AU2006200963A1 - Compositions and vaccines containing antigen(s) of cryptosporidium parvum and of another pathogen - Google Patents

Description

06/03 '06 17:43 FAX 613 8618 4199 F.B. RICE Co.

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AUSTRALIA

@005 Patents Act 1990

MERIAL

COMPLETE

SPECIFICATION

STANDARD PATENT Invention Title: Compositions and vaccines containing antigen(s) of cryptosporidium parvum and of another pathogen The following statement is a full description of this invention including the best method of performing it known to us:- COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:44 FAX 613 8618 4199 F.B. RICE Co. _006 PCT/EP00/13387 VO WO 01/45735 o TITLE OF TlE INVENTION S COMPOSITIONS AND VACCINES CONTAINING ANTIGEN(S) OF CRYPTCSPORIDI U M PARVW AND OF ANOTHER ENTERIC

PATHOGEN

Va FIELD OF THE-INVENTION The invention relates to antigen(s)/epitope(s) of CryptosporidUwn parvnm and/or V enteric pathogens (such as other enteric pathogens), compositions and methods comprising or using the same for eliciting an immune response against, or for prevention, treatment, or O control of Cryptosporidium parvum and/or enteric infections, and uses thereof.

,0 to The invention further relates to methods and/or compositions, and/or uses of such o compositions or components thereof in formulating such compositions, for eliciting an.

immune response against and/or for the prevention and/or treatmnt and/or control of enteric infections in animals, for instance mammals, such as bovines, felines, canines or equines or species thereof.

The invention relates also to methods and/or compositions, and/or uses of such compositions or components thereof in formulating such compositions, for eliciting an immune response against and/or for the prevention and/or treatment and/or control of infection by Cryptosporidimn parvum.

The invention can also relate to the concurrent use of a monovalent Cryptosporidium parvum vaccine with enteric, e.g. bovine enteric rota/coronavirus, E. colt) vaccines and/or use of a combination vaccine containing Cryptosporidium parvum rota/coronavims, E. coli, as well as to preventing, controlling or treating or eliciting an immune response to reduce exacerbation of enteric, bovine enteric, diseases due to co-infection with Cryptosporidium parvum. The immunity induced by vaccination against Cryptosporidium parvt, can significantly reduce the severity of the disease induced by herein mentioned enteric pathogens. A combination vaccine containing Cryptosporidium parvum is useful for a more complete prevention of multietiological enteric disease in newborn animals, such as calves, caused by rota and cororiaviruses and E. coli K99 and F41.

This invention also pertains to the effects of Cryptosporidum parvum co-infection on other enteric, bovine enteric, pathogens. Cryptosporidium parvum is commonly found in the feces of newborn animals such as mammals, calves. Cryptospridinum parvum is able SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:44 FAX 613 8618 4199 F.B. RICE Co. 007 O O 01/45735 PCTIEPOO/133 8 7 o 2 to produce clinical signs of enteric disease by itself, regardless of the presence or absence of IO other potentially pathogenic viruses and bacteria in the gut. Viruses, such as coronavirus, and 0 bacteria, such as E. coli F41, that have been recognized in the field as very pathogenic are not able to cause important clinical signs of disease in experimental challenge models. Thus, S 5 the invention can relate to addressing the co-infection of cattle with Cryptosporidium parvum o as that co-infection can exacerbate the disease caused, by other enteric pathogens such as i coronavirus, rotavirus, and E. coli P41.

SVarious documents are cited in this text. Citations in the text can be by way of a

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0 citation to a document in the reference list, by way of an author(s) and document year citation to a document listed in the reference list, or by full citation in the text to a document that may or may not also be listed in the reference list.

There is no admission that any of the various' documents cited in this text are prior art as to the present invention. Any document having as an author or inventor person or persons named as an inventor herein is a document that is not by another as to the inventive entity herein. All documents cited in this text ("herein cited documents") and all documents cited or referenced in herein cited documents are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION Bovine enteric disease is the result of an enteropathogenic intestinal infection that most often manifests itself in some form of diarrhea. This disease, also commonly referred to as neonatal calf diarrhea, is responsible for substantial economic loss in the faaning industry.

The morbidity of the calves, together with the need for therapeutic intervention and the possible long term detrimental effects on the animals, are the main factors responsible for the economic burden on the farmer. One estimate indicates that neonatal calf diarrhea is responsible for about 75% of the death of dairy calves under 3-weeks of age. Radostits, OM, et al., Herd Health Food Animal Production Medicine, 2" ed., Sounders, Philadelphia, pp.

184-213, 1994. The management of neonatal calf diarrhea is difficult for multiple reasons, some of the most important which include: the involvement of multiple agents in the pathogenesis of the disease; the nonspecificity of clinical signs; the finding that some infections can be asymptomatic; and, the involvement of host factors such as nutrition and endogenous immunity. Moon, HW, et al., JAVMA 173 577 583 (1978). Viring, S. et al., Acta Vet. Scand. 34: 271- 279 (1999).

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:44 FAX 613 8618 4199 F.B. RICE Co. 008 O WO 01/45735 PCT/EP00/13387 0 o0 3 c Developing a strategy to prevent or treat bovine enteric disease has been very difficult since while it is known that multiple enteropathogens are present during the infection, it is not Va o known which pathogen or combination of pathogens is actually responsible for the disease.

Epidemiological studies in the United States as well as in other parts of the world show that C 5 the most prevalent enteropathogens associated with neonatal calf diarrhea include, but are not Va C^ limited to, Cryprosporidium parvum, rotavirus, coronaviras and E. coli. While in most cases O several of these enteropathogens are isolated from outbreaks of the disease, the prevalence of 0O each of the agents is not consistent within a single diseased population or between multiple o infected herds. Traditionally, studies found rotavirus to be the most prevalent enteropathogen in diarrheic calves. For example, in a study of diarrheic calves in Great Britain, rotavirus and Cryptosporidium parvum were detected in 42 and 23% of the population, respectively.

Twenty percent of the calves were infected with more than one pathogen. However, more recent reports indicate Cryptosporidium parvum to be the predominant pathogen in enteric bovine infections. In a recent study evaluating Cryptosporidium parvum and concurrent infections by other major enteropathogens in neonatal calves, Cryptosporidiwn parvum was the only enteropathogen found in 523% of the population, followed by single infections with rotavirus at 42.7%. de la Puente et al., Preventive Veterinary Medicine 36: 145 152 (1998) Concurrent infection with two agents occurred in 21_6% of this study group while infection with three and four pathogens was found in 6% and respectively. The most common mixed infection in this study was a combination of Cryptosporidium-rotavirus. There is limited information available on the role of individual enteric pathogens in neonatal calf diarrhea. Furthermore, combined mechanisms of viral, bacterial and protozoal pathogenesis underlying the bovine enteric disease in neonatal animals are even more poorly understood.

However, irrespective of the lack of understanding of the mechanism of pathogenesis, infection with more than one pathogen tends to lead to a more severe clinical"outcome than infections caused by a single enteropathogen.

At the present time there is no method of treatment that affords adequate protection against neonatal calf diarrhea.' There is no single drug or combination of chemotherapeutic agents useful in the treatment of this disease. While vaccines are available which target bovine enteric disease, they have been met with limited success and acceptance. Presently available are vaccines that contain antigens to three enteropathogens found to be associated SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:45 FAX 613 8618 4199 F.B. RICE Co. 009 NO PCT/EPOO/13387 0 WO 01/45735 S4 with the disease, namely rotavirus, coronavirms and E. coli Efficacy of individual-components NO of these commercially available bovine enteric vaccines (rota/corona, E coli) have been shown to protect in experimental challenge models. Despite the availability of such vaccines, en under field conditions neonatal diarrhea, calf scours and winter dysentery continue to affect NO 5 beef, feedlot and cow calf operations. Producers permanently question the efficacy of current Senteric vaccines containing E. coli K99, rota and coronavirus under field conditions as is reflected by the low usage of the enteric combo vaccinbs in the US market (only 4% of o pregnant animals are vaccinated annually with this product).

CN More recently, a monovalent experimental vaccine against Cryptosporidium parvum has been developed and shown to protect against a Cryptosporidium parvwn experimental .challenge. However, the multiple enteropathogens involved in enteric disease cannot be overcome by treatment with a Cryptosporidiim parvum vaccine alone. Also, enteropathogenic infection appears to be universal; it is found throughout the world and most vertebrates are susceptible to such infection. Therefore, a need to combat enteropathogenic infection is not limited to the bovine species. Furthennore, enteric disease is difficult to control; it is likely multifactoral; Cryptosporidium parvum may be a factor, but heretofore there is no definitive showing that Cryptosporidium parvum indeed enhances enteric disease or that its use in a combination immunogenic, immunological or vaccine composition enhances preverition of enteric disease.

Further, a problem encountered in the preparation and use of combination vaccines is the phenomenon called "efficacy interference" wherein the efficacy of one antigen in the combination is diminished or reduced, believed to be from dominance by another antigen in the combination vaccine; cf Paoletti et al., U.S. Patent No. 5,843,456. This phenomenon has been observed with combination vaccines that employ E coli antigen or antigens; for instance, single or multiple bacterin can interfere with other antigens in combination vaccines.

Thus, it is believed that heretofore the problem of Cryptosporidium parvum contributing to enteric infections and symptoms, or the manner in which this problem is herein addressed, combination compositions including Cryprosporidium parvun antigen(s) or epitope(s) of interest with at least one other antigen or epitope of interest from a pathogen that causes enteric infection and/or symptoms and/or recombinant(s) and/or vector(s) and/or plasmid(s) expressing such antigen(s) or epitope(s) of interest and administration of such SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:45 FAX 613 8618 4199 F.B. RICE Co. [21010 ci ^C compositions to pregnant mammals such as pregnant cows and/or newborn or young mammals such as calves within the first month of birth, and addressing any potential oN issue of efficacy interference, have not been disclosed or suggested.

SUMMARY OF THE INVENTION IsD It is desirable that the present invention provide improved enteric immunological or vaccine compositions, especially those which can be used in the o veterinary field, for instance for mammals such as bovines, canines, felines or equines )0 or species thereof.

S 10 It is also desirable that the present invention provide such immunological or 1 vaccine compositions which can be effectively used to immunize newborn and/or young animals, such as to passively immunize new-born animals, mammals, for instance, bovines, canines, felines or equines or species thereof advantageously bovines.

Still another desirable form, the present invention can provide improved immunological or vaccine compositions against Cryptosporidium parvnum, for instance particular to be used in the veterinary field, such as for use with mammals, e. for canines, felines or equines or species thereof, especially bovines or species thereof.

Yet another desirable form of the invention can be to provide improved methods for immunizing newborns and/or young animals, such as to passively immunize newborn animals, mammals, such as canines, felines or equines or species thereof especially bovines or species thereof.

Even further still, it is desirable that the present invention provide methods for eliciting an immune response against Cryptosporidium parvum or enteric pathogens including Cryprosporidium parvum or for controlling, preventing and/or treating enteric infections and/or symptoms including Cryptosporidium parvum; for instance, comprising administering an inventive composition; as well as methods for preparing such compositions, uses of components of such compositions for formulating such compositions, inter alia.

Thus according to one aspect, the present invention provides a combined enteric immunological, immunogenic or vaccine composition comprising a first antigen or epitope of interest from Cryptosporidium parvum, and a second antigen or epitope of interest from E. coli, rotavirus, coronavirus, Closiridium spp or mixtures thereof, and a pharmaceutically acceptable vehicle.

m:\specificatlons500000502000\50218$dmmrnjc.doc COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:45 FAX 613 8618 4199 F.B. RICE Co. ~011

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o rcl Vaccination or immunization against enteric pathogens, such as enteric pathogens including Cryptosporidium parvum is greatly and unexpectedly improved by using an immunological or vaccine conposition including a combination of at least two Cryptosporidium parvum antigens or epitopes thereof and/or vector expressing at least two Cryptosporidium parvum antigens or epitopes thereof, P21 or an epitope thereof and/or a vector expressing P21 or an epitope thereof or Cp23 or an epitope thereof and/or a vector m:\speeIifcatlons50000\5020001502185climmJ.doc COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:45 FAX 613 8618 4199 F.B. RICE Co. 1012 WO 01/45735 PCT/POO/13387 o 6

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t expressing Cp23 or an epitope thereof and Cpl5/60 or an epitope thereof and/or a vector expressing Cpl5/60 (for instance, a composition containing at least one epitope of Cp23 and at oN least one epitope of CplS/60; and it is noted that the Cp23 antigen or protein can include P21).

The combination of both antigens (or epitope(s) of interest and/or vectors expressing the cn 5 antigens and/or epitope(s)) leads to a synergistic effect with an improved or useful production of an immune response, antibodies, cellular responses or both, against Cryptosporidium o parvum and/or enteric infection or pathogens or symptoms such as a very high production of Santibodies against Cryptosporidium parvum. This also allows for the preparation of efficient o immunological or vaccine compositions, useful to protect newborn or young animals or C i to mammals, for instance, canines, felines or equines or species thereof; especially bovines. For instance, compositions containing antigens and/or epitope(s) of interest may be advantageously employed in inoculating dams or pregnant females, to elicit an immune response that can be passed to the yet born offspring and to new-born or young animals via milk or colostrum during weaning, and, compositions containing vector(s) expressing antigens andlor epitope(s) may advantageously be employed in inoculating males and females of all ages, such as those that are not pregnant and/or are new-born or young animals, and the inoculation of new-bom or young animals can be done alone or advantageously in conjunction with the inoculation of dams or pregnant females, eg., to allow for immune responses to be generated in the young or new-born animals while they also receive antibodies or other immunological agents via milk or colostrum during nursing.

Combining in an immunological or vaccine composition antigen(s) and/or epitope(s) of interest against Cryptosporidium parvnm with at least one other antigen or epitope of interest against at least one other enteric pathogen of the animal species (and advantageously a plurality of antigen(s) and/or epitope(s) of interest from a plurality of pathogen(s), enteric pathogens) can significantly increase protection against enteric pathologies.

