JP2007502292A - Compositions and vaccines comprising Cryptosporidium parvum antigen and other pathogen antigens - Google Patents

Abstract

Cryptosporidium parvum antigen (s) or epitope (s) of interest, and at least one other antigen or subject epitope derived from a pathogen causing intestinal infection and / or intestinal symptoms, and / or Or recombinant (s) and / or vectors (s) and / or plasmids (s) that express such antigen (s) or epitope (s) of interest And a mixture composition comprising: a pregnant mammal and / or a newborn or young mammal, such as a pregnant cow and / or a calf such as within one month of life Administration is disclosed and claimed.
[Selection] Figure 1

Description

  This application is a continuation-in-part of US patent application Ser. No. 09 / 742,512 filed Dec. 20, 2000, claiming priority of U.S. Provisional Application No. 60/171399, filed Dec. 21, 1999. is there. This application also claims priority from US Provisional Application No. 60/495045, filed Aug. 14, 2003.

  Each application and patent cited herein, as well as each document or reference cited in that application and patent (including any pending patents; “application citations”), and any of these applications and patents PCT and foreign applications or patents corresponding to and / or claiming their priority, as well as each document cited or referenced in each application cited document are incorporated herein by reference. More generally, a document or reference is cited in this specification, either in the reference list preceding the claims or in the specification itself, but each such reference or reference (" As well as each reference or reference (including manufacturer's specifications, instructions for use, etc.) cited in each reference cited herein is incorporated herein by reference.

  The present invention relates to Cryptosporidium parvum and / or antigen (s) / epitope (s) of enteropathogens (such as other enteropathogens), and Cryptosporidium parvum and / or intestinal tract Compositions and methods comprising or using the antigen (s) / epitope (s) for inducing an immune response against infection or preventing, treating or controlling them, As well as their use.

  The present invention further elicits an immune response to and / or prevents and / or treats an intestinal infection in an animal, eg, a mammal such as a cow, cat, dog or horse or species thereof. And / or a method and / or composition for controlling and / or the use of such a composition or the use of its components in formulating such a composition.

  The present invention also provides methods and / or compositions and / or for inducing an immune response to and / or preventing and / or treating and / or controlling the infection by Cryptosporidium parvum. It relates to the use of such compositions or the use of ingredients in formulating such compositions.

  The present invention also provides for the simultaneous use of a unitary cryptosporidium parvum vaccine with an enteropathogen, such as a bovine enteropathogen (eg, rota / coronavirus, E. coli) vaccine, and / or cryptosporidium Use of combined vaccines containing parbum and rota / coronavirus or E. coli, and prevention, control or treatment of exacerbations of intestinal diseases such as bovine intestinal diseases due to superinfection with Cryptosporidium parvum, or immunization It may be related to inducing a response and reducing it. Immunity induced by vaccination against Cryptosporidium parvum can significantly reduce the severity of the diseases induced by the enteric pathogens described herein. Combination vaccines containing Cryptosporidium parvum are useful for more complete prevention of multipathogenic intestinal diseases in newborn animals such as calves caused by rota and coronaviruses and E. coli K99 and F41.

  The present invention also relates to the effects of superinfection of Cryptosporidium parvum on other enteric pathogens, such as bovine enteric pathogens. Cryptosporidium parvum is usually present in the faeces of newborn animals such as mammals such as calves. Cryptosporidium parvum can itself cause clinical symptoms of intestinal disease, with or without other potentially pathogenic viruses and bacteria in the intestine. Viruses such as coronaviruses and bacteria such as E. coli, eg F41, are recognized as very pathogenic in the field but cannot cause significant clinical symptoms of the disease in experimental challenge models. Thus, the present invention relates to dealing with co-infection of cattle with Cryptosporidium parvum, as superinfection exacerbates diseases caused by other pathogens such as coronavirus, rotavirus, and E. coli, eg F41. It can be.

Bovine intestinal disease is the result of intestinal infection by enteric pathogens, often manifesting in the form of some form of diarrhea. This disease is also commonly referred to as neonatal calf diarrhea and causes substantial economic losses in agriculture. Calf morbidity is a major factor contributing to the farmer's economic burden along with its therapeutic intervention needs and its long-term adverse effects on animals. One estimate states that approximately 75% of dairy calves younger than 3 weeks of age have neonatal calf diarrhea. Radostits, OM, et al., Herd Health Food Animal Production Medicine, 2 nd ed., Sounders, Philadelphia, pp. 184-213, 1994. Management of neonatal calf diarrhea is difficult for many reasons, the most important of which are (1) involvement of multiple etiologies in the pathogenesis of the disease, (2) nonspecificity of clinical symptoms, ( 3) Findings that infectious diseases can be asymptomatic, and (4) Involvement of host factors such as nutrition and endogenous immunity. Moon, HW, et al., JAVMA 173 (5): 577-583 (1978). Viring, S. et al., Acta Vet. Scand. 34: 271-279 (1999).

  It is known that multiple intestinal pathogens are present during infection, but it is not known which pathogen or combination of pathogens actually causes the disease, thus preventing or treating bovine intestinal diseases Building a strategy to do so has become very difficult. From epidemiological studies in the United States and other countries of the world, the most prevalent enteropathogens associated with neonatal calf diarrhea include but are not limited to Cryptosporidium parvum, rotavirus, coronavirus, and E. coli Is known to be included. In most cases, several of these enteric pathogens are isolated from the outbreak of the disease, but the prevalence of each disease is not consistent within a single affected population or between multiple infected groups.

  Previous studies have found that rotavirus is the most prevalent enteropathogen in diarrheal calves. For example, research on diarrheal calves in the UK detected rotavirus and Cryptosporidium parvum in 42% and 23% of the population, respectively. Twenty percent of the calves in this population were infected with multiple pathogens. More recently, however, Cryptosporidium parvum has become a major pathogen in bovine intestinal infections. In a more recent study evaluating co-infection with Cryptosporidium parvum and other major enteric pathogens in newborn calves, Cryptosporidium parvum is the only enteric pathogen found in 52.3% of the population This was followed by rotavirus alone in 42.7%. de la Fuente et al., Preventive Veterinary Medicine 36: 145-152 (1998). Co-infection with two etiologies occurred in 21.6% of this study group, while infection with 3 and 4 pathogens was seen in 6% and 0.5%, respectively. The most common mixed infection in this study was a combination of Cryptosporidium and rotavirus. Limited information is available on the role of individual enteropathogens in neonatal calf diarrhea. Furthermore, the combined mechanisms of viral, bacterial, and protozoan pathogenesis that underlie the development of bovine intestinal disease in newborns are still poorly understood. However, regardless of lack of understanding of the mechanism of this pathogenesis, infection with multiple pathogens tends to have more serious clinical consequences than infection with a single enteric pathogen.

  There is currently no cure that can adequately protect against neonatal calf diarrhea. There is also no single drug or combination of chemotherapeutic agents useful for the treatment of this disease. Vaccines that target bovine intestinal disease are available, but only a few have been successfully accepted. Currently available vaccines contain three enteric pathogens known to be associated with this disease: rotavirus, coronavirus, and E. coli antigens. Individual components of these commercially available bovine enteric pathogen vaccines (Rota / Corona, E. coli) have been shown to be protective in an experimental challenge model. Despite the availability of such vaccines, under field conditions, neonatal calf diarrhea, calf diarrhea, and winter dysentery will affect the business of beef cattle, feedlots, and dairy calves. Continue to give. E. coli K99, rotavirus, as reflected by the low use of enteric pathogen combination vaccines in the US market (only 4% of pregnant animals vaccinate this product annually) And producers continue to question the effectiveness of current enteric pathogen vaccines, including coronavirus, under practical conditions.

  More recently, experimental unitary vaccines against Cryptosporidium parvum have been developed and shown to protect against Cryptosporidium parvum experimental challenges. However, many enteropathogens involved in intestinal diseases cannot be overcome by treatment with Cryptosporidium parvum vaccine alone. Also, infections caused by enteric pathogens are universal, i.e., found worldwide, and most vertebrates are susceptible to such infections. Thus, the need to combat infections by enteric pathogens is not limited to bovine species. Furthermore, intestinal diseases are difficult to control and are likely to be multifactorial. That is, Cryptosporidium parvum can be a factor, but until now Cryptosporidium parvum actually exacerbates intestinal disease, or Cryptosporidium parvum is mixed immunogenic composition, mixed immune composition, or mixed vaccine There is no definitive indication that use in the composition enhances prevention of intestinal disease.

Furthermore, a problem faced when preparing and using combination vaccines is a phenomenon called “efficacy interference” that reduces or reduces the effectiveness of one antigen of the combination. This is thought to be because one antigen of the mixed vaccine becomes dominant. See US Pat. No. 5,843,456 to Paoletti et al. This phenomenon is observed in combination vaccines that use one or more antigens of E. coli, for example, one or more bacterial antigens interfere with other antigens in the combination vaccine.
US Pat. No. 5,843,456 Radostits, OM, et al., Herd Health Food Animal Production Medicine, 2nd ed., Sounders, Philadelphia, pp. 184-213, 1994 Moon, HW, et al., JAVMA 173 (5): 577-583 (1978) Viring, S. et al., Acta Vet. Scand. 34: 271-279 (1999) de la Fuente et al., Preventive Veterinary Medicine 36: 145-152 (1998)

  Thus, the problem that Cryptosporidium parvum contributes to intestinal infections and symptoms, or how to address this problem here, such as of interest Cryptosporidium parvum antigen (s) Or epitope (s) and at least one other antigen or epitope of interest from a pathogen causing intestinal infection and / or intestinal symptoms and / or such antigen (s) of interest ) Or a recombinant composition (s) expressing epitope (s) and / or a vector (s) and / or a plasmid (s) and a mixed composition, and during pregnancy Such composition for pregnant mammals such as cows and / or new or young mammals such as calves within one month of life Administration, as well as efforts to any potential problems of efficacy interference is considered not disclosed or suggested heretofore.

  One object of the present invention is to use improved immune or vaccine compositions of enteric pathogens, in particular in the field of veterinary medicine, for example in mammals such as cattle, dogs, cats or horses or their species. It can be set as the composition which can be obtained.

  Another object of the present invention is to immunize newborns and / or young animals, such as conferring passive immunity to newborn animals such as mammals such as cows, dogs, cats or horses or their species, preferably cattle. Such an immune composition or vaccine composition can be used effectively.

  Yet another object of the invention is an improved immune or vaccine composition against Cryptosporidium parvum, such as for use in mammals such as dogs, cats or horses or species thereof, in particular cattle or species thereof For example, a composition for use in the field of veterinary medicine can be used.

  Yet another object of the present invention is to provide neonatal animals and / or juvenile animals, such as conferring passive immunity to newborn animals, eg, mammals such as dogs, cats or horses or their species, particularly cattle or their species. It can be an improved method for immunization.

  A further object of the present invention is to control or prevent Cryptosporidium parvum, a method of inducing an immune response against enteric pathogens including Cryptosporidium parvum, or intestinal infections and / or intestinal symptoms including Cryptosporidium parvum And / or methods of treatment, eg, methods comprising administering a composition of the invention, and methods of preparing such compositions, particularly such compositions for formulating such compositions Use of the components.

  Vaccination or immunization against enteric pathogens, including enteropathogens including Cryptosporidium parvum, may include at least two Cryptosporidium parvum antigens or epitopes thereof and / or at least two Cryptosporidium parvum antigens or epitopes thereof. A combination of vector (s) to be expressed, eg, P21 or an epitope thereof and / or a vector expressing P21 or an epitope thereof, or a vector expressing Cp23 or an epitope thereof and / or Cp23 or an epitope thereof, and Cp15 / 60 or an epitope thereof and / or an immune or vaccine composition comprising a combination with a vector expressing Cp15 / 60 (e.g. at least one epitope of Cp23 and Composition comprising at least one epitope of Cp15 / 60; where is unexpectedly improved greatly by using the note should be) that may include P21 to Cp23 antigen or protein.

  By mixing two antigens (or epitope (s) of interest and / or a vector expressing this antigen and / or epitope (s)), antibodies against Cryptosporidium parvum are highly Resulting in a synergistic effect of improving and usefully producing an immune response, such as antibodies, cellular responses, or both, to Cryptosporidium parvum and / or gut infections, or gut pathogens, or gut symptoms It is. This combination also provides an efficient immune or vaccine composition useful for protecting newborns or juveniles, or mammals such as dogs, cats or horses or their species, particularly cattle. Can be prepared. For example, advantageously, a composition comprising the antigen and / or epitope (s) of interest can be applied to, for example, unborn progeny and to newborns or juveniles in weaning milk or colostrum Can be used to inoculate a maternal or pregnant female to induce an immune response that is advantageously transmitted through the host, advantageously with the antigen and / or epitope (s) of interest Use of the composition comprising the vector (s) to be expressed to inoculate males and females of any age, eg, females and / or neonatal or juvenile males and females who are not pregnant. For example, to produce an immune response in the animal while it receives the antibody or other immune agent via lactating milk or colostrum. Alone inoculation into objects, or can preferably be implemented in combination with inoculation of female maternal or during pregnancy.

  In an immune composition or vaccine composition, the antigen (s) and / or epitope (s) of interest to Cryptosporidium parvum are treated with at least other at least one other enteric pathogen of the animal species. Combining a single antigen or epitope of interest (advantageously, multiple pathogens, eg, multiple antigens and / or epitopes of interest from intestinal pathogens) significantly increases protection against intestinal disease Can do.

  A particularly advantageous immune composition or vaccine composition according to the invention can protect against Cryptosporidium parvum, and (i) at least one Cp23 antigen or epitope of interest and / or at least of interest. At least one vector expressing one Cp23 antigen or epitope, or at least one P21 antigen or epitope of interest and / or at least one vector expressing at least one P21 antigen or epitope of interest And (ii) at least one Cp15 / 60 antigen or epitope of interest and / or at least one vector expressing at least one Cp15 / 60. Advantageously, the composition further comprises a vector expressing a desired additional at least one antigen or epitope from another enteric pathogen and / or an additional at least one antigen (which may be a Cp23 of interest Alternatively, it can be the same vector that expresses the P21 antigen or epitope and / or the desired Cp15 / 60 antigen or epitope, eg, the composition is targeted with the desired Cp23 or P21 antigen or epitope Cp15 / 60 antigen or epitope, and optionally additional vectors of any desired antigen or epitope can be included).

  Another Cryptosporidium parvum antigen is the CP41 antigen described in Mark C. Jenkins et al., Clinical and Diagnostic Laboratory Immunology, Nov. 1999, 6, 6: 912-920. The immunizing composition or vaccine composition according to the present invention preferably comprises a target antigen or epitope and / or a vector expressing the antigen or epitope thereof, preferably a Cp23, P21, Cp15 / 60, etc. It can be included in combination with at least one other Cryptosporidium parvum described in the specification, for example, in combination with Cp23 or P21, and / or Cp15 / 60. To express this antigen, a start codon can be added upstream of the nucleotide sequence depicted in FIG. 2 herein and a stop codon downstream of this sequence.

  In addition, as a target Cryptosporidium parvum antigen or epitope, Cp23 or an epitope thereof or a vector expressing this antigen or epitope, P21 or an epitope thereof or a vector expressing this antigen or epitope, or Cp15 / 60 or an epitope thereof Or a vector expressing this antigen or epitope, or only one of CP41 or its epitope or a vector expressing this antigen or epitope, advantageously at least one other Cryptosporidium parvum antigen of interest Alternatively, an immunological composition or vaccine composition effective against enteritis can be produced by using in combination with an epitope or a target antigen or a vector expressing the epitope. It is. The composition may further comprise at least one antigen or epitope of interest from another enteric pathogen and / or a vector expressing the at least one additional antigen (the vector may be an antigen (s)) And / or epitope (s) can be co-expressed).

