EP1654000A1 - Compositions et vaccins contenant un(des) antigene(s) de cryptosporidium parvum et d'un autre pathogene - Google Patents

Compositions et vaccins contenant un(des) antigene(s) de cryptosporidium parvum et d'un autre pathogene

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Publication number
EP1654000A1
EP1654000A1 EP04780910A EP04780910A EP1654000A1 EP 1654000 A1 EP1654000 A1 EP 1654000A1 EP 04780910 A EP04780910 A EP 04780910A EP 04780910 A EP04780910 A EP 04780910A EP 1654000 A1 EP1654000 A1 EP 1654000A1
Authority
EP
European Patent Office
Prior art keywords
antigen
composition according
coli
epitope
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04780910A
Other languages
German (de)
English (en)
Inventor
Frederic Raymond Marie David
Francis William Milward
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merial Ltd
Merial LLC
Original Assignee
Merial Ltd
Merial LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merial Ltd, Merial LLC filed Critical Merial Ltd
Publication of EP1654000A1 publication Critical patent/EP1654000A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/12—Immunoglobulins specific features characterized by their source of isolation or production isolated from milk

Definitions

  • the invention relates to antigen(s)/epitope(s) of Cryptosporidium parvum and/or 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 control of Cryptosporidium parvum and/or enteric infections, and uses thereof.
  • the invention further relates 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 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 Cryptosporidium parvum.
  • the invention can also relate to the concurrent use of a monovalent Cryptosporidium parvum vaccine with enteric, e.g. bovine enteric (e.g., rota/coronavirus, E. coli) vaccines and/or use of a combination vaccine containing Cryptosporidium parvum + rota/coronavirus, E.
  • enteric e.g., bovine enteric
  • the immunity induced by vaccination against Cryptosporidium parvum can significantly reduce the severity of the disease induced by herein mentioned enteric pathogens.
  • a combination vaccine containing Cryptosporidium panmm is useful for a more complete prevention of multietiological enteric disease in newborn animals, such as calves, caused by rota and coronaviruses and E. coli K99 and F41.
  • This invention also pertains to the effects of Cryptosporidum parvum co- infection on other enteric, e.g., bovine enteric, pathogens.
  • Cryptosporidium parvum is commonly found in the feces of newborn animals such as mammals, e.g., calves.
  • Cryptosporidium parvum is able to produce clinical signs of enteric disease by itself, regardless of the presence or absence of other potentially pathogenic viruses and bacteria in the gut.
  • Viruses, such as coronavirus, and bacteria, such as E. coli e.g., 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.
  • the invention can relate to addressing the co-infection of cattle with Cryptosporidium parvum as that co-infection can exacerbate the disease caused by other enteric pathogens such as coronavirus, rotavirus, and E. coli e.g., F41.
  • 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 farming 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.
  • Cryptosporidium panmm was the only enteropathogen found in 52.3% of the population, followed by single infections with rotavirus at 42.7%. de la Fuente 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 0.5%, respectively.
  • the most common mixed infection in this study was a combination of Cryptosporidium-rota . irus. There is limited information available on the role of individual enteric pathogens in neonatal calf diarrhea.
  • 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.
  • enteric disease is difficult to control; it is likely multifactoral; Cryptosporidium paryum may be a factor, but heretofore there is no definitive showing that Cryptosporidium paryum indeed enhances enteric disease or that its use in a combination immunogenic, immunological or vaccine composition enhances prevention of enteric disease.
  • 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.
  • combination compositions including Cryptosporidium paryum 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 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 issue of efficacy interference, have not been disclosed or suggested.
  • An object of the invention can be improved enteric immunological or vaccine compositions, especially those which can be used in the veterinary field, for instance for mammals, such as bovines, canines, felines or equines or species thereof.
  • Another object of the invention can be such immunological or vaccine compositions which can be effectively used to immunize newborn and/or young animals, such as to passively immunize new-born animals, e.g., mammals, for instance, bovines, canines, felines or equines or species thereof; advantageously bovines.
  • Still another object of the invention can be improved immunological or vaccine compositions against Cryptosporidium paryum, for instance particular to be used in the veterinary field, such as for use with mammals, e.g., for canines, felines or equines or species thereof, especially bovines or species thereof.
  • Yet another object of the invention can be improved methods for immunizing newborns and/or young animals, such as to passively immunize newborn animals, e.g., mammals, such as canines, felines or equines or species thereof especially bovines or species thereof.
  • objects of the invention can involve methods for eliciting an immune response against Cryptosporidium parvum or enteric pathogens including Cryptosporidium paryum or for controlling, preventing and/or treating enteric infections and/or symptoms including Cryptosporidium paryum; 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, mter alia.
  • Vaccination or immunization against enteric pathogens is greatly and unexpectedly improved by using an immunological or vaccine composition including a combination of at least two Cryptosporidium parvum antigens or epitopes thereof and/or vector(s) expressing at least two Cryptosporidium paryum antigens or epitopes thereof, e.g., P21 or an eptitope thereof and/or a vector expressing P21 or an eptitope thereof or Cp23 or an epitope thereof and/or a vector expressing Cp23 or an epitope thereof and Cp 15/60 or an epitope thereof and/or a vector expressing Cp 15/60 (for instance, a composition containing at least one epitope of Cp23 and at least one epitope of Cpl5/60; and it is noted that the Cp23 antigen or protein can include P21).
  • antigens or epitope(s) of interest and/or vectors expressing the antigens and/or epitope(s)
  • an immune response e.g., antibodies, cellular responses or both
  • Cryptosporidium paryum e.g., antibodies, cellular responses or both
  • enteric infection or pathogens or symptoms e.g., antibodies, cellular responses or both
  • This also allows for the preparation of efficient immunological or vaccine compositions, useful to protect newborn or young animals or mammals, for instance, canines, felines or equines or species thereof; especially bovines.
  • compositions containing antigens and/or epitope(s) of interest may be advantageously employed in inoculating dams or pregnant females, e.g., to elicit an immune response that can be passed to the yet born offspring and to new- bora or young animals via milk or colostrum during weaning
  • compositions containing vector(s) expressing antigens and/or epitope(s) may advantageously be employed in inoculating males and females of all ages, e.g., such as those that are not pregnant and/or are new-born or young animals, and the inoculation of new-born or young animals can be done alone or advantageously in conjunction with the inoculation of dams or pregnant females, e.g., 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.
