of pseudomembranous colitis and associated diarrhea in humans. The infection commonly occurs after antibiotic therapy, although it also occurs in non-treated populations; for example, infants are also at risk. In particular, elderly individuals undergoing in-patient therapy are highly susceptible to Clostridium difficile infection when given large doses of antibiotics, such as after major surgery. In the past, the treatment for antibiotic associated colitis and diarrhea has been two-fold: withdrawal of the antibiotic that induced the colitis and the administration of vancomycin or metronidazole, present medications of choice in Clostridium difficile infection.

The process of Clostridium difficile infection begins with the ingestion of its bacterial spores. These spores germinate in the digestive tract of the host animal to become vegetative or active toxin-producing Clostridium difficile bacteria. Clostridium difficile produces two toxins, an enterotoxin designated toxin A and a cytotoxin designated toxin B. These toxins are believed to be responsible for the disease pathology, Clostridium difficile colitis, or pseudomembranous colitis.

It has been observed that toxin-neutralizing antibodies can protect experimental animals against Clostridium difficile colitis. Both toxin A and toxin B cross react with Clostridium sordellii HT and LT toxins and can be neutralized by their antisera (Knoop, F.C., et al. (1993) Clin Microbiol Revs 6(3):251-265). Monoclonal antibodies against Clostridium difficile toxin A protect mice against challenge by experimentally induced pseudomembranous colitis (Cormier, G. et al. (1991) 59(3): 1192-1195). Bovine immunoglobulin specific for Clostridium difficile toxoid protected hamsters against antibiotic-associated diarrhea and colitis (Lyerly, D.M., et al. (1991) Infect, and Immun. 59(6):2215-2218). U.S. Patent No. 5,601,823 to Williams et al. claims purified hen egg yolk antibodies to toxin A of Clostridium difficile, which is said to neutralize the toxin in vivo.

SUMMARY OF THE INVENTION According to the invention there is provided hen egg yolk, or a fraction thereof, containing hen egg yolk antibodies having a specificity for at least one spore antigen or germination antigen of Clostridium difficile or a mixture of said antibodies. According to another aspect of the invention there-is provided hen egg yolk comprising hen egg yolk antibodies having a specificity for at least one antigen of Clostridium difficile or a mixture of said antigens, where the lipid content of said egg yolk is reduced at least about 10%. In a preferred embodiment of this aspect of the invention the lipid content of the hen egg yolk is reduced at least about 50%; in a particularly preferred embodiment the lipid content of the hen egg yolk is reduced at least about 90%

According to one embodiment of this aspect of the invention there is provided hen egg yolk comprising hen egg yolk antibodies having a specificity for at least one somatic antigen of Clostridium difficile. Alternatively, the hen egg yolk can comprise hen egg yolk antibodies having a specificity for at least one antigen of Clostridium difficile toxin A or toxin B.

According to another embodiment of this aspect of the invention there is provided a reduced lipid egg yolk product comprising hen egg yolk antibodies having a specificity for at least one spore antigen or germination antigen of Clostridium difficile or a mixture of said antibodies. The invention also comprises isolated hen egg yolk antibodies having a specificity to at least one spore antigen or germination antigen of Clostridium difficile, or a mixture of said antibodies. In a preferred embodiment of the invention the hen egg yolk product or a fraction thereof comprising hen egg yolk antibodies is freeze-dried or lyophilized. The invention also provides pharmaceutical formulations comprising a hen egg yolk product, or a fraction thereof comprising hen egg antibodies against an antigen of the Clostridium difficile bacteria or against a Clostridium difficile spore or germination antigen, or a mixture of these antibodies, in a pharmaceutically acceptable carrier. The invention also provides a pharmaceutical formulation comprising a isolated hen egg antibodies having a specificity for a Clostridium difficile spore antigen or a germination antigen in a pharmaceutically acceptable carrier.

According to another aspect of the invention there are provided methods for treating an intestinal infection caused by Clostridium difficile in a mammal, comprising administering to said mammal, by an enteral route, an effective Clostridium difficile-τieulvsλvήng amount of hen egg yolk, or anjmmunity-conferring fraction thereof, containing hen egg yolk antibodies having a specificity for at least one spore antigen or germination antigen of Clostridium difficile, or a mixture of said antibodies.

In another embodiment of this aspect of the invention, there is provided a method for treating an intestinal infection caused by Clostridium difficile in a mammal, comprising administering to said mammal, by an enteral route, an effective Clostridium difficile-n uixaWvmg amount of hen egg yolk, or an immunity-conferring fraction thereof, comprising hen egg yolk antibodies having a specificity for at least one somatic antigen of Clostridium difficile, or an antigen of Clostridium difficile toxin A or toxin B wherein the lipid content of said hen egg yolk or hen egg yolk fraction is reduced at least about 10%.

According to yet another aspect of the invention there is provided a method for providing a mammal in need thereof with passive immunity against Clostridium difficile infection comprising introducing into the digestive tract of said mammal a protective amount of hen egg yolk, or an immunity-conferring fraction thereof, comprising antibodies having a specificity for at least one spore antigen or germination antigen of Clostridium difficile or a mixture of said antibodies. Alternatively, the mammal in need thereof is provided with passive immunity against

Clostridium difficile infection by introducing into the digestive tract of said mammal a protective amount of hen egg yolk, or an immunity-conferring fraction thereof, containing hen egg yolk antibodies having a specificity for at least one antigen of Clostridium difficile or to a toxin produced by Clostridium difficile, wherein the lipid content of said hen egg yolk or egg yolk fraction is reduced at least about 10%. It therapeutic method according to the invention comprise enteral administration by oral ingestion, gastric intubation, rectal intubation, or direct injection of said hen egg yolk antibodies into the digestive tract of said mammal.

In a preferred embodiment the method treats a mammal that has become infected with Clostridium difficile as a consequence of antibiotic treatment. In particularly preferred embodiments the mammal is a human being, including an immature human being such as a child or infant.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the results of ELISA analyses performed on sera from hens in a first study which were immunized with somatic antigen, toxoid, or somatic antigen plus toxoid.

Figure 2 shows the results of ELISA analyses performed on sera from hens in a second study which were immunized with somatic antigen, toxoid of toxins A and B, or somatic antigen plus toxoid.

DETAILED DESCRIPTION OF THE INVENTION Avian an -Clostridium difficile antibodies can prevent or treat a Clostridium difficile infection in a mammal when they are introduced into the digestive tract of the animal. Egg yolks of avian hens hyperimmunized against an antigen from Clostridium difficile can provide an assured, continuous source of high-titer anti- Clostridium difficile antibodies in substantially unlimited volume. Enteral administration of the hen egg yolk antibody preparations avoids problems of sensitization associated with parenteral injection of foreign proteins into an animal. It also provides a method of anύ-Clostridial therapy or prophylaxis that is suitable for any species of animal. Further, the method of the invention can be applied, preferably as an adjunctive therapy, in the form of an antibody coupled to an antibacterial agent, to direct the agent to the site of Clostridium difficile infection. Clostridium difficile infections are treated or prevented according to the methods of the invention by introducing hen egg yolk preparations containing the egg yolk Clostridium difficile antibodies of the invention having a specificity for a Clostridium difficile somatic antigen, a Clostridium difficile toxin A or B antigen, or a Clostridium difficile spore antigen or germination antigen, or a mixture thereof, into the animal by any enteral route, particularly into the digestive system. Preferably, Clostridium difficile infection in a mammal is treated by the oral administration of the aforesaid anύ-Clostridium difficile hen egg yolk antibodies. Production of HEY Anύ-Clostridium difficile Antibodies Egg yolk anύ-Clostridium difficile antibodies can be raised in the hens of any avian species; however, immunization of hens of the common chicken, which are available in abundant commercial supply, and are adapted to egg production, is preferred. The hens can be immunized intramuscularly, intraperitoneally, or by other routes, according to methods and immunization schedules known to those in the art, for example, as described in Example 3, or as described by Losch, U., Veterinary

Medicine 33:609-619 ( 1986). The co-administration of an adjuvant, such as Freund's (FCA; Sigma, St. Louis, Mo., Cat. No. F5881, 4258) or that of Ribi (Hamilton, Montana) increases and maintains the antibody level in the blood serum and egg yolk of the immunized animal.