  The invention further encompasses methods for inducing or controlling, preventing and / or treating immune responses or defense (vaccine) responses to enteropathogens or intestinal infections or intestinal symptoms including Cryptosporidium parvum For example, the method includes administering a composition of the invention.

  The composition of the present invention can be applied to pregnant mammals such as heifers or cows (hereinafter referred to as cows), dogs, cats, horses, etc. (Generally 9 months or 170 days) can be administered once or twice. For example, a first dose about 1 to about 2.5 or 3 months before childbirth, a second dose or a single dose close to birth, eg 3 weeks before childbirth, preferably about 3-15 days before childbirth . In this way, the female can transmit passive immunity via milk or colostrum to a newborn, for example a post-natal calf. Advantageously, since it is desirable for a pregnant mammal to elicit an antibody response, a composition comprising one or more antigen (s) and / or epitope (s) thereof (vector (s) Or), recombinant (s), and / or compositions containing DNA plasmid (s). Also advantageously, in contrast, to prevent, treat and / or control intestinal conditions, intestinal infections, and / or intestinal symptoms in newborns and / or very young animals. Because cellular responses and / or antibody responses are useful, newborn or very young mammals (eg, mammals susceptible to intestinal disease, such as cattle within about one month of life, and similar) Other mammals in the postnatal period include vectors (one or more), recombinants (one) that express the antigen (s) and / or their epitope (s) in vivo. Or) and / or a composition comprising DNA plasmid (s). Newborn animals and / or very young animals can receive additional administrations of antigenic composition and / or epitope composition and / or vector / recombinant / DNA plasmid composition during a period susceptible to infection, and The dam is also optionally and advantageously as described herein so that newborns and / or very young animals can receive an immune response by direct and passive administration. Can be vaccinated during pregnancy.

  Certain compositions of the present invention may be in the form of one or more E. coli antigens (eg, inactivated E. coli with pilus such as K99, Y, 31A and / or F41, and / or subunits, or recombinant. Such pili expressed in vivo by the body), and / or one or more rotavirus antigens (eg, advantageously inactivated rotavirus), and / or coronavirus antigens (eg, bovine coronavirus antigens, Advantageously inactivated coronaviruses, etc.) may be included in combination with one or more Cryptosporidium parvum antigens such as P21 and / or Cp23 and / or Cp15 / 60 (also as mentioned above) One or more of such antigens may be epitopes contained within the target antigen, and such target antigen or antigen One or more of the pitopes can also be expressed in vivo by recombinants or plasmids).

  Accordingly, certain compositions of the present invention comprise (i) one or more cryptosporidiums such as P21 and / or Cp23 and / or Cp15 / 60 and / or CP41, preferably P21 and / or Cp23 and Cp15 / 60. Parvum antigen and (ii) at least one E. coli antigen (eg K99, Y, 31A, F41, and / or other pili or subunits of inactivated E. coli, or expressed in vivo At least one or all of the other pili; preferably K99 and / or F41 is present, advantageously Y and / or 31A is also present), and / or coronavirus antigen, and / or rota And viral antigens. For example, one or more Cryptosporidium parvum antigens such as P21 and / or Cp23 and / or Cp15 / 60 and / or CP41, preferably P21 and / or Cp23 and Cp15 / 60, and inactivated rotavirus etc. One or more Cryptosporidium comprising one or more rotavirus antigens or such as P21 and / or Cp23 and / or Cp15 / 60 and / or CP41, preferably P21 and / or Cp23 and Cp15 / 60 Comprising a Parbum antigen and one or more coronavirus antigens such as inactivated coronavirus, eg bovine inactivated coronavirus, or P21 and / or Cp23 and / or Cp15 / 60 and / or CP41, preferably One type such as P21 and / or Cp23 and Cp15 / 60 Multiple Cryptosporidium parvum antigens and one of K99, Y, 31A, F41, and / or other pili of inactivated E. coli, or other pili of subunits or expressed in vivo Or a combination of multiple E. coli antigens, eg, K99, Y, 31A, and / or F41. An exemplary E. coli antigen useful in the present invention can be pili, since E. coli pili can avoid efficacy interference. Exemplary compositions comprise at least one Cryptosporidium parvum antigen such as P21 and / or Cp23 and / or Cp15 / 60 and / or CP41, preferably P21 and / or Cp23 and Cp15 / 60, and at least One E. coli antigen, at least one coronavirus antigen, and at least one rotavirus antigen can be included. For example, P21 and / or Cp23 and / or Cp15 / 60 and / or CP41, preferably P21 and / or Cp23 and Cp15 / 60, inactivated rotavirus, inactivated coronavirus, and at least one E. coli antigen , 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 above, One or more of such antigens can be the target epitope contained in the antigen, and one or more of such target antigens or epitopes can be recombinant or plasmid. It can also be expressed in vivo). With regard to potential efficacy interference by one or more bacteria, we can avoid any potential efficacy interference by increasing the amount of other antigens present in the combined vaccine, and the E. coli antigen It was found that the effectiveness interference can be avoided even if pili is used as.

  In such compositions of the invention, a single dose is an E. coli antigen (or multiple antigens) present in an amount normally found in vaccines against enteric pathogens, eg, an amount that provides a serum titer of at least 0.9 log 10 in guinea pigs. In the case of E. coli antigens, each rotavirus antigen can be present in an amount normally found in vaccines against enteric pathogens, such as an amount that provides a serum titer of at least 2.0 log 10 in guinea pigs, The coronavirus antigen can be present in an amount normally found in vaccines against enteric pathogens, eg, an amount that provides a serum titer of at least 1.5 log 10 in guinea pigs. The compositions of the invention can also include an adjuvant, such as aluminum hydroxide, which is present in a single dose, preferably in an amount normally found in vaccines, such as from about 0.7 to about 0.9 mg. can do.

  Accordingly, in one aspect, the present invention provides a first antigen or epitope of interest derived from Cryptosporidium parvum and / or a first vector expressing the first antigen or epitope of interest and other enteric pathogens of interest. A first vector expressing a second antigen or epitope of origin and / or a first antigen or epitope of interest and expressing a second antigen or epitope of interest and / or a second antigen or epitope of interest An intestinal mixed immunity composition, mixed immunogenic composition, or mixed vaccine composition comprising a second vector and a pharmaceutically acceptable vehicle.

  The composition can include an antigen derived from Cryptosporidium parvum and an antigen derived from another enteric pathogen. The composition can include an antigen derived from Cryptosporidium parvum and an antigen derived from other enteric pathogens of bovine, canine, feline, or equine species. The antigen derived from the enteric pathogen can be selected from the group consisting of E. coli, rotavirus, coronavirus, Clostridium spp., And mixtures thereof. This enteric pathogen can be E. coli. The antigen derived from E. coli is selected from the group consisting of E. coli having K99 antigen, E. coli having F41 antigen, E. coli having Y antigen, E. coli having 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, and mixtures thereof. You can choose.

  The enteric pathogen can include bovine coronavirus and / or bovine rotavirus and / or Clostridium perfringens. The enteric pathogen antigen may include C. perfringens type C and D toxoids. In some embodiments, the enteric pathogen can comprise E. coli, bovine rotavirus, bovine coronavirus, and Clostridium perfringens, or can comprise E. coli, bovine rotavirus, and bovine coronavirus.

  Further, in some embodiments, the present invention provides that the enteric pathogen antigen is E. coli having K99 antigen, E. coli having F41 antigen, E. coli having Y antigen, E. coli having 31A antigen, K99 antigen, F41 antigen, Y A composition comprising an E. coli antigen selected from the group consisting of an antigen, 31A antigen, and mixtures thereof, bovine inactivated coronavirus, bovine inactivated rotavirus, and C. perfringens C and D toxoids, or E. coli antigens selected from the group consisting of E. coli having K99 antigen, E. coli having F41 antigen, E. coli having Y antigen, E. coli having 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, and mixtures thereof. A composition comprising bovine inactivated coronavirus and bovine inactivated rotavirus can be included.

  Advantageously, the composition of the present invention comprises a subunit of Cp23 and Cp15 / 60, or a subunit selected from the group consisting of P21, Cp23, Cp15 / 60, CP41, and mixtures thereof, such as P21 and Cp15 / 60. Ptosporidium parvum antigen may be included.

  In a composition of the present invention combining Cryptosporidium parvum and an antigen from at least one other enteric pathogen, the Cryptosporidium parvum antigen is also an inactivated or attenuated live oocyst, or a subunit derived from oocyst Or can be composed of these.

  The composition of the present invention can include an adjuvant such as saponin or aluminum hydroxide, and the composition of the present invention can be in the form of an oil-in-water emulsion.

  The present invention further contemplates an immune composition, immunogenic composition, or vaccine composition against Cryptosporidium parvum, the composition comprising a P21 or Cp23 antigen or epitope thereof, or the first antigen. A first antigen containing a first vector that expresses Cp15 / 60 antigen or epitope thereof, or a first vector that expresses both first and second antigens, or a second vector that expresses a second antigen A second antigen and a pharmaceutically acceptable vehicle. The composition can include Cp23 and Cp15 / 60 antigens, which are in the form of separate fusion proteins. The composition can include a vector that expresses Cp23 and Cp15 / 60. The composition can include a first recombinant vector that expresses Cp23 and a second recombinant vector that expresses Cp15 / 60. The composition can also include P21 and Cp15 / 60. Such recombinants can further include an adjuvant.

  Furthermore, the present invention includes an immune composition, an immunogenic composition, or a vaccine composition against Cryptosporidium parvum, which composition comprises P21, Cp23, Cp15 / 60 or CP41 antigens or their A first antigen comprising an epitope or a first vector expressing this first antigen and a second antigen derived from Cryptosporidium parvum or a first vector or second antigen expressing both the epitope or the first and second antigens A second antigen comprising a second vector that expresses and a pharmaceutically acceptable vehicle. However, the first antigen and the second antigen are different antigens.

  The invention also encompasses a method of immunizing a newborn calf against intestinal disease, such that the newborn calf receives a transit antibody against Cryptosporidium parvum via colostrum and / or milk. Administering the composition of the present invention to a pregnant calf before delivery. The method further includes providing the newborn calf with colostrum and / or milk of the cow (s) administered the composition during pregnancy. The method can include administering the composition to a newborn calf. The composition administered to a pregnant female can include an antigen or epitope thereof, and the composition administered to a calf can include a vector. Accordingly, the present invention also contemplates active immunization of adult and newborn calves, which method comprises administering a composition of the present invention to a calf.

  The present invention also encompasses a method of immunizing a newborn calf, which method comprises providing a newborn calf with colostrum and / or milk from a cow that has been administered this composition during pregnancy. . Similarly, in a broader sense, the present invention encompasses a method of immunizing a newborn mammal, which comprises colostrum and / or female mammals administered the composition during pregnancy. Including feeding the newborn with milk, advantageously, the mammal is a cow, cat, dog or horse. Furthermore, the present invention encompasses a method of preparing the composition of the present invention, which method comprises mixing an antigen or epitope or vector and a carrier.

  Further, the present invention includes a kit for preparing a composition of the present invention comprising an antigen, epitope, or vector, each in separate container (s) (Cryptosporidium parvum antigen, epitope) Or an antigen, epitope, or vector can be placed together in one container, such as a vector, while others can contain an antigen, epitope, or vector in one or more containers, or Carriers, diluents, and / or adjuvants can be in separate containers), the kit can optionally be in one package, and can optionally include instructions for mixing and / or administration.

  In this disclosure, “comprises”, “comprising”, “containing”, “having” and the like have the meanings affixed to them in US Patent Law, respectively. , “Includes”, “including”, and the like. “Consisting essentially of” or “consists essentially” also has the meaning given to it in US patent law, respectively, Are open-ended, and therefore the basic and novel features of what has been described are not altered by the presence of anything other than those described, and so long as they exclude prior art embodiments The existence of something other than is allowed.

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

  The following detailed description is given by way of example and should not be construed as limiting the invention to the specific embodiments, but will be understood in conjunction with the accompanying drawings, which are incorporated herein by reference. Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples with reference to the accompanying drawings.

  Accordingly, one aspect of the present invention preferably provides at least one antigen or epitope of interest from at least one Cryptosporidium protozoa, including Cryptosporidium parvum, and at least one other enteric pathogen, At least one antigen of interest derived from a pathogen that infects species of animals to be protected, such as dogs, cats, horses, cow species, more preferably cow species, and / or Cryptosporidium protozoan antigens or epitopes And / or vector (s) and / or recombinant (one or more) expressing antigen (s) or epitope (s) of at least one other enteric pathogen of interest Immunization of enteropathogens, including multiple) and / or plasmid (s) and a pharmaceutically acceptable vehicle Composition, mixing the immunogenic composition, or a combination vaccine composition. Also, enteric pathogens often infect several (plural) animal species, so a universal immune composition, immunogenic composition, or vaccine composition is envisaged.

  The immune composition elicits a local or systemic immune response. An immunogenic composition likewise elicits a local or systemic immune response. The vaccine composition elicits a local or systemic protective response. Thus, the terms “immune composition” and “immunogenic composition” include “vaccine composition” (since the former two terms can be protective compositions).

  The Cryptosporidium parvum antigen that can be used in the present invention is preferably (1) a 148 amino acid protein called Cp15 / 60 (see, eg, US Pat. No. 5,591,434, which is described in US Pat. No. 5,591,434). Is represented by SEQ ID NO: 2 with an additional 10 amino acids at the 5 ′ end upstream of methionine (Met), whose epitope, advantageously the epitope that induces protection, or the full length of the sequence is immunogenic. A 148 amino acid sequence of CP15 / 60, or a longer amino acid sequence comprising these 148 amino acids, eg, the entire sequence represented by SEQ ID NO: 2 of US Pat. No. 5,591,434, or a fragment of a 148 or 158 amino acid sequence Any polypeptide containing, or essentially And / or (2) Cp23 and / or P21 (Cp23 is an antigen of about 23 kDa. Perryman et al., Molec Biochem Parasitol 80: 137-147 (1996), WO 9807320, and LE Perryman et al., Vaccine 17 (1999) 2142-2149. The major portion of this protein (187 amino acids) is referred to herein as P21. And having an amino acid sequence homologous to the amino acid sequence of protein C7 disclosed as SEQ ID NO: 12 in WO 9807320. For expression, one species such as Met-, Met-Gly-, or similar amino acids Alternatively, two or more amino acids can be added to the end of P21, the epitope, preferably the epitope that induces protection, or the entire sequence is immunogenic. It is within the scope of the invention to use an antigen comprising, consisting essentially of, or consisting of a polypeptide comprising a 187 amino acid sequence, or a longer amino acid sequence, or a fragment of a 187 amino acid sequence, including a tope The entire amino acid sequence of Cp23, and the corresponding nucleotide sequence, are readily available The P21 protein is the major part and the C-terminus of Cp23 The nucleotide sequence of P21 can be found in DNA libraries such as Example 1. Can be used as a probe for screening the libraries disclosed in 1. This method is well known to those skilled in the art. Based on the molecular weight of Cp23, to complete the amino acid sequence of Cp23, it can be asserted that about 25-35 amino acids are missing at the N-terminus of P21. A person skilled in the art has the means to easily find the start codon from this information and thus the 5 'end of the nucleotide sequence of Cp23 and the N-terminus of the amino acid sequence. )including.

  The target antigens or epitopes in the composition of the present invention can be used individually or mixed. For example, the composition of the present invention may comprise (1) or (2) or both (1) and (2).