  • antigen(s) and/or epitope(s) of interest against Cryptosporidium parvum 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), e.g., enteric pathogens) can significantly increase protection against enteric pathologies.
  • An especially advantageous inventive immunological or vaccine composition can be against Cryptosporidium paryum and can comprise (i) 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 Cp 15/60 antigen or epitope of interest thereof and/or at least one vector expressing at least one Cp 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 additional antigen (which can be the same vector that expresses the Cp23 or P21 antigen or epitope of interest and/or the Cp 15/60 antigen or epitope of interest, e.g., the composition can comprise a vector that co-expresses the Cp23 or P21 anti
  • 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 Cryptosporidium panmm as described herein such as Cp23, P21 and Cpl5/60, e.g. in combination with Cp23 or P21 and/or Cpl5/60.
  • 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 Cp 15/60 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 Cryptosporidium paryum; for instance, comprising administering an inventive composition.
  • 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, once or twice during the typical gestation period (for a cow, typically a 9 month or 170 day gestation period), such as a first administration about 1 to about 2.5 or about 3 months before calving and a second or sole administration close to calving, e.g., in the last 3 weeks before calving, preferably about 3 to about 15 days before calving.
  • the female can transfer passive immunity to the newborn, e.g., calves after birth via milk or colostrum.
  • compositions comprising antigen(s) and/or epitope(s) of interest are administered to pregnant mammals as eliciting an antibody response is desired.
  • 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 (e.g., 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.
  • a newborn or very young mammal e.g., 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
  • 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 (e.g., inactivated E.
  • coli bearing pili such as, K99, Y, 31 A, and/or F4 land/or these pili in subunit form or recombinantly expressed in vivo
  • one or more rotavirus antigens e.g., advantageously inactivated rotavirus
  • one or more coronavirus antigen e.g., bovine coronavirus antigen, advantageously such as inactivated coronavirus
  • Cryptosporidium paryum antigens such as P21 and/or Cp23 and/or Cp 15/60.
  • a particular inventive composition can comprise (i) one or more Cryptosporidium panmm antigens, such as P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and Cpl5/60, and (ii) at least one E. coli antigen (e.g., at least one or all of of K99, Y, 31 A, F41 and/or other pili borne by inactivated E.
  • Cryptosporidium panmm antigens such as P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and Cpl5/60
  • E. coli antigen e.g., at least one or all of of K99, Y, 31 A, F41 and/or other pili borne by inactivated E.
  • coli or as subunits or as expressed in vivo 99 and/or F41 are preferably present and Y and/or 31 A are advantageously also present) , and/or coronavirus and/or rotavirus antigen; such as one or more C. parvum antigens, such as P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and Cp 15/60 and one or more rotavirus antigen such as inactivated rotavirus, or one or more C.
  • C. parvum antigens such as P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and Cp 15/60 and one or more rotavirus antigen such as inactivated rotavirus, or one or more C.
  • panmm antigens such as P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and Cp 15/60 and one or more coronavirus antigen such as inactivated coronavirus, e.g., inactivated bovine coronavirus, or one or more C. paryum antigens, such as P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and Cp 15/60 and one or more E. coli antigen such as K99, Y, 31 A, F41 and/or other pili borne by inactivated E.
  • coronavirus antigen such as inactivated coronavirus, e.g., inactivated bovine coronavirus
  • C. paryum antigens such as P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and C
  • 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. panmm antigens, such as P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and Cp 15/60 and at least one E.
  • coli antigen and at least one coronavirus antigen, and at least one rotavirus antigen, e.g., P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and Cp 15/60 and inactivated rotavirus, and inactivated coronavirus, and at least one E coli antigen, advantageously pili or preferably at least one or more of K99, Y, 31 A, and F41, or a combination of K99, Y, 31A and F41.
  • rotavirus antigen e.g., P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23 and Cp 15/60 and inactivated rotavirus, and inactivated coronavirus
  • E coli antigen advantageously pili or preferably at least one or more of K99, Y, 31 A, and F41, or a combination of K99
  • 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.
  • potential efficacy interference by single or multiple bacteria, 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.
  • a single dose can have the E. coli antigen (or each E. coli antigen, in the case of multiple E.
  • the rotavirus antigen can be present in an typically found in vaccines against enteric pathogens, such as an amount to obtain a serum titre in guinea pigs of at least 2.0 log 10
  • the coranovirus antigen can be present in an amount typically found in vaccines against enteric pathogens such as an amount to obtain a serum titre in guinea pigs of at least 1.5 log 10
  • the inventive compositions can include an adjuvant, such as aluminum hydroxide, which can be present in a single dose in an amount typically found in vaccines such as preferably an amount of about 0.7 to about 0.9 mg.
  • the invention provides combined enteric immunological, immunogenic or vaccine composition
  • the composition can comprise antigen, which can be from Cryptosporidium paryum and an antigen from another enteric pathogen.
  • the composition can comprise an antigen from Cryptosporidium 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.
  • the enteric pathogen can comprise bovine coronavirus; and/or bovine rotavirus and/or Clostridium perfringens.
  • the antigen of the enteric pathogen can comprise Clostridium perfringens type C and D toxoids.
  • the enteric pathogen can comprises E. coli, bovine rotavirus, bovine coronavirus and Clostridium perfringen or E. coli, bovine rotavirus, bovine coronavirus.
  • the invention can comprise a composition wherein the antigen of the enteric pathogen comprises E.
  • 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, K99 antigen, F41 antigen, Y antigen, 31A antigen, and mixtures thereof; 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 31 A antigen, K99 antigen, F41 antigen, Y antigen, 31 A antigen and mixtures thereof; inactivated bovine coronavirus; and inactivated bovine rotavirus.
  • the inventive composition advantageously can comprise sub-unit
  • Cryptosporidium parvum antigens selected from the group consisting of P21, Cp23, Cp 15/60, CP41 and mixtures thereof, such as Cp23 and Cp 15/60 or P21 and Cpl5/60.
  • the Cryptosporidium paryum antigen may also comprise or be constituted by, inactivated or live attenuated oocysts, or ' sub-units obtained from oocysts.
  • inventive compositions can include an adjuvant such as saponin or aluminum hydroxide; 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 Cp 15/60 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 Cp 15/60 antigens which are in the form of separate fusion proteins.
  • the composition can comprise a vector expressing Cp23 and Cp 15/60.