The Clostridium difficile antigens used to generate the immune response in hens may be somatic antigens, also referred to herein as whole cell antigens, or they may be toxoids of toxins A or B or combinations thereof. Whole cell or somatic antigens may be prepared as described in Example 1 ; and toxoids of toxins A and B are prepared according to Example 2.

Clostridium difficile Spore/Germination Antigens

In a preferred embodiment, the immunizing agent is a Clostridium difficile spore antigen or an antigen associated with the germination of the bacterium. Clostridium difficile spore antigens comprise all antigens of the inactivated spore, including the activation receptors; Clostridium difficile germination antigens include all antigens expressed in the bacteria during the transition from the time of initiation of germination and the appearance of the mature vegetative cell, including membrane receptors and enzymes. The isolation of these antigens is described in Example 4. In order for Clostridium difficile spores to germinate, spore receptors must be bound by a specific activator. If activation receptors are blocked, germination, toxin production and consequent infection and disease will not occur. According to the invention, the germination process can be blocked by specific hen egg yolk antibodies. Antibodies that prevent Clostridium difficile germination or block or interrupt the germination process are obtained by immunizing a hen fowl with an inactivated spore antigen or a germination antigen of Clostridium difficile, according to procedures of Example 4.

Without being bound by a specific mechanism, it is believed that even though the location of the germination receptors remains to be established, it is accepted that it has to be located outside the spore core and inner membranes, but possessing the resistance properties of the intact spore. There is genetic evidence that suggests the germination receptors are membrane bound multi subunit complexes. The stereospecifity of these receptors demonstrate that this component of the germination trigger mechanism is a protein. Antibodies directed against these proteins (germination receptors) are believed to interfere with the binding of the germinants. This event will keep the spore in the dormancy stage, not allowing the return of the organism to the vegetative state, and blocking its growth and multiplication when nutrients are available. Similar to other Gram-positive spore-formers

(principally Bacillus subtilis), antigens expressed during germination may include the receptor or receptors for taurocholate, NAD biosynthetic enzymes, trypsin-like enzymes and other proteases which degrade proteins in the spore coat, enzymes involved in peptidoglycan synthesis or breakdown, and membrane receptors for amino acids and sugars. [The total number of proteins is substantial, for example, B. subtilis produces about 65 proteins during the first stage of germination (0-10 minutes after induction), about 210 during the next stage (10-20 minutes after induction), and 260 proteins during vegetative growth.] (Setlow, P. (1980) Biochemistry of bacterial forespore development and spore germination, in

Sporulation and Germination. (Levinson, H., et al., eds) pp 13-28. American Society for Microbiology, Washington, D.C.; and Moir, A. et al. (1994) The genetic analysis of bacterial spore germination. J Appl Biotechnol (Symposium Suppl) 76:9S-16S), describe the cross-species processes and mechanisms for spore-forming bacteria.

The hen egg yolk antibodies against spore and germination antigens prevent maturation of the spores into active Clostridium difficile bacteria and the development of the consequent disease in the infected mammal. While not being committed to a specific proposed mechanism, it is believed that the hen egg yolk antibodies having a specificity for a spore antigen of Clostridium difficile binds to spore receptors in vivo, preventing germination and replication of the bacteria. Antibodies to germination antigens similarly interrupt the germination and maturation of the bacterium. The particular advantage of interrupting the Clostridium difficile disease process at the point of spore germination is that this approach is compatible with continued antibiotic therapy. If the passive transfer of immunity is carried out prophylactically with these anti-spore or anti-germination antibodies at the outset of any antibiotic therapy, the antibiotic-associated Clostridium difficile colitis and diarrhea will not occur. Production of HEY Antibody to Clostridium difficile Antigens

Antibody to Clostridium difficile antigens is raised by injecting an immunizing antigenic preparation derived from whole cell antigens or by using at least one toxoid from the A and B toxins of Clostridium difficile as an antigen. Preferably, a

Clostridium difficile spore or germination antigen is used as an immunogen. All isotypes of antibody raised in the hen are transferred from the hen serum into the developing egg yolk and antibody level in the eggs produced peaks in about one week following a serum peak level. Therefore, The titer of immune antibody in egg yolk of the immunized hens closely parallels the titers measured in the serum of the immunized hens. The titer of the antibody in serum or egg yolk can be determined by common immunological methods, for example by an ELISA procedure as described in Example 5, or by Leslie, G. et al. (1969) J Exper Med 130:1337-1352, or by Ouchterlony or rocket immunoelectrophoresis, and can be quantitated against a known antigen, either relatively or absolutely. Once the antibody serum titer of the immunized hens has reached an acceptable level, from at least about 15,000 AU/ml as determined by the ELISA assay, the eggs laid by the hens are harvested and the antibody titer thereof determined by ELISA as for the hen serum. Immune egg yolk having titers expressed anύ-Clostridium difficile activity units (AU) of from at least about 10,000

AU/ml as determined by ELISA as demonstrated in the Examples are useful in treating Clostridium difficile infections. The anύ-Clostridium difficile antibody thus induced is collected by harvesting the yolks of the eggs laid by the hyperimmunized hens. Antibody-containing yolk fractions, or purified anύ-Clostridium difficile antibodies can be prepared as necessary.

Fractions of Egg Yolk containing anύ-Clostridium difficile Antibodies

The immunity-conferring egg yolk antibodies of the invention can be used therapeutically or prophylactically in the form of unpurified native whole egg yolk, or in fractionated or purified preparations isolated from the yolk, provided the native, fractionated, or purified product contains an immunity-conferring amount of HEY anύ-Clostridium difficile antibodies.

Antibody-containing egg yolk fractions can be prepared by various methods known for separating proteinaceous material from natural sources, such as, for example, differential centrifugation, salt fractionation with ammonium sulfate or alcohol, or polyethylene glycol precipitation of proteins. A protein fraction enriched in antibodies can be obtained by solvent extraction of yolk lipids. Preparations of this type are described in U. S. Patents Nos. 4,357,272 and 4,550,019 to Poison. Whole egg yolks or crude fractions thereof can be prepared by spray-drying, freeze- drying to form powdered preparations. The hen egg yolk preparations may also be sanitized by filtration or by heating the egg yolk to a temperature between about

180°F and 212°F for a period of from about 1 to 3 minutes. Methods for reducing lipid content of egg yolk products

In preferred embodiments of the compositions of the invention the relative amount of lipids in the harvested immune egg yolks is reduced. A particularly preferred method is an optimization of a dilution protocol disclosed in U.S. Patent

No. 5,753,228, and application Serial No. 09/089,644, which are hereby incorporated by reference. The preferred method of fractionation by dilution in an aqueous solvent is advantageous because it avoids denaturation and contamination of the hen yolk antibody-containing product. It is also critically important to carry out the procedure at a controlled chilled temperature. We have found that the egg lipids precipitate optimally if the process temperature is between about 0° and 6° C. The objective of the lipid reducing protocol is to remove the greatest quantity of lipid while retaining in the reserved fraction the greatest amount of antibody. Preferably, at least 10% of the lipid content is removed. In preferred embodiments of the reduced lipid egg yolk product of the invention, at least about 50% of the egg yolk lipids are removed. In particularly preferred embodiments of the invention, for example, as described in Example 10, from about 70% to 95% of the lipid content of the antibody-containing egg yolks is removed by dilution of the egg yolks with water and consequent sedimentation of the lipid macromolecule fraction.