  Another usable antigen is the CP41 antigen disclosed above.

  According to a preferred embodiment, these targeted antigens or epitopes are incorporated into the compositions of the invention as proteins, ie subunit antigens. Such antigens can be produced by chemical synthesis or in vitro expression. Methods for obtaining sequences encoding Cp15 / 60 and P21 and methods for constructing vectors that express them are described in the Examples. These sequences can be cloned into an appropriate cloning vector or expression vector. These vectors are then used to transfect suitable host cells. Antigens encoded by the nucleotide sequence inserted into the vector, such as Cp23 and / or P21 and / or Cp15 / 60, are host cells transformed with the expression vector under conditions such that the antigen is produced. It is produced by growing. This method is well known to those skilled in the art. The host cell may be a prokaryotic cell or a eukaryotic cell, and may be, for example, a yeast such as Escherichia coli, Saccharomyces cerevisiae, an animal cell, particularly an animal cell system. Vectors that can be used with a given host cell are known to those of skill in the art. Once a vector is selected that produces a fusion protein, the fusion protein can then be used to easily recover the antigen.

  Furthermore, with regard to the sequence, the nucleic acid sequence useful for expressing the desired Cryptosporidium parvum antigen or epitope is a nucleic acid sequence that can be hybridized under high stringency conditions, or the nucleic acid used in the present invention. Nucleic acid sequences having high homology with the molecule (eg, nucleic acid molecules in the literature described herein) can be included. “Hybridization under high stringency conditions” may be synonymous with the term “stringent hybridization conditions”, which are well known in the art. For example, Sambrook et al., “Molecular Cloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor, 1989, and “Nucleic Acid Hybridisation, A Practical Approach”, Flames and Higgins, both incorporated herein by reference. See eds., IRL Press, Oxford, 1985.

  With respect to nucleic acid molecules and polypeptides that can be used in the practice of the present invention, the nucleic acid molecules and polypeptides include sequences described in the references cited herein (including those subsequences described herein). ) At least about 75% homology or identity, preferably 80% or more homology or identity, more advantageously at least about 85%, about 86%, about 87%, about 88 85% or more homology or identity, such as%, about 89% homology or identity, eg, at least about 90%, such as at least about 91%, about 92%, about 93%, about 94% homology or identity % Homology or identity, more advantageously at least about 95% to 99% homology or identity, such as at least about 95% homology or identity, eg at least about 96%, about 97% , About 98%, about 9%, or even about 100% homology or identity, or about 75% to about 100%, preferably about 85% to about 100%, or about 90% to about 99%, or about 90% to about 100 %, Or about 95% to about 99% or about 95% to about 100% homology or identity. Accordingly, the present invention provides a vector encoding an epitope or epitope region of a Cryptosporidium parvum isolate, or a composition comprising such an epitope or a composition comprising an epitope or epitope region of a Cryptosporidium parvum isolate, as well as this Methods of generating and using such vectors and compositions. For example, the invention also encompasses that such nucleic acid molecules and polypeptides can be used in the same manner as the nucleic acid molecules described herein and fragments and polypeptides thereof.

Nucleotide sequence homologies using the "Alignment" program by Myers and Miller ("Optimal Alignments in Linear Space", CABIOS 4, 11-17, 1988, incorporated herein by reference), available at NCBI. Can be determined. Alternatively or additionally, the term “homology” or “identity”, for example with respect to nucleotide or amino acid sequences, can indicate a quantitative measure of homology between two sequences. This percent sequence homology can be calculated as (N _ref -N _dif ) ^* 100 / N _ref , where N _dif is a non-identical residue when the two sequences are aligned N _ref is the number of residues in one of both sequences. Therefore, the DNA sequence AGTCAGTC has 75% sequence similarity with the sequence AATCAATC (N _ref = 8, N _dif = 2).

  Alternatively or additionally, “homology” or “identity” with respect to a sequence refers to the number of positions having the same nucleotide or amino acid, the number of nucleotides in the shorter of the two sequences, or Divided by the number of amino acids. Here, an alignment of the two sequences is performed according to Wilbur and Lipman's algorithm (Wilbur and Lipman, 1983 PNAS USA 80: 726, incorporated herein by reference), for example, a 20 nucleotide window size, 4 nucleotide word length, and 4 gap penalties can be determined, conveniently using commercially available programs (eg, Intelligents ™ soot, Intelligents, Calif.) Using computer aids for sequence data including alignments Can be analyzed and interpreted. A thymidine (T) in a DNA sequence is considered equal to a uracil (U) in the RNA sequence if the RNA sequence is said to be similar to the DNA sequence or to some extent have sequence identity or sequence homology. An RNA sequence included within the scope of the present invention can be derived from a DNA sequence by assuming that thymidine (T) in the DNA sequence is equal to uracil (U) in the RNA sequence.

  Alternatively, or in addition, amino acid sequence similarity, identity, or homology can be used in determining NCBI (see sequence homology as shown in Appendix I. See also Examples). Can be determined using the BlastP program (Altschul et al., Nucl. Acids Res. 25, 3389-3402, incorporated herein by reference). The following documents (each incorporated herein by reference) also provide algorithms for comparing the relative identities or homologies of the amino acid residues of two proteins, instead of the foregoing, or In addition, the teachings in such literature can be used to determine 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 Biosequences, "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 (1983), Feng DF and Dolittle RF, "Progressive sequence alignment as a prerequisite to correct phylogenetic trees," J. 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, Haeberlie P and Smithies O, "A compreh ensive set of sequence analysis program for the VAX, "Nucl. Acids Res., 12: 387-395 (1984).

  Furthermore, with respect to the nucleic acid molecules used in the present invention (eg in the literature cited in the present invention), the present invention encompasses the use of nucleic acid molecules to which the codons correspond. For example, where the invention includes an “X” protein (eg, P21 and / or Cp23 and / or Cp15 / 60 and / or CP41) having the amino acid sequence “A” and encoded by the nucleic acid molecule “N” The present invention also encompasses a nucleic acid molecule that encodes protein X with one or more codons different from the codons in nucleic acid molecule N.

  Antigens or epitopes of interest for use in practicing the present invention can be obtained from specific antigen (s), such as Cryptosporidium parvum, E. coli, rotavirus, coronavirus, etc., or It can also be obtained from in vitro and / or in vivo recombinant expression of the gene (s) or parts thereof. The method of using and / or creating the expression vector (or recombinant) can be according to the methods disclosed below or similar to them. U.S. Pat. , "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., US Pat. No. 4,745,051 (recombinant baculovirus), Richardson, CD (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, European patent application Publication No. 0370573, U.S. Patent Application No. 920197 filed October 16, 1986, European Patent Application Publication No. 265785, U.S. Pat. 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 applications , "PNA S USA 93: 11371-11377, October 1996, Kitson et al., J. Virol. 65, 3068-3075, 1991, U.S. Pat.Nos. 5591439, 5552143, patents filed on July 3, 1996 Application No. 08/675556 and US Patent Application No. 08 / 675,565 (recombinant adenovirus), Grunhaus et al., 1992, “Adenovirus as cloning vectors,” Seminars in Virology (Vol. 3) p. 237-52, 1993, Ballet et al. EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990, Prevec et al., J. Gen Virol. 70, 429-434, PCT International Publication 91/11525, Felgner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259: 1745-49, 1993, and McClements et al., "Immunization with DNA vaccines encoding glycoprotein. D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease, "PNAS USA 93: 11414-11420, October 1996, and DNA expression U.S. Pat. Nos. 5,591,639, 5,589,466 and 5,580,859 for vectors. WO 98/33510, Ju et al., Diabetologia, 41: 736-739, 1998 (Lentiviral expression system), Sanford et al. US Pat. No. 4,945,050, Fischbach et al. (Intracel) International Publication No. 90 / 01543, Robinson et al., Seminars in IMMUNOLOGY, vol. 9, pp.271-283 (1997) (DNA vector system), Szoka et al. US Pat. No. 4,394,448 (method for inserting DNA into living cells), McCormick US Pat. No. 5,677,178 (use of cytopathic viruses), US Pat. No. 5,928,913 (vectors for gene transfer), and US Pat. No. 5,999,0091 (vectors with enhanced expression) of Tartaglia et al., As cited herein. See also other documents that do. In practicing the present invention, particularly in the case of in vivo expression, for example, a viral vector selected from herpes virus, adenovirus, and pox virus, in particular vaccinia virus, avipox virus, or canarypox virus, and DNA vector It is advantageous to use (DNA plasmids) (for in vitro expression it is advantageous to use bacterial and yeast systems).

  When antigens are produced without excretion by a combination of a host and a vector, it is preferable to make these antigens in the form of fusion proteins (HIS tags) for the convenience of antigen production and recovery. That is, the antigen can include the antigen itself and foreign amino acids.

  In practicing the present invention, techniques for purifying and / or isolating proteins, particularly according to the present disclosure and references cited herein, can be used without undue experimentation by those skilled in the art. Recombinant or vector expression products and / or antigen (s) can be purified and / or isolated, and such techniques are usually of interest, inter alia, at various salt concentrations. Precipitation using protein solubility, precipitation with organic solvents, polymers and other materials, affinity precipitation and selective denaturation; high performance liquid chromatography (HPLC), ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, Or column chromatography including dye-ligand chromatography; immunoprecipitation, alignment Gel filtration, electrophoretic methods, can include the use of ultrafiltration and isoelectric focusing.

  As described herein, according to another aspect, the present invention encompasses expression of an antigen and / or epitope of interest as a subunit in vivo without being incorporated into the composition. For example, the present invention encompasses that the composition comprises recombinant vector (s) that express antigens in vivo when administered to an animal. The vector can include DNA plasmids, herpesviruses, adenoviruses, or poxviruses including vaccinia viruses, avipox viruses, canarypox viruses, swinepox viruses and the like. Vector-based compositions can include vectors that contain and express the antigen to be expressed, eg, Cp15 / 60 and / or Cp23 nucleotide sequences of Cryptosporidium parvum.

  The term plasmid is intended to include any DNA transcription unit in the form of a polynucleotide sequence containing the sequence to be expressed. Advantageously, this plasmid contains the elements necessary for its expression, eg in vivo expression. Whether supercoiled or not, the form of a circular plasmid is advantageous. Linear ones are also included in the scope of the present invention. This plasmid may be a naked plasmid, for example a lipid or a liposome, for example a plasmid incorporated inside a cationic liposome (eg WO90 / 11082, 92/19183, 96/21797 and 95/20660). The plasmid immune composition or vaccine composition can be expressed using a gene gun, or intramuscularly, or nasally, or in vivo, preferably any other means that allows an immune or protective response. Can also be administered. Also, U.S. Patent Application Nos. 09/232278, 09/232468, 09/232477, 09/232279, 09, each filed on Jan. 15, 1999 (incorporated herein by reference). Nos./232478 and 09/232469, and U.S. Patent Application Nos. 60 / 138,352 and 60 / 138,478, each filed June 10, 1999 (incorporated herein by reference). See Since such applications include DNA, and / or vector vaccine compositions, immune compositions, or immunogenic compositions for cats, dogs, cows and horses, the compositions of the present invention include the DNA and / or vectors of such applications. Or vector vaccine compositions, immunogenic compositions, or immunological compositions and / or compositions of the invention may be prepared and / or formulated and / or formulated in a manner similar to the compositions of such applications. Or it can be administered.

  The compositions used in the present invention can be prepared according to standard techniques well known to those skilled in the veterinary, pharmaceutical or medical fields. Such compositions may be administered by doses and techniques well known to those skilled in the veterinary art, taking into account factors such as the age, sex, weight, condition and specific treatment of the animal and the route of administration. it can. The components of the composition of the present invention can be administered alone, or with other compositions, or other prophylactic or therapeutic compositions (eg, other immunogenic compositions, immune compositions) Or vaccine composition) (eg, Cryptosporidium parvum antigen (s) and / or epitope (s) alone) followed by other intestinal tract Pathogen antigen (s) and / or epitope (s) can be administered sequentially or compositions containing enteropathogen antigen (s) or epitope (s) Can also include vectors or recombinants or plasmids that express the same or different enteropathogen antigen (s) or epitope (s)). Accordingly, the present invention provides multivalent compositions, “cocktail” compositions or mixed compositions, and methods of using those compositions. Administration components and administration methods (sequential administration, eg as part of a prime-boost regimen or booster program once a year, seasonally, twice a year, etc.) As part of a booster program to administer the product or vaccine composition regularly throughout the life of the animal, or at the same time) It can also be determined taking into account factors such as the route of administration. In this regard, see rabies and mixed compositions, and US Pat. No. 5,843,456, directed to their use, incorporated herein by reference.

  The compositions of the invention can be used for parenteral or mucosal administration, preferably by intradermal, subcutaneous or intramuscular routes. When using mucosal administration, the oral, nasal, or vaginal route can be used.

  In such compositions, the vector (one or more), antigen (one or more), or epitope (one or more) of the subject (one or more), sterile water, saline, glucose, etc. Can be mixed with a suitable carrier, diluent or excipient. These compositions can also be lyophilized. These compositions can contain adjuvants such as pH buffers, adjuvants, preservatives, polymer excipients used in the mucosal route, depending on the desired route of administration and method of preparation.

  With reference to standard text such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, suitable formulations can be prepared without undue experimentation. Appropriate dosages can also be based on this text and the references cited therein.

  An adjuvant is a substance that enhances the immune response to an antigen. Adjuvants include, among others, aluminum hydroxide and phosphates, saponins such as Quil A, mineral oil emulsions; pluronic polymers including mineral or metabolizable oil emulsions; water-in-oil adjuvants, oil-in-water adjuvants, synthetic polymers (Eg, homopolymers and copolymers of lactic acid and glycolic acid, Eldridge et al., Mel. Immunol. 28: 287-, used to produce antigen encapsulated microspheres, such as biodegradable microspheres. 294 (1993)), nonionic block copolymers, low molecular weight copolymers in oil-based emulsions (Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, DES), 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 for example US Pat. No. 5,334,379), and GM-CSF (granulocyte macrophage-colony stimulating factor; generally US Pat. No. 4,999,291). And Clark et al., Science 1987, 230: 1229, Grant et al., Drugs, 1992, 53: 516), preferably GM-CSF derived from the species to be vaccinated. included. Certain adjuvants, such as cytokines, GM-CSF, can be expressed in vivo with antigen (s) and / or epitope (s) (eg, CR Maliszewski for bovine GM-CSF). et al. Molec Immunol 25 (9): 843-50 (1988), SR Leong, Vet Immunol and Immunopath 21: 261-78 (1989) The plasmid encoding GM-CSF is the bovine according to the invention. DNAs encoding antigens and / or epitopes thereof from, and optionally including, other bovine pathogen antigens and / or epitopes may be modified or expressed. It can also be used in combination with a plasmid.)

  Further examples of adjuvants are compounds selected from polymers of acrylic acid or methacrylic acid, and copolymers of maleic anhydride and alkenyl derivatives. Preferred adjuvant compounds are in particular polymers of acrylic acid or methacrylic acid which are crosslinked with sugars or polyalkenyl ethers of polyhydric alcohols. Such compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art can also refer to U.S. Pat. No. 2,909,462 (incorporated herein by reference) which describes such acrylic polymers, including at least 3, preferably 8 It is crosslinked with a polyhydroxylated compound having the following hydroxyl groups, wherein the hydrogen atoms of at least 3 hydroxyl groups are replaced by unsaturated aliphatic groups having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl groups, allyl groups, and other ethylenically unsaturated groups. This unsaturated group may itself contain other substituents such as a methyl group. In particular, the product marketed under the name Carbopol® (BF Goodrich, Ohio, USA) is suitable. These products are cross-linked with allyl sucrose or allyl pentaerythritol. Examples thereof include Carbopol® 974P, 934P and 971P. For copolymers of maleic anhydride and alkenyl derivatives, the copolymer EMA® (manufactured by Monsanto), which is a copolymer of maleic anhydride and ethylene, linear or cross-linked, for example with divinyl ether, is preferred. Reference may be made to J. Fields et al., Nature, 186: 778-780, 4 June 1960, which is incorporated herein by reference.