  • the composition can comprise a first recombinant vector expressing Cp23 and a second recombinant vector expressing Cpl5/60. And, the composition can comprise P21 and Cpl5/60. These compositions can further comprise an adjuvant.
  • an immunological, immunogenic or vaccine composition against Cryptosporidium parvum 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 antigen or epitope thereof from Cryptosporidium paryum 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.
  • the invention also comprehends a method of bovine immunization of a newborn calf against enteric disease comprising administering an inventive composition to a pregnant female calf before delivering, so that the newborn calf receives maternal antibodies against Cryptosporidium parvum through colostrum and/or milk.
  • the method can further comprise 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 composition to the newborn calf.
  • the composition administered to the pregnant female can comprise antigens or epitopes thereof and the composition administered to the calf can comprise vectors.
  • the invention also envisions a method of active immunization of adult and newborn calves, comprising administering to the calves an inventive composition.
  • the invention also comprehends a method of bovine immunization of a newborn calf, comprising feeding to the newborn calf colostrum and/or milk from cows that have been administered the composition during pregnancy.
  • 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.
  • the invention can encompass a method for preparing an inventive composition comprising admixing the antigens or epitopes or vectors and the carrier.
  • 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 paryum 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.
  • Figure 1 shows a physical and restriction map of plasmid pJCA155
  • Figure 2 shows a physical and restriction map of plasmid pJCA156
  • Figure 3 shows a physical and restriction map of plasmid pJCA157
  • Figure 4 shows a physical and restriction map of plasmid pJCAl58
  • Figure 5 shows a physical and restriction map of plasmid pJCA159;
  • Figure 6 shows a physical and restriction map of plasmid pJCA160
  • Figure 7 shows comparative oocysts count in feces in calves challenged with either
  • 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
  • Figure 12 shows comparative anorexia status for calves according to example 12.
  • Figure 13 shows comparative rectal temperature evolution in calves according to example 12.
  • Figure 14 depicts average P21 (P21) colostrum antibody levels per vaccine group.
  • Figure 15 shows the average CP 15/60 colostrum antibody levels per vaccine group.
  • Figure 16 shows the average P21 (P21) serum antibody levels per vaccine group.
  • Figure 17 depicts average CP 15/60 antibody levels per vaccine group.
  • Figure 18 depicts the hematocrit levels comparing challenged and unchallenged animals.
  • Figure 19 illustrates the daily differences in % fecal dry matter by group and by daily collection time points.
  • Figure 20 is a graph showing the results of a P21 indirect ELISA antibody-detection assay.
  • Figure 21 shows the results from a CP 15/60 ELISA antibody detection assay.
  • Figure 22 is a score chart depicting overall sickness of animals for all vaccines over time.
  • Figure 23 is a chart depicting the overall sickness of animals for the GST- 15/60 and placebo vaccines only.
  • Figure 24 is a cloud diagram showing the diarrhea score for all vaccines.
  • Figure 25 is a cloud diagram showing the anorexia score for all vaccines.
  • Figure 26 is a cloud diagram showing the depression score for all vaccines.
  • Figure 27 is a cloud diagram showing the fecal dry matter for all vaccines.
  • Figure 28 depicts oocyst shedding for all vaccines used in this study.
  • Figure 29 is a graph showing the mean evolution of rectal temperatures.
  • Figure 30 shows the average local reaction to the first vaccination (crypto + combo; combo alone).
  • Figure 31 shows the average local reaction to the second vaccination (crypto + combo; combo alone).
  • Figure 32 is a graph showing the mean ELISA CP 15/60 antibody titers.
  • Figure 33 shows the ELISA antibody titers to bovine coronavirus.
  • Figure 34 shows the virus neutralizing antibody titers to bovine coronavirus.
  • Figure 35 illustrates the ELISA antibody titers to bovine rotavirus.
  • Figure 36 illustrates the virus neutralizing antibody titers to bovine rotavirus.
  • Figure 37 depicts the ELISA antibody titers to E. coli K99 antigen.
  • Figure 38 depicts the ELISA antibody titers to E. coli F41 antigen.
  • An aspect of the invention is thus a combined enteric immunological, immunogenic or vaccine composition
  • Universal immunological, immunogenic or vaccine compositions are also envisioned as enteric pathogens are often infecting several (more than one) animal species.
  • An immunological composition elicits an immunological response - local or systemic.
  • An immunogenic composition likewise elicits a local or systemic immunological response.
  • a vaccine composition elicits a local or systemic protective response.
  • the terms "immunological composition” and “immunogenic composition” include a "vaccine composition” (as the two former terms can be protective compositions).
  • Cryptosporidium paryum antigens which can be used in this invention comprise preferably: (1) A protein of 148 amino acids called Cpl5/60 (See, e.g., 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 5' 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 Cp 15/60 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 (2) Cp23 and/or P21.
  • P21 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, such as, Met-, or Met-Gly- or similar amino acids.
  • an antigen comprising or consisting essentially of or consisting of the 187 amino acid sequence or a longer amino acid sequence, or a polypeptide comprising a fragment of the 187 amino acid sequence that comprises an epitope thereof, advantageously a protection-eliciting epitope or an epitope that has the immunogenicity of the full length sequence.
  • the whole amino acid sequence of Cp23 and the 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 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.
  • the antigens or epitopes of interest can be used individually or in combination in compositions of the invention, e.g., an inventive composition can include (1) or (2) or both (1) and (2).
  • an inventive composition can include (1) or (2) or both (1) and (2).
  • 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.
  • Host cells can be produced by chemical synthesis or by expression in vitro.
  • the examples describe how to obtain the sequences encoding Cp 15/60 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 Cp 15/60, 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 (E.
  • 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.
  • nucleic acid sequences useful for expressing the C are also known.
  • paryum 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 (e.g., 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 example, Sambrook et al., “Molecular Cloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor, 1989; “Nucleic Acid Hybridisation, A Practical Approach”, Hames and Higgins eds., IRL Press, Oxford, 1985; both incorporated herein by reference.