The amount of water added in the dilution step in the procedure of Example 10 is not critical, but is at least about 1: 1; typically the yolk: water ratio is about 1:9.

Dilution of the yolks is necessary to free the antibodies from association with solid or liquid lipids and to avoid trapping these proteins in the viscous, fat-rich pellet of the precipitation step. The antibodies can be prepared from whole egg by the same process; however, this approach is disadvantageous, because it adds neutral albumin to the protein fraction, and increases the caloric value of the preparation unnecessarily.

It should be understood that the invention includes lipid-reduced preparations of egg yolk comprising any of the anύ-Clostridium difficile antibodies disclosed herein and combinations thereof. A suitable lipid-reducing method will remove at least about 10% of the total lipid content while preserving the integrity and potency of the constituent antibodies and avoiding the introduction of toxic material. Preferably, the lipid reducing procedure removes at least about 30%, and most preferably, at least about 50%, of the constituent lipids of the native egg yolk. The antibody proteins in the yolks are preferably substantially retained, but at least are not reduced to the same extent as the yolk lipid. To increase the recovery of immunoglobulin, the yolk lipids present in the crude product can be precipitated and washed of protein according to the invention most preferably by dilution with water or phosphate-buffered saline optionally followed by either centrifugation or filtration of the crude product as described in Example 10.

Alternative methods can also be employed to reduce both the solid and liquid fatty substances of the yolks. According to yet another approach, both the solid and liquid fatty substances of the yolk can be removed by filtration through a fractionating filter, for example, a polyamide, polysulfone or other synthetic membrane conventionally used to isolate protein from whole cell contents (Millipore, Bedford, MA). In a similar approach, the solid and liquid fatty substances can be removed by passage of a diluted or otherwise appropriately prepared yolk suspension by adsorption/partition on a chromatographic column having a packing material selected, for example, from among alumina, cellulose, silica, hydroxyapatite, or specifically, Chromosorb supports known to those skilled in the pharmaceutical and food product arts as adapted to separate fractions of differing polarity by direct or gradient elution. Those separation procedures that are rapid and can be scaled to the production of food products are preferred. Other lipid reducing procedures applicable in extracting lipids from whole egg yolks in preparing the reduced lipid antibody preparations are supercritical carbon dioxide extraction, tangential flow ultrafiltration, alternate and successive freezing and thawing to disrupt the lipid emulsion of the yolk. Methods by which antibodies are exposed to denaturing conditions or procedures that introduce toxic or unpalatable materials into the product are disadvantageous.

The lipid removal procedure can be monitored by HPLC analysis as it proceeds as is known to those skilled in the art. The efficiency of the lipid-removal can be measured by lipid extraction of the processed and unprocessed egg yolk product according to the procedure of Bligh and Dyer (1957) or that of Folch-Pi, J. et al. (1959) J. Biol. Chem. 226:494-509. Antibody concentration in the yolk product can be monitored by determination of gross protein concentration by UV absorption method, by chemical colorimetric assay, or by using the ELISA procedure of Example 5 or 6. Antibody potency can be determined in combination with conventional immunological assays, for example, the Ouchterlony method, double diffusion in agar or immunoelectrophoresis or by the same ELISA method.

Following lipid reduction, the egg yolk preparation can be cleared of bacterial contamination by any procedure either that known to be bactericidal, or to reduce the bacterial population, as long as the process does not denature the effective antibodies.

It is not necessary to remove all bacteria from the preparation, but only to sanitize the material to make it suitable for oral ingestion. Preferably, the reduced lipid preparation is sanitized by passage through a filter that excludes particles on the basis of size. The filtered material can be prepared immediately as a liquid formulation, or it can be lyophilized and stored indefinitely. Lyophilization carried out to minimize protein denaturation is known to those skilled in the pharmacy ,arts. The sweeteners and other agents to promote palatability can be added before or after filtration and before or after lyophilization. Hyperimmune whole egg yolks and crude egg yolk fractions containing anti-

Clostridium difficile antibodies are effective to confer immunity even when partially cooked by heating to the consistency of soft boiled egg yolk. If desired, the egg yolks may be prepared by heating to a temperature of about 200°F, preferably at the boiling point of water, for a period of from about one to three minutes. The procedure can be carried out conveniently by placing the intact egg in boiling water for this period of time. The heating procedure can be carried out to "pasteurize" the eggs to eliminate any infectious organisms or to provide a more palatable preparation for oral ingestion. Pasteurization is described in standard texts on food processing, and typically comprises heating the eggs or fractions thereof to about 145-150°F for about 30 min, lowering the temperature to about 138°F for several minutes, followed by cooling to about 50°F. Hyperimmune egg yolks of fractions thereof can be stabilized against decomposition caused by changes in acidity or alkalinity by the use of buffering agents.

Alternatively, the egg yolk products may be diluted in water, centrifuged and frozen. Thereafter, the egg yolk products may be prepared for administration by irradiating them. Preferably, the irradiation is at levels between about 1.5 and about 2.5kGy.

Highly purified hen egg yolk antibodies or antibodies of distinct classes can be obtained by means of conventional protein purification procedures known for the isolation of immunoglobulins; for example, gel filtration, ion-exchange chromatography, ion-exchange chromatography, affinity chromatography, or isoelectric focusing Candidates for Passive Immunization Therapy:

The passive immunization methods of the invention can be beneficially applied to either prevent infection by Clostridium difficile to an individual at risk for such infection, or to eliminate Clostridium difficile from an infected individual. The recipient can be of any age, either newborn, a developing child, or an adult. The effectiveness of the present passive immunization methods is independent of the immune status of the recipient, that is, immunocompetent, immunocompromised, or immunotolerant.

Enteral Administration of HEY Antibodies:

The dosage of passively immunizing antibodies for a vertebrate to be treated, including a human, may vary depending upon the extent and severity of the condition that is treated and the antibody titer of the administered immunoglobulin fraction. Thus the dose of egg yolk product administered per day can range from about 0.1 mg/kg to about 1500 mg/kg, containing from at least about 100,000 to about 5,000,000 AU/day for an adult individual of 70 kg. The egg yolk, or fraction thereof, containing antibodies against Clostridium difficile can be prepared as described herein and administered to a 70 kg human at a preferred dose of at least about 100 to 200 mg/hr over a 24 hour period to provide a total daily egg yolk dose of about 2400 to

4800 mg comprising from about 2 x 10⁴ to 5 x 10⁶ AU/gm of specific antibody. The period of treatment is at least about 3 days or until the symptoms abate. Preferably, the egg yolks are administered at a dose of approximately 860 mg kg/day. If desired, the egg yolks may be mixed with sucrose prior to administration in order to enhance the flavor. Where sucrose is added to enhance the flavor, the sucrose may comprise

15-25% of the weight of the egg yolk preparation. The prophylactic concentration of maternal antibodies found in bovine colostrum and human milk provides some guidance as to the optimal effective dose. The dosage of the hen egg yolk antibody fraction is determined by reference to these factors, bearing in mind that, in selecting the appropriate dosage in any specific case, consideration must be given to the patient's weight, general health, metabolism, age and other factors which influence response to the immunizing agent.