  In view of their structure, polymers of acrylic acid or methacrylic acid, and copolymers EMA® are basic units of the formula

で形成することが好ましく、上式で、Ｒ_１及びＲ_２は、同一であるか又は異なっており、Ｈ又はＣＨ_３を表し、
ｘ＝０又は１、好ましくはｘ＝１であり、且つ
ｙ＝１又は２であり、但しｘ＋ｙ＝２である。

Wherein R ₁ and R ₂ are the same or different and represent H or CH ₃ ,
x = 0 or 1, preferably x = 1, and y = 1 or 2, provided that x + y = 2.

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

When such a polymer is dissolved in water, it becomes an acidic solution, which is preferably neutralized to a physiological pH to make an adjuvant solution, which includes the immunogenic composition, the immune composition, or the vaccine composition itself. At this time, a part of the carboxyl group of the polymer is in the form of COO ⁻ .

  Preferably, a solution of an adjuvant according to the invention, in particular a carbomer, is prepared in distilled water, preferably in the presence of sodium chloride, and the resulting solution has an acidic pH. This stock solution is added in several portions at once (in order to obtain the desired final concentration) to the desired or nearly desired amount of NaCl-added water, preferably saline (NaCl 9 g / l). Dilute and, at the same time or later, neutralize preferably with NaOH (pH 7.3-7.4). This solution of physiological pH can be used as is and mixed with the vaccine, which can be stored in particular in lyophilized, liquid or frozen form.

  The polymer concentration in the final vaccine composition is 0.01% -2% (w / v), for example 0.06-1% (w / v), 0.1-0.6% (w / v), etc. It can be.

  Immunogenic compositions and vaccine compositions containing adjuvants according to the present invention are also emulsions, especially oil-in-water emulsions such as “Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell, M. Newman, Plenum Press. They can be produced by blending them in the form of an emulsion such as the SPT emulsion described on page 147 of 1995, or the emulsion MF59 described on page 183 of the same document. Specifically, oil-in-water emulsions are light liquid paraffin oils (according to European Pharmacopoeia); isoprenoids such as squalane, squalene; oils obtained by oligomerization of alkenes, especially isobutylene or decene; linear Acids or alcohol esters having an alkyl group of, in particular, vegetable oil, ethyl oleate, di (caprylic / capric) propylene glycol, tri (caprylic / capric) glycerol, or propylene glycol dioleate; branched fatty acids or alcohols Of these esters, in particular isostearic acid esters. This oil is used in combination with an emulsifier to form an emulsion. Emulsifiers are nonionic surfactants, especially sorbitan esters, mannide esters, glycerol esters, polyglycerol esters, propylene glycol esters or esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid Some are preferably ethoxylated, block copolymers such as polyoxypropylene-polyoxyethylene, especially a product called Pluronic, ie Pluronic L121.

  With the present disclosure and knowledge in the art, those skilled in the art are suitable for use in an immune composition, immunogenic composition, or vaccine composition according to the present invention as needed without undue experimentation. An adjuvant and its amount can be selected.

  An immune composition, immunogenic composition, or vaccine composition according to the present invention is converted to at least one immunity of interest derived from at least one live attenuated vaccine, inactivated vaccine, or subunit vaccine, or other pathogen Recombinant vaccines that express the original, antigen, or epitope (eg, poxvirus as a vector or DNA plasmid) can be combined.

Compositions in forms for various routes of administration are contemplated by the present invention. Furthermore, the effective dose and route of administration will depend on known factors such as age and weight. The dose of each active agent, for example, each Cryptosporidium parvum antigen or epitope of interest, and / or each antigen or epitope derived from each enteric pathogen can be found in the literature cited herein, or other And / or in the range of 1 or several micrograms to hundreds or thousands of micrograms, for example, immunogenic compositions, immune compositions, or vaccine compositions of subunits In the case of a product, 1 μg to 1 mg, in the case of an inactivated immunogenic composition, an inactivated immunity composition, or an inactivated vaccine composition, 10 ⁴ to 10 ¹⁰ TCID ₅₀ , preferably 10 it can be in the range of ^{6 ~10 8} TCID _50.

The recombinant or vector can be administered in an amount suitable to obtain in vivo expression corresponding to the administration described herein and / or references cited therein. For example, a suitable range for a virus suspension can be determined empirically. The viral vector or recombinant of the present invention is at least about 10 ³ pfu, more preferably from about 10 ⁴ pfu to about 10 ¹⁰ pfu, such as from about 10 ⁵ pfu to about 10 ⁹ pfu, such as from about 10 ⁶ pfu to about 10 ^8. Animals can be administered in an amount of pfu, or can be infected or transfected into cells. The dose is usually in the unit dosage range of about 10 ⁶ to about 10 ¹⁰ , preferably about 10 ¹⁰ pfu, advantageously about 10 ⁸ pfu per dosage of about 1 ml to about 5 ml, advantageously about 2 ml. It is. When multiple gene products are expressed by multiple recombinants, each recombinant can be administered in such amounts, or when combined, the total amount of recombinants will be such amounts. Each recombinant can also be administered. The dosage of the plasmid composition used in the present invention can be as described in the literature cited herein or as described herein. Advantageously, this dosage elicits a response similar to a composition in which the antigen (s) or epitope of interest is directly present, or is similar to the administration of such a composition The amount of plasmid should be sufficient to produce an expression of or similar to that obtained in vivo by the recombinant composition. For example, an appropriate amount of each plasmid DNA in the plasmid composition can be 1 μg to 2 mg, preferably 50 μg to 1 mg. A person skilled in the art can determine other suitable administrations for the DNA plasmid vector composition of the present invention without undue experimentation, with reference to the DNA plasmid vector literature cited herein. Can do.

  However, the dose of the composition (s), the concentration of its components, and the timing of administering the composition (s) that elicit an appropriate immune response can be determined by serum antibody titers, eg, It can be determined by methods such as ELISA and / or seroneutralization and / or seroprotection assay analysis. Given the knowledge of those skilled in the art, the present disclosure, and the references cited herein, such determination does not require undue experimentation. In addition, the time for sequential administration can be determined without undue experimentation by methods that can be determined from the present disclosure and knowledge in the art as well.

  It is preferred that the mixed immune composition, mixed immunogenic composition, or mixed vaccine composition of enteric pathogens comprises both Cryptosporidium parvum antigens as defined above.

  Cryptosporidium antigen (s) or epitope (s) (preferably P21 and / or Cp23 and / or Cp15 / 60 and / or CP41, such as P21 or Cp23 and Cp15 / 60, or epitopes thereof Intestinal pathogen antigens or epitopes that are advantageously combined with (one or more) are one or more of the genus Clostridium such as E. coli and / or rotavirus and / or coronavirus and / or Clostridium perfringens It preferably contains an antigen or epitope of interest. Preferably, the antigen from E. coli comprises one, preferably several (plural), more preferably all of the antigens called K99, F41, Y, 31A and / or their epitopes. Preferred antigens are K99 and F41. Thus, the composition advantageously comprises one of K99 and F41, preferably both. It is preferred that the composition also comprises Y and / or 31A, advantageously Y and 31A. For example, such an antigen may be incorporated as a subunit or possessed by E. coli. Preferably, the composition according to the present invention comprises E. coli having K99 antigen, E. coli having F41 antigen, E. coli having Y antigen, E. coli having 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, and any of them. At least one antigen selected from the group consisting of:

  As described herein, E. coli can be used to produce Cryptosporidium parvum antigens or epitopes. The Cryptosporidium parvum antigen or epitope can be expressed in an E. coli strain that expresses at least one of the various E. coli antigens so that the E. coli antigen and Cryptosporidium parvum antigen are co-expressed. In the case of in vitro expression, the cells can then be divided as usual and the E. coli antigen and Cryptosporidium parvum antigen, or epitope can be recovered. Advantageously, if the antigen or epitope is expressed internally, other than on the surface, the antigen or epitope is expressed as a fusion protein or with a tag, such as a HIS tag. For in vivo expression, it is advantageous to link a nucleic acid molecule that expresses the expressed antigen or epitope with a signal sequence so that the antigen or epitope is expressed extracellularly, and this E. coli is non-pathogenic It is advantageous. Thus, in some embodiments, E. coli can be a vector and an antigen or epitope thereof.

  The antigen derived from C. perfringens is preferably a C-type and / or D-type toxoid, more preferably a C-type and D-type toxoid.

  One specific aspect of the present invention is a mixed immunity composition, mixed immunogenic composition, or mixed vaccine composition of bovine enteric pathogens, which composition preferably comprises Cryptosporidium parvum At least one antigen or epitope from at least one Cryptosporidium protozoa, preferably P21 and / or Cp23 and / or Cp15 / 60 and / or CP41 and / or its subject, such as P21 or Cp23 and Cp15 / 60 An epitope and preferably at least one antigen or epitope from each pathogen of E. coli, bovine rotavirus, bovine coronavirus and Clostridium perfringens, or at least one antigen or epitope from E. coli, rotavirus, and coronavirus Including these pathogens or combinations thereof Senado, and at least one antigen or epitope from additional at least one calf intestinal pathogens. Regarding the epitope of the desired antigen of interest and the method of determining which part of the antigen is the epitope of interest, U.S. Patent No. 5990091, and U.S. Patent Application Nos. 08 / 675,565, 08 / 675,556. As well as other references cited herein. With the disclosure of the present specification and knowledge in the art, such as references cited herein, the epitope of interest is determined, or the antigen (s) and / or epitope (s) And / or undue experimentation is not required to formulate the composition of the invention comprising the vector (s) expressing the antigen (s) and / or the epitope (s).

  According to a preferred embodiment, the present invention provides an immune composition, immunogenic composition, or vaccine composition of bovine enteric pathogens comprising the antigens K99, F41, Y, and 31A, as well as the E. coli antigens described herein, such as bovine inactivated coronavirus, bovine inactivated rotavirus. The composition may further comprise C. perfringens type C and D toxoids. Preferably, the E. coli valency is inactivated E. coli with K99 antigen, inactivated E. coli with F41 antigen, inactivated E. coli with Y antigen, and inactivated E. coli with 31A antigen, or K99 antigen, F41 antigen, Y Any one of the antigen and 31A antigen.

  Another aspect of the present invention is an immune composition, immunogenic composition, or vaccine composition against Cryptosporidium parvum, which composition comprises Cp23 or P21 and Cp15 / 60 antigens or epitopes thereof, And an acceptable vehicle.

  According to an advantageous embodiment, such antigens are incorporated into the composition as protein or subunit antigens. Such antigens can be produced by chemical synthesis or in vitro expression. For convenience in production by expression in a suitable host and recovery of the antigen, it is preferred that such antigen be in the form of a fusion protein (eg, with a HIS tag). That is, the antigen can include the antigen itself and foreign amino acids.

  According to other embodiments, such antigens are not incorporated into the composition as subunits, and the composition expresses the antigen (s) or epitope (s) in vivo when administered to an animal. A recombinant vector expressing Cp23 or P21 and Cp15 / 60 or an epitope thereof, or a recombinant vector expressing Cp23 or P21 or an epitope thereof, and a recombinant vector expressing Cp15 / 60 or an epitope thereof including. The composition can include a vector that expresses one antigen or epitope and another antigen or epitope.

  A further aspect of the present invention provides for vaccination of administering to a target animal a mixed immunity composition, mixed vaccine composition, mixed immunity composition, or mixed vaccine composition of enteric pathogens against Cryptosporidium parvum according to the present invention. Is the method. The present invention can relate to a method of immunizing a newborn calf against intestinal disease, which method comprises Cp23 or P21 and Cp15 / 60 Cry such that the newborn calf has a transition antibody against Cryptosporidium parvum. Administering an immune or vaccine composition comprising a Ptosporidium parvum antigen or epitope thereof and a pharmaceutically acceptable vehicle to a pregnant or pre-natal heifer before delivery. Preferably, the method comprises providing colostrum and / or milk to a newborn calf from a cow so vaccinated, such as a dam. In vaccination or immunization against intestinal diseases, not only mixed vaccine compositions, mixed immunogenic compositions, or mixed immunogenic compositions containing various titers are used, but also vaccine compositions, immunogenic compositions Alternatively, the immune composition can be divided and administered separately, eg, sequentially, or mixed prior to use.

  Antigens and epitopes of interest useful in the compositions and methods of the present invention can be obtained using any method available to those skilled in the art, for example, US Pat. No. 5,591,434 and WO 98/07320. Can be produced using the method in the number. In addition, antigens of other enteric pathogens can be obtained from commercial sources such as TRIVACTON® 6. For example, Cp23 and / or P21 and / or Cp15 / 60 or epitopes thereof, such as P21 or Cp23 and Cp15 / 60 or epitopes thereof, or such antigen (s) or epitope (s) The expression vector can be added to TRIVACTON® 6 in the amount specified herein. Advantageously, Clostridium perfringens type C and D toxoids can be added to TRIVACTON® 6. Further, in TRIVACTON (registered trademark) 6, inactivated Escherichia coli having pili can be replaced with the separated pili. Also, such a vaccine composition to which Cryptosporidium parvum antigen (s) or epitope (s) or epitope (s) and / or Clostridium perfringens antigen (s) or epitope (s) are added Also included in the present invention are methods, immunogenic compositions, or immune compositions (having inactivated E. coli or isolated pili) and methods of making and using such compositions and kits thereof.

  Further, E. coli valency and / or antigen (s) and / or epitope (s) useful for practicing the present invention are described in European Patent Application Publication No. 80412, European Patent Application Publication No. No. 60129, British Patent Application No. 2094314, and U.S. Pat.Nos. 4,298,597, 5,804,198, 4,787,056, 3,975,517, 4,237,115, 3,907,987, 4,338,298, 4,443,547, 4,343,792, See 4788056 and 4311797. For rotavirus antigen (s) and / or epitope (s), see PS Paul and YS Lyoo, Vet Microb 37: 299-317 (1993) and US Pat. Nos. 3,914,408 and 5,620,896. See See WO 98/40097, WO 96/41874, and US Pat. Nos. 3,914,408 and 3,919,413 for coronavirus antigen (s) and / or epitope (s) That. For Clostridium spp., For example, Clostridium perfringens antigen (s) and / or epitope (s), WO 94/22476, European Patent Application Publication No. 734731, WO 98/27964 UK Patent Application Publication No. 20508830, UK Patent Application Publication No. 1128325, 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. Pat. Nos. 5,178,860, 4,981,684 and 4,292,307, and IMOTOXAN (registered trademark) (manufactured by MERIAL, Lyon, France) (types B, C, D-type C. perfringens, Cl. Septicum), See Cl. Novyi, Cl. Tetani, and Cl. Chauvoei culture toxoids). Furthermore, in addition to TRIVACTON® 6, other commercially available combination vaccines that can add Cryptosporidium parvum valency according to the present invention, such as bovine inactivated rotavirus and coronavirus, K99 E. coli Bacterin (bacterin) And SCOURGUARD 3 (K) / C (registered trademark) (manufactured by SmithKline Beecham) containing Clostridium perfringens type C toxoid can also be used.

