Cryptosporidiosis vaccines

Abstract

To provide safe and effective vaccines against cryptosporidiosis.SOLUTION: Provided herein is a Cryptosporidium gp40 protein or immunogenic moiety thereof where the gp40 protein or immunogenic moiety thereof comprises one or more alkylated amino acids.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to the field of parasitological vaccines. More specifically, the present invention relates to the use of Cryptococcus pneumoniae in parasitological vaccines. Sporidiosis gp40 protein, method for preparing said protein, and The medical use of the protein as a vaccine against the and the manufacture of vaccines therefor, as well as methods for protection against cryptosporidiosis do. [Background technology]

[0002] Cryptosporidium (Cryptosporidia) is a member of the phylum Apicomplexa ( It is a parasitic protozoan of the phylum Apicomplexa. Sporidiosis is primarily found in the intestines and is characterized by diarrhea accompanied by cramps, dehydration, and secondary infections. The disease can affect immunocompetent humans or non-humans. The disease ranges from mild in animals to severe in more vulnerable hosts, such as young or immunocompromised individuals. In human medicine, the disease is seen in malnourished children and AIDS patients. In veterinary medicine, for animal welfare and economic reasons, neonatal ruminant Disease in sheep, goats and especially calves is of great concern. Cryptosporidium parvae occurring in common and human-infectious genotypes The human pathogen Cryptosporidium parvum Many other cryptosporidium species, such as C. hominis, Species of Bacillus species also cause cryptosporidiosis.

[0003] The parasitic stage released by infected hosts is the oocyst, which is highly infectious. They are resistant to environmental conditions and many disinfectants. Thus, infections can occur, e.g. Through fecal material, either by close contact or through fecal contamination of food or drinking water It can spread rapidly.

[0004] Several drugs have been approved for the therapeutic treatment of cryptosporidiosis. Tazoxanide (thiazolide) is indicated for use in immunocompetent humans. Halocure® (halofuginone, MSD Animal Health lth) can be used on newborn calves. Ideally, A suitable vaccine should also be developed, but as a result of the complex life cycle of this parasite, However, this proved extremely difficult as in vitro culture of this parasite is not possible. As a result, there are currently no registered vaccines against cryptosporidiosis. Does not exist.

[0005] Live attenuated, inactivated, lysate and subunit vaccines A wide variety of experimental vaccines against cryptosporidiosis have been tested over time, including This is not for lack of trials, as subunit vaccines have been tried and tested. Many proteins from different parasite stages have been tried. Lemieux et al.(2018,Pathogens,vol.7,DOI: Please refer to the National Institute of Infectious Diseases (NIID) 10.3390 / pathogens7010002.

[0006] One of many candidate antigens for a subunit vaccine is Cryptosporidium:gp 40 kDa mucin-type glycoprotein derived from the motile parasite It is present on the surface of the cytoplasm and is highly glycosylated.

[0007] The nomenclature of Cryptosporidium proteins is confusing, with gp40 (or its coding region) being the most common. gene or gene product) is Cpgp40 / 15 (Cevallos et al. ,2000,Inf.&Imm.,vol.68,p.4108-4116);gp15 / 45 / 60(Strong et al.,2000,Inf.&Imm.,vol. 68, pp. 4117-4134); or S60 (Winter et al., 200 0,Funct.Integr.Genomics,vol.1,p.207-217) It should be noted that the Cp17 protein is also related (Pr iest et al.,2000 Mol.Biochem.Parasit.,vo The differences in molecular weights shown are due to sequence and glycosylation levels. The antigens designated "Cp15 / 60" are distinct proteins. It should also be noted that d Imm., vol.61, pp.2377-2382;GenBank accession The same can be said for "CP15" (GenBank accession number L 34568) or the antigen designated "cp41" (WO 01 / 040439). The same applies to.

[0008] Expression of gp40 in a recombinant expression system and its use for (passive) vaccination For many years, e.g., WO 93 / 024649, WO 01 / 0402 48, WO 01 / 077293 and U.S. Patent Application Publication No. 2002 / 0081 However, functional vaccines are still not available. do not have.