The prophylactic or therapeutic unit dose of hen egg yolk anύ-Clostridium difficile antibodies can be administered by introduction into the digestive tract at any point and by any means so as to most effectively target the antibodies to the site of infection. Enteral administration of a unit dose of antibody of this type has been carried out by coating the mouth (buccal swab), by encouraging voluntary ingestion by the recipient, through delivery by gastric intubation, by injection directly into a selected site in the intestinal tract, or by intubation into the intestine through the anus. Oral administration is preferred. Formulations of Immune Egg Yolk Products

One formulation of the invention comprises as an active ingredient immune egg yolk wherein the lipids therein have been reduced by at least about 10% while the amount and concentration of egg yolk antibody is retained. Alternatively stated, the relative amount of antibody protein in the egg yolk preparation is increased after lipid reduction. An acceptable preferred final product will be one in which the lipid concentration has been reduced, according to the procedure of Example 10, for example, to about 10% to 25% w/v and the protein content is from about 25% to 35%. The proteimlipid ratio in the lipid-reduced product is preferably from about 3: 1 to about 1: 1. The formulation can also include non-immune active agents, for example drugs such as antibiotics or analgesics. The formulations can also include other nutritive materials, such as protein or carbohydrates, or therapeutic materials such as vitamins.

The formulation can further comprise other agents which contribute to palatability and encourage consumption, for example, sweeteners and flavorings, as well as agents to preserve the taste and efficacy of the formulation, for example, anti-oxidants such as the (α-tocopherols, or parahydroxybenzoic acid or other preservatives or synergists. Preferred sweeteners are sugars such as sucrose, dextrose and the milk sugars, lactose and galactose. Sucrose is particularly preferred. Artificial sweeteners can also be used. Any flavoring, nutritive or non-nutritive, which increases palatability is useful. Formulations for oral ingestion are in the form of tablets, capsules, pills, ampoules of powdered antibody preparation, lyophilized antibody preparations, freeze dried antibody preparations, or oily or aqueous suspensions or solutions. Oral pharmaceutical formulations that are specific for immune globulins are disclosed in U.S. Patent No. 4,477,432 to Hardie. Tablets or other non-liquid oral compositions may contain acceptable excipients, vehicles, diluents, fragrances, or flavors known to the art for the manufacture of pharmaceutical compositions, to make the medication palatable or pleasing to use. The formulation can therefore include diluents, such as lactose or calcium carbonate, binding agents such as gelatin or starch; and one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring or preserving agents to provide a palatable preparation. Moreover, such oral preparations may be coated by known techniques to further delay disintegration and absorption in the intestinal tract.

Aqueous suspensions may contain the active ingredient in admixture with pharmacologically acceptable excipients, comprising suspending agents, such as methyl cellulose; and wetting agents, such as lecithin or long-chain fatty alcohols.

The aqueous suspensions may also contain preservatives, coloring agents, flavoring agents and sweetening agents in accordance with industry standards. The preparations may further comprise antioxidants, such as ascorbic acid or tocopherol, and preservatives, such as p-hydroxybenzoic acid esters. The antibodies, immune egg yolk or egg yolk fraction can be administered undiluted or combined with other anti-infective or nonactive ingredients in solution or in capsules or otherwise incorporated in a matrix that provides a delayed release of antibodies at the site of infection.

Formulations comprising hyperimmune egg fractions or antibodies isolated therefrom can also contain other therapeutically active agents; for example, anti- diarrheal medications, anti-spasmodics, anti-bacterial agents, or antacids. The anύ-Clostridium difficile hen egg yolk antibodies of the invention can be also administered in a formulation including an anti-bacterial drug that is effective against Clostridium difficile. The present invention is described below in detail using the following examples, but the procedures described are disclosed in terms of their general application to the preparation of the hen egg yolk antibodies of the invention.

Occasionally, the procedure may not be applicable as described to each immune preparation included within the disclosed scope of the invention. The preparations for which this occurs will be readily recognized by those skilled in the art. In all such cases, either the procedures can be successfully performed by conventional modifications known to those skilled in the art, e.g., by changing to alternative conventional reagents, or by routine modification of procedural conditions. Alternatively, other procedures disclosed herein or otherwise _^conventional will be applicable to the preparation of the corresponding preparations of the invention. In all preparative methods, all starting materials are known or readily prepared from known starting materials; all temperatures are set forth uncorrected in degrees

Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

It is believed that one skilled in the art can, using the preceding description, utilize the invention to its fullest extent. The following preferred embodiments are, therefore, to be construed as merely illustrative and not limitative for the remainder of the disclosure in any way whatsoever.

EXAMPLE 1 Preparation of Whole Cell Clostridium difficile Antigen

Sterile BHI/YE/cys medium was prepared by autoclaving the following composition. The following amount of constituent was added per liter BHIAΕ/cys medium: 37.0 g BHI (Difco)

5.0 g Yeast Extract (Difco)

0.5 g Cysteine HC1 (Fisher Scientific)

Water to 1000 ml

An overnight culture of Clostridium difficile J9 (ATCC/Virginia Polytechnic Institute) was used to inoculate 250 ml of the BHI/YE/cys medium. The culture was grown at 37°C for 48 hours without shaking. If desired, an aliquot of the culture was removed to determine the purity of the culture and the cell count. Thereafter 50 ml of 10% buffered formalin was added to the culture and the resulting mixture was incubated at 37°C for 72 hours without shaking. To confirm that all the cells in the culture of Clostridium difficile had been killed, a 1 ml aliquot of the above mixture was added to 10 ml of fresh BHI/YE/cys medium. The medium was incubated for up to 7 days at 37°C. A lack of growth confirmed that all the cells in the culture used to inoculate the fresh medium had been killed. The cells in the 250 ml culture were harvested by centrifugation at 15,000 φm in a Sorvall® SS4 rotor for 10 minutes at 4°C. The cells were then washed twice with distilled water to remove residual formalin and lyophilized. Cells prepared in this manner were used to immunize hens as described below.

EXAMPLE 2

Preparation of Clostridium difficile Toxoid

For each 4 liter culture of Clostridium difficile, toxoid was prepared as follows. BHI YE/cys medium was prepared as above, inoculated with Clostridium difficile J9, and grown at 37°C for 5 to 7 days. The culture was centrifuged at 15,000 in a Sorvall SS34 rotor for 20 minutes at 4°C. The supernatant containing the bacterial toxins was filtered through a 0.45μm filter to remove remaining bacteria.

An aliquot of the toxin was removed. To the remainder, 10% buffered formalin was added to a final concentration of 0.4%. The mixture was incubated at

37°C for 72 hours with gentle rocking, followed by dialysis overnight against several changes of PBS at 4°C in a dialysis bag having a cutoff of 10,000 molecular weight.

The dialyzed mixture containing the inactivated toxins (toxoids) was filtered through a 0.45μm. BCA protein assays (Pierce Chemical Co.) and CHO cell toxicity assays, performed as described below, were conducted to determine the amount of toxoid present and to confirm that the toxins had been inactivated. The toxoid was stored at 4°C in an unfrozen state prior to its use for immunizing hens as described below. EXAMPLE 3 Generation of Immune Response Against Clostridium difficile in Avian Hens

I. Immunization of Hens Four groups of White Leghorn hens (3 hens/group) were immunized with the following:

• Group 1: somatic antigen 5 mg + toxoid antigen 5 mg.

• Group 2: somatic antigen 5 mg.

• Group 3: toxoid antigen 5 mg.

• Group 4: Saline control

All groups were prime immunized with Clostridium difficile antigens emulsified in Freund's Complete Adjuvant (FCA). Booster immunizations used Freund's Incomplete Adjuvant (FIA) (Sigma). Clostridium difficile (somatic or toxoid) antigens were diluted to the desired concentrations with sterile saline and mixed with equal volumes of FCA into 10 ml all-plastic syringes (Air-Tite, Virginia).