  • nucleic acid molecules and polypeptides which can be used in the practice of the invention, the nucleic acid molecules and polypeptides advantageously have at least about 75% or greater homology or identity, advantageously 80% or greater homology or identity, more advantageously 85% or greater homology or identity, such as at least about 85% or about 86% or about 87% or about 88% or about 89% homology or identity, for instance at least about 90% or homology or identity or greater, such as at least about 91%, or about 92%, or about 93%o, or about 94% identity or homology, more advantageously at least about 95% to 99% homology or identity or greater, such as at least about 95% homology or identity or greater e.g., 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
  • 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 Miller, ("Optimal Alignments in Linear Space", CABIOS 4, 11-17, 1988, incorporated herein by reference) and available at NCBI.
  • 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 (N re/ - Nr f , / )*100 N re/ , wherein N ⁇ is the total number of non-identical residues in the two sequences when aligned and wherein N,- e / is the number of residues in one of the sequences.
  • homology or “identity” with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the 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 (Wilbur and Lipman, 1983 PNAS USA 80:726, incorporated herein by reference), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer- assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics TM Suite, Intelligenetics Inc. CA).
  • RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
  • RNA sequences within the scope of the invention can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.
  • 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
  • nucleic acid molecules used in this invention e.g., as in herein cited documents
  • the invention comprehends the use of codon equivalent nucleic acid molecules.
  • X protein
  • the invention comprehends "X" protein (e.g., P21 and/or Cp23 and/or Cpl5/60 and/or CP41) having amino acid sequence "A” and encoded by nucleic acid molecule "N”
  • the invention comprehends nucleic acid molecules that also encode protein X via one or more different codons than in nucleic acid molecule N.
  • the antigen or epitope of interest used in the practice of the invention can be obtained from the particular pathogen(s), e.g., C.
  • parvum E. coli, rotovirus, coronavirus, and the like or can be obtained from in vitro 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.
  • 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).
  • these antigens are preferably under the form of fusion proteins (e.g, a HIS tag).
  • 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, mter 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 precipitation and selective denaturation; column chromatography, including high performance liquid chromatography (HPLC), ion-exchange, affinity, immunoaffinity or dye-ligand chromatography; immunoprecipitation and the use of gel filtration, electrophoretic methods, ultraf ⁇ ltration and isoelectric focusing, inter alia.
  • HPLC high performance liquid chromatography
  • the invention comprehends that the antigens and/or epitopes of interest are not incorporated as subunits in the composition, but rather that they are expressed in vivo; e.g, the invention comprehends that the composition comprises recombinant vector(s) expressing the antigens in vivo when administered to the animal.
  • the vector can comprise a DNA vector plasmid, a herpesvirus, an adenovirus, a 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, e.g, Cpl5/60 and/or Cp23 for Cryptosporidium parvum.
  • the word plasmid is intended to include any DNA transcription unit in the form of a polynucleotide sequence comprising the sequence to be expressed.
  • 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, e.g, cationic liposomes (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 allows for expression in vivo, and advantageously an immunological or protective response.
  • a gene gun or intramuscularly, or nasally, or by any other means that allows for expression in vivo, and advantageously an immunological or protective response.
  • 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.
  • compositions can be administered in dosages and by techniques well known to those skilled in the veterinary arts taking into consideration such factors as the age, sex, weight, condition and particular treatment of the animal, and the route of administration.
  • the components of the inventive compositions can be administered alone, or can be co-administered or sequentially administered with other compositions (e.g, the C.
  • panmm antigen(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 recombinants or plasmids that also express enteric antigen(s) or epitope(s) of the same or different pathogens) or with other prophylactic or therapeutic compositions (e.g, other immunogenic, immunological or vaccine compositions).
  • the invention provides multivalent or "cocktail" or combination compositions and methods employing them.
  • ingredients and manner can be •determined, taking into consideration such factors as the age, sex, weight, condition and particular treatment of the animal, e.g, cow, and, the route of administration.
  • compositions of the invention may be used for parenteral or mucosal administration, preferably by intradermal, subcutaneous or intramuscular routes.
  • mucosal administration it is possible to use oral, nasal, or vaginal routes.
  • 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 lyophilized.
  • compositions can contain auxiliary substances such as pH buffering agents, adjuvants, preservatives, polymer excipients used for mucosal routes, and the like, depending upon the route of administration and the preparation desired.
  • auxiliary substances such as pH buffering agents, adjuvants, preservatives, polymer excipients used for mucosal routes, and the like, depending upon the route of administration and the preparation desired.
  • Standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. Suitable dosages can also be based upon the text herein and documents cited herein.
  • Adjuvants are substances that enhance the immune response to antigens.
  • Adjuvants can include aluminum hydroxide and aluminum phosphate, saponins e.g, Quil A, mineral oil emulsions, pluronic polymers with mineral or metabolizable oil emulsion, the water-in-oil 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), e.g, biodegradable microspheres), nonionic block copolymers, low molecular weight copolymers in oil-based emulsions (see Hunter et al.
  • saponins e.g, Quil A, mineral oil emulsions, pluronic polymers with mineral or metabolizable oil emulsion
  • the water-in-oil adjuvant e.g, mineral oil emulsion
  • GM-CSF from the animal species to be vaccinated, mter alia.
  • Certain adjuvants can be expressed in vivo with antigen(s) and/or epitope(s); e.g, cytokines, GM-CSF (see, e.g., 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 antigen and/or epitope of another bovine pathogen, or can be used in conjunction with such a plasmid)
  • an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers 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.
  • the polymers of acrylic or methaciylic acid and the copolymers EMA® are preferably formed of basic units of the following formula:
  • 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 immunogenic, immunological or vaccine composition itself will be incorporated.
  • the carboxyl groups of the polymer are then partly in COO " form.
  • a solution of adjuvant according to the invention is prepared in distilled water, preferably in the presence of sodium chloride, the solution 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 with NaCl, preferably physiological saline (NaCl 9 g/1) all at once in several portions with concomitant or subsequent neutralization (pH 13 to 7.4), preferably with NaOH.
  • This solution at physiological pH will be used as it is for mixing with the vaccine, which may be especially stored in freeze-dried, liquid or frozen form.
  • the polymer concentration in the final vaccine composition can be 0.01% to
  • 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 pi 83 in the same book.
  • the oil-in-water emulsion may be based on light liquid paraffin 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 / caprate), glycerol tri(caprylate / caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular esters of isostearic acid.
  • light liquid paraffin oil accordinging 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
  • 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, block-copolymers such as polyoxypropylene-polyoxyethylene, in particular the products called Pluronic, namely Pluronic L121.
  • Pluronic namely Pluronic L121.