[0009] In neonatal ruminants, who are usually infected at a very early age, active vaccination is practically It may only provide protection after 3-4 weeks, which is too late. For example, by feeding colostrum from vaccinated cows to calves, Cryptosporidium can be prevented. A simple method of passive vaccination against polydiosis is possible. Such vaccination of newborn calves against neonatal diarrhea, also known as calf diarrhea, is e.g. For example, those described in WO 01 / 045735 and WO 2011 / 056175 This method of protection has also been shown to be effective against Escherichia coli (Escherichia coli). coli), bovine coronavirus, bovine rotavirus, and Clostridium This would be consistent with current administration for other causes of neonatal calf diarrhea. Passive vaccination of humans by giving them antibodies from Lemi has also been effective. See eux et al., 2018 (cited above).

[0010] Thus, many publications have been published on the isolation or expression of Cryptosporidium antigens and on the diagnosis of Cryptosporidium. These studies describe their use in immunohistochemistry or antigenic studies. It has also been suggested that it may be used for active or passive vaccination of dogs or animals. However, no commercial vaccine has been approved to date. Therefore, there is a need in the art for an effective vaccine against cryptosporidiosis.

[0011] Aziridines are organic chemical compounds containing an aziridine ring. Due to their tendency to damage acids and cause cross-linking and chain scission, these compounds are not suitable for use as anti-cancer agents. In biotechnology, this property of aziridines is It is used for the inactivation of microorganisms such as bacteria and viruses.

[0012] Next to nucleic acids, aziridines bind to proteins by alkylation of nucleophilic sites on amino acids. can react with sulfur-containing groups, such as sulfhydryl or thioether groups. Reaction with side chains containing nitrogen, such as amino groups, or oxygen, such as hydroxy groups By making them do so.

[0013] These reactions can occur with one or more of the amino acids of a protein, but It is not fully understood how an amino acid (or amino acids) can be alkylated by reacting with an aziridine. Not yet.

[0014] The aziridine that is commonly used for virus inactivation is ethyleneimine (EI). It is mainly used in the form of binary ethyleneimine (BEI). According to Ahnemann (1990, Vaccine, vol. 8, pp. 299-303) This is the general description.

[0015] Although a dangerous chemical, the use of EI is beneficial due to its more predictable, i.e. linear, response. Due to its kinetics, it is preferred over traditional viral inactivation with formalin. or nucleic acids, causing less damage to the immunogenicity of protein antigens than formalin. No (Blackburn & Besselaar, 1991, J.of Virol.M ethods,vol.33,p.367-374;Hulskotte et al. , 1997, Vaccine, vol. 15, p. 1839-1845).

[0016] It has been described that incubation of protein antigens with chemicals increases antigenicity. There have been several reports of this, but these were based on treatment with formalin and / or heat. only about protein cross-linking reactions (Grovit-Ferbas et al. ,2000,J.of Virol.,vol.74,pp.5802-5809). Other incubations with inactivators of unit proteins include formalin and bacterial It has only been described for the detoxification of toxins. Aziridines have not been shown to inactivate microorganisms. Outside the context of use for cleavage, incubation with subunit proteins has not been described. Incubation with aziridine has not been shown to inhibit Cryptosporidium gp40. Protein is not described.

[0017] Several papers have reported on the effects of aziridines on proteins during inactivation reactions. described negative effects on immunogenicity and recommended caution, Chen et al. 999, J. of Exp. Med., vol. 189, pp. 1757-1764) They described the loss of antigenicity due to S-alkylation of cysteine ​​in proteins. To reduce such reactivity, WO 98 / 51660 further provides a method for treating nucleic acids with hydroxybenzoates. The development of more specific EI polymers is described in WO 98 / 45415. The issue is based on EI at acidic pH levels to reduce adverse reactions with viral proteins. describes the inactivation of [Prior art documents] [Patent documents]

[0018] [Patent Document 1] International Publication No. 01 / 040439 [Patent Document 2] International Publication No. 93 / 024649 [Patent Document 3] International Publication No. 01 / 040248 [Patent Document 4] International Publication No. 01 / 077293 [Patent Document 5] U.S. Patent Application Publication No. 2002 / 0081312 [Patent Document 6] International Publication No. 01 / 045735 [Patent Document 7] International Publication No. 2011 / 056175 [Patent Document 8] International Publication No. 98 / 51660 [Patent Document 9] International Publication No. 98 / 45415 [Non-patent literature]