Emulsification needles were used to emulsify the mixture. Emulsion was tested for completeness by placing a drop in cold water. If observation showed it to hold its form, then emulsion was complete. The syringes were then assembled with 18 G x 1/2" needles (Becton Dickinson) and were kept at 4°C until utilized at the farm. Each hen received the above described doses of antigen in a total volume of

1.0 ml. The different antigen preparations were injected subcutaneously in the dorsal region of the upper third of the cervical area (back of the upper third of the neck). The booster immunization, utilizing FIA, was carried out 4 weeks after the prime immunization using the same methods of antigen preparation and immunogen emulsification.

Within 2 weeks of the booster injection, several of the hens showed signs of chronic inflammatory reactions at the injection site, but overall health and egg production were not affected. Serum and yolk samples were collected and assayed by ELISA as described below. Study II

A second study to further define appropriate doses of antigen was performed as follows. The hens of Study II received antigen preparations in two different adjuvants, Ribi adjuvant (RAS; RIBI Immunochem Research Inc., Montana) and Freund's Complete adjuvant. The hens in this study received a prime immunization followed by a booster immunization eight weeks later.

For the hens receiving antigen in Freund's Complete Adjuvant, Clostridium difficile (somatic or toxoid) antigen was diluted with sterile 0.025 M PBS and mixed with equal volumes of Freund's Complete Adjuvant (Sigma Chemical Co., Missouri) into 10 ml all-plastic syringes (Air-Tite, Virginia). Emulsification needles were used to emulsify the mixture. Emulsion was tested for completeness by placing a drop in cold water; if observation showed it to hold its form, then emulsion was complete. The syringes were then assembled with 18 G x 1/2" needles (Becton Dickinson) and were kept at 4°C until utilized on the same day at the farm. For the Main Flock group, repetitive syringes were utilized instead of 10 ml disposable syringes.

For the hens receiving antigen in Ribi adjuvant, Clostridium difficile (somatic or toxoid) antigen was diluted with sterile 0.025 M PBS and mixed as per the manufacturer's recommendations. Briefly, the antigen was placed in 15 ml sterile tissue culture tubes in the proportion of 3 volumes of antigen in saline to 1 volume of RAS. The tubes were vortexed until a milky emulsion was formed, and subsequently loaded in 10 ml all plastic syringes. The preparations were kept at 4°C until utilized later that day at the farm. A total of 16 immunization groups was tested. One large group of birds

("Main Flock") consisting of 384 hens was immunized with 25 μg/hen of toxoid A and B mixtures. In the additional 15 study groups, 4 hens each were immunized as follows:

Group 1 20μg somatic antigen in FCA/hen Group 2 lOμg somatic antigen in FCA hen Group 3 lμg somatic antigen in FCA/hen Group 4 lOμg toxoid antigen in FCA/hen Group 5 lμg toxoid antigen in FCA/hen Group 6 lOμg somatic + lOμg toxoid antigen in FCA/hen Group 7 lμg somatic + lμg toxoid antigen in FCA/hen Group 8 20μg somatic antigen in RIBI/hen Group 9 lOμg somatic antigen in RIBI/hen

Group 10: lμg somatic antigen in RIBI/hen

Group 1 1 : 20μg toxoid antigen in RIBI/hen Group 12 lOμg toxoid antigen in RLB I/hen Group 13 lμg toxoid antigen in RIBI/hen Group 14 lOμg somatic + lOμg toxoid antigen in RIBI/hen Group 15 lμg somatic + lμg toxoid antigen in RIBI/hen

White Leghorn hens were prime immunized with the above described doses of antigen in a total volume of 0.5 ml. Each antigen preparation was injected subcutaneously in the dorsal region of the upper third of the cervical area (back of the upper third of the neck). The booster immunization was carried out eight weeks after the initial immunization using the same methods of antigen preparation and immunogen emulsification described above, with the exception that Freund's Incomplete Adjuvant (FIA) (Sigma) was substituted for FCA in all booster preparations.

EXAMPLE 4

Passive Protection of Hamsters against Clostridium difficile Infection with Antibodies Against Spores or Germination Antigens

(a) Preparation of Clostridium difficile Spores

Clostridium difficile strain JGSX (a nontoxigenic isolate which does not produce toxins A or B, from J. Glenn Songer, Department of Veterinary Science, The University of Arizona, Tucson, AZ 85721) is cultivated on brain heart infusion (BHI, a commercially-available bacteriologic culture medium) agar with yeast extract (0.5%), cysteine (0.05%) and 5% bovine blood (collected with sodium citrate as an anticoagulant), incubated for 7 days at 37°C in an atmosphere of 10%H₂: 10%CO_2:go%N2 (a standard culture method). Colonies are harvested in sterile phosphate buffered saline (0.1 M, pH 7.4, PBS), centrifuged (3000 x g, 10 min), and resuspended in PBS. After incubation at 55°C for 15 min (this kills vegetative cells, leaving only spores), the suspension is chilled on ice. It is then layered onto a 3 step

(Percoll) gradient (80, 70, and 60% of Percoll or sodium bromide, equilibrated with PBS) and centrifuged at 3000 x g for 20 min to separate vegetative cells and cell debris from spores. The pellet, which contained approximately 98% spores, is washed once and stored at 4°C in sterile water until use. The number of viable spores in the final preparation is determined by plating dilutions on taurocholate fructose agar (TFA, a standard plating medium for cultivation of Clostridium difficile; germination of spores is stimulated by taurocholate). Plates are incubated anaerobically at 37°C for 24 to 48 h, as described, prior to counting of colonies,

(b) Antigen Preparation

Spore antigen is prepared by UV-irradiation, or by formalin- or iodine-inactivation (standard methods for inactivation of bacteria or spores), of Clostridium difficile spore stocks which are free of vegetative cells (as determined by phase-contrast microscopy).

For UV-irradiation, the spore stock is diluted to an optical density of 1, measured by spectrometry in a Beckman® DU-6. This diluted suspension is then pipetted into a plastic Petri dish with the lid removed and placed beneath a Mineralight® Model UVG-11 ultraviolet source. UV irradiation (wavelength 254 nanometers) is then delivered at 1000 Joules per minute, for 10 minutes, for a total dose of 10,000 Joules. Stirring, at about 30 φm, is continual during UV irradiation.

For formalin- or iodine-inactivation, spore stocks are treated with concentrations of the respective reagents and for sufficient time periods (at 4°C or

37°C) to achieve 100% inactivation. Efficacy of inactivation is tested by plating increasing dilutions of treated spores on TFA.

Antigens expressed during germination are prepared by inactivating spores at different stages of germination. (The theory is that the bacteria are producing molecules which sense the surrounding environment, or which perhaps serve the organism by transferring vital nutrients into the germinating cell.) Germination is induced by suspending spores in BHI broth containing 0.05% to 1.0% taurocholate, or to another appropriate germination medium. Germination is followed by phase contrast microscopy, and germinating cells are inactivated at varying times after induction by the methods described. The degree of germination is determined in parallel, as loss of resistance to heat or oxygen over time. Efficacy of inactivation is tested as described. (The idea is to collect germinating cells which possess all the antigens expressed by clostridium difficile between the time of initiation of germination and the appearance of the mature vegetative cell).