An especially advantageous inventive immunological or vaccine composition can be against Cryptosporidium parvum and can comprise at least one Cp23 antigen or epitope of interest thereof and/or at least one vector expressing at least one Cp23 antigen or epitope of interest thereof or at least one P21 antigen or epitope of interest thereof and/or at least one vector expressing at least one P21 antigen or epitope of interest thereof and (ii) at least one Cpl5/60 antigen or epitope of interest thereof and/or at least one vector expressing at least one SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:46 FAX 613 8618 4199 F.B. RICE Co. I013 WO 01/45735 PCTIEP00/13387 0 7 0 cCp 15/60 The composition can advantageously further comprise at least one additional antigen or epitope of interest from another enteric pathogen and/or a vector expressing at least one O additional antigen (which can be the same vector that expresses the Cp23 or P21 antigen or epitope of interest and/or the Cpl5/60 antigen or epitope of interest, the composition can c 5 comprise a vector that co-expresses the Cp23 or P21 antigen or epitope of interest and the I0 Cpl5/60 antigen or epitope of interest, and optionally the optional additional antigen or Sepitope of interest).

S- Another Cryptosporidium parvum antigen is the CP41 antigen described in Mark C.

SJenkins et al., Clinical and Diagnostic Laboratory Immunology, Nov. 1999, 6, 6 912-920.

CN 10 The immunological or vaccine compositions according to the invention may comprise this antigen or epitope of interest thereof and/or a vector expressing said antigen or epitope thereof, possibly and preferably in association with at least one other Cryptosporidiumparvum as described herein such as Cp23, P21 and Cp15/60, e.g. in combination with Cp23 or P21 and/or Cp 15/60. For expression of this antigen, one may add a start codon upstream the nucleotide sequence appearing on Figure 2 of this publication, and a stop codon downstream this sequence.

An efficient immunological or vaccine composition against enteritis is also produced by using only one of: the Cp23 or an epitope thereof or a vector expressing the antigen or epitope, or P21 or an epitope thereof or a vector expressing the antigen or epitope, or Cp15160 or an epitope thereof or a vector expressing the antigen or epitope thereof, or CP41 or an epitope thereof or a vector expressing the antigen or epitope, as a Cryptosporidium parvum antigen or epitope of interest, advantageously in combination with at least one other Cryptosporidium parvum antigen or epitope of interest or vector expressing such an antigen or epitope of interest; and, this composition can further comprise at least one additional antigen or epitope of interest from another enteric pathogen and/or a vector expressing the at least one additional antigen (and this vector can co-express antigen(s) and/or epitope(s)).

The invention further comprehends methods for eliciting an immunological or protective (vaccine) response against or for controlling, preventing and/or treating enteric pathogens or enteric infections or enteric symptoms, including Cryptosporidiuw parvum; for instance, comprising administering an inventive composition.

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:46 FAX 613 8618 4199 F.B. RICE Co. 1014 WO 01/45735 PCT/EP00/13387

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o8 t An inventive composition can be administered to a pregnant mammal, such as a heifer or a cow (hereinafter called cow), dog, cat, or horse during the gestation period; for instance, NO once or twice during the typical gestation period (for a cow, typically a 9 month or 170 day 0 gestation period), such as a first administration about 1 to about 2.5 or about 3 months before C 5 calving and a second or sole administration close to calving, in the last 3 weeks before -calving, preferably about 3 to about 15 days before calving. In this way, the female can Stransfer passive immunuiry to the newbom, calves after birth via milk or colostrum.

t C Advantageously, compositions comprising antigen(s) and/or epitope(s) of interest (as opposed Va 0 to compositions comprising vector(s), recombinant(s) and/or DNA plasmid(s)) are CN 10 administered to pregnant mammals as eliciting an antibody response is desired And, in contrast, such compositions that comprise vector(s), recombinant(s) and/or DNA plasmid(s) that express the antigen(s) and/or epitope(s) of interest in vivo are advantageously administered to a newborn or very young mammal a mammal that is susceptible to enteric disease, such as a bovine during about its first month of life and other mammals during analogous periods in their life), as a cellular and/or antibody response can be useful to prevent, treat, and/or control enteric conditions, infections or symptoms in such newborn and/or very young animals- The newborn and/or very young animals can receive a booster of an antigenic and/or epitopic and/or vector/recombinant/DNA plasmid composition during the period of susceptibility, and, its mother, optionally and advantageously, can also have been vaccinated during pregnancy, as herein described, such that the newborn and/or very young animal can be receiving an immunological response by way of the administration directly to it and passively.

A particular inventive composition can comprise one or more E. coli antigens inactivated E. coli bearing pill, such as, K99, Y, 31A, and/or F41and/or these pili in subunit form or recombinantly expressed in vivo) and/or one or more rotavirus antigens advantageously inactivated rotavirus), and/or one or more coronavirus antigen bovine coronavirus antigen, advantageously such as inactivated coronavirus), in combination with one or more Cryptosporidium parvim antigens, such as P21 and/or Cp23 and/or Cp15/60. (And, as mentioned previously, one or more of these antigens can be an epitope of interest contained within the antigen; and, one or more of these antigens or epitopes of interest can be expressed in vivo by a recombinant or a plasmid.) SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:47 FAX 613 8618 4199 F.B. RICE Co. [1015 WO 01/45735 PCTEP 13387

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o9 SThus, a particular inventive composition can comprise one or more Cryptosporidium parvun antigens, such as P21 and/or Cp23 and/or Cp15/60 and/or CP41 and VO advantageously P21 and/or Cp23 and Cp15160, and (ii) at least one E. coli antigen at 0 least one or all of of K99, Y, 31A, F41 and/or other pili borne by inactivated E. coli or as S 5 subunits or as expressed in vivo; K99 and/or F41 are.preferably present and Y and/or 31A are O advantageously also present) and/or coronavirus and/or rotavirus antigen; such as one or 0 more C. parvum antigens, such as P21 and/or Cp23 and/or Cpl5/60 and/or CP41 and C1 advantageously P21 and/or Cp23 and Cpl5/60 and one or more rotavirus antigen such as o inactivated rotavirms, or one or more C. parvum antigens, such as P21 and/or Cp23 and/or CN 10 CplS/ 6 0 and/or CP41 and advantageously P21 and/or Cp23 and Cp15/60 and one or more coronavirus antigen such as inactivated coronavirus, inactivated bovine coronavirus, or one or more C, parvum antigens, such as P21 and/or'Cp23 and/or Cpl5/60 and/or CP41 and advantageously P21 and/or Cp23 and Cp15/60 and one or more E. coli aitigen such as K99, Y, 31A, F41 and/or other pili borne by inactivated E. coli or as subunits or as expressed in vivo, a combination of K99, Y, 31A and/or F41. An- exemplary E. coli antigen useful -in the invention can be pili as E. coli pili can avoid efficacy interference. An exemplary composition can comprise one or more C. parvum antigens, such as P21 and/or Cp23 and/or Cpl5/60 and/or CP41 and advantageously P21 and/or Cp23 and Cpl5/60 and at least one E.

coli antigen, and at least one coronavirus antigen, and at least one rotavirus antigen, P21 and/or Cp23 and/or Cpl5/60 and/or CP41 and advantageously P21 and/or Cp23 and Cp15/60 and inactivated rotavirns, and inactivated coronavirus, and at least one E coli antigeu, advantageously pili or preferably at least one or more of K99, Y, 31A, and F41, or a combination of K99, Y, 31A and F41. (And, as mentioned previously, one or more of these antigens can be an epitope of interest contained within the antigen; and, one or more of these antigens or epitopes of interest can be expressed in vivo by a recombinant or a' plasmid.) In regard to potential efficacy interference by single or multiple bacterin, the inventors have found that by increasing the amount of other antigens present in a combination vaccine, any potential efficacy interference is avoided; and, that the use of pili as an E. coli antigen also avoids efficacy interference.

In these inventive compositions, a single dose can have the E. coli antigen (or each E.

coli antigen, in the case of multiple E. coli antigens) present in an amount usually found in SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:47 FAX 613 8818 4199 F.B. RICE Co. [1016 WO 01/45735 PCT/EPO0113387

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010 ci k vaccines against enteric pathogens such as an amount to obtain a serum titre in guinea pigs of at least 0.9 log 10; the rotavirus antigen can be present in an typically found in vaccines \D against enteric pathogens, such as an amount to obtain a serum titre in guinea pigs of at least log 10, and the coranovirus antigen can be present in an amount typically found in s vaccines against enteric pathogens such as an amount to obtain a serum titre in guinea pigs of N0 at least 1.5 log 10; and, the inventive compositions can include an adjuvant, such as aluminum 0 hydroxide, which can be present in a single dose in an amount typically found in vaccines Ci such as preferably an amount of about 0.7 to about 0.9 mg o Accordingly, in an aspect the invention provides combined enteric immunological, immunogenic or vaccine composition comprising a first antigen or epitope of interest from Cryptosporidiam parvum and/or a first vector that expresses the first antigen or epitope of interest, and a second antigen or epitope of interest from another enteric pathogen and/or the first vector that expresses the first antigen or epitope of interest also expresses the second antigen or epitope of interest and/or a second vector that expresses the second antigen or epitope of interest, and a pharmaceutically acceptable vehicle.

The composition can comprise antigen which can be from Cryptosporidin parvum and an antigen from another enteric pathogen. The composition can comprise an antigen from Cryposporidium and an antigen from another enteric pathogen of a bovine species; or of a canine species; or of a feline species; or of an equine species. The antigen from the enteric pathogen can be chosen from the group consisting of the antigens from E. coli, rotavirus, coronavirus, Clostridium spp. and mixtures thereof. The enteric pathogen can be E. coli. The antigen from E. coli can be selected from the group consisting of E. coli bearing K99 antigen, E. coli. bearing F41 antigen, E. coli bearing Y antigen, E. coli bearing 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, and mixtures thereof.

The enteric pathogen can comprise bovine coronavirus; and/or bovine rdtavirus and/or Clostridium perfingens. The antigen of the enteric pathogen can comprise Clostridium perfringens type C and D toxoids. In certain embodiments, the enteric pathogen can comprises E coli, bovine rotavirms, bovine coronavirus and Closrridiun perfringen or E. coli, bovine rotavirus, bovine coionavirus.

Yet further, in certain aspects the invention can comprise a composition.wherein the antigen of the enteric pathogen comprises E. coli antigens selected from the group consisting SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:47 FAX 613 8618 4199 F.B. RICE Co. 017 WO 01/45735 PCT/EP(O/13387

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0 ^c of E. coli bearing K99 antigen, E. coli. bearing F41 antigen, E. coli bearing Y antigen, E coli bearing 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, and mixtures thereof; V inactivated bovine coronavirus; inactivated bovine rotavirus and Clostridium perfringens type C and D toxoids; or E, coli antigens selected from the group consisting of E. coli bearing K99 antigen, E. coli. bearing F41 antigen, E. coli bearing Y antigen, E. coli bearing 31A antigen, 0N K99 antigen, F41 antigen, Y antigen, 31A antigen and .mixtures thereof; inactivated bovine coronavirus; and inactivated bovine rotavirus.

hThe inventive composition advantageously can comprise sub-uit Cryptosporidium o parvum antigens selected from the group consisting of P21, Cp23, CpI5/60, CP41 and C 10 mixtures thereof, such as Cp23 and Cp15/60 or P21 and Cp 15/60.

In the inventive compositions associating antigens from Cryptosporidium parvum and at least one other enteric pathogen, the Cryptosporidi m parvum antigen may also comprise or be constituted by, inactivated or live attenuated oocysts, or sub-units obteined from oocysts.

Inventive compositions can include an adjuvant such as saponin or aluminum hydroxyde; and, inventive compositions can be in the form of an oil-in-water emulsion.

The invention further envisions an immunological, immunogenic or vaccine composition against Cryptosporidium parvum, which comprises a first antigen comprising a P21 or Cp23 antigen or an epitope thereof or a first vector that expresses the first antigen and a second antigen comprising Cp15/60 antigen or epitope thereof or the first vector wherein the first vector expresses both the first and second antigens or a second vector that expresses the second antigen, and a pharmaceutically acceptable vehicle. The composition-can comprise Cp23 and Cpl5160 antigens which are in the form of separate fusion proteins. The composition can comprise a vector expressing Cp23 and Cp15/60. The composition can comprise a first recombinant vector expressing Cp23 and a second recombinant vector expressing Cp15/60. And, the composition can comprise P21 and Cp15/60. These compositions can further comprise an adjuvant.

Still further, the invention comprehends an immunological, immunogenic or vaccine composition against Cryptosporidium parvwn, which comprises a first antigen comprising a P21 or Cp23 or Cp 15/60 or CP41 antigen or an epitope thereof or a first vector that expresses the first antigen and a second antigen comprising a second aiitigen or epitope thereof from Cryptosporidiuz parvum or the first vector wherein the first vector expresses both the first and SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:48 FAX 613 8618 4199 F.B. RICE Co. 1. 018 WO 01/45735 PCT/EP00/13387 o 12 0 Ssecond antigens or a second vector that expresses the second antigen, wherein the first and second antigens are different from each other, and a pharmaceutically acceptable vehicle.

NO The invention also comprehends a method of.bovine immunization of a newborn calf against enteric disease comprising administering an inventive composition to a pregnant M 5 female calf before delivering, so that the newborn calf receives materal antibodies against SCryptosporiditan parvum through colostrum and/or milk. The method can further comprise o the feeding to the newborn calf colostrum and/or milk from cow(s) which has (have) been administered the composition during pregnancy. The method can comprise administering the Scomposition to the new-born calf. The composition administered to the pregnant female can Cl 10 comprise antigens or epitopes thereof and the composition administered to the calf can comprise vectors. Thus, the invention also envisions a method of active imThunization of adult and newborn calves, comprising administering to the icalves an inventive composition.

The invention also comprehends a method of bovine immunization of a newborn calf, comprising feeding to the new-bor calf colostrum and/or milk from cows which have been administered the composition during pregnancy. Similarly, in a broader sense, the invention comprehends a method of immunization of a new-born mammal comprising feeding to the new-born colostrum and/milk from a female mammal which has been administered the composition during pregnancy; and, the mammal is advantageously, a bovine, a feline, a canine, or an equine. Still further, the invention can encompass a method for preparing an inventive composition comprising admixing the antigens or epitopes or vectors and the carrier.

And, the invention can include a kit for preparing an inventive composition comprising the antigens, epitopes or vectors, each in separate container or containers (some antigens, epitopes or vectors may be together in one container, such as the Cryptosporidium parvwn antigens, epitopes or vectors may be together in one container, and the other antigens, epitopes or vectors in one or more other containers, or the carrier, diluent and/or adjuvant may be in separate containers), optionally packaged together and further optionally with instructions for admixture and/or administration.

The term "comprising" in this disclosure can mean "including" or can have the meaning commonly given to the term "comprising" in U.S. Patent Law.

Other aspects of the invention are described in or are obvious from (and within the ambit of the invention) the following disclosure.