[0019] [Non-Patent Document 1] Lemieux et al.,(2018,Pathogens,vol.7,DOI:10.3390 / pathogens7010002) [Non-patent document 2] Cevallos et al.,2000,Inf.&Imm.,vol.68,p.4108-4116 [Non-patent document 3] Strong et al.,2000,Inf.&Imm.,vol.68,p.4117-4134 [Non-patent document 4] Winter et al.,2000,Funct.Integ.Genomics,vol.1,p.207-217 [Non-Patent Document 5] Priest et al.,2000 Mol.Biochem.Parasit.,vol.106,p.261-271 [Non-patent document 6] Jenkins et al.,1993,Inf.and Imm.,vol.61,p.2377-2382 [Non-Patent Document 7] H.Bahnemann(1990,Vaccine,vol.8,p.299-303) [Non-patent document 8] Blackburn&Besselaar,1991,J.of Virol.Methods,vol.33,p.367-374 [Non-Patent Document 9] Hulskotte et al.,1997,Vaccine,vol.15,p.1839-1845 [Non-Patent Document 10] Grovit-Ferbas et al.,2000,J.of Virol.,vol.74,p.5802-5809 [Non-Patent Document 11] Chen et al.(1999,J.of Exp.Med.,vol.189,p.1757-1764) Summary of the Invention [Problem to be solved by the invention]

[0020] The object of the present invention is to overcome the shortcomings of the prior art and to provide a safe and effective cryptosporidiamine derivative. To address this need in the art by providing a vaccine against rhidiopathic ... This is what we should do. [Means for solving the problem]

[0021] The present inventors have developed a method for the detection of cryptosporidiosis based on recombinantly expressed gp40 protein. When attempts were made to develop a vaccine, early results were promising: gp40 It can be expressed in Escherichia coli as described in the literature and used in pregnant unborn children. It was used with standard adjuvants to vaccinate heifers. Colostrum containing high levels of gp40-specific antibodies was obtained. Cattle were protected from disease caused by severe Cryptosporidium parvum challenge infection. Unfortunately, the level of recombinant expression is not high and the expressed gp40 protein is immunogenic. Because the antigen concentration was low, a relatively high antigen dose had to be applied. The production of a cryptosporidiosis vaccine based on recombinantly expressed gp40 is currently underway for commercialization. It was not thought to be feasible for this purpose.

[0022] A further problem is that the gp40 protein appears to be highly reactive upon vaccination. That is, immunologically effective doses of gp40 were administered in standard emulsions. When administered to cattle as cutin, the overall safety profile is good and there are no effects on pregnancy. Although no vaccines were found, unacceptable levels of local vaccination were observed, particularly using the subcutaneous route of administration. Local reactions occurred, and these local reactions increased after booster vaccinations.

[0023] The use of lower antigen doses reduces local vaccination side effects to an acceptable level. Unfortunately, these doses did not provide effective passive vaccination of calves. However, the antibody levels in the colostrum were not sufficient to provide a satisfactory result.

[0024] The present inventors have developed an affordable, safe, and effective vaccine against cryptosporidiosis. I have no clue how to overcome these problems to develop a It was.

[0025] Surprisingly, the gp40 protein was isolated before it was used as a vaccine. The objectives of the present invention can be met by incubating the substance with aziridine. It has been found that one or more of the drawbacks of the prior art can be overcome. This incubation was found to significantly increase the immunogenicity of gp40. This results in the vaccinated mammalian target Higher titers of gp40-specific antibodies were also produced in the colostrum obtained from the target animals. compared with immunization with the same amount of gp40 that was not incubated with aziridine. This is what was done.

[0026] This finding was consistent with the results of the analysis of gp40 incubated with aziridine compared to gp40 that was not incubated with aziridine. It is possible to reduce the antigen mass of gp40 used for vaccination by 10-20 fold. but still induce sufficient antibody levels in the colostrum to protect against severe Cryptosporidium challenge It provides effective passive immune protection in offspring against infection. Next, to provide an effective vaccine against gp40 per possible vaccine dose, The significant reduction in antigen mass solved two additional problems: the ability to deliver the vaccine at an effective dose; Furthermore, recombinantly expressed Making gp40-based cryptosporidiosis vaccines economically feasible.

[0027] gp40-specific antibodies can inhibit the entry of Cryptosporidium parasites into host cells. As a result, vaccination against cryptosporidiosis is Passively, for example by feeding colostrum from vaccinated mammals , the increased antibody levels in the colostrum result in increased local levels of antibodies in the gastrointestinal tract, Provides protection against parasitic infestation.