(c) Polyclonal Antibody Production

(1) Immunization of Goats for Production of Reference Antibodies to Clostridium difficile spores or germination receptors

Goats (intact or castrated Nubian males, 6-12 months of age) are immunized intramuscularly or subcutaneously with the equivalent of 10⁸ colony forming units

(CFU) of inactivated Clostridium difficile spores in Ribi adjuvant, according to the manufacturer's instructions. After 4 weeks, each animal is given a booster identical to the primary immunization. Serum collected following the booster immunization is examined for antibodies against spore or germination antigens by dot blotting (see below). Those animals not producing useful titers in response_^to an initial and one booster immunization are given a second booster, identical to the first. (2) Immunization of Hens for Production of Antibodies to Clostridium difficile spores or germination receptors

Animals and Immunization Procedure

Leghorn chicken hens or pullets hens are immunized intramuscularly or subcutaneously with 10¹ to 10¹⁰ inactivated spores of Clostridium difficile, emulsified in Freund's complete adjuvant (FCA); booster doses are administered 2-6 weeks apart. In the booster immunizations FCA is replaced by Freund's incomplete adjuvant where applicable.

Egg and blood collection Eggs are collected every two weeks and stored at 4° C until tested. Blood samples are collected from the wing vein at 2-4 week intervals, centrifuged at 200 g within 24 hours and sera stored at -20° C until analyzed. Serum and yolks samples are analyzed by enzyme immune assays such as ELISA, IFA, dot blot, electro immuno transfer blot (western blot) or immuno gold labelling. Antibody Specificity

Antibodies are screened for binding to vegetative cells or spores of Clostridium difficile. Vegetative cells or spores (n = 10⁶) from several strains are applied to a nitrocellulose membrane using a dot blot apparatus, followed by incubation with the prepared polyclonal antibodies. Reactivity is detected with a rabbit-anti-goat IgG-horseradish peroxidase conjugated or rabbit anti-chicken IgG-horseradish peroxidase conjugate. Color development is with4-chloro-l-naphthol and H₂O₂. Binding of the antibodies to the bacteria gives an indication that they may be useful in interfering with the normal physiologic processes of the organism.

Assay of Antibody-Efficacy

Antibody batches are screened for their ability to prevent germination of

Clostridium difficile spores. Spores (n = 10³) are incubated at 37°C, with shaking, with dilutions of the antibody to be tested. Spores are then plated on TFA and incubated anaerobically at 37°C for 24 to 48 h (as described), prior to counting colonies. (Binding is important, but inhibition of germination is more important).

EXAMPLE 5 Determination of Antibody Concentration in Yolk Preparations by an ELISA

Procedure

A quantity of Clostridium difficile spores was diluted to a protein concentration of 0.1 to 10.0μg/100 μl in buffer (0.048 M carbonate, pH 9.6), and lOOμl of the solution are deposited into each of the sample wells of a 96- well microtiter plate (Nunc Maxisorb⁰ Immunoplates). A quantity of lOOμL of carbonate buffer (0.048M, pH 9.6) was deposited in negative control wells. The covered plates were then incubated at 4°C for 12 to 24 h. The wells of the plates were then drained of fluid and washed 3x each with an ELISA wash buffer (0.1% v/v Tween-20° in phosphate buffer, 0.85% NaCl, 0.15M PO₄, pH 7.2). A quantity of 100 μL of blocking solution (1% casein or BSA) in ELISA dilution buffer (phosphate buffered saline) was delivered into each well and the plate was then incubated at 4°C for 12 to 24 h, and then the contents were aspirated and the plate wells washed as in the previous step. A sample of 1.0 ml immune hen egg yolk to be tested was dissolved or suspended in 1.0 ml of phosphate buffer. The stock solution was diluted 1:1000, and lOOμl of the diluted sample was serially diluted in two-fold increments and the diluted samples pipetted into each sample well. 100 μL of non-immune hen egg yolk sample was added to negative control wells; 100 μL of dilution buffer were added to non-primary antibody control wells, and lOOμl of known immune yolk preparation were added to positive control cells. The plates were covered and incubated at 37°C for 1.0 to 1.5 h. Contents of the wells were then aspirated and the wells washed with

ELISA wash buffer as in the prior incubations.

Peroxidase-labeled goat anti-chicken IgG antibody (Kirkegaard & Perry, Gaithersburg, MD 20879, Cat. No. 14-24-06, 0.5 mg) was diluted to a concentration of 0.025 μg/100 μL in phosphate buffer and an aliquot of lOOμl was delivered into each well of the microtiter plates, including the control wells. The covered plate was again incubated at 37°C for 1.0 to 1.5 h. After incubation with the second antibody, the plates were aspirated and washed as before.

The peroxidase label was developed by adding lOOμl of TMB® microwell peroxidase substrate system (Kirkegaard & Perry, Product code 50-76-00) to each well. The reaction was allowed to proceed for one minute and then stopped by the addition of 100 μL of 10% phosphoric acid.

The optical density (OD) of each well was read at 450nm using a Dynatech® ELISA plate reader. The concentration of primary antibody was calculated in activity units (A.U.) wherein 1 A.U. is equivalent to the OD at 2 standard deviations from the mean of the negative control wells, and using the known immune hen egg yolk sample to establish a standard calibration curve. These studies were followed by determination of the efficacy of antibodies against spores or germination antigens in preventing Clostridium difficile-induced disease in the hamster model.

EXAMPLE 6

Treatment of Hamster Model of Antibiotic-Associated Pseudomembranous

Colitis

Outbred female Syrian hamsters (Mesocricetus auratus, Charles River), approximately eight weeks old, were treated with 5-15 mg clindamycin (Upjohn) in

250μl of water using an 18-gauge, 2" gastric feeding needle (Popper and Sons).

Twenty four hours later, the animals were challenged orally with 10² to 10⁸ Clostridium difficile strain J9 spores in 250 μl using a gastric feeding needle. (The spores were harvested as described above).

Following challenge, the hamsters were monitored 2-3 times daily for the onset of symptoms of colitis. The onset of symptoms occured approximately 1-5 days after challenge and the animals were monitored more frequently during this time, as death is rapid after symptoms appear, often occurring within 24 h.

Passive protection from infection was tested by feeding egg yolk preparations from hens immunized with Clostridium difficile strain JGSX at daily intervals, for up to 5 days before challenge and up to 14 days after challenge.

EXAMPLE 7 ELISA Measurement of Antibody Titers

Preparation of Egg Yolk Samples for Assay

Eggs were collected weekly and stored at 4°C until processed. Blood samples were collected from the wing vein every other week, centrifuged at 200 g within 24 hours and sera stored at -20°C until evaluated.

Egg yolks were mechanically separated from whites, placed on soft paper towels, and rolled until the yolk sacks were dried. The yolk sacks were torn with a plastic tip and the contents carefully poured into sterile 50ml tubes and diluted 1 : 1 with sterile PBS. These yolk preparations were stored at -20°C until analyzed by an enzyme-linked immunosorbent assay (ELISA) as described below. Preparation of Hen Serum Samples

Serum samples collected as described above were diluted as required in PBS- T and lOOμl volumes were dispensed into each well of the 96-well plate and incubated at 37°C for 1 hour. Unbound antibodies were removed by washing three times with PBS-T. Assay Procedure

Clostridium difficile antigen solution was diluted to a protein concentration of 100-200 micrograms/ml of antigen in buffer (0.048 M carbonate, pH 9.6) and 100 μl of antigen suspension was dispensed into each if the sample and control wells of a 96-well flat-bottomed ELISA plate (Nunc Maxisorb Immunoplates. A quantity of 100 microliters of carbonate buffer (0.048 M, pH 9.6) was deposited in negative control wells. The antigen suspension was incubated on the covered plate for 12 to 24 h at 4°C. Unbound antigen was removed by washing three times with ELISA wash buffer, PBS containing 0.1% v/v Tween 20 in phosphate buffer, 0.85% NaCl, 0.15 M PO₄, pH 7.2 (PBS-T).

Thereafter, 200μl of 1% casein blocking solution (Fisher Scientific) in PBS was added to each well, incubated 12 to 24 h at 4°C, and the contents aspirated and the wells washed in PBS containing 0.1% Tween as described above.