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:48 FAX 613 8618 4199 F.B. RICE Co. a o01 .3 WO 01/45735 PCT/EP00/13387 cN t BRIEF DESCRIPTION OF FIGURES The following Detailed Description, given by way of example, and not intended to IN0 limit the invention to specific embodiments described, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which: S 5 Figure 1 shows a physical and restriction map of plasmid aO Figre 2 shows a physical and restriction map of plasmid pJCA156; SFigure 3 shows a physical and restriction map of plasmid pJCA157; C, Figure 4 shows a physical and restriction map of plasmid pJCA158; O Figure 5 shows a physical and restriction map of plasmid pJCA159; N 10 Figure 6 shows aphysical and restriction map of plasmidpJCA160; Figure 7 shows comparative oocysts count in feces in calves challenged with either C.

parvum, or bovine rotavirus, or both, or non challenged (example 12); Figure 8 shows comparative rotavirus excretion in feces in calves according to example 12; Figure 9 shows comparative animal general condition for calves according to example 12; Figure 10 shows comparative animal dehydration status in calves according to example 12; Figure 11 shows comparative count of liquid feces for calves according to example 12; Figure 12 shows comparative anorexia status for calves according to example 12; and Figure 13 shows comparative rectal temperature evolution in calves according to example 12.

DETAILED DESCRIPTION An aspect of the invention is thus a combined enteric immunological, imunogenic or vaccine composition comprising at least one an antigen or epitope of interest from at least one Cryptosporidium spp., preferably including Cryptosporidium parvum, and at least one antigen from at least one other enteric pathogen, advantageously a pathogen infecting the animal species to be protected, such as canine, feline, equine or bovine species and more advantageously bovine species; and/or a vector or vectors and/or a recombinant or recombinants and/or a plasmid or plasmids that expresses the Cryptosporidium spp antigen or SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:48 FAX 613 8618 4199 F.B. RICE Co. 21020 WO01145735 PCT/EPO0/13387 14 t epitope of interest and/or at least one of the antigen(s) or epitope(s) of interest of the other enteric pathogen; and a pharmaceutically acceptable -vehicle. Universal immunological, Simmunogenic or vaccine compositions are also envisioned as enteric pathogens are often infecting several (more than one) animal species.

Mn 5 An immunological composition elicits an immunological response local or systemic.

Va An immunogenic composition likewise elicits a local or systemic immunological response. A Svaccine composition elicits a local-or systemic protective response. Accordingly, the terms S"immunological composition" and "immunogenic composition" include a 'vaccine o composition" (as the two former terms can be protective compositions).

Cryptosporidiwn parvum antigens which can be used in this invention comprise preferably A protein of 148 amino acids called Cpl5/60 (See, U.S. Patent No. 5,591,434. This protein is represented in US-A-5,591,434 in SEQ ID NO:2 with 10 further amino acids at the end, upstream the methionine (Met). It is within the scope of the present invention to use an antigen comprising or consisting essentially of the 148 amino acid sequence of Cpl5160 or of a longer amino acid sequence including these 148 amino acids, e.g. the whole sequence represented in SEQ ID NO:2 in US-A-5,591,434 or any polypeptide comprising a fragment of the 148 or 158 amino acid sequences that comprises an epitope thereof, advantageously a protection-eliciting epitope or an epitope that has the immumogenicity of the full length sequence.) and/or Cp23 and/or P21. (Cp23 is an antigen of about 23 kDa; see Perryman et al., Molec Biochem Parasitol 80:137-147-(1996); WO-A-9807320 and L. E. Perryman et al., Vaccine-17 (1999) 2142-2149. The major part of this protein (187 amino acids) is herein termed P21 and has an amino acid sequence homologous to the amino acid sequence of protein C7 which is disclosed as SEQ ID NO. 12 in WO-A-98 07320 To be expressed, one or two or more amino acids can be added at the end of P21, sich as, Met-, or Met-Gly- or similar amino acids. It is within the scope of the present invention to use an antigen comprising or consisting essentially of or consisting of the 187 amino acid sequence or a longer amino acid sequence, or a polypeptide compiising a fragment of the 187 amino acid sequence that comprises an epitope thereof, advantageously a protection-eliciting epitope or an epitope that has the irmmunogenicity of the full length sequence. The whole amino acid sequence of Cp23 and the SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:49 FAX 613 8618 4199 F.B. RICE Co. l021 WO 01/45735 PCT/EPOO/13387 t

IN

t corresponding nucleotide sequence is easily obtainable. The P21 protein represents the major part and the C-terminal end of Cp23. The P21 nucleotide sequence may be used as a probe to IND screen a DNA library, e.g. a library as disclosed in Example 1, This methodology is well known to the one skilled in the art. On the basis of the molecular weight of Cp 2 3, it can be asserted that about 25-35 amino acids are missing at the N-terminal end of P21 to have the Ic complete Cp23 amino acid sequence. This information gives those skilled in the art the means o to easily find the start codon and thus the 5' end of the Cp23 nucleotide sequence and the CI N-terminal amino acid sequence.

o The antigens or epitopes of interest can be used individually or in combination in 0 C1 10 compositions of the invention, an inventive composition can include or or both (1) and Another possible antigen is the CP41 antigen as disclosed supra.

According to the preferred embodiment, these antigens or epitopes of interest are incorporated into the composition as proteins or sub-unit antigens. They can be produced by chemical synthesis or by expression in vitro. The examples describe how to obtain the sequences encoding Cpl5/60 and P21 and how to construct vectors expressing them. These sequences can be cloned into suitable cloning or expression vectors. These vectors are then used to transfect suitable host cells. The antigens encoded by the nucleotide sequence which is inserted into the vector, e.g. Cp23 and/or P21 and/or Cpl5/60, are produced by growing the host cells transformed by the expression vectors under conditions whereby the antigen is produced. This methodology is well known to the one skilled in the art. Host cells may be either procaryotic or eucaryotic, e.g. Escherichia coli coli), yeasts such as Saccharomyces cerevisiae, animal cells, in particular animal cell lines. The one skilled in the art knows the vectors which can be used with a given host cell. The vectors may be chosen such that a fusion protein is produced which can be used then to easily recover the antigen. Furthermore, with respect.to sequences, nucleic acid sequences useful for expressing the C. parvum antigen or epitope of interest can include nucleic acid sequences that are capable of hybridizing under high stringency conditions or those having a high homology with nucleic acid molecules employed in the invention nucleic acid molecules in documents mentioned herein); and, "hybridizing under high- stringency conditions" can be synonymous with "stringent hybridization conditions", a term which is well known in the art; see, for SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:49 FAX 613 8618 4199 F.B. RICE Co. 022 WO 01/45735 PCT/EPOO/13387 o16 Mc example, Sambrook et aL, "Molecular Cloning, A Laboratory Manual" second ed., CSH Press, SCold Spring Harbor, 1989; "Nucleic Acid Hybridisation, A Practical Approach", Hames and o Higgins eds., IRL Press, Oxford, 1985; both incorporated herein by reference.

With respect to nucleic acid molecules and polypeptides which can be used in the cr 5 practice of the invention, the nucleic acid molecules and polypeptides advantageously have at Q, least about 75% or greater homology or identity, advantageously 80% or greater homology or Sidentity, more advantageously 85% or greater homology or identity, such as at least about Sor about 86% or about 87% or about 88% or about 89% homology or identity, for instance at Sleast about 90% or homologyor identity or greater, such as at least about 91%, or about 92%, Ci 10 or about 93%, or about 94% identity or homology, more advantageously at least abott 95% to 99% homology or identity or greater, such as at least about 95% homology or identity or greater at least about 96%, or about 97%, or about 98%, or about 99%, or even about 100% identity or homology, or from about 75%, advantageously from about 85% to about 100% or from about 90% to about 99% or about 100% or from about 95% to about 99% or about 100% identity or homology, with respect to sequences set forth in herein cited documents (including subsequences thereof discussed herein); and thus, the invention comprehends a vector encoding an epitope or epitopic region of a C. parvum isolate or a composition comprising such an epitope, compositions comprising an epitope or epitopic region of a C. parvu isolate, and methods for making and using such vectors and compositions, the invention also comprehends that these nucleic acid molecules and polypeptides can be used in the same fashion as the herein mentioned nucleic acid molecules, fragments thereof and polypeptides.

Nucleotide sequence homology can be determined using the "Align" program of Myers and Miler, ("Optimal Alignments in Linear Space", CABIOS 4, 11-17, 1988, incorporated herein by reference) and available at NCBL Alternatively or additionally, the term "homology" or "identity", for instance, with respect to a nucleotide or amino acid sequence, can indicate a quantitative measure of homology between two sequences. The percent sequence homology can be calculated as Nd)*100/N wherein Ndy is the total number of non-identical residues in the two sequences when aligned and wherein Nf is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence similarity of 75% with the sequence AATCAATC 8; Ndip2).

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:50 FAX 613 8618 4199 F.B. RICE Co. [1023 WO 01/45735 PCTIEP00/13387

O

17

IN

c3 Alternatively or additionally, "homology" or "identity" with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the IN number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm S 5 (Wilbur and Lipman, 1983 PNAS USA 80:726, incorporated herein by reference), for

\O

instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap Spenalty of 4, and computer-assisted analysis and interpretation of the sequence data including Salignment can be conveniently performed using commercially available programs SIntelligenetics TM Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar, SI 10 or have a degree of sequence identity or homology with DNA sequences, thymidine in the DNA sequence is considered equal to uracil in the RNA sequence. RNA sequences within the scope of the invention can be derived from DNA sequences, by thymidine in the DNA sequence being considered equal to uracil in RNA sequences.

Additionally or alternatively, amino acid sequence similarity or identity or homology can be determined using the BlastP program (Altschul et al., Nucl. Acids Res. 25, 3389-3402, incorporated herein by reference) and available at NCBI (used in determining sequence homology, as shown in Appendix I; see also the Examples). The following references (each incorporated herein by reference) also provide algorithms for comparing the relative identity or homology of amino acid residues of two proteins, and additionally or alternatively with respect to the foregoing, the teachings in these references can be used for determining percent homology or identity: Needleman SB and Wunsch CD, "A general method applicable to the search for similarities in the amino acid sequences of two proteins," J. MoL Biol. 48:444-453 (1970); Smith TF and Waterman MS, "Comparison of Bio-sequences," Advances in Applied Mathematics 2:482-489 (1981); Smith TF, Waterman MS and Sadler JR, "Statistical characterization of nucleic acid sequence functional domains," Nucleic Acids Res., 11:2205- 2220 Feng DF and Dolittle RF, "Progressive sequence alignment as a prerequisite to correct phylogenetic trees," of Molec. Evol, 25:351-360 (1987); Higgins DG and Sharp PM, "Fast and sensitive multiple sequence alignment on a microcomputer," CABIOS. 5: 151- 153 (1989); Thompson JD, Higgins DG and Gibson TJ, "ClusterW: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, positions-specific gap penalties and weight matrix choice, Nucleic Acid Res., 22:4673-480 (1994); and, Devereux J, SUBSTITUTE SHEET (RULE 28) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:50 FAX 613 8618 4199 F.B. RICE Co. Z024 WO 01145735 PCT/EP00/13387 o 18 0 SHaeberlie P and Smithies 0, "A comprehensive set of sequence analysis program for the VAX," Nucl. Acids Res., 12: 387-395 (1984).

SFurtihermore, as to nucleic acid molecules used in this invention as in herein cited documents), the invention comprehends the use of codon equivalent nucleic acid molecules.

For instance, if the invention comprehends protein P21 and/or Cp23 and/or Cpl5/60 N0 and/or CP41) having amino acid sequence and encoded by nucleic acid molecule the O invention comprehends nucleic acid molecules that also encode protein X via one or more CN different codons than in nucleic acid molecule N.

SThe antigen or epitope of interest used in the practice of the invention can be obtained (C 10 from the particular pathogen(s), C, parvwn, E. coli, rotovirus, coronavirus, and the like or can be obtained from in iitro and/or in vivo recombinant expression of gene(s) or portions thereof. Methods for making and/or using vectors (or recombinants) for expression can be by or analogous to the methods disclosed in: U.S. Patent Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, PCT publications WO 94/16716, WO 96/39491, Paoletti, "Applications of pox virus vectors to vaccination: An update," PNAS USA 93:11349-11353, October 1996, Moss, "Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety," PNAS USA 93:11341-11348, October 1996, Smith et al., U.S. Patent No- 4,745,051 (recombinant baculovirus), Richardson, C.D. (Editor), Methods in Molecular Biology 39, "Baculovirus Expression Protocols" (1995 Humana Press Inc.), Smith et al., "Production of Huma Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector," Molecular and Cellular Biology, Dec., 1983, Vol. 3, No. 12, p. 2156-2165; Pennock et al., "Strong and Regulated Expression of Escherichia coli B- Galactosidase in Infect Cells with a Baculovirus vector," Molecular and Cellular Biology Mar.

1984, Vol. 4, No. 3, p. 399-406; EPA 0 370 573, U,S. application Serial No. 920.197, filed October 16, 1986, EP Patent publication No. 265785, US. Patent No. 4,769,331 (recombinant herpesvirus), Roizman, "The function of herpes simplex'virus genes: A primer for genetic engineering of novel vectors," PNAS USA 93:11307-11312, October 1996, Andreansky et al., "The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors," PNAS USA 93:11313-11318, October 1996, Robertson et al.

"Epstein-Barr virus vectors for gene delivery to B lymphocytes," PNAS USA 93:11334- 11340, October 1996, Frolov et al., "Alphavirus-based expression vectors: Strategies and SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 08/03 '06 17:50 FAX 613 8618 4199 F.B. RICE Co. 0025 WO 01/45735 PCT/EP0113387 O 19 0 Sapplications," PNAS USA 93:11371-11377, October 1996, Kitson et J. Virol. 65, 3068- 3075, 1991 U.S. Patent Nos. 5,591,439, 5,552,143, allowed U.S. applications Serial Nos.

ND 08/675,556 and 08/675,566, filed July 3, 1996 (recombinant adenovirus), Grunhaus et al., 0 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3) p. 237-52, 1993, Ballay et al EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990, Prevec et aL, ID J. Gen Virol. 70, 429-434, PCT W091/11525, Felgner et al. (1994), J. BioL Chem. 269, 2550- 0 2561, Science, 259:1745-49, 1993 and McClements et al., "Immunization with DNA vaccines CI encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective Simmunity in animal models of herpes simplex virus-2 disease," PNAS USA 93:11414-11420, C 10 October 1996, and U.S. Patents Nos 5,591,639, 5,589,466, and 5,580,859 relating to DNA expression vectors, inter alia. See also WO 98/33510; Ju et al., Diabetologia, 41:736-739, 1998 (lentiviral expression system); Sanford et al., U.S. Patent No. 4,945,050; Fischbach et al.