  • 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 recombinant vaccine (e.g. poxvirus as vector or DNA plasmid) expressing at least one immunogen, antigen or epitope of interest from another pathogen.
  • Compositions in forms for various administration routes are envisioned by the invention. And again, the effective dosage and route of administration are determined by known factors, such as age, weight. Dosages of each active agent e.g, of each C.
  • paryum antigen or epitope of interest and/or of each antigen or epitope from each enteric pathogen can be as in herein cited documents or as otherwise mentioned herein and/or can range from one or a few to a few hundred or thousand micrograms, e.g, 1 ⁇ g to lmg, for a subunit immunogenic, immunological or vaccine composition; and, 10 4 to 10 10 TCID 50 advantageously 10 6 to 10 8 TCID 50 , 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 and/or in herein cited documents.
  • 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 10 4 pfu to about 10 10 pfu, e.g, about 10 5 pfu to about 10 9 pfu, for instance about 10 6 pfu to about 10 8 pfu, with doses generally ranging from about 10 6 to about 10 10 , preferably about 10 10 pfu/dose, and advantageously about 10 pfu per dose of about 1 ml to about 5 ml, advantageously about 2 ml.
  • 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.
  • dosages can be as described in documents cited herein or as described herein.
  • 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.
  • suitable quantities of each plasmid DNA in plasmid compositions can be 1 ⁇ g to 2 mg, preferably 50 ⁇ g to lmg.
  • Documents cited herein regarding DNA 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.
  • the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable immunological response can be determined by methods such as by antibody titrations of sera, e.g, by ELISA and/or seroneutralization and/or seroprotection assay analysis.
  • the combined enteric immunological, immunogenic or vaccine composition comprises both Cryptosporidium parvum antigens as defined above. Antigens or epitopes of enteric pathogens advantageously combined with
  • Cryptosporidium antigen(s) or epitope(s) comprise preferably one or more antigen or epitope of interest from E. coli, and/or rotavirus, and/or coronavirus, and/or Clostridium spp., such as CI. perfringens; for instance, at least one antigen or epitope of interest from E. coli, rotavirus, and coronavirus.
  • 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. coli bearing Y antigen, E.
  • E. coli bearing 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen and any mixtures thereof.
  • E. coli may be used to produce Cryptosporidium parvum antigens or epitopes.
  • the Cryptosporidium parvum 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 parvum antigens is performed. For in vitro expression, the cells may then be disrupted as usual and the E.
  • tags e.g. HIS tags.
  • the nucleic acid molecules encoding the antigens or epitopes are linked to a signal sequence so that there is extracellular expression of the antigens or epitopes; and, advantageously, the E. coli is non-pathogenic.
  • E. coli can, in certain embodiments, be the vector and the antigen or epitope of interest.
  • Antigens from Clostridium perfringens are preferably type C and/or D toxoids, more 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 panmm, advantageously P21 and/or Cp23 and/or Cp 15/60 and/or CP41 such as P21 or Cp23 and Cp 15/60 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.
  • the invention provides a bovine enteric immunological, immunogenic or vaccine composition comprising E. coli antigens as discussed herein such as antigens K99, F41, Y and 31 A, as well as inactivated bovine coronavirus, inactivated bovine rotavirus.
  • This composition can further include Clostridium perfringens type C and D toxoids.
  • the E. coli valency comprises either inactivated E. coli bearing K99 antigen, inactivated E. coli. bearing F41 antigen, inactivated E. coli bearing Y antigen and inactivated E. coli bearing 31A antigen, or, K99 antigen, F41 antigen, Y antigen and 31A antigen.
  • Another aspect of the present invention is an immunological, immunogenic or vaccine composition against Cryptosporidium parvum, which comprises Cp23 or P21 and Cp 15/60 antigens or epitopes thereof, and a pharmaceutically acceptable vehicle.
  • these antigens are incorporated in the composition as proteins or sub-unit antigens. They can be produced by chemical synthesis or by expression in vitro.
  • these antigens are preferably under the form of fusion protein (e.g, with HIS tag).
  • the antigen can comprise the antigen er se and foreign amino acids.
  • these antigens are not incorporated as subunits in the composition, but the composition comprises either a recombinant vector expressing Cp23 or P21 and Cp 15/60 or an epitope thereof or a recombinant vector expressing Cp23 or P21 or an epitope thereof and a recombinant vector expressing Cp 15/60 or an epitope thereof, wherein these vectors express the antigen(s) or epitope(s) in vivo when administered to the animal.
  • composition can contain an antigen or 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
  • the invention can concern a method of immunization of a newborn calf against enteric disease, comprising administering an immunological or vaccine composition comprising Cp23 or P21 and Cp 15/60 Cryptosporidium paryum 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 panmm.
  • 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.
  • compositions and methods 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.
  • 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 methods in US-A-5,591,434 and WO-A-9807320.
  • antigens of other enteric pathogens from commercially available sources, such as TRIVACTON®6; for instance, Cp23 and/or P21 and/or Cp 15/60 or an epitope thereof, e.g, P21 or Cp23 and Cp 15/60 or an epitope thereof, or a vector expressing these antigen(s) or epitope(s) can be added to TRIVACTON®6, in herein specified amounts.
  • Clostridium perfringens toxoids C and D may advantageously be added to TRIVACTON®6.
  • the inactivated E. coli bearing pili may be replaced in TRIVACTON®6 by the isolated pili.
  • Such a vaccine, 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.
  • 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.
  • E. coli valency and/or antigen(s) and/or epitope(s) useful in the practice of the invention, reference is made to ⁇ P-A-80,412, EP-A- 60,129, GB-A-2,094,314, and U.S. Patents Nos.
  • Fusion plasmids e.g, that express the antigen(s) or epitope(s) with a tag such as a His tag
  • Fusion plasmids are preferred as they allow one to recover easily the produced antigen. Suitable plasmids are described in the examples. Production of antigens by chemical synthesis is also within the scope of the invention.
  • the invention further comprehends methods for using herein discussed antigens or epitopes or vectors expressing such antigens or epitopes for the preparation of a vaccine, immunological, or immunogenic composition, e.g, against C.
  • 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 ingredients 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) (e.g., C. paryum composition or combined enteric composition according to the invention).
  • inventive composition(s) e.g., C. paryum composition or combined enteric composition according to the invention.
  • 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, e.g, 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 to prevent or treat C. paryum and/or enteric infection in a young animal, such as a newborn; for instance, a calf.