A sample of 1.0 grams of lyophilized immune hen egg yolk or unprocessed hen serum to be tested was dissolved or suspended in 4.0 ml of phosphate buffer to a protein concentration of between about 25-35 mg/ml. The stock solution was diluted 1:1000, and 100 microliters of the diluted sample was serially diluted in twofold increments and the diluted samples dispensed into a series of sample wells on the test plates. 100 microliters of non-immune hen egg yolk or serum sample, diluted to a comparable protein concentration, was added to negative control wells. 100 microliters of dilution buffer were added to non-primary antibody control wells, and 100 microliters of appropriately diluted known immune yolk preparation or hen serum were added to positive control wells. The plates were covered and incubated at 37 degrees C for 1.0 to 1.5 h. Contents of the wells were then aspirated and the wells washed with ELISA wash buffer as in the prior incubations.

Peroxidase conjugated goat anti-chicken IgG immunoglobulins (Kirkegard and Perry Laboratories, Gaithersburg, Maryland 20879, Cat. No. 14-24-06, 0.5 mg) were diluted to a concentration of 0.025 micrograms/100 microliters in PBS and 100 μl volumes were dispensed into each well and the plates again incubated at 37°C for 1 hour. Unbound secondary antibodies were removed by aspirating the wells of the plates and washing five times with PBS-T.

The peroxidase label was developed by adding 100 μl of TMB substrate solution (Kirkegard and Perry Laboratories, Product Code 50-76-00) to each well and allowing the reaction to proceed for 1-3 minutes. The reaction was stopped by the addition of 100 μl of 0.1 N phosphoric acid. The optical density (OD) of each well was read at 450nm using a Dynatech MR600 ELISA Microplate Reader. The concentration of primary antibody was calculated in activity units (AU) wherein 1 AU is equivalent to the OD at 2 standard deviations from the mean of the negative control wells, and using the known immune hen egg yolk sample or hen serum sample to establish a standard calibration curve.

Results of ELISA Assays

In Study I above, the hens showed detectable antibody production against the antigens used for immunization. Figure 1 shows the results of the ELISA assays on the hens of Study I. Bar T shows the average ELISA results from the hens of Group 3, which were immunized with 5mg of toxoid. Bar S shows the average ELISA results from the hens of Group 2, which were immunized with 5mg of somatic antigen. The bar labeled ST shows the average ELISA results from the hens of Group 1 , which were immunized with 5mg of somatic antigen plus 5 mg of toxoid antigen. The bar labeled Neg Control shows the average ELISA results from the control hens of Group 4, which were immunized with a saline control. As shown in

Figure 1, the average ELISA analyses of the antibodies produced by the hens of Groups 1 , 2, and 3 in Study I yielded optical densities of about 1.0.

The hens of Study II also mounted an immune response against the Clostridium difficile antigens. Average ELISA OD results from the Main Flock of Study II were as follows:

3 weeks post prime 0.797 - • 0.974

6 weeks 0.943

9 weeks 0.910

12 weeks 0.909

4.5 months 0.882

Peaks in antibody titers in all groups were detected 2-4 weeks from the booster immunization (ELISA OD values around 1.0). High Elisa OD values were still detectable 4 months post peak. The titers did not significantly increase after the booster, but were maintained at almost the same level for an additional 4 months.

The results from the 15 additional groups of Study II were are shown in Figure 2. The results obtained with hens immunized with antigen in Freund's Complete Adjuvant were as follows. The average optical densities in the ELISA assays for Group 1 of Study π, depicted in the bar labeled S20(F), were 0.713, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 2 of Study II, depicted in the bar labeled S 10(F), were 0.418 as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 3 of Study II, depicted in the bar labeled S 1(F), were 0.372, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 4 of Study II, depicted in the bar labeled T 10(F), were 1.008, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 5 of Study II, depicted in the bar labeled T1(F), were 1.106, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 6 of Study II, depicted in the bar labeled S10T 10(F), were 1.001, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 7 of Study II, depicted in the bar labeled S 1T1(F), were 0.840, as indicated in the table adjacent to the graph. The results obtained with hens immunized with antigen in Ribi Adjuvant were as follows. The average optical densities in the ELISA assays for Group 8 of Study II, depicted in the bar labeled S20(R), were 0.362, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 9 of Study II, depicted in the bar labeled S10(R), were 0.406, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group

10 of Study II, depicted in the bar labeled S1(R), were 0.454, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 11 of Study II, depicted in the bar labeled T20(R), were 0.790, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 12 of Study II, depicted in the bar labeled T10(R), were 0.622, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 13 of Study II, depicted in the bar labeled T1(R), were 0.493, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 14 of Study II, depicted in the bar labeled S 10T10(R), were 0.578, as indicated in the table adjacent to the graph. The average optical densities in the ELISA assays for Group 15 of Study II, depicted in the bar labeled S1T1(R), were 0.354, as indicated in the table adjacent to the graph.

Although concentrations of somatic antigen of 20μg/hen/dose had a mild adverse effect on the hens, such concentrations still yielded antibody titers suitable for passive immunization. No adverse effects were observed in hens which were immunized with the toxoid preparations.

The above results demonstrate that the hens mounted a significant immune response against the Clostridium difficile antigens when doses between lμg/hen/dose and 25 μg/hen/dose were administered. In hens receiving antigen in Freund's adjuvant, high levels of antibody response were detected at doses of 10 μg/hen/dose and 1 μg/hen/dose of toxoid either alone or in combination with lOμg of somatic antigen.

EXAMPLE 8 Chinese Hamster Ovary (CHO) Cell Tissue Culture Assay for Determination of Clostridium difficile Toxicity

Chinese hamster ovary cells were grown to confluence in Iscove's Modified Eagle's medium (IMEM, Gibco-BRL), supplemented with 10% fetal bovine serum (FBS, Omega Scientific) and 100 μg/gentamicin (Sigma) at 37°C and 5% CO₂ in a humidified incubator. The cells were trypsinized and 100 μL of the suspension containing 5 x 10³ cells were dispensed into well of a 96-well microtiter plate. The plate was incubated at 37°C and in an atmosphere of 5% CO₂ in a humidified incubator for 18 h to allow the cells to attach to the surface of the wells. Two-fold dilutions of toxin and toxoid were prepared in IMEM- 10% FBS and lOOμL of the various dilutions were added to marked wells in duplicate.

The endpoint ot titer for toxicity was defined as the lowest toxin or toxoid dilution that causes rounding of <100% of cells (100% cytopathic effect, CPE) compared to a control sample which has not been exposed to toxin or toxoid. 100% CPE is therefore that dilution point that causes 100% rounding of cells compared to the control. Optionally, the cells were then fixed with methanol for 5 min, following by staining with crystal violet solution for 10 min.

Neutralizing antibody titers are determined by incubating 2-fold serial dilutions of the antibody to be tested with a 2X volume of the 100% CPE dose for 1 h at room temperature before adding lOOμL of each of the mixtures to the wells containing attached CHO cells above. The neutralizing antibody titer is defined as the dilution that prevents all cells from rounding as compared to the control.

EXAMPLE 9 Evaluation of Antibodies in CHO Assay

The ability of the antibodies generated as described above to neutralize Clostridium difficile toxin was assessed in an in vitro assay using Chinese Hamster Ovary (CHO) cells. The CHO cells were grown to confluence in Iscove's Modified Eagle's medium (IMEM, Gibco-BRL) supplemented with 10% fetal bovine serum (FBS, Omega Scientific) and lOOμg/ml gentamicin (Sigma) at 37°C and 5% CO₂ in a humidified incubator.