(Intracel), WO 90/01543; Robinson et aL, seminars in IMMUNOLOGY, vol. 9, pp.271-283 (1997) (DNA vector systems); Szoka et al., U.S. Patent No. 4,394,448 (method of inserting DNA into living cells); McCormick et al., U.S. Patent No. 5,677,178 (use of cytopathic viruses); U.S. Patent No. 5,928,913 (vectors for gene delivery), and Tartaglia et al. U.S. Patent No. 5,990,091 (vectors having enhanced expression), as well as other documents cited herein.

A viral vector, for instance, selected from herpes viruses, adenoviruses, poxviruses, especially vaccinia virus, avipox virus, canarypox virus, as well as DNA vectors (DNA plasmids) are advantageously employed in the practice of the invention, especially for in vivo expression (whereas bacterial and yeast systems are advantageously employed for in vitro expression).

If the host-vector combination leads to the production of antigen without excretion, for the convenience of their production, and their recovering, these antigens are preferably under the form of fusion proteins a HIS tag). In other words, the antigen can comprise the antigen per se and foreign amino acids.

Techniques for protein purification and/or isolation from this disclosure and documents cited herein, inter alia, and thus within the ambit of the skilled artisan, can be used, without undue experimentation, to purify and/or isolate recombinant or vector expression products and/or antigen(s), in the practice of the invention, and such techniques, in general, can include: precipitation by taking advantage of the solubility of the protein of interest at.varying salt concentrations, precipitation with organic solvents, polymers and other materials, affinity SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:51 FAX 613 8618 4199 F.B. RICE Co. 1026 WO 01/45735 PCT/EP00113387 O 020 precipitation and selective denaturation; column chromatography, including high performance liquid chromatography (HPLC), ion-exchange, affinity, imnunoaffnity or dye-ligand \NO chromatography; immunoprecipitation and the use of gel filtratin, .electrophoretic methods, ultrafiltration and isoelectric focusing, inter alia.

As mentioned herein, according to another aspect, thp invention comprehends that the O antigens and/or epitopes of interest are not incorporated as subunits in the composition, but o rather that they are expressed in vivo; the invention comprehends that the composition c comprises recombinant vector(s) expressing the antigens in vivo when administered to the N animal. The vector can comprise a DNA vector plasmid, a herpesvirus, an adenovins, a S 10 poxvirus, including a vaccinia virus, an avipox virus, a canarypox virus, and a swinepox virus, and the like. The vector-based compositions can comprise a vector that contains and expresses a nucleotide sequence of the antigen to be expressed, Cpl5/60 and/or Cp23 for Cryprosporidiun parvun.

The word plasmid is intended to include any DNA transcription unit in the form of a polynucleotide sequence comprising the sequence to be expressed- Advantageously, the plasmid includes elements necessary for its expression; for instance, expression in vivo. The circular plasmid form, supercoiled or otherwise, is advantageous; and, the linear form is also included within the scope of the invention. The plasmid can be either naked plasmid or plasmid formulated, for example, inside lipids or liposomes, cationic liposornes (see, e.g., WO-A-90 11082; WO-A-92 19183; WO-A-96 21797; WO-A-95 20660). The plasmid immunological or vaccine composition can be administered by way of a gene gun, or intramuscularly, or nasally, or by any other means that alows for expression in vivo, and advantageously an immunological or protective response. Reference is also made to U.S.

applications Serial Nos. 09/232,278, 09/232,468, 09/232,477, 09/232,279, 09/232,478, and 09/232,469, each filed January 15, 1999 (and incorporated herein by reference), and to U.S.

applications Serial Nos. 60/138,352 and 60/138,478, each filed June 10, 1999 (and incorporated herein by reference), as these applications involve DNA and/6r vector vaccines or nimmnogenic or immunological compositions for felines, canines, bovines, and equines, and inventive compositions can include DNA and/or vector vaccines or immunogenic or immunological compositions from these applications and/or inventive compositions can be SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:51 FAX 613 8618 4199 F.B. RICE Co. 0027 SWO 01/45735 PCT/EP00/13387 o 21 0 k prepared and/or formulated and/or administered in a fashion analogous to the compositions of these applications.

IN Compositions for use in the invention can be prepared in accordance with standard techniques well known to those skilled in the veterinary or pharmaceutical or medical arts.

Such compositions can be admi-nstered in dosages and by techniques well known to those IN skilled in the veterinary arts taking into consideration such factors as the age, sex, weight, 0 condition and particular treatment of the animal, and the route of administration. The Cl components of the inventive compositions can be administered alone, or can be co- O administered or sequentially administered with other compositions the C. parvum C I 10 atigen(s) and/or epitope(s) can be administered alone, and followed by the administration sequentially of antigen(s) and/or epitope(s) of other enteric pathogens, or compositions comprising a enteric antigen(s) or epitope(s) can include vectors or recombinaats or plasmids that also express enteric antigen(s) or epitope(s) of the same or different pathogens) or with other prophylactic or therapeutic compositions other immunogenic, immunologiqal or vaccine compositions). Thus, the invention provides multivalent or "cocktail" or combination compositions and methods employing them. The ingredients and manner (sequential, as part of a prime-boost regimen, or as part of a booster program wherein immunogenic, immunological or vaccine composition is administered periodically during the life of the animal such as an annual, seasonal, biannual and the like booster program; or coadministration) of administration, as well as dosages, can be determined, taking into consideration such factors as the age, sex, weight, condition and particular treatment of the animal, cow, and, the route of administration. In this regard, reference is made to U.S.

Patent No. 5,843,456, incorporated herein by reference, and directed to rabies compositions and combination compositions and uses thereof.

Compositions of the invention may be used for parenteral or mucosal administration, preferably by intradermal, subcutaneous or intramuscular routes. When mucosal administration is used, it is possible to use oral, nasal, or vaginal routes.

In such compositions, the vector(s), or antigen(s) or epitope(s) of interest(s) may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions can also be lyopbilized. The compositions can contain auxiliary substances such as pH buffering agents, adjuvants, preservatives, polymer SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:51 FAX 613 8618 4199 F.B. RICE Co. @1028 WO 01/45735 PCT/EP0013387

O

o 22.

c3 excipients used for mucosal routes, and the like, depending upon the route of administration and the preparation desired.

O Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable C<c 5 preparations, without undue experimentation. Suitable dosages can also be based upon the \0 text herein and documents cited herein.

SAdjuvants are substances that enhance the immune response to antigens. Adjuvants, O can include aluminum hydroxide and aluminum phosphate, saponins Quil A. mineral oil emulsions, pluronic polymers with mineral or metabolizable oil emulsion, the water-in-oil Ci 10 adjuvant, the oil-in-water adjuvant, synthetic polymers (e.g.,homo- and copolymers of lactic and glycolic acid, which have been used to produce microspheres that encapsulate antigens, see Eldridge et al., MoL Immunol. 28:287-294 (1993), biodegradable microspheres), nonionic block copolymers, low molecular weight copolymers in oil-based emulsions (see Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull John Wiley and Sons, NY, pp51-94 (1995)), high molecular weight copolymers in aqueous formulations (Todd et al., Vaccine 15:564-570 (1997)), cytokines such as IL-2 and IL-12 (see, U.S. Patent No. 5,334,379), and GM-CSF (granulocyte macrophage-colony stimulating factor; see, generally, U.S. Patents Nos. 4,999,291 and 5,461,663, see also Clark et al., Science 1987, 230:1229; Grant et al., Drugs, 1992, 53:516), advantageously GM-CSF from the animal species to be vaccinated, inter alia. Certain adjuvants can be expressed in vivo with antigen(s) and/or epitope(s); cytokines, OM-CSF (see, C. R- Maliszewski et al.

Molec Immunol 25(9):843-50 (1988); S.R Leong, Vet Immunol and Immunopath 21:261-78 (1989) concerning bovine GM-CSF. A plasmid encoding GM-CSF can be modified to contain and express DNA encoding an antigen from a bovine pathogen according to the instant invention and/or an epitope thereof optionally also with DNA encoding an intigen and/or epitope of another bovine pathogen, or can be used in conjunction with such a plasmid) A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copoIymers of maleic anhydride and alkenyl derivative.

Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:52 FAX 613 8618 4199 F.B. RICE Co. R029 WO 01/45735 PCT/EP00/13387 S 23 0 the art can also refer to U.S. Patent No. 2,909,462 (incorporated herein by reference) which describes such acrylic polym cross-inked with pol lind wi olyhydroxylated compound having at \least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The prefered radicals are those containing from 2 to 4 carbon atoms,-e.g. vinyls, allyls and other V0 ethylenically unsaturated groups. The unsaturated radicals may themselves contain other O substiteunts, such as methyl. The products sold under the name Carbopol® (BF Goodrich, C-I Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with Va Sallyl pentaerythritol. Among then, there may be mentioned Carbopol® 974P, 934P and 971P.

10 Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA® (Monsanto) which are copolymers of maleic anhydride and ethylene, linear or cross-linked, for example cross-linked with divinyl ether, are preferred Reference may be made to J. Fields et al., Nature, 186 778-780, 4 June 1960, incorporated herein by reference.

From the point of view of their structure, the polymers of acrylic or methacrylic acid and the copolymers EMA® are preferably formed of basic units of the following formula:

R,

c-o in which:

R

1 and R 2 which are identical or different, represent H or CH 3 x 0 or 1, preferably x 1; and y 1 or 2, with x y 2.

For the copolymers EMA®, x 0 and y 2. For the carbomers, x y =1.

The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the imrmunogenic, immunological or vaccine composition itself will be incorporated. The carboxyl groups of the polymer are then partly in COO" formx SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06.17:52 FAX 613 8618 4199 F.B. RICE Co. 030 SWO 01/45735 PCT/EP0O/13387 o 24 0 ci cPreferably, a solution of adjuvant according to the invention, especially of carbomer, is prepared in distilled water, preferably in the presence of sodium chloride, the solution qD obtained being at acidic pH. This stock solution is diluted by adding it to the desired quantity (for obtaining the desired final concentration), or a substantial part thereof, of water charged S 5 with NaCI, preferably physiological saline (NaCl 9 g/1) all at once in several portions with 0O concomitant or subsequent neutralization (pH 7.3 to preferably with NaOH. This solution Sat physiological pH will be used as it is for mixing with the vaccine, which may be especially Ct stored in freeze-dried, liquid or frozen form.

O The polymer concentration in the final vaccine composition can be 0.01% to 2% w/v, Ci 10 0.06 to 1% w/v, such as 0.1 to 0.6% w/v.

Adjuvanting immunogenic and vaccine compositions according to the invention may also be made with formulating them in the form 'of emulsions, in particular oil-in-water emulsions, e.g. an emulsion such as the SPT emulsion described p 147 in Vaccine Design, The Subunit and Adjuvant Approach edited by M. Powell, M. Newman, Plenum Press 1995, or the emulsion MF59 described p183 in the same book. In particular, the oil-in-water emulsion may be based on light liquid parafin oil (according to European Pharmacopoeia) isoprenoid oil such as squalane, squalene oil obtained by oligomerisation of alkenes, in particular of isobutylene or of decene acid or alcohol esters with linear alkyl groups, particularly vegetable oils, ethyl oleate, propylene glycol di(caprylate caprata), glycerol tri(caprylate caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular esters of isostearic acid. The oil is used in combination with emulsifiers t form the emulsion. Emulsifiers are preferably non-ionic surfactants, in particular sorbitan esters, mannide esters, glycerol esters, polyglycerol esters, propylene glycol esters or esters of oleic acid, of isostearic acid, of ricinoleic acid, of hydroxystearic acid, possibly ethoxylated, blockcopolymers such as polyoxypropylene-polyoxyethylene, in particular the products called Pluronic(, namely Pluronic( L121.

From this disclosure and the knowledge in the art, the skilled artisan can select a suitable adjuvant, if desired, and the amount thereof to employ in an immunological, immunogenic or vaccine composition according to the invention, without undue experimentation.

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:52 FAX 613 8618 4199 F.B. RICE Co. [1031 WO 01/45735 PCTIEP00/13387

(N

o c The immunological, immunogenic or vaccine compositions according to the invention may be associated to at least one live attenuated, inactivated, or sub-unit vaccine, or IN recombinant vaccine poxvirus as vector or DNA plasmid) expressing at least one immunogen, antigen or epitope of interest from another pathogen.

c 5 Compositions in forms for various administration routes are envisioned by the Sinvention. And again, the effective dosage and route of administration are determined by o known factors, such as age, weight. Dosages of each active agent of each C. parvum C antigen or epitope of interest and/or of each antigen or epitope from each enteric pathogen can 0 be as in herein cited documents or as otherwise mentioned herein and/or can range from one or i 10 a few to a few hundred or thousand micrograms, 1 pg to 1mg, for a subunit immunogenic, immuological or vaccine composition; and, 10 to 10'0 TCIDso advantageously 106 to, 10i TCID 5 o, before inactivation, for an inactivated immunogenic, immunological or vaccine composition.

Recombinants or vectors can be administered in a suitable amount to obtain in vivo expression corresponding to the dosages described herein andlor in herein cited documents.

For instance, suitable ranges for viral suspensions can be determined empirically. The viral vector or recombinant in the invention can be administered to the animal or infected or transfected into cells in an amount of about at least 10 3 pfu; more preferably about 104 pfu to about 10'0 pfu, about 105 pfu to about 109 pfu, for instance about 10 6 pfu to about 10 8 pfu, with doses generally ranging from about 10 6 to about 1010, preferably about 1010 pfu/dose, and advantageously about 10 pfu per dose of about 1 ml to about 5 ml, advantageously about 2 ml. And, if more than one gene product is expressed by more than one recombinant, each recombinant can be administered in these amounts; or, each recombinant can be administered such that there is, in combination, a sum of recombinants comprising .these amounts. In plasmid compositions employed in the invention, dosages can be as described'in documents cited herein or as described herein. Advantageously, the dosage should be a sufficient amount of plasmid to elicit a response analogous to compositions wherein the antigen(s) or epitope(s) of interest are directly present; or to have expression analogous to dosages in such compositions; or to have expression analogous to expression obtained in vivo by recombinant compositions. For instance, suitable quantities of each plasmid DNA in plasmid compositions can be 1 pg to 2 mg, preferably 50 pg to lmg. Documents cited herein regarding DNA SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:53 FAX 613 8618 4199 F.B. RICE Co. [I032 WO 01/45735 PCT/EP00/13387 o 26 O k plasmid vectors may be consulted by the skilled artisan to ascertain other suitable dosages for DNA plasmid vector compositions of the invention, without-undue experimentation.