  • the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
  • oocysts are then stored in distilled water at +4°C.
  • 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. paryum DNA is constituted by the EcoRI genomic libraries for the Cryptosporidium paryum Iowa (A), Iowa (I), KSU-1 and KSU-2 isolates available from the American Tissue Culture Collection (ATCC numbers 87667, 87668, 87439 and 87664 respectively).
  • the specific P21and Cp 15/60 genes are isolated as follows: The sequence encoding the P21 protein is amplified by a polymerase chain reaction (PCR) using C. parvum DNA and the following primers: oligonucleotide JCA295 (35 mer) SEQ ID NO: 1 5' TTT TTT CCA TGG GGC TCG AGT TTT CGC TTG TGT TG 3' and oligonucleotide JCA296 (33 mer) SEQ ID NO: 2 5' TTT TTT GAA TTC TTA GGC ATC AGC TGG CTT GTC 3' This PCR generates a fragment of about 585 bp PCR fragment.
  • PCR polymerase chain reaction
  • 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 Cp 15/60 protein and to add convenient restriction sites in 5' and 3' for further cloning. The PCR is done using C.
  • oligonucleotide JCA297 (35 mer) SEQ ID NO: 3 5 ' TTT TTT CTC GAG ATG GGT AAC TTG AAA TCC TGT TG 3 ' and oligonucleotide JCA298 (42 mer) SEQ ID NO: 4
  • This PCR generates a fragment of about 465 bp.
  • This fragment is purified and then digested with Xhol and EcoRI in order to get, after agarose gel electrophoresis and recovery with the GeneClean kit (BIOIOI Inc.), the 453 bp
  • 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
  • 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 plasmid 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-Pharmacia Biotech) and the following primers: oligonucleotide JCA299 (35 mer) SEQ ID NO: 5 5' TTT TTT CCA TGG GGT CCC CTA TAC TAG GTT ATT GG 3' and oligonucleotide JCA300 (45 mer) SEQ ID NO: 6
  • This PCR generates a fragment of about 720 bp encoding the GST moiety with the addition of a Ncol restriction site at the 5' end for cloning purposes into pBAD/HisA; this modification adds a Glycine codon to the GST-P21 fusion protein).
  • the second PCR is done using C. parvum DNA and the following primers: oligonucleotide JCA301 (33 mer) SEQ ID NO: 7
  • Example 3 Construction of plasmid pJCA156 (His6-P21 fusion protein in vector pBAD/HisA)
  • a PCR is done to amplify the sequence encoding the His6-P21 fusion and to add the Ncol 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 DNA and the following primers: oligonucleotide JCA302 (65 mer) SEQ ID NO: 8
  • Example 4 Construction of plasmid pJCA157 (P21 protein alone in pBAD/HisA vector)
  • the pBAD/HisA vector Cat # V430-01, InVitrogen Corp.
  • a PCR is done to amplify the sequence encoding the P21 protein and to add the Ncol 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.
  • Example 5 Construction of plasmid pJCA158 (GST-Cp 15/60 fusion protein in pBAD HisA vector) A PCR is done to amplify the sequence encoding the GST protein and to add convenient restriction sites in 5' and 3' in order to subclone the PCR fragment into the final pBAD/HisA plasmid vector.
  • Fragments C, E and B 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-Cp 15/60 fusion protein.
  • Example 6 Construction of plasmid pJCA159 (His6-Cpl5/60 fusion protein in pBAD/HisA vector) The pBAD/HisA vector (Cat # V430-01, InVitrogen Corp.) is digested with
  • a PCR is run to amplify the sequence encoding the His6-Cp 15/60 fusion and to add convenient restriction sites in 5' and 3' in order to subclone this PCR fragment into the pB AD/HisA plasmid vector.
  • the PCR is done using either C. panmm DNA and the following primers: oligonucleotide JCA303 (64 mer) SEQ ID NO: 9 5' TTT TTT CCA TGG GGG GTT CTC ATC ATC ATC ATC ATC ATC ATG GTA
  • a PCR is run to amplify the sequence encoding the Cp 15/60 protein 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 C. parvum DNA and the following primers: oligonucleotide JCA304 (31 mer) SEQ ID NO: 10
  • Example 8 Culture of E. coli recombinant clones and induction of recombinant proteins Plasmid DNA (Examples 2 to 7) is transformed into Escherichia coli DH5 ⁇ (or any other suitable E. coli K12 strain well known to those skilled in the art, such as E. coli TOP 10 (Cat # C4040-03 InVitrogen Corp.)) and grown on Luria-Bertani (LB) medium agar plates with 50 ⁇ 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) for overnight growth.
  • Escherichia coli DH5 ⁇ or any other suitable E. coli K12 strain well known to those skilled in the art, such as E. coli TOP 10 (Cat # C4040-03 InVitrogen Corp.)
  • LB Luria-Bertani
  • Example 9 Extraction and purification of the recombinant fusion proteins
  • cells are harvested by centrifugation (3000 g, 10 minutes, +4°C) and resuspended in lysis buffer (50 mM Tris pH 8.0, 1 mM EDTA, 1 ⁇ M PMSF, 1 mg/ml lysozyme) and sonicated 25 times for 30 seconds bursts with 1 -minute pauses between bursts.
  • Triton X-100 is added to a final concentration of 0.1%. Debris is removed by centrifugation. If necessary, alternative techniques (known to those of skill in the art) may be used for the lysis of bacterial cells. 9J .
  • GST-fusion recombinant 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
  • His6-fusion recombinant proteins Recombinant His6-fusion proteins have all been prepared and purified using the ProBondTM 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 pJCAl 59) are harvested by centrifugation (3000 g for 5 minutes).
  • the pellet is resuspended in 200 ml of Native Binding Buffer (20 mM phosphate, 500 mM NaCl, pH 7.8).
  • the resuspended pellet is then incubated with egg lysozyme at a final concentration of 100 ⁇ g/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 completing the lysis and the insoluble debris are finally removed by centrifugation at 3000 g for 15 minutes.
  • the lysate is cleared by passage through a 0.8 ⁇ m filter and stored on ice or at -20°C until purification.