The cells were trypsinized and aliquots of 5 X 10³ cells/well were distributed into the wells of a 96 well tissue culture plate. The cells were incubated overnight at 37°C and 5% CO, in a humidified incubator. Two fold dilutions of toxin and toxoid were prepared in IMEM- 10% FBS. lOOμl of each of the dilutions was added to duplicate wells containing the CHO cells. The cells were incubated for 18 hours at 37°C and 5% CO₂ in a humidified incubator. Optionally, the cells were then fixed with methanol for minutes, followed by staining with crystal violet for 10 minutes.

100% CPE was defined as the lowest toxin dilution that caused rounding of 100% of the cells when compared to the controls.

Neutralizing antibody titers of serum samples was defined as the lowest antibody titer determined as follows. Two fold serial dilutions of the antibody to be tested in IMEM-10% FBS were incubated with an equal volume of the 100% CPE dose for 1 hour at room temperature. lOOμl of each of these mixtures was added to wells containing 5 X 10³ cells/well. The neutralizing titer was defined as the antibody dilution which prevents 100% of the cells from rounding as compared to a control to which no antibody was added.

In the hens of Study I above, hens immunized with somatic and toxoid showed the highest in-vitro neutralization activities, with titers up to 65,536. The toxin control was titrated at 81,920.

EXAMPLE 10

Partial Removal of Lipids from Immune Egg Yolks by Dilution (Low Lipid Lyophilized (LLL) Product)

Egg yolks were obtained from the eggs of Salmonella-free ISA brown chicken hens that had been hyperimmunized with Clostridium difficile,, sporozoite antigens. The yolks were separated from the whites, using a Seymour Egg Breaker (Sanovo Seymour, Topeka, Kansas) pooled, and then diluted at a ratio of about 1:9

(yolk:water) with purified water. The water was precooled to about 2°C to 5°C prior to use and the mixture was maintained at this temperature overnight for batch preparations. Approximately 50% of the fat solids, together with other macromolecules, spontaneously separated as solids from the diluted yolk phase of the supernate. The anti-Cryptosporidium parvum antibodies contained in the hen egg yolks were found in the supernatant phase. A quantity of sugar or other sweetening agent (preferably about 0.4% w/v) was added to the supernatant phase containing the antibodies, and optionally, the solution was sterilized by filtration, for example, through a 0.22 micron filter (Amicon, Beverly, Massachusetts). The antibody containing supernate fraction is optionally concentrated by cross-flow filtration and then optionally, it is lyophilized and placed in clean or sanitized containers. The material is also preferably analyzed for nutrient content.

The improved egg yolk anti-Cryptosporidium parvum formulation is a lyophilized product having at least about 50%, and preferably at least about 70% of the yolk lipids removed. Over about 90% of the lipids were removed in some batches of the procedure. The greater fraction of protein, including antibody protein, remains in the supernate. Summary

It should be apparent from the foregoing that other avian animals or antibody preparations can be substituted in the Examples to obtain similar results of passively immunizing a vertebrate animal effectively through the oral route. In particular, it will be appreciated that yolk preparations made according to the procedures disclosed in the U.S. Application entitled "Lipid Reduced Oral Formulation for Egg Yolk Derived Therapeutic Protein," Serial No. 60/089,644, filed June 4, 1998, the disclosure of which is incoφorated herein by reference, may also be used in the present invention. It should be further emphasized that the present invention is not limited to the use of any particular antigenic agent in producing the antibody preparations of the invention. Thus, regardless of whether a specific Clostridium difficile strain or antigen is presently known, or whether it becomes known in the future, the methods of forming the presently contemplated HEY antibody preparations therefrom are based on established chemical techniques, as will be apparent to those of skill in the art, and therefore these preparations are broadly enabled by the preceding disclosure. It should be emphasized again that the present methods are broadly applicable to formation of antibody preparations from essentially all Clostridium difficile antigens, and the immunization of a vertebrate to Clostridium difficile may be improved by preparing an avian antibody oral form for use in the practice of the invention.

Accordingly, the invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive, and the scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All modifications which come within the meaning and range of the lawful equivalency of the claims are to be embraced within their scope. All references and patent applications cited herein are incoφorated herein by reference in their entirety.

Claims

1. Hen egg yolk, or a fraction thereof, containing hen egg yolk antibodies having a specificity for at least one spore antigen or germination antigen of Clostridium difficile or a mixture of said antibodies.

2. Hen egg yolk comprising hen egg yolk antibodies having a specificity for at least one antigen of Clostridium difficile or a mixture of said antigens, where the lipid content of said egg yolk is reduced at least about 10%.

3. Hen egg yolk according to Claim 2 wherein the lipid content of said egg yolk is reduced at least about 50%

4. Hen egg yolk according to Claim 2 wherein the lipid content of said egg yolk is reduced at least about 90%

5. Hen egg yolk according to Claim 2 comprising hen egg yolk antibodies having a specificity for at least one somatic antigen of Clostridium difficile.

6. Hen egg yolk according to Claim 2 comprising hen egg yolk antibodies having a specificity for at least one antigen of Clostridium difficile toxin

A or toxin B.

7. Hen egg yolk according to Claim 2 comprising hen egg yolk antibodies having a specificity for at least one spore antigen or germination antigen of Clostridium difficile or a mixture of said antibodies.

8. Isolated hen egg yolk antibodies having a specificity to at least one spore antigen or germination antigen of Clostridium difficile, or a mixture of said antibodies.

9. A hen egg yolk product or hen egg yolk antibodies according to Claim 1 or 2 that is freeze-dried or lyophilized.

10. A pharmaceutical formulation comprising a hen egg yolk product according to Claim 1 or 2 in a pharmaceutically acceptable carrier.

11. A pharmaceutical formulation comprising a hen egg yolk product according to Claim 7 in a pharmaceutically acceptable carrier.

12. A pharmaceutical formulation comprising a hen egg yolk product according to Claim 8 in a pharmaceutically acceptable carrier._^

13. The use of immune hen egg yolk, or an immunity-conferring fraction thereof, containing hen egg yolk antibodies having a specificity for at least one spore antigen or germination antigen of Clostridium difficile, or a mixture of said antibodies in the treatment of an intestinal infection caused by Clostridium difficile in a mammal.

14. The use of immune hen egg yolk, or an immunity-conferring fraction thereof, comprising hen egg yolk antibodies having a specificity for at least one somatic antigen of Clostridium difficile, or having specificity for an antigen of Clostridium difficile toxin A or toxin B wherein the lipid content of said hen egg yolk or hen egg yolk fraction is reduced at least about 10% for treating an intestinal infection caused by Clostridium difficile in a mammal.

15. The use of immune hen egg yolk, or an immunity-conferring fraction thereof, comprising antibodies having a specificity for at least one spore antigen or a germination antigen of Clostridium difficile or a mixture of said antibodies to provide passive immunity against Clostridium difficile in a mammal.

16. The use of immune hen egg yolk, or an immunity-conferring fraction thereof, containing hen egg yolk antibodies having a specificity for at least one antigen of Clostridium difficile or to a toxin produced by Clostridium difficile, wherein the lipid content of said hen egg yolk or egg yolk fraction is reduced at least about 10% in providing passive immunity against Clostridium difficile to said mammal.

17. A use of said immune hen egg yolk according to any one of Claims 13-16 wherein said enteral administration comprises oral ingestion, gastric intubation, rectal intubation, or direct injection of said hen egg yolk antibodies into the digestive tract of said mammal.

18. A use of said immune hen egg yolk according to any one of Claims 13 or 14 in treating a mammal that has become infected with Clostridium difficile as a consequence of antibiotic treatment.

19. A use of said immune egg yolk according to any one of Claims 13-16 wherein said mammal is a human being.

20. A use of said immune egg yolk according to Claims 13-16 wherein said mammal is a juvenile.