  A preferred method of obtaining the antigen or epitope of interest is to clone the DNA sequence encoding the antigen or epitope of interest into a fused or non-fused plasmid and express it in E. coli. A fusion plasmid (eg, expressing a tagged antigen (s) or epitope (s) such as a His tag) is preferred because it allows for easy recovery of the produced antigen. Suitable plasmids are described in the examples. Production of antigens by chemical synthesis is also within the scope of the present invention.

  The present invention further uses the antigens or epitopes described herein or vectors expressing such antigens or epitopes, eg, the antigens, epitopes, or vectors, suitable or acceptable carriers. Or a method of preparing a vaccine composition, immune composition, or immunogenic composition, for example against Cryptosporidium parvum or intestinal disease, by mixing with a diluent and optionally an adjuvant. The composition can also be lyophilized and dissolved. The present invention further includes kits for preparing the compositions of the present invention. The kit can include the antigen (s), epitope (s), and / or vector (s), carrier, and / or diluent, and optionally an adjuvant. The components can be placed in separate containers. The container containing the components may be in one or more packages, and the kit may further mix the components and / or vaccine composition, immunogenic composition, or immune composition. Instructions for administering can be included.

  Other aspects of the invention provide, for example, the composition (s) of the invention (eg, a Cryptosporidium parvum composition or a mixed enteropathogen composition according to the invention) at least once, advantageously Hyperimmune colostrum and / or milk is produced by hyperimmunization of a pregnant female animal (such as a cow) by administration at least twice, more advantageously at least three times. In some cases, however, advantageously, the colostrum and / or milk thus produced is then processed to concentrate the immunoglobulins to produce the desired immune response, immunogenic response, and / or vaccine response. Alternatively, components of colostrum or milk that do not contribute to the nutritional value of colostrum or milk can be removed. Advantageously, this treatment may comprise coagulating the colostrum or milk using, for example, rennet and a liquid phase containing the recovered immunoglobulin. The invention also encompasses compositions comprising hyperimmune colostrum or milk or mixtures thereof, and / or hyperimmune colostrum or milk or mixtures thereof. Furthermore, the present invention relates to hyperimmune colostrum or milk or a mixture thereof, or a composition comprising the same, using young animals such as neonates, such as calves Cryptosporidium parvum and / or intestinal infections. Is supposed to prevent or treat.

The following examples are set forth to provide a person skilled in the art with a complete disclosure and description of how to make and use the cells of the present invention, and the scope of what the present inventors regard as the present invention. It is not intended to limit.
[Example]

Sequence list:
SEQ ID NO: 1 Oligonucleotide JCA295
SEQ ID NO: 2 Oligonucleotide JCA296
SEQ ID NO: 3 oligonucleotide JCA297
SEQ ID NO: 4 Oligonucleotide JCA298
SEQ ID NO: 5 Oligonucleotide JCA299
SEQ ID NO: 6 Oligonucleotide JCA300
SEQ ID NO: 7 oligonucleotide JCA301
SEQ ID NO: 8 Oligonucleotide JCA302
SEQ ID NO: 9 Oligonucleotide JCA303
SEQ ID NO: 10 oligonucleotide JCA304

All plasmid constructs, Sambrook J. et al. (Molecular Cloning:..... A Laboratory Manual 2 nd Edition Cold Spring Harbor Laboratory Cold Spring Harbor New York 1989) as described in the standard molecular biology Made using techniques (cloning, restriction digestion, polymerase chain reaction (PCR)). All DNA restriction fragments and PCR fragments generated and used for the present invention were isolated and purified using the “Geneclean®” kit (BIO101, La Jolla, Calif.).

Cloning of Cryptosporidium parvum P21 and Cp15 / 60 genes
Cryptosporidium parvum oocysts are isolated from infected calves and purified from bovine fecal samples as described by Sagodira S. et al. (Vaccine. 1999. 17. 2346-2355). The purified oocysts are then stored at + 4 ° C. in distilled water. Genomic DNA is released from purified oocysts as described by Iochmann S. et al. (Microbial Pathogenesis 1999. 26. 307-315) for use as a template for PCR reactions.

  Alternative sources of Cryptosporidium parvum DNA are Cryosporidium parvum Iowa (A), Iowa (I), KSU-1, and KSU available from ATCC (American Tissue Culture Collection) -2 isolates (ATCC numbers 87667, 87668, 87439 and 87664, respectively). Specific P21 and Cp15 / 60 genes are isolated as follows.

Cryptosporidium parvum DNA, as well as primers, ie oligonucleotide JCA295 (35mer) SEQ ID NO: 1
5 'TTT TTT CCA TGG GGC TCG AGT TTT CGC TTG TGT TG 3'
And oligonucleotide JCA296 (33mer) SEQ ID NO: 2
5 'TTT TTT GAA TTC TTA GGC ATC AGC TGG CTT GTC 3'
Is used to amplify the sequence encoding the P21 protein by polymerase chain reaction (PCR).

  This PCR generates a fragment of a PCR fragment of about 585 bp. Next, this PCR fragment is digested with restriction enzymes NcoI and EcoRI, and after agarose gel electrophoresis, a 575 bp NcoI-EooRI restriction fragment (= fragment A) is isolated and recovered using a GeneClean kit (manufactured by BIO101). The sequence of this fragment encodes a protein homologous to the sequence set forth as SEQ ID NO: 12 in WO 98/07320 (PCT / US97 / 14834).

A second PCR is performed to amplify the sequence encoding the Cp15 / 60 protein and add restriction sites at the 5 ′ and 3 ′ ends for later cloning. Cryptosporidium parvum DNA, as well as primers, ie oligonucleotide JCA297 (35mer) SEQ ID NO: 3
5 'TTT TTT CTC GAG ATG GGT AAC TTG AAA TCC TGT TG 3'
And oligonucleotide JCA298 (42mer) SEQ ID NO: 4
5 'TTT TIT GAA TTC TTA GTT AAA GTT TGG TTT GAA TTT GTT TGC 3'
Perform this PCR using

  This PCR generates a fragment of about 465 bp. This fragment is purified and then digested with XhoI and EcoRI. After agarose gel electrophoresis, a 453 bp XhoI-EcoRI fragment (= fragment B) is collected and collected using a GeneClean kit (manufactured by BIO101). This amplified sequence is homologous to the sequence defined by nucleotides # 31 to # 528 of SEQ ID NO: 1 in US Pat. No. 5,591,434, and the sequence deposited with GenBank as accession numbers U22892 and AAC47447.

Construction of plasmid pJCA155 (GST-P21 fusion protein in vector pBAD / HisA)
The sequences necessary to express the GST-P21 fusion protein are amplified by PCR and easily cloned into the pBAD / HisA expression plasmid vector (Cat. No. V430-01, InVitrogen, 92008 Carlsbad, CA, USA). Generate two types of fragments. Plasmid pGEX-2TK (Catalog No. 27-4587-01, manufactured by Amersham-Pharmacia Biotech), and primer, ie, oligonucleotide JCA299 (35mer) SEQ ID NO: 5
5 'TTT TTT CCA TGG GGT CCC CTA TAC TAG GTT ATT GG 3'
And oligonucleotide JCA300 (45mer) SEQ ID NO: 6
5 'TTT TTT CTC GAG CCT GCA GCC CGG GGA TCC AAC AGA TGC ACG ACG 3'
Is used to perform the first PCR.

  This PCR generates an approximately 720 bp fragment encoding the GST moiety with an NcoI restriction site added to the 5 'end for cloning into pBAD / HisA. This modification adds a glycine codon to the GST-P21 fusion protein). Next, this PCR fragment is digested with NcoI and XhoI, and after agarose gel electrophoresis, a 710 bp NcoI-XhoI fragment (= fragment C) is collected and collected using a GeneClean kit (manufactured by BIO101).

Cryptosporidium parvum DNA, as well as primers, oligonucleotide JCA301 (33mer) SEQ ID NO: 7
5 'TTT TTT CTC GAG TTT TCG CTT GTG TTG TAC AGC 3'
And oligonucleotide JCA296 (33mer) SEQ ID NO: 2
A second PCR is performed using

  This PCR generates an approximately 580 bp fragment encoding the P21 portion with XhoI and EcoRI restriction sites added to the 5 'and 3' ends, respectively. Next, this PCR fragment is digested with Xhol and EcoRl, and after agarose gel electrophoresis, a 572 bp XhoI-EcoRI fragment (= fragment D) is collected and collected using a GeneClean kit (manufactured by BIO101).

  The plasmid pBAD / HisA (catalog number V430-01, manufactured by InVitrogen) is digested with NcoI and EcoRI. This digested fragment is separated by agarose gel electrophoresis, and a # 3960 bp NcoI-EcoRI restriction fragment (= fragment E) is recovered (GeneClean kit, manufactured by BIO101).

  Fragments C, D and E are then ligated to create plasmid pJCA155. This plasmid has a total size of 5243 bp (FIG. 1) and encodes a 425 amino acid GST-P21 fusion protein.

Construction of plasmid pJCA156 (His6-P21 fusion protein in vector pBAD / HisA)
The vector pBAD / HisA (catalog number V430-01, manufactured by 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. .

  PCR is performed to amplify the sequence encoding the His6-P21 fusion, add NcoI and EcoRI restriction sites to the 5 ′ and 3 ′ ends, respectively, and subclon this PCR fragment into the plasmid vector pBAD / HisA. .

Cryptosporidium parvum DNA, as well as primers, ie oligonucleotide JCA302 (65mer) SEQ ID NO: 8
5 'TIT TTT CCA TGG GGG GTT CTC ATC ATC ATC ATC ATC ATG GTC TCG AGT TTT CGC TTG TGT TGT AC 3'
And oligonucleotide JCA296 (33mer) SEQ ID NO: 2
Perform PCR using.

  This PCR generates a fragment of about 610 bp. This fragment is purified and then digested with NcoI and EcoRI. After agarose gel electrophoresis, a 600 bp NcoI-EcoRI fragment (= fragment F) is isolated and recovered using a GeneClean kit (manufactured by BIO101).

  Fragments E and F are ligated to create plasmid pJCA156. This plasmid is 4562 bp in total size (FIG. 2) and encodes a 199 amino acid His-6 / P21 fusion protein.

Construction of plasmid pJCA157 (single P21 protein in vector pBAD / HisA)
The vector pBAD / HisA (catalog number V430-01, manufactured by 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 3. .

PCR is performed to amplify the sequence encoding the P21 protein, add NcoI and EcoRI restriction fragments to the 5 ′ and 3 ′ ends, respectively, and subclone this PCR fragment into the plasmid vector pBAD / HisA. Cryptosporidium parvum DNA, as well as primers, ie oligonucleotide JCA295 (35mer) SEQ ID NO: 1
And oligonucleotide JCA296 (33mer) SEQ ID NO: 2
Is used to obtain a 575 bp NcoI-EcoRI fragment (fragment A) as described in Example 1.

  Fragments E and A are ligated to create plasmid pJCA157. This plasmid is 4535 bp in total size (FIG. 3) and encodes 189 amino acids including the P21 protein.

Construction of plasmid pJCA158 (GST-Cp15 / 60 fusion protein in vector pBAD / HisA)
PCR is performed to amplify the sequence encoding the GST protein, add convenient restriction sites at the 5 ′ and 3 ′ ends, and subclone this PCR fragment into the final plasmid vector pBAD / HisA. In this PCR, the DNA of plasmid pGEX-2TK (Catalog No. 27-4587-01, manufactured by Amersham-Pharmacia Biotech) as a template, and a primer, ie, oligonucleotide JCA299 (35mer) SEQ ID NO: 5
And oligonucleotide JCA300 (45mer) SEQ ID NO: 6
Is used to obtain a 710 bp NcoI-XhoI fragment (= fragment C) as described in Example 2.

  The vector pBAD / HisA (catalog number V430-01, manufactured by 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 to create plasmid pJCA158. This plasmid is 5132 bp in total size (FIG. 4) and expresses a 388 amino acid GST-Cp15 / 60 fusion protein.

Construction of plasmid pJCA159 (His6-Cp15 / 60 fusion protein in vector pBAD / HisA) Vector pBAD / HisA (catalog number V430-01, manufactured by InVitrogen) was digested with NcoI and EcoRI and # 3960 bp as described in Example 2 The NcoI-EcoRI restriction fragment (= fragment E) is recovered and isolated.

PCR is performed to amplify the sequence encoding the His6-Cp15 / 60 fusion, add convenient restriction sites at the 5 ′ and 3 ′ ends, and subclone the PCR fragment into the plasmid vector pBAD / HisA. Cryptosporidium parvum DNA, as well as primers, ie oligonucleotide JCA303 (64mer) SEQ ID NO: 9
5 'TTT TTT CCA TGG GGG GTT CTC ATC ATC ATC ATC ATC ATG GTA TGG GTA ACT TGA AAT CCT GTT G 3'
And oligonucleotide JCA298 (42mer) SEQ ID NO: 4
Perform PCR using any of the above.

  This PCR generates a fragment of about 495 bp. This fragment is purified and then digested with NcoI and EcoRI to obtain a 483 bp NcoI-EcoRI fragment (= fragment G) after agarose gel electrophoresis, which is recovered with the GeneClean kit (manufactured by BIO101).

  Fragments E and G are ligated to create plasmid pJCA159. This plasmid is 4445 bp in total size (FIG. 5) and expresses a 159 amino acid His-6 / Cp15 / 60 fusion protein.

Construction of plasmid pJCA160 (single Cp15 / 60 protein in vector pBAD / HisA) Vector pBAD / HisA (catalog number V430-01, manufactured by InVitrogen) was digested with NcoI and EcoRI and # 3960 bp as described in Example 2. NcoI-EcoRI restriction fragment (= fragment E) is recovered and isolated.

  PCR is performed to amplify the sequence encoding the Cp15 / 60 protein, add convenient restriction sites at the 5 'and 3' ends, and subclone this PCR fragment into the plasmid vector pBAD / HisA.

Cryptosporidium parvum DNA, as well as primers, ie oligonucleotide JCA304 (31mer) SEQ ID NO: 10
5 'TTT TTT CCA TGG GTA ACT TGA AAT CCT GTT G 3'
And oligonucleotide JCA298 (42mer) SEQ ID NO: 4
Perform PCR using.

  This PCR generates a fragment of about 460 bp. This fragment is purified and then digested with NcoI and EcoRI to obtain a 450 bp NcoI-EcoRI fragment (= fragment H) after agarose gel electrophoresis, and this fragment is recovered with the GeneClean kit (manufactured by BIO101).

  Fragments E and H are ligated to create plasmid pJCA160. This plasmid is 4412 bp in total size (FIG. 6) and expresses a 148 amino acid Cp15 / 60 protein.

Cultivation of E. coli recombinant clone and induction of recombinant protein Plasmid DNA (Examples 2 to 7) is used for other well known to those skilled in the art such as E. coli DH5α (or E. coli TOP10 (Catalog No. C4040-03, InVitrogen)). Any suitable E. coli strain K12) is transformed and grown on LB (Luria-Bertani) agar plates containing 50 μg / ml ampicillin. One colony is picked for each plasmid transformed with the E. coli population, placed in 10 ml LB medium containing ampicillin (or other suitable antibiotic) and grown overnight. 1 ml of the overnight culture is added to 1 liter of LB medium and grown at + 30 ° C. until the OD _{600 nm} reaches about 3.0.

  DL-arabinose (catalog number A9524, Sigma, St. Louis, MO) at various final concentrations (determining the concentration for optimal yield in the range of 0.002% to 0.2%) to the culture Add and incubate at + 30 ° C. for 4-6 hours to induce protein production.