0 However, the dosage of the composition(s), concentration of components therein and timing of administering the- composition(s), which elicit a suitable immunological response, S 5 can be determined by methods such as by antibody titrations of sera, by ELISA and/or -N seroneutralization and/or seroprotection assay analysis. Such determinations do not require o undue experimentation from the knowledge of the skilled artisan, this disclosure and the Ci documents cited herein. And, the time for sequential administrations can be likewise Va o ascertained with methods ascertainable from this disclosure, and the knowledge in the art, CN 10 without undue experimentation.

Preferably, the combined enteric immunological, immunogenic or vaccine composition comprises both Cryptosporidium parvwn antigens as defined above.

Antigens or epitopes of enteric pathogens advantageously combined with Cryptosporidiwn antigen(s) or epitope(s) (advantageously P21 and/or Cp23 and/or Cpl5/60 and/or CP41 such as P21 or Cp23 and Cp15/60, or epitope(s) thereof) comprise preferably one or more antigen or epitope of interest from E. coli, and/or rotavimus, and/or coronavirus, and/or Clostridium spp., such as CL perfringens; for instance, at least one antigen or epitope of interest from E. coli, rotavirus, and coronavirus. Antigens from E. coli. include preferably one, preferably several (more than one), more preferably all, of the antigens called K99, F41, Y and 31A and/or epitopes therefrom. Preferred antigens are K99 and F41. A composition thus advantageously comprises one of K99 and F41, and preferably both. It is also preferred for a composition to comprise also Y and/or 31A, advantageously Y and 31A. For instance, these antigens may be incorporated as subunits or can be borne by E. coli bacteria. Preferably the compositions according to the invention comprise at least one antigen chosen from the group consisting of E. coli bearing K99 antigen, E- coli. bearing F41 antigen, E. boli bearing Y antigen, E. coli bearing 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen and any mixtures thereof.

As mentioned herein, E. coli may be used to produce Cryptosporidium parvun antigens or epitopes. The Cryptosporidium parvwm antigens or epitopes can be expressed in an E. coli strain expressing at least one of the E. coli antigens so that simultaneous expression of E. coli and Cryptosporidium parvirm antigens is performed. For in vitro expression, the SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:53 FAX 613 8618 4199 F.B. RICE Co. R1033 WO 01/45735 PCT/EPOO/13387

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o27 j cells may then be disrupted as usual and the E. colt and Cryptosporidium parvun antigens or epitopes recovered; advantageously, if there is internal or non-surface expression of the INO antigens or epitopes, the antigens or epitopes are expressed as fusion proteins or with tags, e.g.

HIS tags. For in vivo expression, advantageously the nucleic acid molecules encoding the antigens or epitopes is linked to a signal sequence so that there is exracellular expression of QO the antigens or epitopes; and, advantageously, the E. coliis non-pathogenic. Thus, E. coli can, o in certain embodiments, be the vector and the antigen or epitope of interest C Antigens from Clostridium perfringens are preferably type C and/or D toxoids, more o preferably type C and D toxoids.

A particular aspect of the invention is a combined enteric immunological, immunogenic or .vaccine composition for bovine species, comprising at least one antigen or epitope from at least one Cryptosporidium spp., preferably including Cryptosporidium parvum, advantageously P21 and/or Cp23 and/or Cpl5/60 and/or CP41 such as P21 or Cp23 and Cpl5/60 and/or an epitope of interest thereof, and at least one antigen or epitope from at least one additional bovine enteric pathogen such as E. coli, bovine rotavirus, bovine coronavirus and Clostridium perfringens, or combinations thereof, and preferably including at least one antigen or epitope from each of these pathogens or at least one antigen or epitope from ~E coli, rotavirns, and coronavirs. With respect to an epitope of interest of a desired antigen and how to determine what portion of an antigen is an epitope of interest, reference is made to U.S. Patent No. 5,990,091 and U.S. applications Serial Nos. 08/675,566 and 08/675,556, as well as other documents cited herein. From the disclosure herein and the knowledge in. the art, such as in herein cited documents, there is no undue experimentation needed to ascertain an epitope of interest, or to formulate a composition within the invention comprising antigen(s) and/or epitope(s) and/or vector(s) expressing antigen(s) and/or epitope(s).

According to a preferred embodiment, the invention provides a bovine enteric inmnological, immunogenic or vaccine composition comprising E. coli antigens as discussed herein such as antigens K99, F41, Y and 31A, as well as inactivated bovine coronavirus, inactivated bovine rotavirus. This composition can further include Clostridizon perfringens type C and D toxoids. Preferably the E. coli valency comprises either inactivated E. coli bearing K99 antigen, inactivated E. colL. bearing F41 antigen, inactivated E. coli bearing Y SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:54 FAX 613 8618 4199 F.B. RICE Co. 1034 WO 01/45735 PCT/EPOO/13387

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o 28 c antigen and inactivated E. coli bearing 31A antigen, or, K99 antigen, F41 antigen, Y antigen and 31A antigen.

IN0 Another aspect of the present invention is an immunological, immunogenic or vaccine composition against Cryptosporidium parvan, which comprises Cp23 or P21 and Cp15/60 antigens or epitopes thereof, and a pharmaceutically acceptable vehicle.

IND According to an advantageous embodiment, these antigens are incorporated in the o composition as proteins or sub-unit antigens. They can be produced by chemical synthesis or tC by expression in vitro. For the convenience of production by expression in a suitable host, and O their recovery, these antigens are preferably under the form of fusion protein with HIS Cl 10 tag). In other words, the antigen can comprise the antigenper se and foreign amino acids.

According to another embodiment, these antigens are not incorporated as subunits in the composition, but the composition comiprises eithbr a recombinant vector expressing Cp23 or P21 and. Cpl5/60 or an epitope thereof or a recombinant vector expressing Cp23 or P21 or an epitope thereof and a recombinant vector expressing Cpl5/60 or an epitope thereof, wherein these vectors express the antigen(s) or epitope(s) in vivo when administered to the animal. The composition can contain an antigen at epitope and a vector expressing the other antigen or epitope.

A still further aspect of the present invention is the methods of vaccination wherein one administers to a target animal a combined enteric immunological or vaccine composition or an immunological or vaccine composition against Cryptosporidium parvum according to the invention. The invention can concern a method of immunization of a new-born calf against enteric disease, comprising administering an immunological or vaccine composition comprising Cp23 or P21 and Cpl5/60 Cryptosporidium parvum antigens or epitopes thereof and a pharmaceutically acceptable vehicle, to the pregnant cow or pregnant heifer before delivering, so that the newborn calf has maternal antibodies against Cryptosporidium parvum.

Preferably, the method comprises the feeding of the newborn calf with colostrum and/or milk coming from a cow, e.g. the mother, which has been so vaccinated. For vaccination or immunization against enteric disease, one may not only use a combined vaccine, immunogenic or immunological composition, containing the various valencies, but also separate vaccine, immunogenic or immunological compositions which can be administered separately, e.g., sequentially, or which can be mixed before use.

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:54 FAX 613 8618 4199 F.B. RICE& Co. 1035 WO 01/45735 PCT/EP0/13387 o 29 0 Antigens and epitopes of interest useful in inventive compositions and methods may be produced using any method available to the one skilled in the art and for instance using the O methods in US-A-5,591,434 and WO-A-9807320. Further, one can obtain antigens of other enteric pathogens from commercially available sources, such as TRIVACTON@6; for instance, Cp23 and/or P21 and/or CplS/60 or an epitope thereof, P21 or Cp23 and C p Cpl5/60 or an epitope thereof, or a vector expressing these antigen(s) or epitope(s) can be 0 o added to TRIVACTON®6, in herein specificed amounts. Clostridium perfringens toxoids C Va Oand D may advantageously be added to TRIVACTON®6. Also, the inactivated E. coli bearing pili may be replaced in TRIVACTON®6 by the isolated pili- Such a vaccine, To immunogenic or immunological composition (with inactivated E. coli or isolated pili) to which C. parvum antigen(s) or epitope(s) and/or Clostridium perfringens antigen(s) or epitope(s) is/are added and methods of making and using.such a composition and kits therefor are also within the invention.

Furthermore, as to the E. coli valency and/or antigen(s) and/or epitope(s) useful in the practice of the invention, reference is made to EP-A-80,412, EP-A-60,129, GB-A-2,094,314, and U.S. Patents Nos. 4,298,597, 5,804.,198,; 4,788,056, 3,975,517, 4,237,115, 3,907.987, 4,338,298, 4,443,547, 4,343,792, 4,788,056, and 4,311,797. As to rotavirs antigen(s) and/or epitope(s), reference is made to P.S. Paul and Y.S. Lyoo, Vet IMicrob 37:299-317 (1993) and U.S. Patents Nos. 3,914,408 and 5,620,896. With respect to coronavirus antigen(s) and/or epitope(s), reference is made to WO-A-98 40097, WO-A-96 41874, and U.S. Patents Nos.

3,914,408 and 3,919,413. For Closridimwn, CL perfringens, antigen(s) and/or epitope(s), reference is made to WO-A-94 22476, EP-A-734,731, WO-A-98 27964, GB-A-2,050,830, GB-A-1,128,325, D. Calmels and Ph. Desmettre, IV Symposium of the Commission for the study of animal diseases caused by anaerobes, Paris, Nov. 16-18, 1982, U.S. Patents Nos.

5,178,860, 4,981,684, and 4,292,307; and, to IMOTOXAN (MIERIAL Lyon, France) (containing types B, C, D, C1. perfringens, toxoids from CL septictrni, CL. novyi, CL. tetani and culture of CL. chauvoei). And, in addition to TRIVACTON®6, one may use other commercial combined vaccines to which C. parvun valency can be added, in accordance with this invention; for instance, SCOURGUARD 3 (SmithKline Beecham) containing inactivated bovine rotavirus and coronavirus, K99 E. coli bacterin and CL. perfringens type C toxoid.

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:54 FAX_613 8618 4199 F.B. RICE Co. o036a WO 01/45735 PCT/EP00/13387

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A preferred method to obtain antigens or epitopes of interest is to clone the DNA sequence encoding the antigen or epitope of interest into a fusion or non-fusion plasmid and to \O have its expression in E. coli. Fusion plasmids that express the antigen(s) or epitope(s) with a tag such as a His tag) are preferred as they allows one to recover easily the produced S 5 antigen. Suitable plasmids are described in the examples. Production of antigens by chemical VO synthesis is also within the scope of the invention.

o The invention further comprehends methods for using herein discussed antigens or C, epitopes or vectors expressing such antigens or epitopes for the preparation of a vaccine, Simmunological, or immunogenic composition, against C parvum or against enteric 10 disease; for instance, by admixing the antigens, epitopes or vectors with a suitable or acceptable carrier or diluent and optionally also with an adjuvant. The compositions may be lyophilized for reconstitution. The invention further comprehends a kit for the preparation of an inventive composition. The kit can comprise the antigen(s), epitope(s) and/or vector(s), carrier and/or diluent and optionally adjuvant; the ingredients can be in separate containers.

The containers containing the ingredients can be within one or more than one package; and, the kit can include instructions for admixture of ingedients and/or administration of the vaccine, immunogenic or immunological composition composition.

Another aspect of the invention is the production of hyperimmune colostrum and/or milk; for instance, by hyperimmunization of the pregnant female mammal (such as a cow) by at least 1, advantageously at least 2, and more advantageously at least 3, administrations of inventive composition(s) C. parvum composition or combined enteric composition according to the invention). Optionally, but advantageously, the colostrum and/or milk so produced can then be treated to concentrate the immunoglobulins and to eliminate components of the colostrum or milk that do not contribute to the desired immunological, immunogenic and/or vaccine response or to the nutritional value of the colostrum or milk, That treatment can advantageously comprise coagulation of the colostrum or milk, with rennet, and the liquid phase containing the immunoglobins recovered. The invention also comprehends the hyperimmune colostrum or milk or mixture thereof and/or compositions comprising the hyperimmune colostrum or milk or mixture thereof. Further, the invention envisions the use of the hyperimmune colostrum or milk or mixture thereof or composition comprising the same SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:55 FAX 613 8618 4199 F.B. RICE Co. 1037 WO 01/45735 PCT/EP00113387 31 to prevent or treat C. parvum and/or enteric infection in a young animal such as a new-born; for instance, a.calf.

Accordingly, the invention shall be further described by way of the following Examples, provided for illustration and not to be considered a limitation of the invention.

S EXAMPLES List of sequences: SEQ ID NO: 1 SEQ lD NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ D NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 oligonucleotide JCA295 oligonucleotide JCA296 oligonucleotide JCA297 oligonucleotide JCA298 oligonucleotide JCA299 oligonucleotide JCA300 oligonucleotide JCA301 oligonucleotide 3CA302 oligonucleotide JCA303 oligonucleotide JCA304 All plasmid constructs have been done using standard molecular biology techniques (cloning, restriction digestion, polymerase chain reaction (PCR)) as described in Sambrook J.

et al. (Molecular Cloning: A Laboratory Manual. 2 nd Edition. Cold Spring Harbor Laboratory.

Cold Spring Harbor. New York. 1989). All DNA restriction fragments generated and used for the present invention, as well as PCR fragments, have been isolated and purified using the "Geneclean®" kit (IO0101 Inc. La Jolla, CA).

Example 1: Cloning of the C. parvum P21 and Cp15/60 genes Oocysts of Cryptosporidium parvwn are isolated from an infected calf and are purified from bovine fecal samples as described by Sagodira S. et al. (Vaccine. 1999. 17. 2346-2355).

Purified oocysts are then stored in distilled water at For use as a template for PCR reactions, genomic DNA is released from the purified oocysts as described by lochmann S. et al (Microbial Pathogenesis 1999.26. 307-315).