[0028] However, it remains unclear how aziridine incubation affects the immunogenicity of gp40. It is not known exactly why this increases.

[0029] The inventors are not bound by any theory or model that may explain these findings. Although it is not desirable to do so, incubation with aziridine, especially with one or more amines, Chemical changes to the gp40 protein induced by alkylation of aziridine The immune response to gp40 that was not alkylated by vaccinating It is expected that the resulting antibodies will provoke different responses from the immune system of the selected human or non-human animal target. The observed increase in the level of anti-gp40 antibodies produced was followed by an increase in the binding activity and activity of the produced antibodies. and / or specificity of one or more of the generated antibody profiles. Other changes are likely present. In addition, the type or level of cellular immune response that is activated There may be a change in

[0030] This was not at all obvious from any disclosure in the prior art. A number of publications have reported damage to immunogenic epitopes following aziridine incubation. The positive effect of the antibody on immunogenicity was completely unexpected. Needless to say, this reaction was completely unexpected. Similarly, when incubated with ton X-100 or after gamma irradiation, No effect was observed.

[0031] Thus, in one aspect, the present invention provides a Cryptosporidium gp40 protein or or immunogenic portions thereof, wherein said gp40 protein and said portions thereof are one or more Cryptosporidium gp is characterized by containing multiple alkylated amino acids. 40 protein or immunogenic portions thereof.

[0032] A "protein" is a molecular chain of amino acids as defined herein. Peptides, peptides, and oligopeptides are included within the definition of protein. can be of natural or synthetic origin and can be a native or mature protein, a preprotein Or it may be a proprotein or part of a protein.

[0033] The term "Cryptosporidium" refers to the phylum Apicomplexa and the subphylum Coccidia. These microorganisms are characterized by morphological, genomic, and biochemical characteristics. The characterizing features of each taxonomic class, as well as physiological, immunological or pathological behavior Many species of Cryptosporidium parasites are known. They are capable of infecting a wide variety of non-human animals as well as humans.

[0034] A well-known species of cryptosporidium parasite is Cryptosporidium parvum, It has two genotypes: one that is thought to be infectious only to humans, and one that is thought to be infectious only to humans. It occurs in genotype I and genotype II, which is a proven zoonotic agent. Both genotypes of C. parvum cause cryptosporidiosis in particularly vulnerable targets. References on the characteristics and impact of Cryptosporidium parvum in veterinary medicine The references are "The Merck veterinary manual" (11th ed., 2016, ISBN-10:9780911910612).

[0035] As is known in the art, the classification of a microorganism in a particular taxonomic group is determined by its characteristics. Therefore, the present invention is based on the combination of other species of Cryptosporidium classified in this genus. Similarly, this includes subspecies, strains, isolates, genotypes, variants, subtypes, etc. Refers to parasites that are in some way subclassified from this genus, such as as a type or subgroup.

[0036] Furthermore, certain parasites of the present invention may not currently be assigned to such species or genus. Such classifications are important because new insights may lead to reclassification into new or different taxa. It will be apparent to those skilled in the art that the assignment is a taxonomic classification that may change over time. However, this does not alter the parasite itself or its antigenic repertoire. Such reclassifications are not intended to be a substitute for scientific names or classifications, as they merely change the species' scientific names or classifications. Parasites remain within the scope of the present invention.

[0037] Samples of Cryptosporidium parasites for use in the present invention can be obtained from a variety of sources. from, for example, humans or wild isolates from wild or farmed non-human animals. These can be obtained commercially or from various research institutes, (depository) institutions or (veterinary) universities. Additionally, a wealth of genetic information on Cryptosporidium and gp40 is available at the NCBL. Digitally available in public sequence databases such as GenBank and EMBL's EBI A public database specifically for sequences from Cryptosporidium is available at Cr yptoDB, available online at cryptodb.org.

[0038] In the present invention, "gp40" refers to a protein comprising the amino acid sequence of SEQ ID NO: 1 or The same sequence is also shown in Figure 1 using the single-letter IUPAC code. The specific sequence of is from GenBank accession number AAF78345.1 The sequence corresponds to the core sequence of the gp40 protein from amino acid number 31 to number 220 (Str (G et al., 2000, supra). This GenBank entry is a bovine isolate from the United States. from the Iowa strain of Cryptosporidium parvum, which is of genotype II The amino acid sequence of the 60 kDa precursor protein is shown.