Extraction and purification of recombinant fusion protein At the end of this induction (Example 8), cells were harvested by centrifugation (3000 g, 10 min at + 4 ° C.) and lysis buffer (50 mM Tris pH 8.0, 1 mM EDTA). 1 μM PMSF, 1 mg / ml lysozyme), and disrupted by sonication for 30 seconds, with a 1 minute rest and 25 times. Add Triton X-100 to a final concentration of 0.1%. Remove debris by centrifugation.

  If desired, alternative techniques (known to those skilled in the art) can be used to lyse the bacterial cells.

9.1. GST-fusion recombinant protein Glutathione-agarose (Catalog No. G4510, Sigma) or Glutathione-Sepharose 4B (Catalog No. 17-0756-01, Amersham-Phharmacia Biotech) as described in Example 8. Recombinant GST-fusion proteins (produced by E. coli transformed with plasmids pJCA155 or pJCA158) were affinity purified from bacterial lysates prepared as described. Bacterial lysate and glutathione-agarose were incubated at + 4 ° C. for 4 hours. Subsequently, GST-fusion protein was eluted batchwise from agarose using 10 mM reduced glutathione (catalog number G4705, Sigma) (K. Johnson and D. Smith Gene. 1988. 67. 31-40) (Anonymous GST gene fusion system :... technical manual 3 rd edition Arlington Heights, IL: see Amersham-Pharmacia Biotech, 1997). Those skilled in the art will be able to scale up this method of purifying large quantities of GST-fusion proteins without undue experimentation with the present disclosure and knowledge in the art.

9.2. His6-fusion recombinant protein
All recombinant His6-fusion proteins were prepared and purified using ProBond ™ nickel-chelate resin (Cat. No. R801-15, InVitrogen) according to the manufacturer's instructions.

Preparation of native E. coli cell lysate (soluble recombinant protein)
E. coli cells are recovered from 1 liter E. coli (transformed with plasmids pJCA156 or pJCA159) by centrifugation (3000 g, 5 min). The pellet is resuspended in 200 ml 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 μg / ml for 15 minutes on ice. The mixture is then crushed for 2-3 seconds, 10 seconds each, with moderate intensity by sonication, leaving the suspension on ice. The mixture is then subjected to a series of freeze / thaw cycles to complete lysis and finally insoluble debris is removed by centrifugation at 3000 g for 15 minutes. The lysate is clarified by filtration through a 0.8 μm filter and stored on ice or at −20 ° C. until purification.

  Using two 100 ml aliquots of the clarified lysate, the soluble recombinant His6-fusion protein present in the lysate was pre-equilibrated in a 50 ml ProBond ™ resin column (Catalog Nos. R640-50 and R801). -15, manufactured by InVitrogen) in a batch manner. The column is gently shaken for 10 minutes to resuspend the resin and allow the polyhistidine tagged protein to bind well. The resin is precipitated by gravity or low speed centrifugation (800 g) and the supernatant is carefully aspirated. Repeat the same cycle with a second aliquot.

Column washing and elution 4 consecutive steps according to manufacturer's instructions (Anonymous. Xpresss ™ System Protein Purification-A Manual of Methods for Purification of Polyhistidine-Containing Recombinant Proteins. InVitrogen Corp. Editor. Version D. 1998) To implement.
1. After washing the column with 100 ml Native Binding Buffer, pH 7.8, the resin is resuspended, shaken for 2 minutes, and then separated into resin and supernatant by gravity or centrifugation. Repeat this procedure two more times (total 3 washes).
2. After washing the column with 100 ml of Native Wash Buffer, pH 5.5, the resin is resuspended, shaken for 2 minutes, and then separated into resin and supernatant by gravity or centrifugation. This procedure is repeated at least three times until OD ₂₈₀ is less than 0.01.
3. After washing the column with 100 ml of Native Wash Buffer, pH 5.5, the resin is resuspended, shaken for 2 minutes, and then separated into resin and supernatant by gravity or centrifugation. Repeat this procedure one more time (total 2 washes).
4). The column is then secured in the vertical position and its lower end cap is removed. The recombinant protein is eluted using 150 ml Native pH Elution Buffer. Collect 10 ml fractions. Elution is monitored by measuring the OD ₂₈₀ value of the fraction.
If desired, the eluted recombinant protein can be concentrated either by dialysis or precipitation with ammonium sulfate.

The final concentration of the recombinant protein batch is measured by the _OD280 value.

  Those skilled in the art will be able to scale up this method of purifying large quantities of His 6 -fusion protein without undue experimentation with the present disclosure and knowledge in the art.

Extraction and purification of Cryptosporidium parvum P21 and Cp15 recombinant non-fusion proteins E. coli cells (transformed with plasmids pJCA157 or pJCA160) were OD _{600 nm in} 4 liters of M9 minimal medium (supplemented with appropriate amino acids). There was incubated at 30 ° C. until reached approximately 3.0 (Sambrook J. et al (Molecular Cloning:...... a Laboratory Manual 2 nd Edition Cold Spring Harbor Laboratory Cold Spring Harbor New York 1989), example 8 The bacterial cells are then ground through a high-pressure RANNIE homogenizer Mini-Lab, model 8.30H, with a maximum flow rate of 10 liters per hour and a working pressure of 0 to 1000 bar. Clarify by filtering with CUNO filter Zeta plus, LP type, then ultrafilter PALL Filtron (reference number OS010G01) UF (ultrafiltration amount ) Concentrate this protein suspension 50 times at 10 kDa using gel filtration chromatography using a High Resolution Sephacryl S-100 gel in an amount corresponding to 2-3% of the column volume. Load onto the chromatography column, elute with PBS buffer, collect the recovered fraction corresponding to the expected molecular weight of the subunit vaccine protein, hollow fiber cartridge A / G Technology, Midge cartridge type, UFP-10-B- Concentrate 10 times with MB01 model (or UFP-10-C-MB01 model or UFP-10-E-MB01 model) Store this concentrated sample at -70 ° C. until use. Parvum recombinant protein can be mixed in an appropriate ratio to the final combined vaccine (see Example 11).

Vaccine formulation, vaccination of pregnant cows, and passive immunization and challenge experiments in newborn calves Products (with or without adjuvant) can be intramuscular (IM), subcutaneous (SQ) or intradermal (ID) to induce a serum antibody response against Cryptosporidium parvum. When administered twice to pregnant animals, a serum antibody response is induced, which is passively transmitted to the newborn through colostrum and milk. Vaccination protocols for pregnant animals are subject to treatment practices (but the following schedule favors maximum efficacy), between the time of pregnancy diagnosis and delivery, for example one month of delivery 2 doses before and about 3-5 days before childbirth, or 2 months before childbirth (corresponding to the dry season of dairy cows) and before childbirth (for example, sometime 1-3 weeks before childbirth ) Can be boosted. In current treatment practice, it is preferred to administer the product during the three months prior to childbirth. The amount of product can be 1-5 ml, for example 2 ml. A combination vaccine has a lyophilized portion and a liquid portion that can be mixed prior to injection. Cryptosporidium antigen can be added as a component of the E. coli / Rota / Corona combination vaccine to provide maximum protection under field conditions.

  Perform the following test.

Test A: Cryptosporidium parvum exacerbates enterovirus and / or enterobacterium pathogenicity.
The experimental challenge uses 3 newborn calves for each group below.
1. 1. Coronavirus only 2. Coronavirus + Cryptosporidium parvum E. coli F41 only 4. 4. E. coli F41 + Cryptosporidium parvum Only Cryptosporidium parvum 6. Untreated control group

  Challenge oral calves within 24 hours after birth. The amount of challenge material used should be the amount necessary to cause clinical symptoms (decreased function, diarrhea, dehydration, etc.) and the type of animal (normally raised by feeding from a purely isolated artificial calf or mother) It can vary depending on the calf). Collect general clinical symptoms (body temperature, appearance, dehydration, clinical score of diarrhea, etc.). In addition, serum and excretion information is collected.

Results Experimental challenges with coronavirus or E. coli F41 unit prices do not produce clinical symptoms of intestinal disease in newborn calves. Double administration of coronavirus or E. coli F41 and Cryptosporidium parvum at a dose of Cryptosporidium parvum that usually does not cause clinical disease causes significant clinical symptoms of intestinal disease.

Test B: Combined vaccine containing Cryptosporidium parvum (E. coli K99 / F41, rotavirus and coronavirus) enhances protection against pathogenesis of intestinal disease by co-infection with multiple enteroviruses and / or enterobacteria in newborn calves To do.
The treatment group is 30 pregnant cows vaccinated as follows.
1. Mixed (Rota and coronavirus, E. coli K99 and F41), 8 heads 2. Mix + Cryptosporidium, 8 heads Untreated control group, 14 experimental challenges are as follows.
1. Multiple challenges (Coronavirus and F41 + asymptomatic Cryptosporidium parvum)
2. Sentry animals Untreated

The calves are fed colostrum (by artificial feeding or fed from the mother) and the calves to be challenged are challenged by oral route within 24 hours of birth. The amount of challenge material is the amount necessary to cause clinical symptoms (eg, as determined in Test A) and, as described in Test A, the type of animal used (pure isolated artificial calf or mother Normal calves that are fed by breastfeeding) Collect general clinical symptoms (body temperature, appearance, diarrhea score, etc.). In addition, serum and excretion information is collected.
Planning 6 calves born from vaccinated (mixed, mixed + cryptosporidium) or control cows were challenged with 3 ingredients (coronavirus and F41 + cryptosporidium parvum) Three calves (born from an unvaccinated control cow) are left as sentinel animals.

Results In the context of multiple challenges (Coronavirus and E. coli F41 + asymptomatic dose of Cryptosporidium parvum), a combined vaccine containing Cryptosporidium parvum provides better protection than the mixed vaccine alone.

Impact of double infection with Cryptosporidium parvum and bovine rotavirus in an experimental challenge model of newborn calves Severity of calf clinical symptoms and feces after unitary challenge with Cryptosporidium parvum or bovine rotavirus This study is designed to compare excretion with calves after double challenge with bovine rotavirus and Cryptosporidium parvum.

  Four groups of six calves are used to collect sufficient data to detect differences in the incidence of clinical symptoms between the four groups.

  Cows are housed individually in barns or small pastures. Newborn calves are pulled away from their dams as soon as possible after birth, inspected to remove feces or dirt attached to the calves, and the umbilical cord is immersed in about 7% iodine tincture. These calves are then immediately transferred to a housing facility and individually housed in a metabolic crate. Challenge calves within 6 hours of birth.

  Calves are given a commercial calf milk replacer containing 30% colostrum substitute, twice daily, in an amount of 1-2 quarts or 10% of body weight per lactation for all tests. Be especially careful to avoid giving milk within 2 hours before and after the challenge.

  Since the natural route of infection is oral, all challenge substances are administered orally using the esophageal tract.

Group A: untreated control calves.
Group B: 1-3 × 10 ⁵ Cryptosporidium parvum oocysts (Beltsville strain) diluted with 60 ml of commercially available soy milk without antibiotics.
Group C: 1-3 × 10 ⁵ Cryptosporidium parvum oocysts (Beltsville strain) diluted with 60 ml of commercial soymilk without antibiotics and 10 ml of bovine rotavirus inoculum (IND BRV G6P5 strain) diluted with PBS 40 ml Simultaneous inoculation with.
Group D: 10 ml of stool filtrate from a calf infected with bovine rotavirus (IND BRV G6P5 strain) diluted with 40 ml of PBS.

  The stool sample is collected once a day after being thoroughly mixed so that a representative sample can be obtained.

  Oocysts are separated from calf stool by centrifugation on a sucrose cushion and counted under a microscope using a cell counter (cytometer). For excretion of rotavirus, the stool is diluted with buffer and the rotavirus antigen is quantified using an ELISA kit from Le Center d'Economie Rurale (CER), 1 rue du Carmel, B6900 Marloie, Belgium.

  The clinical symptoms of the calves are observed twice daily before challenge and then for 10 days after challenge. Observation items include rectal temperature, general condition, loss of appetite, diarrhea, dehydration, and death.

  Hypofunction, diarrhea, and dehydration are classified as follows.

  Anorexia is determined based on whether the calf drinks less than 2 liters of milk. During the first 48 hours after birth, calves can be fed via the esophageal canal. Each calf score is derived daily based on whether each disease category has clinical symptoms (defined as 1) or not (defined as 0). Rectal temperature is recorded at Fahrenheit temperature.

  Two calves died in Group C on days 7 and 8, 2 in Group B on Day 7, 0 in Group D, and 1 on Day 3 in Group A. The results are shown in FIGS. When two microorganisms are administered, a synergistic effect on clinical symptoms and fecal microbial excretion is observed compared to one administration.

Production of bovine colostrum containing antibodies to Cryptosporidium parvum subunit protein C7 (P21) and / or CP15 / 60 expressed by E. coli 6 vaccinated pregnant dairy calves from 4 different herds Randomly assigned to one of the groups: GST-P21, 6His-P21, GST-CP15 / 60, 6His-CP15 / 60, GST-P21 + GST-CP15 / 60, and placebo control. Upon entering the dry period, each cow was given 3 doses of the assigned vaccine subcutaneously every 14 days at a dose of 5 ml. Colostrum was collected from each cow three times during the first 24-36 hours after delivery and labeled. Ten to 20 ml samples were collected and the remainder was frozen in individual containers at each collection. Colostrum total IgG levels were tested by RIDA. Antibodies of P21 and Cp15 / 60 subunit proteins were tested by ELISA. Serum analysis by ELISA was performed on the same subunit protein antibody both immediately after vaccination and at birth. Feces were collected before vaccination and tested for the presence of Cryptosporidium parvum using the ProSpect test kit. All samples tested were negative for Cryptosporidium parvum.

Colostrum antibody against P21 An antibody response specific for P21 was detected in all groups inoculated with P21 antigen. In contrast, no antibody response to P21 could be detected in the group inoculated with CP15 / 60 and in the placebo group (see FIG. 14). Interestingly, the “mixed” group (vaccinated with 0.25 mg GST-P21 in combination with 0.25 mg GST-CP15 / 60) is the GST-P21 unit price group (0.5 mg GST-P21). It showed a very similar P21 response when compared to vaccination). The group receiving 0.5 mg His-P21 had a slightly but always lower P21 response than the group receiving 0.5 mg GST-P21, with the greatest difference being seen at the second milking. Further analysis of the individual values revealed that in the His-P21 group, two non-responding cows (G418 and M26) and abnormal values at the second milking (first milking = 0.055, second milking = 0.296, It was shown to contain a cow J54 showing the third milking = 0.055). Note also that all non-responding cows have very low total IgG levels. Excluding values corresponding to non-responding animals and abnormal values from this analysis, the group mean of the His-P21 group is very similar to the GST-P21 group.

Colostrum antibody against CP15 / 60 Antibody responses specific for CP15 / 60 were detected in all groups containing CP15 / 60 antigen (FIG. 15). In contrast, no antibody response to Cp15 / 60 could be detected in the group inoculated with P21 or in the placebo group. The His-CP15 / 60 group and the group containing 0.25 mg GST-P21 in combination with 0.25 mg GST-CP15 / 60 (mixed group) showed very similar responses. The GST-CP15 / 60 unit price group had twice as much GST-CP15 / 60 antigen (0.5 mg vs. 0.25 mg) as the mixed group, but the response was always lower than the mixed group. Cows inoculated with unit cost GST-CP15 / 60 produce lower quality colostrum compared to mixed groups, but this unexpected result is assumed to be due to genetic differences between these two groups .