An alternative source for C. parvenz DNA is constituted by the EcoRI genomic libraries for the Cryptosporidiztm parvum Iowa Iowa KSU-1 and KSU-2 isolates SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:55 FAX 613 8618 4199 F.B. RICE Co. @038 WO 01/45735 PCT/EPOO/13387

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S32 available from the American Tissue Culture Collection (ATCC numbers 87667, 87668, 87439 Sand 87664 respectively). The specific P21and Cpl5/ 6 0 genes are isolated as follows: IsO The sequence encoding the P21 protein is amplified by a polymerase chain reaction

O

(PCR) using C. parvum DNA and the following primers: oligonucleotide JCA295 (35 mer) SEQ ID NO: 1 V0 5' TIT TTT CCA TGG GGC TCG AGT TIT CGC TTG TGT TG 3' 0 and oligonucleotide JCA296 (33 mer) SEQ ID NO: 2 N 5' TTT TIT GAA TTC TTA GGC ATC AGC TGG CTT GTC 3' o This PCR generates a fragment of about 585 bp PCR fragment. This PCR fragment is C 10 then digested with NcoI and EcoRI restriction enzymes to isolate, after agarose gel electrophoresis and recoveiy with the GeneClean kit (BI0101 Inc.), a 575 bp NeoI-EcoRI restriction fragment fragment The sequence of this fragment encodes a protein homologous to the sequence described as SEQ ID NO: 12 in patent application WO 98/07320 (PCT/US97/14834).

A second PCR is run to amplify the sequence encoding the Cpl5/ 6 0 protein and to add convenient restriction sites in 5' and 3' for further cloniig. The PCR is done using C. parvum DNA and the following primers: oligonucleotide JCA297 (35 mer) SEQ ID NO: 3 TTT TTT CTC GAG ATO GGT AAC TTG AAA TCC TGT TG 3' and oligonucleotide JCA298 (42 mer) SEQ ID NO: 4 TTT TTT GAA TTC TTA GTT AAA GTT TGG TTT GAA TTT GTT TGC 3' This PCR generates a fragment of about 465 bp. This fragment is purified and then digested with Xol and EcoRI in order to get, after agarose gel electrophoresis and recovery with the GeneClean kit (BIO101 Inc.), the 453 bp Xho-EcoRI fragment fragment The amplified sequence is homologous to be similar to the sequence defined from nucleotide #31 to #528 of SEQ ID NO: 1 in US Patent 5,591,434 and to the sequences deposited in GenBank under Accession Numbers U22892 and AAC47447.

Example 2: Construction of plasmid pJCA155 (GST-P21 fusion protein in vector pBAD/HisA) The sequences required to express the GST-P21 fusion protein are amplified by PCR in order to generate 2 fragments that can be cloned easily into the pBAD/HisA.expression SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:55 FAX 613 8618 4199 F.B. RICE Co. 1039 WO 01/45735 PCTIEP00/13387 o 33 0 plasnmid vector (Cat V430-01 InVitrogen Corp., Carlsbad, CA 92008, USA). The first PCR is done using the pGEX-2TK plasmid (Cat 27-4587-01 Amersham-Phanacia Biotech) and V.O the following primers: oligonucleotide JCA299 (35 mer) SEQ ID NO: 5' TT TT CCA TGG GGT CCC CTA TAC TAG OTT ATT GG 3' and oligonucleotide JCA300 (45 mer) SEQ ID NO: 6 O 5' TTT TTT CTC GAG CCT GCA GCC.CGCG GGA TCC AAC AGA TGC ACG ACG 3' \O c1 This PCR generates a fragment of about 720 bp encoding the GST moiety with the o addition of a Ncol restriction site at the 5' end for cloning purposes into pBAD/isA; this c to modificatioh adds a Glycine codon to the GST-P21 fusion protein). This-PCR fragment is then digested with NcoI and XhoI in order to get, after agarose gel electrophoresis and recovery with the GeneClean kit (BIO101 Inc.), the 710 bp Nco-XhoI fragment fragment C).

The second PCR is done using C. parvzmz DNA and the following primers: oligonucleotide JCA301 (33 mer) SEQ ID NO: 7 5' T TT ITC GAG TT TCG CTT GTG TTG TAC AGC 3' and oligonucleotide JCA296 (33 mer) SEQ ID NO: 2 This PCR generates a fragment of about 580 bp encoding the P21 moiety with the addition of XhoI and EcoRI restriction sites at the 5' and 3' ends respectively. This PCR fragment is then digested with Xhot and EcoRI in order to get, after agarose gel electrophoresis and recovery with the GeneClean kit (BIO101 Inc.), the 572 bp XhoI-EcoRI fragment fragment D).

The pBAD/HisA plasmid (Cat V430-01, InVitrogen Corp.) is digested with Ncol and EcoR The digested fragments are separated by agarose gel electrophoresis in order to recover (GeneClean kit, BI101 Inc.) the 3960 bp Ncol-EcoRI restriction fragment fragment E).

Fragments C, D and E are then ligated together to generate plasmid pXCA155. This plasmid has a total size of 5243 bp (Figure 1) and encodes a 425 amino acids GST-P21 fusion protein.

Example 3: Construction of plasmnid pJCA156 (His 6-P21 fusion protein in vector pBAD/HisA) SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:56 FAX 613 8618 4199 F.B. RICE Co. 1040 WO 01/45735 PCT/EP00113387 cN 34 S"The pBAD/IisA vector (Cat V430-01, InVitrogen) is digested with NcoI and EcoRI and the 3960 bp NcoI-EcoRI restriction fragment fragment E) is recovered and isolated as O described in Example 2.

A PCR is done to amplify the sequence encoding the His6-P21 fusion and to add the C 5 NcoI and EcoRI restriction sites respectively in 5' and 3' in order to subclone this PCR O, fragment into the pBAD/HisA plasmid vector.

o The PCR is done using C. parumn DNA and the following primers: Q oligonucleotide JCA302 (65 mer) SEQ ID NO: 8 TTT TTT CCA TGG GGG GTT CTC ATC ATC ATC ATC ATC ATG GTC TCG AGT C 10 TTT COC TTGTGT TGT AC 3' and oligonucleotide JCA296 (33 mer) SEQ ID NO: 2 This PCR generates a fragment of about 610 bp. This fragment is purified, and then digested with NcoI and EcoRI in order to isolate, after agarose gel electrophoresis and recovery with the GeneClean kit (BI0101 Inc.), the 600 bp NcoI-EcoRI fragment fragment

F).

Fragments E and F are ligated together to generate plasmid pJCA156. This plasmid has a total size of 4562 bp (Figure 2) and encodes a 199 amino acids His-6/P21 fusion protein.

Example 4: Construction of plasuid pJCA157 (P21 protein alone in pBAD/HisA vector) The pBAD/HisA vector (Cat V430-01, InVitrogen Corp.) is digested with Ncol and EcoRI and the 3960 bp NcoI-EcoRI restriction fragment fragment E) is recovered and isolated as described in Example 3.

A PCR is done to amplify the sequence encoding the P21 protein and to add the NcoI and EcoRI restriction sites respectively in 5' and 3' in order to subclone this PCR fragment into the pBAD/HisA plasmid vector. The PCR is done using C. parvum PNA and the following primers: oligonucleotide JCA295 (35 mer) SEQ ID NO: 1 and oligonucleotide JCA296 (33 mer) SEQ ID NO: 2 to get, as described in Example 1, a 575 bp NcoI-EcoRI fragment (fragment A).

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:56 FAX 613 8618 4199 F.B. RICE Co. [1041 WO 01/45735 PCT/EPOO/13387

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035 ct Fragments E and A are ligated together in order to generate plasmid pJCA157. This Splasmid has a total size of 4535 bp (Figure 3) and encodes 189 amino acids including the P21 No protein.

Example 5: Construction of plasmid pJCA158 (GST-Cpl5/60 fasion protein in pBAD/HIsA vector) V.0 A PCR is done to amplify the sequence encoding the GST protein and to add 0 convenient restriction sites in 5' and 3' in order to subclone the PCR fragment into the final SpBAD/IHisA plasmid vector. The PCR uses the DNA of plasmid pGEX-2TK (Cat 27-4587cI 0 01, Amersham-Pharmacia Biotech) as a template and the following primers; C 10 oligonucleotide JCA299 (35 mer) SEQ ID NO: and oligonucleotide JCA300 (45 mer) SEQ ID NO: 6 to get, as described in example 2, a 710 bp NcoI-XhoI fragment fragment C).

The pBAD/IfisA vector (Cat V430-01, InVitrogen) is digested with NcoI and EcoRI and the 3960 bp NcoI-EcoRI restriction fragment fragment E) is recovered and isolated as described in Example 2.

Fragments C, E and B (Example 1) are ligated together in order to generate plasmid pJCA158. This plasmid has a total size of 5132 bp (Figure 4) and expresses a 388 amino acids GST-Cpl5/60 fusion protein.

Example 6: Construction of plasmid pJCA159 (His6-Cpl5/60 fusion protein in pBAD/JisA vector) The pBAD/HisA vector (Cat V430-01, InVitrogen Corp.) is digested with NcoI and EcoRI and the 3960 bp NcoI-EcoRI restriction fragment fragment E) is recovered and isolated as described in Example 2.

A PCR is ran to amplify the sequence encoding the His6-Cpl5/60 fusion and to add convenient restriction sites in 5' and 3' in order to subclone this PCR fragment into the pBAD/HisA plasmid vector. The PCR is done using either C. parvum DNA and the following primers: oligonucleotide JCA303 (64 mer) SEQ ID NO: 9 TIT TT CCA TGG GGG GTT CTC ATC ATC ATC ATC ATC ATG GTA TGG GTA ACT TGA AAT CCT GTT G 3' and oligonucleotide JCA298 (42 mer) SEQ ID NO: 4 SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:56 FAX 613 8618 4199 F.B. RICE Co. 1042 WO 01/45735 PCT/EP00/13387

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o36 c This PCR generates a fragment of about 495 bp. This fragment is purified and then digested with Ncol and EcoRI in order to get, after agarose gel electrophoresis and recovery Va with the GeneClean kit (BI0101 Inc.), the 483 bp NcoI-EcoRI fragment fragment G).

Fragments E and G are ligated together in order to generate plasmid pJCA159. This c 5 plasmid has a total size of 4445 bp (Figure 5) and expresses a 159 amino acids His-6/Cpl5/60 sO fusion protein.

o Example 7: Construction of plasmid pJCA160 Ci (Cpl5/60 protein alone in pEAD/BsA vector) O The pBAD/HisA vector (Cat V430-01, InVitrogen Corp.) is digested with Ncol and EcoRI and the 3960 bp NcoI-EcoRI restriction fragment fragment E) is recovered and isolated as described in Example 2.

A PCR is run to amplify the sequence encoding the Cpl5/60 protein and to add convenient restriction sites in 5' and 3' in order to subelone this PCR fragment into the pBAD/HisA plasmid vector.

The PCR is done using C. parvum DNA and the following primers: oligonucleotide JCA304 (31 mer) SEQ ID NO: TTT TTT CCA TGG GTA ACT TGA AAT CCT GTT G 3' and oligonucleotide JCA298 (42 mer) SEQ ID NO: 4 This PCR generates a fragment of about 460 bp. This fragment is purified and then digested with NcoI and EcoRI in order to get, after agarose gel electrophoresis and recovery with the GeneClean kit (BI0101 Inc.), the 450 bp Neol-EcoRI fragment fragment H).

Fragments E and H are ligated together in order to generate plasmid pJCA160. This plasmid has a total size of 4412 bp (Figure 6) and expresses a 148 amino acids Cpl5/60 protein.

Example 8: Culture of E. coli recombinant clones and induction of recombinant proteins Plasmid DNA (Examples 2 to 7) is transformed into Escherichia coli DH5c (or any other suitable E. coli K12 strain well known to those skilled in the art, such as E. coli (Cat C4040-03 InVitrogen Corp.)) and grown on Luria-Bertani (LB) medium agar plates with 50p g/ml of ampicillin. One colony is picked for each plasmid transformed E. coli population and placed in 10 ml of LB medium with ampicillin (or other appropriate antibiotic) SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:57 FAX 613 8618 4199 F.B. RICE Co. 21043 WO 01/45735 PCT/EP00/13387

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S37 Sfor overnight growth. One ml from the overnight culture is added to one liter of LB medium and grown at 4-30°C until ODeoo m reaches approximately o Protein production is induced with different final concentrations of DL-arabiose (Cat# A9524, Sigma, St Louis, MO) (range of 0.002% to 0.2% for determining the concentration for C C 5 optimal yield) added to the culture and incubated at +30 0 C for 4-6 hours.

SExample 9: Extraction and purification of the recombinant fusion proteins o At the end of the induction (Example cells are harvested by centrifugation (3000 g, C- 10 minutes, and resuspended in lysis buffer (50 mM Tris pH 8.0, 1 mM EDTA, 1 gM O PMSF, 1 mg/ml lysozyme) and sonicated 25 times for 30 seconds bursts with 1 minute pauses C 10 i between bursts. Triton X-100 is added to a final concentration of Debris are removed by centrifugation.

If necessary, alternative techniques (known to'those of skill in the art) may be used for the lysis of bacterial cells.

9.1. GST-fusion recombinant proteins: Recombinant GST-fusion proteins (produced by E. coli transformed with plasmids pJCA155 or pJCA158) were affinity purified from the bacterial lysates, prepared as described in Example 8, using a glutathione-agarose (Cat# G4510, Sigma) or glutathione-Sepharose 4B (Cat# 17-0756-01, Amersham-Pharmacia Biotech). Bacterial lysates and the glutathioneagarose were incubated for 4 hours at +40C. GST-fusion proteins were then eluted from the agarose in a batch format with 10 mM reduced form glutathione (Cat# G4705, Sigma) under mild conditions Johnson and D. Smith Gene. 1988. 67. 31-40). (Reference Anonymous.

GST gene fusion system technical manual. 3 edition. Arlington Heights, IL: Amersham- Pharmacia Biotech, 1997). Anyone skilled in the art can achieve scaling up of this process for purifying large quantities of GST-fusion proteins, from this disclosure and the knowledge in the art, without undue experimentation.

9.2. His6-fusion recombinant proteins: Recombinant His6-fusion proteins have all been prepared and purified using the ProBond T m Nickel-Chelating resin (Cat# R801-15, InVitrogen Corp.) following the manufacturer's instructions.

Preparation of native E. coli cell lysate (soluble recombinant protein) the bacterial cells from a 1 liter culture of E. coli (transformed with plasmids pJCA156 or pJCA159) are SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:57 FAX 613 8618 4199 F.B. RICE Co. 0044 WO 01/45735 PCT/EPOO/13387

\N

o 38 t harvested by centrifugation (3000 g for 5 minutes). The pellet is resuspended in 200 ml of Native Binding Buffer (20 mM phosphate, 500 mM NaC1, pH The resuspended pellet is NO then incubated with egg lysozyme at a final concentration of 100 jpg/ml, for 15 minutes on ice.