  • the soluble recombinant His6-fusion protein present in the clear lysate is batch bound to a 50 ml pre-equilibrated ProBondTM 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. XpressTM System Protein Purification - A Manual of Methods for Purification of Polyhistidine - Containing Recombinant Proteins. InVitrogen Corp. Editor. Version D. 1998) :
  • Example 10 Extraction and purification of the C. panmm P21 and Cpl5 recombinant non-fusion proteins
  • the bacterial cells of E. coli transformed with plasmids pJCA157 or pJCA160
  • the bacterial cells of E. coli are 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 nd Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York. 1989) at 30°C until OD 60 onm 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-MBO 1 (or model UFP- 10-C- MB01 or model UFP-10-E-MBOl).
  • the concentrated samples are then stored at - 70°C until use.
  • the specific C. panmm 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 immunization and challenge experiment in newborn calves Product (adjuvanted or not) is administered intramuscular (IM), subcutaneous (SQ) or intradermal (ID) to elicit serum antibody responses against C. paryum.
  • IM intramuscular
  • SQ subcutaneous
  • ID intradermal
  • Vaccination protocol for pregnant animals can comprise 2 doses given between when pregnancy is diagnosed and calving, such as about 1 month before calving and about 3 to 5 days before calving; or, 2 months prior to calving (which coincides with dry-off in dairy cows) and a boost prior to calving (e.g, anywhere from 3 weeks to 1 week prior to calving), depending on management practices (however, these schedules favor maximum efficacy) .
  • Current management practices favor that are products 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 to injection.
  • the Cryptosporidium antigen can be added as a component of an E. co/t/Rota/Corona combination vaccine.
  • Study A C. paryum enhances the pathogenicity of enteric virus and/or bacteria
  • Combo rota and coronavirus, E. coli K99 and F41), 8 animals;
  • Example 12 Effect of dual infection with C. paryum and bovine rotavirus 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. paryum or bovine rotavirus and after a dual challenge with bovine rotavirus plus C. parvum.
  • Four groups of six calves are used in order to yield sufficient data to be able to detect differences in incidence of clinical signs between groups.
  • Cows are individually housed in pens or paddocks. Newborn calves are separated from their dams as soon as possible after birth, inspected to eliminate feces or dirt on the calf and their ombilical cord dipped in approximate 7% iodine solution. They are then immediately transferred to containment accomodations and housed individually in metabolic crates. Calves are challenged within 6 hours after birth.
  • Calves are fed 1 to 2 quarts per feeding or at 10% body weight, twice daily for the entire trial using a commercial calf milk replacer with 30% colostrum substitute. Special care will be given to avoid the administration of milk within 2 hours pre or post challenge.
  • 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 ⁇ C. paryum oocysts (strain Beltsville), diluted in 60 ml of commercial antibiotics free soy milk.
  • Group C Coinoculation of 1-3x10 ⁇ C.
  • parvum oocysts (strain Beltsville), diluted in 60 ml of commercial antibiotics free soy milk, and of 10 ml bovine rotavirus inoculum (strain END BRV G6P5) diluted in 40 ml PBS.
  • Group D 10 ml fecal filtrate from bovine rotavirus infected calves (strain IND BRV G6P5) 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.
  • Anorexia is determined based on whether the calf nurses less than 2 liters of milk. During the 1 st 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. Rectal temperature is recorded in degrees Fahrenheit. 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. A synergistic effect on clinical signs and microorganisms excretion in feces is observed when both microorganisms are administered compare to single administrations.
  • Example 13 Production of Bovine Colostrum Containing Antibodies to the E. coli expressed C. paryum Subunit Proteins C7 (P21) and/or CP15/60
  • Pregnant dairy cows from 4 different herds were randomly assigned to one of 6 vaccinate groups: GST-P21; 6His-P21; GST-CP15/60; 6His-CP15/60; GST-P21 + GST-CP 15/60, and placebo controls.
  • each cow Upon entering dry-off, each cow received three 5 ml doses of the assigned vaccine subcutaneously, with each dose given fourteen days apart.
  • Colostrum from each cow was collected 3 times during the first 24-36 hours post-calving and labeled; a 10-20 ml sample was withdrawn, -and the balance frozen in individual containers at each collection. Colostrum was assayed for total IgG levels by RIDA. ELISA assayed for P21 and CP 15/60 subunit protein antibodies. Serology analysis by ELISA was conducted for the same subunit protein antibodies, both immediately prior to vaccination, and at the time of calving. Feces were collected pre-vaccination and were tested with the ProSpect test kit for the presence of C. pan m; all samples tested were negative for C. paryum.
  • Colostrum antibodies to P21 A P21 -specific antibody response was detected in all groups vaccinated with the P21 antigen. In contrast, groups vaccinated with CP 15/60 and the placebo group had no detectable antibody response to P21 (see Figure 14). Interestingly, the "combo" group (vaccinated with 0.25 mg of GST-P21 in combination with 0.25 mg of GST-CP15/60) had a very similar P21 response as compared to the monovalent GST-P21 group (vaccinated with 0.5 mg of GST-P21). The group receiving 0.5 mg of His-P21 had a P21 response that was slightly, but consistently, lower than the groups receiving 0.5 mg of GST-P21, the greatest difference found at the second milking.
  • Example 14 Experimental Challenge of C. parvum in Newborn Calves Eight colostrum-deprived beef calves obtained by induced labor were divided evenly into 2 groups and each group was placed in an isolation room for the 6-day study period. Each calf occupied a metabolism crate. Each group was bottle- fed 2 pints (-960 ml) of colostrum at 3 and 12-15 hours post-partum. At 24 hours post-partum, all calves had blood IgG levels >1000 mg/dL as detected by RIDA (Radial Immunodiffusion Assay). Each calf in the challenge group was orally challenged with 10 8 oocysts of C. parvum. Blood samples were collected daily and were tested for serum antibodies to C.
  • Challenged calves started at 6.4 on Day -1 and reached their highest level at Day 2 (7.0) and remained higher than the control calves throughout the study period. Fecal dry matter content, as a % of volume, remained fairly constant in the control calves, while the challenged calves began a downward trend (lower % dry matter equaling diarrhea) on Day 2, which continued through the end of the study. Challenged calves had consistently lower dry matter content, by 6 - 39%, than control calves. Mean fecal diy matter content in unchallenged calves ranged from 39.9%) at the 24-hour post-partum time point to 51.7%) at the Day 2 morning sample collection.
  • Mean fecal dry matter content in challenged calves ranged from 28.4% at the 24 hour post-partum time point to 41.0% at the Day 2 morning sample collection, steadily decreasing thereafter to a mean low of 9.6% at the Day 4 evening sample collection.