Serum antibodies to P21 On day 0, all groups were seronegative. The group vaccinated with P21 all showed similar antibody responses specific for P21, except for the His-P21 group, whose antibody levels were significantly lower on day 14 than the GST-P21 group (see FIG. 16). . In contrast, no antibody response to P21 could be detected in the CP15 / 60 inoculated group and the placebo group. Similar to what was seen in the case of colostrum, the combined vaccine containing 0.25 mg GST-P21 showed exactly the same results as the GST-P21 monovalent vaccine containing 0.5 mg GST-P21. . Each of those groups that received each GST-P21 vaccine reached a stable state with very similar values on days 14 and 28 after the first injection. However, the His-P21 group did not reach its maximum value until the 28th day. Similar to that seen with colostrum, further analysis of individual values indicated that cows G418 and M24 were poorly responsive animals. If the corresponding value is excluded from this analysis, the group mean of the His-P21 group will be very similar to the GST-P21 group. Finally, a similar decrease in antibody titer was observed at birth in all P21 groups. This is likely due to the active export of immunoglobulins in colostrum and perinatal immunosuppression.

Serum antibodies to Cp15 / 60 On day 0, all groups were seronegative. Placebo controls remained negative throughout this test. The group vaccinated with P21 was weakly positive on days 14 and 28. This is likely to reflect experimental error (non-specific background). The group vaccinated with Cp15 / 60 caused seroconversion after the first vaccination (FIG. 17). The second injection enhanced the antibody response. His-Cp15 / 60 group showed similar antibody response. Finally, a similar decrease in antibody titer was observed at birth in all Cp15 / 60 groups. This is likely due to the active export of immunoglobulins in colostrum and perinatal immunosuppression.

Experimental challenge of Cryptosporidium parvum in newborn calves Eight beef calves with no colostrum obtained by inducing calving were equally divided into two groups, Groups were placed in an isolation room. Each calf lived in one metabolic box. Each group received 2 pints (about 960 ml) of colostrum in baby bottles at 3 hours and 12-15 hours after delivery. 24 hours after delivery, all calves had blood IgG levels detected by RIDA (radioimmunodiffusion) greater than 1000 mg / dL. Each calf in the challenge group was orally administered 10 ⁸ oocysts of Cryptosporidium parvum. Blood samples were taken daily and tested for serum antibodies, hematocrit, and total protein against Cryptosporidium parvum P21 and Cp15 / 60 antigens. Feces were collected three times a day (from the rectum) and the dry matter content was measured. Fecal oocyst excretion was measured daily by ELISA kit ProSpecT. Clinical symptoms including body temperature, general condition (such as reduced function), loss of appetite, dehydration, stool hardness (such as diarrhea), and death were assessed daily (see Example 12 for how to score clinical symptoms) . All dead or euthanized calves have autopsy, intestinal analysis, and intestinal bovine rotavirus, bovine coronavirus, E. coli, Salmonella spp., And Cryptosporidium parvum content. Analyzed.

Oocyst Excretion Table 1 shows the detection of Cryptosporidium parvum oocyst excretion by ELISA for all oocysts.

Clinical symptoms All untreated controls remained healthy during the study period. All calves in the challenge group showed clinical symptoms consistent with cryptosporidiosis by day 4 after challenge. Three calves in the challenge group died before the end of the study (one on day 4 and two on day 5). The fourth calf in the challenge group was euthanized at the end of the study (day 6) because it met previously established euthanasia criteria. Body temperature, hypofunction, diarrhea, anorexia, and dehydration were monitored and characterized as described in Example 15.

  The mean body temperature of each group is less than 1 ° F. at any time point, ranging from 101.57 to 103.5 ° F. for the challenge group and 101.53 to 102.78 ° F. for the untreated group. All were within the clinically normal range.

  All untreated calves remained healthy and agile. The challenged calves began to show decline on day 2 (2/4) and the remaining 3 calves in the challenge group continued to show decline for days 2-5. The remaining one calf was still dysfunctional on day 6 which was the end of the study.

  All calves in the challenge group showed symptoms of diarrhea consistent with cryptosporidiosis, such as stool yellowing, foaming, high water content and high volume. This group of diarrhea began on day 2 and continued until the end of the experiment. One of the untreated calves had temporary diarrhea again on days 3 and 5, which resolved by the end of the study. The calf did not show any other symptoms of cryptosporidiosis, so the diarrhea was likely a result of nutrition.

  All calves in the untreated group, except for one on the third day, were very appetizing and were breastfeeding actively (feeding bottles). On the third day (corresponding to the first day of the diarrhea), the calves that lost appetite were fed only on that day using an esophageal feeder and then returned to normal feeding. All calves in the challenge group had anorexia by day 3 and remained unappetizing until the trial was over.

  None of the untreated calves showed clinical dehydration. One calf in the challenge group began to show clinical dehydration on day 2 and by day 3 all calves in this group were clinically dehydrated and remained in that state until the end of the study. The results of blood parameters (hematocrit) indicating dehydration are shown in FIG.

  The hematocrit level of untreated calves remained constant after day 1, but the hematocrit of challenged calves increased on day 2, and on days 4, 5, and 6 It remained higher than the control calf. The average hematocrit of untreated calves on the day of birth (day -1) was 41.0%. It dropped on day 0 and remained at a level of 32.0-37.5% for the remainder of the study. The calves challenged had an average hematocrit of 37.8% on the day of birth (day -1), then dropped to 29.5-35.0% on days 0-3. On the fourth day after challenge, the average hematocrit increased to a clinically significant 41.7%. FIG. 18 shows the difference between the daily groups of hematocrit. Values on day 5 and day 6 represent the results for one calf.

  Total plasma protein (TP) in untreated calves remained constant throughout the study, averaging 6.45 on day -1 (pre-challenge) to 5 on day 0 (time point 24 hours after challenge). The range was as low as 85. Challenged calves started at 6.4 on day -1 and reached the highest level on day 2 (7.0) and remained higher than control calves throughout the study period.

  Fecal dry matter content, expressed as% of volume, remained nearly constant in control calves but decreased on day 2 in challenged calves (lower than comparable to diarrhea) Dry matter content) started and continued until the end of the test. Challenged calves always had a dry matter content of 6-39% lower than control calves. The average dry matter content of feces of untreated calves ranged from 39.9% at 24 hours after birth to 51.7% at the time of day 2 sample collection. The average dry matter content of feces of the calves challenged ranged from 28.4% at 24 hours after birth to 41.0% at the time of sample collection on the second day, and on the fourth day thereafter. It gradually decreased to an average value as low as 9.6% at the time of evening sample collection. FIG. 19 shows the difference between daily groups in fecal dry matter content.

  Control calves remained negative for Cryptosporidium parvum infection throughout the study period. Challenged calves excreted Cryptosporidium parvum oocysts, and calves challenged with Cryptosporidium parvum showed clinical symptoms of cryptosporidiosis. Untreated controls remained healthy.

Demonstration of efficacy of various Cryptosporidium parvum subunit protein vaccines by challenge to calves observed in calves fed colostrum from inoculated cows versus calves fed colostrum from control cows Based on a significant reduction in the severity of clinical symptoms, there were six groups: 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) calves were selected. Approximately 8 animals were assigned to each treatment group. Prior to the first colostrum intake, blood of a newborn calf is collected to obtain serum and observed for body weight, body temperature, feces, and other clinical observation items (ie, loss of appetite, reduced function, diarrhea, etc.) did. At about 3 hours of age, the first colostrum was fed via a baby bottle or esophageal tube. About 12 hours later, the second colostrum was administered. We were challenged with Cryptosporidium parvum (10 ⁷ oocysts) for about 24 hours of age. Calves were observed four times a day, during which blood samples were collected, body temperature and clinical observation items were monitored, and feces were collected.

All calves 24 hours after birth were challenged by oral administration of 10 ⁸ oocysts of Cryptosporidium parvum. Immediately prior to challenge, the calf was administered 60-100 ml of calf milk replacer using a clean baby bottle or a clean esophageal tube. Following this, challenge material was administered and then rinsed with 40-100 ml water or calf milk replacer.

  The clinical signs of the calves were observed immediately before the challenge and then four times daily at approximately the same time for 6 days after the challenge. Clinical observations included rectal temperature, general condition, loss of appetite, diarrhea, dehydration, and death.

Serum The ELISA for detecting the P21 antibody used to generate the data in the graph shown in FIG. 20 is an indirect competitive ELISA, where the higher the OD value, the lower the antibody level and the lower the OD value. Increases antibody levels. The calves of this study were sensitized on day -1 but showed seroconversion after feeding test colostrum containing P21 antibodies (GST-P21, His-P21, and combination vaccine). . Calves fed colostrum containing all GST-15 / 60, His-15 / 60, and placebo antibodies remained positive for P21 antibody throughout the 6 day observation period.

  The ELISA for detecting Cp15 / 60 antibodies is a direct ELISA, where high OD values correspond to high antibody levels and low OD values correspond to low antibody levels (FIG. 21). All calves were naive on day-1. Calves fed colostrum with all 15/60 antibodies (GST-15 / 60, His-15 / 60, and combination vaccine) showed rapid seroconversion. A slight increase in the value of cows fed colostrum with P21 and placebo antibody is common but is assumed to be background due to cross-reaction and is a limitation of ELISA. Nevertheless, calves that fed colostrum containing P21 and placebo antibody remained negative throughout the 6-day observation period.

Total Disease Score Graph The total disease score is the cumulative total of all clinical symptoms (diarrhea, loss of appetite, and loss of function) observed over a 6 day period (4 observations per day) in this study. Combined with other data, this graph showed that the GST-P21 and His-P21 vaccines had no protective effect. However, the GST-15 / 60 vaccine shows a slight but significant reduction in clinical symptoms. This defense can be seen more clearly in FIG. Excluding other vaccine data, calves fed colostrum containing GST-15 / 60 antibody throughout the six-day observation period may always be less susceptible to disease (ie, have fewer clinical symptoms). Obviously (FIG. 23).

Diarrhea A score for the type of diarrhea observed in calves was given 4 times a day. At observation point 10, there were 4 calves in the placebo group with a diarrhea score of 1, so the total score for this observation was 4. Regardless of which calf group, all calves showed symptoms of diarrhea, but for most of the 6 day study, the GST-15 / 60 group had a lower score than the placebo group. became. Differences between groups were evident especially at observation time points 6-11.

  FIG. 24 is a cloud diagram showing the relative distribution of diarrhea for all calves in this study. This cloud diagram shows the relative distribution of all calves and averages 24 disease scores for each calf (each black circle represents one calf in the treatment group), then The values were generated by averaging the values to obtain an average for the treatment group (represented by a purple square). When multiple data points occupy the same space, the number of overlapping data points is represented by a superscript number. The GST-15 / 60 average is lower than the placebo, and the GST-15 / 60 values are more closely clustered (4 data points overlap the group average). Also, the His-15 / 60 group performed well, although the total collection of values was not as dense as GST-15 / 60, but the average was much lower than the placebo or other groups.

Anorexia After the second day of the study, any calf that fed less than 2 liters of milk and required an esophageal tube was scored as anorexia (observation of anorexia during the first 2 days of life) Was recorded but not analyzed). Calves in the GST-15 / 60 group showed no anorexia throughout most of the study, in contrast to the placebo group, which often contained two or three anorexic calves. FIG. 25 shows a cloud diagram showing the relative distribution of the total anorexia score for all calves for all vaccines. GST-15 / 60 has the densest assemblage as well as the lowest average of all groups in this study.

Hypofunction Calves were observed 4 times a day and scored for their condition. There were more healthy calves in the GST-15 / 60 group than in the placebo group. Note that none of the calves in any group showed a score higher than state score 1 (insensitive) in any observation. That is, the score 3 at the observation time point 21 in the placebo group indicates not three calves with score 3 but three calves with score 1. FIG. 26 shows the distribution of total general condition scores for each calf. The GST-15 / 60 group shows a much closer set than the other vaccine groups and has a much lower average than the placebo. Interestingly, the results for the GST-P21 vaccine alone are similar to the placebo, but the mixed vaccine group (consisting of GST-P21 and GST-15 / 60) performed well. This result suggests that GST-15 / 60 vaccine improves the general condition.

Fecal dry matter Whole feces were collected (four times daily), pooled, and dried during the day. Fecal dry matter in the GST-15 / 60 group was slightly higher than the placebo group for most of the study and all animals developed symptoms, but the incidence of diarrhea in the GST-15 / 60 group was reduced. It has been shown. FIG. 27 is a cloud diagram showing the average fecal dry matter score for each calf for all vaccines. All vaccine groups, including 15/60, show a dense population and higher average amounts than the placebo group.

FIG. 28 shows oocyst excretion determined by ELISA kit ProSpect (not the number of oocysts observed directly with a microscope). Symptoms appeared in all animals in this study, similar to those seen in other clinical symptoms (such as diarrhea). However, oocyst excretion in the His-15 / 60 group appears to be delayed compared to the placebo group.

Immunogenicity and safety of vaccines containing rotavirus, coronavirus, and Escherichia coli K99 and F41, and the GST-CP15 / 60 antigen of Cryptosporidium parvum. It was to evaluate the safety and antibody response elicited by the two combined vaccines. The specific purpose of this study was to determine whether the addition of Cryptosporidium parvum subunit antigens would interfere with immune responses against bovine rotavirus, bovine coronavirus, and other antigens such as E. coli antigens K99 and F41. It was to judge. To answer these questions, two vaccines were tested. Both included an aluminum / saponin adjuvant and included inactivated antigens, namely bovine rotavirus, bovine coronavirus, E. coli K99 and E. coli F41. In addition, one of the vaccines contained live Cryptosporidium parvum GST-CP15 / 60 subunit antigen produced in E. coli. Two groups of calves were vaccinated twice at 28 day intervals, each with 5 ml treatment of the vaccine. The other two calves served as environmental controls.

Rectal Temperature FIG. 29 shows the change in mean rectal temperature following the first and second vaccinations in the vaccinated and control groups. A hyperthermic transition phase is observed in the two vaccination groups, with peaks within 24 hours after the first and second vaccinations. The mean maximum rise in rectal temperature after the first vaccination (Δmax1 = T ° of peak 1−T ° of D0) was 1.4 ° C. and 1.3 ° C. for mixed + cryptospodium and mixed alone, respectively. It was. There were no calves with Δmax ≧ 2.0 ° C. The mean maximum increase in rectal temperature after the second vaccination (Δmax2 = T ° of peak 2−T ° of D28) was 1.4 ° C. and 1.1 ° C. There were no calves with Δmax ≧ 2.0 ° C.

  Interestingly, the control calf also increased in body temperature following vaccination. This increase was limited (average 0.4-0.5 ° C.) and was likely due to animal handling. From this, the maximum hyperthermia particularly attributed to the vaccine was approximately 1.0 ° C.

Local reaction (in vivo)
FIG. 30 shows the change in the average size of the local reaction following the first vaccination. FIG. 31 shows the change in the average size of the local reaction that occurs following the second vaccination. A local reaction was seen immediately after both injections in all vaccinations, except for the first vaccination of calves administered mixed + Cryptosporidium. The local response was maximal approximately 24-48 hours after vaccination and remained strong for 1 week. A rapid reduction in response size was then observed. In all cases, local reactions disappeared or became very limited 3 weeks after vaccination. Local reactions could be accompanied by a temporary slight increase in drained lymph nodes. The vaccinated groups were compared for local response at different time points with ANOVA (first injection D1, D21; second injection D29, D49). There was no significant difference.

The average antibody titer of serum Cryptosporidium parvum is shown in FIG. As expected, all calves vaccinated that were not Cryptosporidium remained negative for antibodies to Cryptosporidium parvum. After the first vaccination, seroconversion was observed in 3 out of 6 animals inoculated with Cryptosporidium. At 14 days after the second vaccination, strong seroconversion was observed in all vaccinated calves. The average antibody titer of Cp15 / 60 was 2.66 at D42. One calf was a weak responder with an antibody titer of 1.4.