[0039] In nature, gp40 is expressed as a precursor glycoprotein, and the N-terminal two-thirds of the precursor is g The precursor is p40, and the C-terminal one-third is gp15. Glycerin chains (both in the gp40 portion), and C-terminal GPI anchor (in gp15) After cleavage of the precursor, the two proteins interact and form the Cryptosporidium membrane. The sporozoite and sporozoite stages attach to the apical surface region where they mediate attachment to host cells and In nature, gp40 is primarily composed of threonine and serine amino acids. It is heavily glycosylated with O-linked GalNac structures.

[0040] A gene encoding the precursor protein of gp40 from Cryptosporidium parvum The child is registered in the CryptoDB database (see above) under gene number: cgd6_1080. It is written.

[0041] A "homologue" of the Cryptosporidium gp40 polyprotein of the present invention is the homologue of SEQ ID NO: 1 When aligned over the entire length, it has at least 60% amino acid sequence identity with SEQ ID NO: 1 Proteins containing amino acid sequences. Sequence alignments were performed using blast.ncbi.nlm.nih.gov / blast.ncbi.nlm.nih / . NCBI BLAST™ suite available online at nlm.nih.gov The alignment software was used with the "blastp" algorithm using standard parameters. It must be done rhythmically.

[0042] This spread in sequence identity levels is well known to be highly heterogeneous. To encompass natural variations in the amino acid sequence of the Cryptosporidium gp40 protein (Strong et al., 2000, see also supra). For example, Cryptosporidium Even among the gp40 proteins from U. parvum genotype II isolates, SEQ ID NO: 1 There is a variation in amino acid sequence identity levels of up to 23% across the sequences. On the one hand, some scattered amino acid sequence variation, and on the other hand, variation in the length of the polyserine chain. This polyserine chain in Cryptosporidium gp40 consists of 6 to 25 consecutive Although these may have serines present, they are all functional gp40 proteins. For example, GenBank accession number AOA32955.1 (6 serines) and Cryptosporidium parvum gp40 in ACR78128.1 (25 serines) See genotype II homologs. When aligned longitudinally, they exhibit 77 and 96% amino acid sequence identity, respectively. .

[0043] Further variation in the gp40 amino acid sequence is associated with Cryptosporidium parvum genotype I occur in isolates as listed in Table 1 of Strong et al. (2000, supra). As shown, the correlation between the gp15 / 45 / 60 precursor proteins from type I and type II isolates Amino acid identity can be as low as 67% and still be a functional gp40 protein is.

[0044] For a discussion of the diversity of Cryptosporidium parvum gp40, see Leav et al. al.,2002,Inf.and Imm.,vol.70,p.3881-389 0; and: Wu et al., 2003, Appl. Environ. Microb See also iol., vol. 69, pp. 4720-4726.

[0045] A further level of variability in the gp40 amino acid sequence is found in the amino acid sequences of SEQ ID NO:1. The sequence identity of closely related strains, such as Cryptosporidium hominis, decreases to approximately 60%. It is derived from gp40 homologs from different species.

[0046] However, all of these homologous gp40 proteins are suitable for use in the present invention. It is possible.

[0047] In the present invention, alkylation can be achieved by incubation with aziridine. To be functional, the Cryptosporidium gp40 homologue contains cysteine, methionine, Serine, threonine, tyrosine, lysine, arginine, valine, glutamic acid and aspartic acid It has at least one amino acid selected from paragic acid.

[0048] As used herein, the terms "comprise" (as well as "includes") "comprises" and "com Variations such as "prised" are used even if such elements or combinations are explicitly listed. Even if not listed, the section, paragraph, or claim in which the term is used All elements encompassed by or contained within, etc., and to the present invention The term "combination" refers to any possible combination that can be envisaged for such element(s) or elements. does not mean to exclude any of the combinations.

[0049] Accordingly, any such section, paragraph, claim, etc. of the text shall be deemed to "contain" The term "consisting" (or variations thereof) is used to refer to "of," "consisting of," or "consisting essentially of" replaced by terms such as "consist essentially of" The present invention may also relate to one or more embodiments in which

[0050] In the present invention, an "alkylated amino acid" is an amino acid in which the sulfur, nitrogen, or oxygen atom is azido. It is an amino acid that has been alkylated by reaction with dimethylaminomethylpropional, so that the amino acid The amino acid will have an additional alkyl group compared to its natural structure. For example, the sulfhydryl group of the side chain of an amino acid is converted to an alkylene thioether. and alkylated cysteine ​​amino acids, or the thioether in its side chain is a tertiary thioether. Methionine amino acid alkylated to a ter; the amino group of the side chain is an alkylamine group lysine or arginine alkylated to ; or Serine, threonine, tyrosine, glutamic acid or relates to aspartic acid.