This mixture is then sonicated with 2-3 10-second bursts at medium intensity while holding the suspension on ice. The mixture is then submitted to a series of freezing/thawing cycles for O completing the lysis and the insoluble debris are finally removed by centrifugation at 3000 g o for 15 minutes. The lysate is cleared by passage through a 0.8 gm filter and stored on ice or at -20 0 C until purification.

SThe soluble recombinant His6-fusion protein present in the clear lysate is batch bound i 10 to a 50 ml pre-equilibrated ProBond T resin column (Cat R640-50 and R801-15, InVitrogen Corp.) with two 100 ml lysate aliquots. The column is gently rocked for 10 minutes to keep the resin resuspended and allow the polyhistidine-tagged protein to fully bind. The resin is settled by gravity or low speed centrifugation (800 g) and the supernatant is carefully aspirated. An identical cycle is repeated with the second aliquot Column washing and elution: 4 successive steps are done according to the manufacturer's instructions (Anonymous.

Xpress M System Protein Purification A Manual of Methods for Purification of Polyhistidine Containing Recombinant Proteins. InVitrogen Corp. Editor. Version D. 1998) 1. The column is washed with 100 ml of Native Binding Buffer pH 7.8, by resuspending the resin, rocking for 2 minutes and then separating the resin from the supernatant by gravity or centrifugation. This procedure is repeated 2 more times (total of 3 washes) 2. The column is washed with 100 ml of Native Wash Buffer pH 6.0 by resuspending the resin, rocking for 2 minutes and then separating the resin from the superatant by gravity or centrifugation, This procedure is repeated at least 3 more times until OD2.o.is less than 0.01.

3. The column is washed with 100 ml of Native Wash Buffer pH 5.5 by resuspending the resin, rocking for 2 minutes and then separating the resin from the supernatant by gravity or centrifugation. This procedure is repeated once (total of 2 washes).

4. The column is then clamped in vertical position and the cap is snapped off on the lower end.

The recombinant protein is. eluted with 150 ml of the Native pH Elution Buffer. 10 ml fractions are collected_ Elution is monitored by taking OD2so readings of the fractions.

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:57 FAX 613 8618 4199 F.B. RICE Co. 1045 WO 01/45735 PCT/EP00/13387

\N

o 39 bIf needed, the eluted recombinant protein can be concentrated either by dialysis, or by precipitation with ammonium sulfate.

IO Final concentration of the recombinant protein batch is measured by OD 2 s 0 readings.

Anyone skilled in the art can achieve scaling up of this process for purifying large quantities of His6-fusion proteins, from this disclosure and the knowledge in the art, without INO undue experimentation.

o Example 10: Extraction and purification of the C. parvwn SP21 and Cpl5 recombinant non-fusion proteins NO The bacterial cells of E. coli (transformed with plasmids pJCA157 or pJCA160) are O 10 cultured in 4 liters of the M9 minimum medium (supplemented with the appropriate amino acids) (Sambrook J. et al. (Molecular Cloning: A Laboratory Manual. 2 d Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor. New York. 1989) at 30°C until ODoo m reaches approximately 3.0 and are induced as described in Example 8. The bacterial cells are then disrupted by passing through a high pressure RANNIE homogeneizer Mini-Lab type 8.30 H with a maximum flow of 10 liters per hour and working pressure between 0 and 1000 bars.

The lysate is cleared by filtration through a CUNO- filter Zeta plus, LP type, and then concentrated 50 times on an ultrafilter PALL Filtron (reference OS010G01) UF 10 kDa. The protein suspension concentrate is loaded on a size-exclusion chromatography column with High Resolution Sephacryl S-100 gel under a volume corresponding to 2-3% of the column volume. Elution is done with a PBS buffer. The collected fractions corresponding to the expected molecular weight for the subunit vaccine proteins are concentrated 10 times on a hollow fibers cartridge A/G Technology type Midgee cartridge model UFP-10-B-MB01 (or model UFP-10-C-MB01 or model UFP-10-E-MB01). The concentrated samples are then stored at -70C until use. The specific C. parvum recombinant proteins can be then mixed in the appropriate proportions to the final associated vaccine (see Example 11).

Example 11: Formulation of vaccines; vaccination of pregnant cows; passive immunizition and challenge experiment in newborn calves Product (adjuvanted or not) is administered intramuscular subcutaneous (SQ) or intradennal (ID) to elicit serum antibody responses against C. parvum. When administered twice to pregnant animals it elicits a serum antibody response that will be passively transferred to the newborn via colostrum and milk. Vaccination protocol for pregnant animals can comprise 2 doses given between when pregnancy is diagnosed and calving, such as about 1 SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:58 FAX 613 8618 4199 F.B. RICE Co. Q046 -WO 01/45735 PCTIEP00/13387 Va c month before calving and about 3 to 5 days before calving; or, 2 months prior to calving S(which coincides with dry-off in dairy cows) and a boost prior to calving anywhere from 3 weeks to I week prior to calving), depending on management practices (however, these schedules favor maximum efficacy) Current management practices favor that are products cn 5 administered in the last trimester. Volume of the product can be from 1 ml to 5 ml, such as 2 ml. Combination vaccines can have a lyophylized and a liquid portion that can be mixed prior Sto injection. To afford maximum protection under field conditions the Cryptosporidium n antigen can be added as a component of an E. colilRota/Corona combination vaccine.

SThe following studies are conducted: Ci 10 Study A: C. parvum enhances the pathogenicity of enteric virus and/or bacteria Experimental challenge utilizing 3 newborn calves per group as follow: 1. Coronavirus only 2. Coronavirus plus C. parvum 3. E. coli F41 only 4. E coli F41 plus C. parvun C. parvum only 6. Unchallenged controls Calves are challenged within 24 hours of being bor, by the oral route. The amount of challenge material used is that which is necessary to produce clinical signs (depression, diairhea, dehydration) and may depend on the type of animal (gnotobiotic artificially raised or conventional calve nursing its dam). Common clinical signs (temperature, demeanor, hydration, diarrhea scores, etc.) are collected. Additional serological and shedding information is collected- Outcome Coronavirus or E. coli F41 monovaleit experimental challenges do not produce clinical signs of enteric disease in newborn calves. Dual challenge with coronavirus or E coli F41 with C parvum, at a C. parvum dose that normally does not cause clinical disease, will produce significant clinical signs of enteric disease.

Study B: A combo vaccine col K99/F41, rota and coronavirus) containing C. parvum provides enhanced protection against enteric disease cause by concurrent infection of multiple enteric virus and/or bacteria in newborn calves.

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:58 FAX 613 8618 4199 F.B. RICE Co. 1047 WO 01/45735 'PCT/EP00/13387 STreatment groups are 30 pregnant cows vaccinated with: 1. Combo (rota and coronavirus. E. coli K99 and F41), 8 animals; o 2. Combo plus Crypto, 8 animals; 3. Unvaccinated controls, 14 animals.

eC 5 Experimental challenge as follow: Va 0 1. Multiple challenge (coronavirus and F41 plus C. parm at subclinical level); o 2. Sentinel animals O 3. unchallenged.

o Calves receive colostrum (manually fed or allowing the calve to nurse from the dam) and those that are challenged are challenged within 24 hours of being born, by the oral route.

The amount of challenge material is an amount necessary to produce clincal signs as determined in Study A, and as mentioned under Studj A, can vary depending upon the type of animal used gnotobiotic artificially raised or conventional calves nursing their dams).

Common clinical signs (temperature, demeanor, diarrhea scores) are collected. Additional serological and shedding information is collected.

Design: 6 calves born from vaccinated (combo and combo plus Crypto) or control cows are challenged with a challenge containing 3 components (coronavirus and F41 plus C. parvum), and 3 calves (from unvaccinated control cows) remain as sentinels.

Outcome Use of a combo vaccine containing C. parvum produces a better protection than a combo vaccine alone under a multiple challenge situation (coronavirus and E coli V41 with C.

parvum at a subclinical dose).

Example 12 Effect of dual infection with C. parvum and bovine rofavirus in an experimental challenge model in newborn calves This study is designed to compare the severity of clinical signs and fecal excretion in calves after monovalent challenge with C. parvum or bovine rotavirus and after a dual challenge with bovine rotavirus plus C parvwn.

Four groups of six calves are used in order to yield sufficient data to be able to detect SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:58 FAX 613 8618 4199 F.B. RICE Co. 1048 WO 01/45735 PCT/EP00/13387 042 ic differences in incidence of clinical signs between groups.

SCows are individually housed in pens or paddocks. Newborn calves are separated from V.0 their dams as soon as possible after birth, inspected to eliminate feces or dirt on the calf and 0 their ombilical cord dipped in approximate 7% iodine solution. They are then immediately transferred to containment accomodations and housed individually in metabolic crates. Calves 0N are challenged within 6 hours after birth.

SCalves are fed 1 to 2 quarts per feeding or at 10% body weight, twice daily for the C, entire trial using a commercial calf milk replacer with 30% colostrum substitute. Special care 1 will be given to avoid the administration of milk within 2 hours pre or post challenge.

cN 10 The route of natural infection is oral therefore, all the challenges will be administered orally using an esophageal tube.

Group A non-challenged control calves.

Group B 1-3x10 5 C. parvum oocysts (strain Beltsville), diluted in 60 ml of commercial antibiotics free soy milk.

Group C Coinoculation of 1-3x10 5 C. parvum oocysts (strain Beltsville), diluted in ml of commercial antibiotics free soy milk, and of 10 ml bovine rotavirus inoculum (strain IND BRV G6P5) diluted in 40 ml PBS.

Group D 10 ml fecal filtrate from bovine rotavirus infected calves (strain IND BRV diluted in 40 ml PBS.

Fecal samples are collected from the collection pan once a day after thoroughly mixing to ensure a representative sample is obtained.

Oocysts are separated from calves feces by centrifugation on sucrose cushions and counted using a cell counting chamber (hemocytometer) under a microscope. For rotavirus shedding, the feces are diluted in buffer and the rotavirus antigen is quantified using an ELISA kit from Le Centre d'Economie Rurale (CER) 1 rue du Carmel, B6900 Marloie, Belgium.

Calves are observed for clinical signs prior to challenge and then twice daily for days post-challenge. Observations include rectal temperature, general condition, anorexia, diarrhea, dehydration and death.

Depression, diarrhea, and dehydration are categorized as follows General condition: SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:59 FAX 613 8618 4199 F.B. RICE Co. 049 PCT/EPOO/13387 WO 01145735 Good The calf is bright, alert and responsive Apathic The calf is quiet, alert and responsive Depression The calf is lying aside, reluctant to rise, and slow to respond Prostration The calf is curled up or prostrate and not responsive Dehydration: None No dehydration Moderate Persistent skin fold, dry mouth and depressed eyeballs Shock State of shock Diarrhea: Anorexia is detennined based on whether the calf nurses less than 2 liters of milk. During the 1' 48 hours of life, calves may be fed via an esophageal tube.

The score is derived for each calf on each day based on the presence of clinical signs (rated 1) or absence (rated 0) for each sickness category.

i.

Rectal temperature is recorded in degrees Farhenheit Two calves died in Group C on days 7 and 8, two in Group B on day 7, none in Group D and one in Group A on day 3.

Results are shown on Figures 7 to 13.

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:59 FAX 613 8618 4199 "ri WO 01/45735 F.B. RICE Co. [1050 PFCT/PO0/13387 44 A synergistic effect on clinical signs and microorganisms excretion in feces is observed when both microorganisms are administered compare to single administrations.

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

REFERENCES

Tzipori S. The relative importance of enteric pathogens affecting neonates of domestic animals. Adv Vet Sci Comp Med, 290:103-206. 1985 Angus K W. Cryptosopridiosis in Ruminants. In: Cryptosopridiosis of man and animals (Edited by Dubey J P, Speer C A Fayer pp 83-103. CRC Press, Boston. 1990.

De la Fuente R; Luz'on M; Ruiz-Santa-Quiteria JA; Garclia A; Cid D; Orden JA; Garcia S; Sanz R; G'orez-Bautista M. Cryptosporidium and concurrent infections with other major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain. Vet Parasitol, 80(3):179-85. 1999.

Moon HW; McClurkin AW; Isaacson RE; Pohlenz J; Skartvedt SM; Gillette KG; Baetz AL.

Pathogenic relationships of rotavirus, Escherichia coli, and other agents in mixed infections in calves. J Am Vet Med Assoc, 173(5 Pt 2):577-83 1978 Sep 1 Viring S; Olsson SO; Aleni'us S; Emanuelsson U; Jacobsson SO; Larsson B; Linde N; Uggla A. Studies of enteric pathogens and gamma-globulin levels of neonatal calves in Sweden. Acta Vet Scaud, 34(3):271-9 1993 Perryman LE, Kapil SJ, Jones ML, Hunt EL, "Protection of calves against cryptosporidiosis with immune bovine colostrum induced by a Cryptosporidium parvum recombinant protein" Vaccine 17(17):2142-9 (1999, April 23).

Wakelin, D. "Immune response to intestinal parasites: protection, pathology and prophylaxis" Parassitologia 39(4):269-74 (1997, December).

Iochmann, S. et al., "Comparison of the humoral and cellular immune responses to two preparations of Cryptosporidium parvum CP15/60 recombinant protein" Microb. Pathog.

26(6):307-15 (1999 June).

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:59 FAX 613 8618 4199 F.B. RICE Co. [1051 WO 01/45735 PCT/EP00/13387 c Sagodira, S. et al., "Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA" Vaccine 17(19):2346-55 (1999, May 14).

o1 Enriquez FJ et al., ."Role of immunoglobulin A monoclonal antibodies against P23 in controlling murine Cryptosporidiumf parvum infection" Infect Immun 66(9):4469-73 (1998 September).

V.Q Harp JA et at, "Strategies for the control of Cryptosporidium parvum infection in calves" J, SDairy Sci 81(1):289-94 (1998 January).

i Sreter T. et aL, "Attempts to immunize chickens against Cryptosporidium baileyi with C.

NO parvum oocysts and Paracox vaccine" Folia Parasitol (Praha) 44(1):77-80 (1997).

C, Harp JA et al., "Field testing of prophylactic measures against Cryptosporidium parvum infection in calves in a California dairy herd" Am Vet Res 57(11):1586-8 )1996 November).