  • Figure 19 illustrates the daily differences in %> fecal dry matter by group. Control calves remained negative to C parvum infection throughout the study period. Challenged calves shed C. panmm oocysts and calves challenged with C. parvum developed clinical signs of cryptosporidiosis. Unchallenged controls remained healthy.
  • Example 15 Demonstration of efficacy of various C. parvum subunit protein vaccines via calf challenge Based upon the significantly less sever clinical signs observed in calves fed colostrums from vaccinated cows versus calves fed colostrums from control cows, six groups of calves were selected: GST-P21 (group 1); His-P21 (group 2); GST- 15/60 (group 3); His-15/60 (group 4); GST-C& + GST-C15/60 (group 5); Placebo vaccine (group 6). Approximately eight animals were in each treatment group. Prior to the first colostrum intake, newborn calves were bled for serology, and observed for body weight, body temperature, fecal matter, and other clinical observations (i.e.
  • the first colostrum was fed at approximately 3 hours of age by calf nurser or esophageal tube.
  • the second colostrum was administered approximately 12 hours later.
  • the C. parvum challenge (10 7 oocysts) was provided at approximately 24 hours of age. Observation of the calves occurred four times daily, during which time blood samples were obtained, body temperature and clinical observations were monitored and feces collection occurred. All calves were challenged by oral administration of 10 8 oocysts of C. paryum 24 hours after time of birth. Sixty to 100 ml of calf milk replacer was administered to the calf via clean calf nurser or clean esophageal tube immediately prior to challenge.
  • the P21 antibody-detection ELISA used to generate the data for the chart shown in Figure 20 is an indirect competitive ELISA, meaning that higher OD's correspond with lower antibody levels, and lower OD's correspond with higher antibody levels.
  • the calves in this study were nai e at day -1, but showed seroconversion after receiving test colostrums containing P21 antibodies (GST-P21, His-P21 , and the combo).
  • the calves that received colostrum containing GST- 15/60, His-15/60, and placebo antibodies all remained negative for P21 antibodies throughout the 6-day observation period.
  • the CP 15/60 antibody-detection ELISA is a direct ELISA, so high OD's correspond with high antibody levels, and low OD's correspond with low antibody levels ( Figure 21). All calves were naive at Day -1.
  • Figure 24 is a cloud diagram that shows the relative distribution of diarrhea for all the calves in the study.
  • the cloud diagram shows the relative distribution of all the calves and was generated by averaging the 24 sickness scores for each calf (each filled black circle represents one calf in that treatment group), and then averaging those values to obtain an average for the treatment group (represented by a filled purple square). If more than one data point occupies the same space, the number of overlapping data points is indicated by the superscript.
  • the average for GST-15/60 is lower than that of the placebo and the GST-15/60 values are more closely grouped (four of the data points overlap with the average for the group).
  • the His-15/60 group also did well, having an average much lower than the placebo or other groups, although the overall grouping of the values is not as close as GST- 15/60.
  • Anorexia After the second day of study, any calf nursing less than 2 liters of milk and requiring an esophageal tube was scored as anorexic (anorexia observations during the first two days of life were recorded, but not analyzed).
  • the calves in the GST- 15/60 group had no anorexia throughout most of the study, in contrast with the placebo group, which often contained two or three anorexic calves.
  • Figure 25 shows a cloud diagram depicting the relative distribution of all the calves' total anorexia scores, for all vaccines.
  • the GST-15/60 has the closest grouping as well as the lowest average of all the groups in the study. Depression Four times a day, calves were observed and given a score correlating to their condition. The number of healthy calves in the GST-15/60 group was greater than that of the placebo group. It should be noted that none of the calves in either group scored higher than a 1 (apathetic) condition score at any observation. Thus, the score of 3 on observation 21 for the placebo group indicates three calves with scores of 1, not one calf with a score of 3.
  • Figure 26 shows the distribution of the total general condition scores for each calf.
  • the GST-15/60 group shows a much closer grouping than the other vaccine groups, as well as having a very low average occurrence as compared to the placebo. Interestingly, the combo vaccine group
  • FIG. 27 is a cloud diagram showing the average fecal dry matter score for each calf, for all vaccines.
  • the 15/60-containing vaccine groups all show close grouping and a higher average amount than the placebo group.
  • Oocyst Shedding Figure 28 shows the oocyst shedding as dete ⁇ nined by the ProSpect ELISA kit (not direct microscopic oocyst counts). As seen before in other clinical signs (such as diarrhea), all animals in the study became symptomatic. However, oocyst shedding in the His-15/60 group appears to be delayed as compared to the placebo group.
  • Example 16 Immunogenicity and Safety of Vaccines Containing Rotavirus. Coronavirus, E. coli K99 and F41 and Containing the C. paryum GST-CP 15/60 antigen
  • the objective of this study was to assess, in susceptible calves, the safety and the antibody response induced by two combination vaccines.
  • a specific objective of the study was to determine if addition of a C. paryum subunit antigen interferes with the immune response to other antigens, such as bovine rotavirus, bovine coronavirus, and E. coli antigens K99 and F41.
  • a transient phase of hyperthermia was observed in the two vaccinated groups, with a peak within 24 hours after the first and second vaccinations.
  • the control calves also had an increase of temperature following vaccinations.
  • Figure 30 shows the evolution of the average size of local reactions following first vaccination.
  • Figure 31 shows the evolution of average size of local reactions following the second vaccination.

Abstract

L'invention concerne des compositions de combinaison comprenant un ou des antigènes ou un ou des épitopes d'intérêt du C. parvum avec au moins un autre antigène ou épitope d'intérêt d'un pathogène induisant une infection entérique et/ou des symptômes et/ou un(des) recombinant(s) et/ou un(des) vecteur(s) et/ou un(des) plasmide(s) exprimant un(des) antigène(s) ou un(des) épitope(s) d'intérêt de ce type. L'invention concerne également l'administration de compostions de ce type à des mammifères en gestation et/ou à des mammifères nouveau-nés ou à de jeunes mammifères, par exemple à des vaches en gestation et/ou à des veaux d'un mois.
EP04780910A 2003-08-14 2004-08-12 Compositions et vaccins contenant un(des) antigene(s) de cryptosporidium parvum et d'un autre pathogene Withdrawn EP1654000A1 (fr)

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