[0051] The alkylation reaction that occurs upon incubation with aziridine is It depends on the availability of amino acids such as: two cysteines in a sulfur bridge; When connected by , the two cysteines are under non-reducing conditions and are suitable for alkylation. On the other hand, amino acids in which one of the groups is normally unreactive are not available for such a group, e.g. For example, when exposed at the N- or C-terminus of a protein fragment, the amino acid sequence of such a group may be The alkylation can be on the carboxyl or carboxyl groups of the gp40 protein or If a part of the valine starts with valine, the exposed amino group at the N-terminus of the valine is aziridin It can be alkylated by reaction with dimethylsilyl.

[0052] As will be appreciated by those skilled in the art, one or more alkylated amino acids according to the present invention may be A portion of the Cryptosporidium gp40 protein, including It can equally be used to induce specific antibodies in immunized targets. Some of the quality gp can be used to protect against cryptosporidiosis gp40 protein, in the sense that it must be able to induce 40-specific antibodies. The antigen-binding protein must be an "immunogenic portion" of the antigen-binding protein, which is, among other things, as described herein. As such, the amino acid sequence is provided by a moiety having one or more alkylated amino acids.

[0053] Those skilled in the art will appreciate that gp40-specific antibodies can be produced by immunizing a target and testing whether they are produced. Therefore, it is possible to determine whether a portion of the gp40 protein or its homologues is such an immunogenic portion. It can be easily determined whether

[0054] To ensure immunogenic efficacy for the present invention, one or more alkylated amino acids may be used. The immunogenic portion of the Cryptosporidium gp40 protein containing amino acids is SEQ ID NO: 1. and having at least 50 consecutive amino acids from SEQ ID NO: 1 (aa.no. .) glutamic acid corresponding to glutamic acid at positions 88 or 94, and amino acid sequence of SEQ ID NO: 1 At least one aspartic acid selected from the group consisting of aspartic acid number 129 Contains alkylated amino acids of

[0055] In the present invention, the feature "amino acid corresponding to the amino acid of the amino acid number" "Corresponding to" refers to a sequence that is at an equivalent position in the amino acid sequence, etc., of the indicated sequence identifier, and / or Or refers to an amino acid in the context of an equivalent amino acid.

[0056] The Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention is Each can be modified in different ways, as is well known in the art. The polypeptide may be acylated, lipidated, or PEGylated. Also, the polypeptide may be provided with a carrier, a hapten, a signal sequence, an anchor sequence, or the like. Further moieties of the protein, such as sequences, markers or tags, may be added. Any of these may be useful in the expression, detection and / or purification of proteins. Such modifications may also serve to adapt or even increase immunogenicity.

[0057] For example, the examples provided herein below contain a C-terminal 6x-histidine tag. Cryptosporidium gp40 protein containing one or more alkylated amino acids This tag did not affect immunogenicity, but the use of e.g. Metal affinity chromatography was performed when proteins were present in the harvest of recombinant expression systems. This enabled the purification of gp40 protein by ELISA.

[0058] The N-terminus of the gp40 protein used in the vaccination experiments described in the Examples the coding sequence of which is extended with a portion of the native signal sequence, It encodes an N-terminal methionine to initiate its transcription. Improved expression levels in baculovirus-insect cell expression systems without inducing secretion of ribosomal proteins It was good.

[0059] The amino acid sequence of the Cryptosporidium gp40 protein used in the examples is The protein is shown in SEQ ID NO: 3, along with additional N- and C-terminal sequences as indicated. 1, containing the entire amino acid sequence of SEQ ID NO: 1, a His tag, an additional methionine and a partial sigma Furthermore, gp40 for the present invention was expressed from a recombinant gene providing the null sequence. This gene, which encodes the polyhedrin gene of the baculovirus AcMNPV, follows the codon preference of the polyhedrin gene. The coding nucleotide sequence is provided in SEQ ID NO:2. It has been done.