Jenkins M. et al., "Serum and colostrum antibody responses induced by jet-injection of sheep with DNA encoding a Cryptosporidium parvum antigen" .Vaccine 13(17):1658-64 (1995 December).

Tatalick LM et al., "Attempts to protect severe combined immunodeficient (scid) mice with antibody enriched for reactivity to Cryptospoidium parvum surface antigen-l." Vet Parasitol 58(4):281-90 (1995 July).

Harp JA et al., "Protection of calves with a vaccine against Cryptosporidium parvum" J Parasitol 81(1):54-7 (1995 February).

Dellert SF et al., "Diarrheal disease. Established pathogens, new pathogens, and progress in vaccine development" Gastroenterol Cin North Am 23(4):637-54 (1994 December).

Bellinzoni RC et al., "Efficacy of an inactivated oil-adjuvanted rotavirus vaccine in the control of calf diarrhoea in beef herds in Argentina" Vaccine 7(3):263-8 (1989 June).

Harp JA et al., "Field testing of prophylactic measures against Crytosporidium parvum infection in calves in a California dairy herd" Am J Vet Res 57(11):1586-8 (1996 November).

Harp JA et al, "Resistance of calves to Cryptosporidium patvum: effects of age and previous exposure" 58(7):2237-40 (1990 July).

Payer R. et al., "Efficacy of hyperimmune bovine colostrum for prophylaxis of cryptospoidiosis in neonatal calves" J Parasitol 75(3)393-7 (1989 June).

Mosier DA et al., "Bovine humoral immune response to Cryptosporidium parvumn" J. Clinical Microbiol 30(12):3277-9 (1992 December).

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 17:59 FAX 613 8618 4199 F.B._RICE Co. 1052 WO 01/45735 PCT/BPOO/13387 S46 4c Sagodira S. et aL, "Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA" 17(19):2346-55 (1999 May 14) o Finch GR et al., "Dose response of Cryptosporidium parvum in outbred neonatal CD-1 mice" Appl Environ Microbiol 59(11):3661-5 (1993 November).

C Jenkins MC et al., "Hyperimmune bovine colostrum specific for recombinant I Cryptosporidium parvum antigen confers partial protection against cryptosporidiosis in o immunosuppressed adult mice" Vaccine 17(19):2453-60 (1999 May).

Ci Avila FA et al., "A comparative study of the efficiency of a pro-biotic and the anti-K99 and Santi-Al4 vaccines in the control of diarrhea in calves in Brazil" Rev Elev Med Vet pays Trop S48(3):239-43 (1995).

Castrucci "Field trial evaluation of an inactivated rotavirus vaccine against neonatoal diarrhea of calves" Eur J Epidemiol 3(1):5-9 (1987 March).

Perryman LE et al., "Immunotherapy of cryptosporidiosis in immunodeficient animal models" J. Protozool 38(6):98S-100S (1991 Nov-Dec).

Yano T. et al., "Determination of the efficiency of K99-F41 fimbrial antigen vaccine in newborn calves" Braz J. Med. Biol. Res. 28(6):651-4 (1995 June).

Kadel WL et al., "Field-trial evaluation of a Pasteurella vaccine in preconditioned and nonpreconditioned lightweight calves" Am. J. Vet Res. 46(9);1944-8 (1985 September).

Kharalambivev KhE et aL, "Attenuated vaccine against rota- and coronavirus enteritis in calves" Vet. Med. Nauki 23(10):26-31 (1986).

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Comp Med 41(2):131-6 (1977 April).

Jeff B- Wilson et al., "A Case-control Study of Selected Pathogens Including Verocytotoxigenic Escherichia coli in Calf Diarrhea on an Ontario Veal Farm" Can J. Res 56:184-188 (1992).

J. De Rycke et al., "Prevalence of Various Enteropathogens in the Feces Of Diarrheic And healthy Calves" Ann. Rech. Vet. 17(2):159-168 (1986).

D.J. Reynolds, et al., "Microbiology of Calf diarrhoea in Southern Britain" Veterinary Record 119:34-39 (1986).

Jorge W. Lopez et al., "Rotavinis and Cryptosporidium Shedding in Dairy Calf Feces and Its Relationship to Colostrum Immune Transfer" J. Dairy Science 71:1288-1294 (1988).

SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 18:00 FAX 613 8618 4199 F.B. RICE Co. 1053 0 47 H. W. Moon, DVM, PhD. et al, "Pathogenic Relationships of Rotavirus, D Escherichia coli, and Other Agents in Mixed Infections in Calves" JAVMA (September S1, 1978).

S. Virigng et al., "Studies of Enteric Pathogens and-Globulin Levels of Neonatal en 5 Calves in Sweden" Acta Vet. Scand. Vol. 34: 271-279 (1993).

I

N R. de la Fuenta et al., "Cryptosporidium and concurrent infections with other 0 major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain" 0 Veterinary Parasitology 80:179-185(1999).

IN F. Biirki et al, "Reduction of Rotavirus-, Coronavirus-and E. coli-Associated 0 10 Calf-Diarrheas in a Large-Size Dairy Herd" J. Vet. Med. B. 33,241-252 (1986).

R. de la Fuente et aL, "Proportional morbidity rates of enteropathogens among diarrheic dairy calves in central Spain" Preventive Veterinary Medicine 36; 145-152 (1998).

Saul Tzipori, "The Relative Importance of Enteric Pathogens Affecting Neonates of Domestic Animals" Advances Veterinary Science and Comparative Medicine, Vol.29.

Kenneth W. Angus, "Cryptosporidiosis in Ruminants" Cryptosporidiosis of Man and Animals, Vol. Robert E. Holland, "Some Infectious Causes of Diarrhea in Young Farm Animals" Clinical Microbiology Reviews, p. 345-375 (1990 October)- Perryman et al, "Neutralization-Sensitive Epitopes of Cryptosporidium parvum" PCT WO 98/07320.

Jenkins et aL, "DNA Sequence Encoding Surface Protein of Cryptosporidium parvum" U. S. Patent Number 5,591,434.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

m:specifications\500000502000\5021 COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06

Claims (27)

1. A combined enteric immunological, immunogenic or vaccine composition sNO comprising a first antigen or epitope of interest from Cryptosporidiwn and/or a first vector that expresses the first antigen or epitope of interest, and a second antigen or epitope of interest from another enteric pathogen and/or the first vector that expresses the first antigen or V0 epitope of interest also expresses the second antigen or epitope of interest and/or a second 0 vector that expresses the second antigen or epitope of interest, and a pharmaceutically 0 acceptable vehicle. o 2. The composition according to claim 1 comprising an antigen from 0 10 Cryptosporidium parvm and an antigen from another enteric pathogen.

3. The composition according to claim 2 comprising an antigen from Cryptosporidium and an antigen from another enteric pathogen of a bovine, canine, feline or equine species.

4. The composition according to any one of claims 1 to 3, wherein the antigen from the enteric pathogen is selected from the group consisting of the antigens from E. coli, rotavirus, coronavirus, Clostridium spp. and mixtures thereof. The composition according to any one of claims 1 to 3, wherein the enteric pathogen comprises E. coli.

6. The composition according to claim 5, wherein the antigen from E. coli comprises an antigen selected from the group consisting of inactivated E. coli bearing K99 antigen, inactivated E. coli. bearing F41 antigen, inactivated E. coli bearing Y antigen, inactivated E. coli bearing 31A.antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, and mixtures thereof.

7. The composition according to claim 6 wherein the E. coli antigen comprises a K99 antigen selected from the group consisting of inactivated E. coli bearing the K99 antigen, K99 antigen, and mixtures thereof; and/or a F41 antigen selected from the group consisting of inactivated E. coli bearing the F41 antigen, F41 antigen, and mixtures thereof.

8. The composition according to any one of claims 1, 2, 3, wherein the enteric pathogen comprises bovine coronavirus.

9. The composition according to any one of claims 1, 2, 3, wherein the enteric pathogen comprises bovine rotavirus. SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 18:00 FAX 613 8618 4199 F.B, RICE_& Co. [a055 WO 01145735 PCT/EP00/13387 O o 49 The composition according to any one of claims 1, 2, 3, wherein the enteric pathogen comprises Clostridiwm perfringens. V. 11. The composition according to claim 10, wherein the antigen of the enteric pathogen comprises Clostridium perfringens type C and/or D toxoids.

12. The composition according to claim 1, 2 or 3, wherein the enteric pathogen \O comprises E coli, bovine rotavirus, bovine coronavirus and Clostridimn perfringens or E. coli, Sbovine rotavirus, bovine coronavirus. 0 C1 13. The composition according to claim 12, wherein the antigen of the enteric \V 0 pathogen comprises E. coli aatigens selected from the group consisting of inactivated E coli 10 bearing K99 antigen, inactivated E. coli. bearing F41 antigen, inactivated E. coli bearing Y antigen, inactivated E. coli bearing 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, and mixtures thereof; inactivated bovine coronavirus; inactivated bovine rotavirus and Clostridiun perfringens type C and/or D toxoids; or E. coli antigens selected from the group consisting of inactivated E coli bearing K99 antigen, inactivated E. coli. bearing F41 antigen, inactivated E. coli bearing Y antigen, inactivated E. coli bearing 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen and mixtures thereof; inactivated bovine coronavirus; and inactivated bovine rotavirus.

14. The composition according to claim 13 wherein the E. coli antigen comprises a K99 antigen selected from the group consisting of inactivated E. coli bearing the 199 antigen, K99 antigen, and mixtures thereof; and/or a F41 antigen selected from the group consisting of inactivated E. coli bearing the F41 antigen, F41 antigen, and mixtures thereof. The composition according to any one of claims 1 to 14, comprising sub-unit Cryptosporidium parvna antigens selected from the group consisting of P21, Cp23, Cp15/60, CP41 and mixtures thereof.

16. The composition according to claim 15, comprising Cp23 and Cpi15/60.

17. The composition according to claim 15, comprising P21 and Cp15/60.

18. The composition according to any one of claims 1 to 17, which comprises an adjuvant

19. The composition according to claim 18, wherein the adjuvant comprises saponin or aluminum hydroxyde or is in the form of an oil-in-water emulsion. SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 18:01 FAX 613 8618 4199 F.B. RICE Co. 0056 WO 01/45735 PCT/EP00/13387 WO 01/45735 o 0 ON ¢ti An immunological, immunogenic or vaccine composition against Cryptosporidiwn parvum, which comprises a first antigen comprising a P21 or Cp23 antigen NO or an epitope thereof or a first vector that expresses the first antigen and a second antigen 0 comprising Cpl5/60 antigen or epitope thereof or the first vector wherein the first vector expresses both the first and second antigens or a second vector that expresses the second \O antigen, and a pharmaceutically acceptable vehicle. 0 21, The composition according to claim 20, wherein P21 or Cp23 and Cpl5/60 0, antigens are in the form of separate fusion proteins. S22. The composition according to claim 20, which comprises a recombinant vector S to expressing P21 and Cpl5/60.

23. The composition according to claim 20, which comprises a recombinant vector expressing P21 and a recombinant vector expressing Cpl5/ 60

24. The composition according to claim 20, which comprises Cp23 and Cp15160. The composition according to any one of claims 20 to 24, which frther 1i comprises an adjuvant.

26- An immunological, immunogenic or vaccine composition against Cryptosporidium parvum, which comprises a first antigen comprising a P21 or Cp23 or Cpl5160 or CP4I antigen or an epitope thereof or a first vector that expresses the first antigen and a second antigen comprising a second antigen or epitope thereof from Cryptosporidium parvum or the first vector wherein the first vector expresses both the first and second antigens or a second vector that expresses the second antigen, wherein the first and second antigens are different from each other, and a pharmaceutically acceptable vehicle.

27. A method of bovine immunization of a new-born calf against enteric disease comprising administering the composition according to any one of claims 1 to 26 to a pregnant cow before calving, so that the new-born calf has maternal antibodies against Cryptosporidian parvuma

28. The method according to claim 27, which comprises further the feeding to the newborn calf colostrum and/or milk from the cow which has been administered the composition during pregnancy.

29. A method of active immunization of adult and new-born bovines, comprising administering to the bovines a composition as claimed in any one of claims 1 to 26. SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 18:01 FAX 613 8618 4199 F.B. RICE Co. I057 WO 01/45735 PCT/EP00/13387 O 51 The method of claim 27 or 28 further comprising administering the composition to the new-born calf.

31- The method of claim 30 wherein the composition administered to the cow O comprises antigens or epitopes thereof and the composition administered to the calf comprises vectors. M 32. A method for preparing a composition according to any one of claims 1 to 26 Scomprising admixing the antigens or epitopes or vectors and the carrier. S33. A kit for preparing a composition according to any one of claims 1 to 26 O comprising the antigens, epitopes or vectors each in separate container or containers, o 10 optionally packaged together; and further optionally with instructions for admixture and/or adminstration.

34. A hyperimmunized colostrum and/or milk composition obtained by administering a composition according to any one of claims 1 to 26 to a pregnant cow and thereafter removing colostrum and/or milk from the cow.

35. The composition of claim 34 wherein the composition comprises concentrated immunoglobulins obtained by coagulation of the colbstrum and/or milk and recovery of immunoglobulins.

36. A method for preventing, treating and/or controlling enteric disease, symptom(s) and/or condition(s) and/or pathogen(s) responsible for such disease, symptom(s) and/or condition(s) and/or C. parvwu comprising administering to a new-bor calf the composition of claim 34 or

37. The method of claim 36 wherein the administering is oral administration.

38. The method of claim 37 wherein the oral administration is by the new-born calf nursing from the cow.

39. A method for preparing a hyperimmunized colostrum and/or milk composition comprising administering a composition according to any one of claims 1 to 26 to a pregnant cow and'thereafter removing colostrum and/or milk from the cow. The method of claim 39 further comprising concentrating immunoglobulins in the milk and/or colostrum obtained from the cow by coagulation of the colostrum and/or milk and recovery of immunoglobulins, whereby the composition comprises said immunoglobulins. SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06 06/03 '06 18:01 FAX 613 8618 4199 F.B. RICE Co. 058 WO 01/45735 PCT/EP00/13387 52

41. A use of a first antigen or epitope from Cryptosporidium and/or a vector that expresses such antigen or epitope, and of a second antigen or epitope from another enteric pathogen and/or a vector that expresses such antigen or epitope, for the preparation of an immunogenic or vaccine composition against enteric infections. SUBSTITUTE SHEET (RULE 26) COMS ID No: SBMI-02877509 Received by IP Australia: Time 18:02 Date 2006-03-06