  FIG. 33 shows the ELISA results of antibody responses against bovine coronavirus (BCV). On the 14th day after the first vaccination, seroconversion was observed in all vaccinated calves. All vaccinated calves had very high ELISA antibody titers for D42 (14 days after additional vaccination). In the serum neutralization test, almost all calf sera neutralized the virus at all dilutions tested (antibody titer> 3.84 (log CCID 50 / ml) (Figure 34). ELISA mean for BCV Antibody titers were approximately 10% lower at each time point for the combined plus Cryptosporidium vaccine compared to the combined vaccine alone, and this difference at D49 was significant (p = 0.01).

  FIG. 35 shows the ELISA results of the antibody response to bovine rotavirus (BRV). At D28, seroconversion could be detected at 4/6 and 5/5 respectively in the mixed + Cryptosporidium group and the vaccinated group. All vaccinated calves had high D49 ELISA antibody titers. The ELISA antibody titer of D49 was more uniform in the mixed group (StD = 0.12) than in the mixed + cryptosporidium group (StD = 0.46), and the average ELISA antibody titer was approximately 15% higher. Differences between groups (measured by repeating ANOVA) were significant (p = 0.05). FIG. 36 shows the change in neutralizing antibody titer for BRV. In all calves vaccinated, antibody titers increased abnormally at D14. These antibody titers decreased to more normal values at D28, at which time all calves had undergone seroconversion. The average antibody titer of D49 (log CCID 50 / ml) was 1.9 in the mixed + cryptospodium group and 2.2 in the mixed group alone. This approximately 18% difference was significant (p = 0.01).

  The change in ELISA antibody titer for E. coli F5-K99 is shown in FIG. At D28, seroconversion could be detected at 4/6 and 3/5 respectively in the mixed + cryptosporidium group and the vaccinated group. However, the calf-caused calf ELISA antibody titer was much higher than the mixed group. The average antibody titer of D49 (log OD 50%) was 2.56 in the mixed + cryptospodium group and 3.91 in the mixed group alone. This approximately 40% difference was very significant (p = 0.002).

  The change in ELISA antibody titer for E. coli F41 is shown in FIG. At D28, seroconversion could be detected in all vaccinated animals in both groups. The antibody titer of D49 became more uniform and higher only in the mixed group. The average antibody titer of D49 (log OD50%) was 2.57 in the mixed + cryptospodium group and 3.67 in the mixed group alone. This approximately 40% difference was very significant (p = 0.005).

  On average, both vaccines induced transient and moderate hyperthermia after their respective injections. There were no other systemic reactions observed. Both vaccines elicited a strong local response but reduced to a fairly acceptable size within 2 weeks. Regardless of the nature of the vaccine, the reaction was more pronounced after the second vaccination.

  Both vaccines induced the production of antibodies against all the respective antigen components. With the exception of antibodies against Cryptosporidium parvum, the antibody response was always higher for the combined vaccine alone than for the mixed + Cryptosporidium vaccine. In particular, this difference is evident when looking at antibodies against E. coli K99 and E. coli F41, and therefore the addition of Cryptosporidium antigen in the vaccine is associated with a 40% (log) reduction in antibody response.

  These results clearly suggest that Cryptosporidium antigen interference (especially against E. coli K99 and F41, and in some cases against the BRV antibody response) is important and may affect protection. This addition may therefore require redefinition of the antigen dose of E. coli K99, F41 and BRV fractions.

The foregoing invention has been described in detail for purposes of clarity and understanding by way of illustration and example, and it will be apparent to those skilled in the art that several modifications and variations can be made. Let's go. Accordingly, the description and examples should not be construed as limiting the scope of the invention described by the appended claims.
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US Patent Number 5,591,434.

FIG. 3 shows a physical restriction map of plasmid pJCA155. FIG. 3 shows a physical restriction map of plasmid pJCA156. FIG. 3 shows a physical restriction map of plasmid pJCA157. FIG. 3 shows a physical restriction map of plasmid pJCA158. FIG. 3 shows a physical restriction map of plasmid pJCA159. FIG. 3 is a diagram showing a physical restriction map of plasmid pJCA160. It is a figure which shows the comparison (Example 12) of the number of oocysts in the stool of the calf which challenged either the Cryptosporidium parvum or bovine rotavirus, or both, or is untreated. FIG. 10 shows a comparison of rotavirus excretion in calf stool according to Example 12. FIG. 14 shows a comparison of calf general conditions according to Example 12. FIG. 14 shows a comparison of calf dehydration according to Example 12. FIG. 10 shows a comparison of the number of calves liquid feces according to Example 12. FIG. 14 shows a comparison of calf anorexia status according to Example 12. FIG. 10 shows a comparison of calf rectal temperature changes according to Example 12. It is a figure which shows the average P21 (P21) colostrum antibody level for every vaccine group. It is a figure which shows the average CP15 / 60 colostrum antibody level for every vaccine group. It is a figure which shows the average P21 (P21) serum antibody level for every vaccine group. It is a figure which shows the average CP15 / 60 antibody level for every vaccine group. FIG. 6 shows hematocrit levels comparing challenged and untreated animals. It is a figure which shows the daily difference of the dry matter content rate of the stool for every collection time between groups and every day. It is a graph which shows the result of the antibody detection assay of P21 by indirect ELISA. It is a figure which shows the result of the antibody detection assay of CP15 / 60 by ELISA. FIG. 6 is a score graph showing total animal disease over time for all vaccines. FIG. 2 is a graph showing total animal disease for GST-15 / 60 and placebo vaccine alone. It is a cloud diagram showing diarrhea scores for all vaccines. It is a cloud diagram showing anorexia scores for all vaccines. It is a cloud diagram which shows the functional decline score about all the vaccines. It is a cloud diagram which shows the stool dry matter about all the vaccines. It is a figure which shows the oocyst excretion about all the vaccines used by this test. It is a graph which shows the average of the change of rectal temperature. It is a figure which shows the average local reaction (Cryptosporidium + mixing, mixing only) with respect to the 1st vaccination. It is a figure which shows the average local reaction (Cryptosporidium + mixing, mixing only) with respect to the 2nd vaccination. It is a graph which shows the average ELISA antibody titer of CP15 / 60. It is a figure which shows the ELISA antibody titer with respect to a bovine coronavirus. It is a figure which shows the virus neutralization antibody titer with respect to a bovine coronavirus. It is a figure which shows the ELISA antibody titer with respect to a bovine rotavirus. It is a figure which shows the virus neutralization antibody titer with respect to a bovine rotavirus. It is a figure which shows the ELISA antibody titer with respect to colon_bacillus | E._coli K99 antigen. It is a figure which shows the ELISA antibody titer with respect to colon_bacillus | E._coli F41 antigen.

Claims (55)

  Express first antigen or epitope derived from Cryptosporidium and / or first vector expressing first antigen or epitope and second antigen or epitope derived from other enteric pathogen and / or express first antigen or epitope Intestinal mixed immunity composition, mixed immunogenicity, comprising first vector that also expresses second antigen or epitope and / or second vector that expresses second antigen or epitope and pharmaceutically acceptable vehicle Composition, or mixed vaccine composition.   The composition according to claim 1, comprising an antigen derived from Cryptosporidium parvum and an antigen derived from another enteric pathogen.   The composition according to claim 2, comprising an antigen derived from Cryptosporidium and an antigen derived from another enteric pathogen of bovine species.   The composition according to claim 2, comprising an antigen derived from Cryptosporidium and an antigen derived from a canine intestinal pathogen.   The composition according to claim 2, comprising an antigen derived from Cryptosporidium and an antigen derived from an enteric pathogen of a feline species.   The composition according to claim 2, comprising an antigen derived from Cryptosporidium and an antigen derived from an equine enteropathogen.   2. The composition of claim 1, wherein the enteric pathogen-derived antigen is selected from the group consisting of E. coli, rotavirus, coronavirus, Clostridium, and mixtures thereof.   The composition of claim 1, wherein the enteric pathogen comprises E. coli.   E. coli-derived antigen is inactivated E. coli having K99 antigen, inactivated E. coli having F41 antigen, inactivated E. coli having Y antigen, inactivated E. coli having 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, 9. The composition of claim 8, comprising an antigen selected from the group consisting of and mixtures thereof.   E. coli antigen is selected from the group consisting of inactivated E. coli having K99 antigen, K99 antigen, and mixtures thereof; and / or the group consisting of inactivated E. coli having F41 antigen, F41 antigen, and mixtures thereof 10. The composition of claim 9, comprising an F41 antigen selected from.   4. The composition of claim 3, wherein the enteric pathogen comprises bovine coronavirus.   4. The composition of claim 3, wherein the enteric pathogen comprises bovine rotavirus.   4. The composition of claim 3, wherein the enteric pathogen comprises C. perfringens.   14. The composition of claim 13, wherein the enteric pathogen antigen comprises a C. perfringens type C and / or D type toxoid.   The composition according to claim 3, wherein the enteric pathogen comprises Escherichia coli, bovine rotavirus, bovine coronavirus, and Clostridium perfringens, or contains Escherichia coli, bovine rotavirus, and bovine coronavirus.   Intestinal pathogen antigen is inactivated E. coli having K99 antigen, inactivated E. coli having F41 antigen, inactivated E. coli having Y antigen, inactivated E. coli having 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, And an E. coli antigen selected from the group consisting of these and mixtures thereof, bovine inactivated coronavirus, bovine inactivated rotavirus, and Clostridium perfringens C and / or D toxoids, or a K99 antigen. Selected from the group consisting of activated E. coli, inactivated E. coli with F41 antigen, inactivated E. coli with Y antigen, inactivated E. coli with 31A antigen, K99 antigen, F41 antigen, Y antigen, 31A antigen, and mixtures thereof 16. The composition of claim 15, comprising an E. coli antigen, bovine inactivated coronavirus, and bovine inactivated rotavirus.   E. coli antigen is selected from the group consisting of inactivated E. coli having K99 antigen, K99 antigen, and mixtures thereof, and / or the group consisting of inactivated E. coli having F41 antigen, F41 antigen, and mixtures thereof 17. The composition of claim 16, comprising an F41 antigen selected from.   4. The composition of claim 3, comprising the subunit Cryptosporidium parvum antigen selected from the group consisting of P21, Cp23, Cp15 / 60, CP41, and mixtures thereof.   16. The composition of claim 15, comprising the subunit Cryptosporidium parvum antigen selected from the group consisting of P21, Cp23, Cp15 / 60, CP41, and mixtures thereof.   17. The composition of claim 16, comprising the subunit Cryptosporidium parvum antigen selected from the group consisting of P21, Cp23, Cp15 / 60, CP41, and mixtures thereof.   19. A composition according to claim 18 comprising Cp23 and Cp15 / 60.   20. A composition according to claim 19 comprising Cp23 and Cp15 / 60.   21. The composition of claim 20, comprising Cp23 and Cp15 / 60.   19. A composition according to claim 18 comprising P21 and Cp15 / 60.   The composition of claim 1 further comprising an adjuvant.   The composition of claim 15 further comprising an adjuvant.   27. The composition of claim 26, wherein the adjuvant comprises saponin.   27. The composition of claim 26, wherein the adjuvant comprises aluminum hydroxide.   27. The composition of claim 26, wherein the composition is in the form of an oil-in-water emulsion.   A first vector that expresses a P21 or Cp23 antigen or epitope thereof, or a first antigen that expresses a first antigen and Cp15 / 60 antigen or an epitope thereof, or both a first antigen and a second antigen Or an immunogenic composition, an immunogenic composition, or a vaccine composition against Cryptosporidium parvum, comprising a second antigen comprising a second vector that expresses the second antigen, and a pharmaceutically acceptable vehicle.   31. The composition of claim 30, wherein the P21 or Cp23 antigen and the Cp15 / 60 antigen are in the form of separate fusion proteins.   32. The composition of claim 30, comprising a vector that expresses P21 and Cp15 / 60.   31. The composition of claim 30, comprising a recombinant vector that expresses P21 and a recombinant vector that expresses Cp15 / 60.   32. The composition of claim 30, comprising Cp23 and Cp15 / 60.   32. The composition of claim 30, further comprising an adjuvant.   A P21, Cp23, Cp15 / 60 or CP41 antigen or an epitope thereof or a first antigen comprising a first vector expressing this first antigen, a second antigen derived from Cryptosporidium parvum or an epitope thereof or a first antigen and A second antigen comprising a first vector that expresses both two antigens or a second vector that expresses a second antigen, and a pharmaceutically acceptable vehicle, wherein the first and second antigens are different antigens, An immune composition, an immunogenic composition, or a vaccine composition against Cryptosporidium parvum.   A method of bovine immunization of a newborn calf against intestinal disease, wherein the composition of claim 1 is administered to a pregnant cow prior to birth so that the newborn calf has a transfer antibody against Cryptosporidium parvum A method involving that.   38. The method of claim 37, further comprising providing a newborn calf with colostrum and / or milk from a cow administered the composition during pregnancy.   A method for active immunization of adult and newborn calves comprising administering to said cow the composition of claim 1.   38. The method of claim 37, further comprising administering the composition to a newborn calf.   40. The method of claim 38, further comprising administering the composition to a newborn calf.   41. The method of claim 40, wherein the composition administered to a cow comprises an antigen or epitope thereof, and the composition administered to a calf comprises a vector.   42. The method of claim 41, wherein the composition administered to a cow comprises an antigen or epitope thereof and the composition administered to a calf comprises a vector.   A method for preparing the composition of claim 1 comprising mixing an antigen or epitope or vector and a carrier.   A kit for preparing the composition of claim 1, comprising antigens, epitopes or vectors, each in separate container (s), optionally together in a package, Further kits optionally including instructions for mixing and / or administration.   Hyperimmune colostrum and / or milk composition obtained by administering the composition according to claim 1 to a pregnant cow and then collecting colostrum and / or milk from said cow.   47. The composition of claim 46, wherein the composition comprises concentrated immunoglobulin obtained by coagulating colostrum and / or milk and recovering the immunoglobulin.   Intestinal disease, intestinal symptom (s), and / or intestinal condition (s), and / or such disease, symptom (s) and / or condition (s) 47. A method for preventing, treating and / or controlling enteropathogen (s) and / or Cryptosporidium parvum which is responsible for the development of the composition of claim 46 A method comprising administering to a calf.   Intestinal disease, intestinal symptom (s), and / or intestinal condition (s), and / or such disease, symptom (s) and / or condition (s) 48. A method for preventing, treating and / or controlling enteropathogen (s) and / or Cryptosporidium parvum which is responsible for the development of the composition of claim 47 A method comprising administering to a calf.   49. The method of claim 48, wherein the administration is oral administration.   52. The method of claim 49, wherein the administration is oral administration.   51. The method of claim 50, wherein the oral administration is by lactation from a cow to a newborn calf.   A method for preparing a hyperimmune colostrum and / or milk composition comprising administering the composition of claim 1 to a pregnant cow and then collecting colostrum and / or milk from said cow A method involving that.   Further comprising concentrating the milk obtained from the cow by coagulating the colostrum and / or milk and recovering the immunoglobulin and / or immunoglobulin in the colostrum, the composition comprising said immunoglobulin by this concentration, 54. The method of claim 53.   A first antigen or epitope derived from Cryptosporidium and / or a vector expressing such an antigen or epitope and a second antigen or epitope derived from another enteric pathogen and / or a vector expressing such an antigen or epitope Use of a method of preparing an immunogenic composition or vaccine composition against intestinal infection, comprising mixing a first antigen or epitope and / or vector and a second antigen or epitope and / or vector Including methods.

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