[0060] of Cryptosporidium gp40 protein or its immunogenic portions by aziridine Alkylation results in an increase in the immunogenicity of that portion of the protein. The term is well known in the art and refers to the ability of a compound to induce a protective immune response in a target. The immune response induced can be of the humoral and / or cellular type, and protection is This can be achieved in a variety of ways, as described below. Immunogenicity for Vaccination is also called "efficacy."

[0061] Increased immunogenicity of a protein can be determined by one skilled in the art by performing comparative immunization experiments. In the present invention, this can be easily determined by one or more of the Immunization with Cryptosporidium gp40 protein containing alkylated amino acids The immune response in non-human animals was evaluated by the use of a g This may mean comparing the immune response of animals immunized with p40, where all other The parameters and conditions are substantially the same. In accordance with the present invention, increased immunogenicity is achieved by: or compared with a similar dose of gp40 that did not contain multiple alkylated amino acids. Cryptosporidium gp40 protein containing one or more alkylated amino acids The protein results in higher amounts of gp40-specific antibodies being produced in the serum of the target. The level and specificity of the serological response can be determined by using a variety of methods, such as ELISA, IFT, or AlphaLisa. The effects of the present invention can be easily measured by any suitable serodiagnostic technique. For comparison, gp40 that did not contain one or more alkylated amino acids was used as a pseudotype. should be incubated, i.e., in the absence of aziridine. The incubation should be carried out under the same conditions as the dNTP incubation.

[0062] For example, 10 μg of alkylated or sham-treated mice were administered with a standard oil-based adjuvant. A single immunization of cattle with modified gp40 results in the production of one or more alkylated gp40s according to the present invention. Cryptosporidium gp40 protein containing amino acids is used to coat gp40. As measured by standard antibody ELISA using 2-4 Log2 units higher gp4 titers compared to titers generated by gp40 containing no gp4 0-specific antibody titers, which result from alkylation with aziridine. 0-specific antibody titers up to 16-fold increase.

[0063] In the present invention, a gp40-specific antibody recognizes gp40 in a "specific" manner, In the art, this refers to antibodies that bind to gp40. As a result, in the case of specific binding, the concentration of the antigen or antibody is correlated with the Any dilution of the antibody will result in binding that is detectable in a standard serological assay. It should show a gradual decrease in levels. [Brief explanation of the drawings]

[0064] [Figure 1] 1 is the amino acid sequence of the Cryptosporidium gp40 protein of the present invention in single letter IUPAC code, corresponding to SEQ ID NO:1. [Figure 2]1 is a graphical representation of the measured titers of gp40-specific antibodies in the serum of vaccinated cattle, shown in Log2 Elisa units. Details are provided in Examples 3 and 4. Test groups were as follows: rgp40His = vaccine with unalkylated Cryptosporidium gp40 protein; rgp40His+BEI = vaccine with Cryptosporidium gp40 protein containing one or more aziridine-alkylated amino acids according to the present invention; RC vaccine = Rotavec Corona vaccine; rgp40His+RC = double-vaccinated group, received both the Rotavec Corona vaccine and the vaccine with unalkylated Cryptosporidium gp40 protein. [Figure 3] 1 shows the results of a vaccination-challenge experiment in calves regarding diarrhea severity. The horizontal axis shows the number of days after challenge. The vertical axis shows the mean daily diarrhea score for the rgp40His vaccine-treated group and the rgp40His+BEI vaccine-treated group. Experimental details are described in Examples 3 and 4. [Figure 4] 1 shows the results of mass spectrometry analysis of Cryptosporidium gp40 protein incubated with aziridine. The graph shows the amino acid sequence of gp40 (shown herein as SEQ ID NO: 3) on the horizontal axis and the number of times a particular amino acid from gp40 was found to be alkylated by incubation with aziridine on the vertical axis. Experimental details are described in Example 6. [Figure 5] Health scoring results of a vaccination-challenge experiment using very low doses of gp40 protein to generate colostrum with large amounts of antibodies. The horizontal axis represents days after challenge. The vertical axis represents health scores according to the Wisconsin-Madison scale. Details are provided in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0065] Details of embodiments and further aspects of the invention are set forth below.

[0066] In one embodiment of a Cryptosporidium gp40 protein according to the invention, the homologue is When aligned against the full length of SEQ ID NO: 1 as defined herein, It is a protein containing an amino acid sequence that has at least 60% amino acid sequence identity.

[0067] More preferably, a homologue of the Cryptosporidium gp40 protein for the purposes of the present invention is , and when aligned against the entire length of SEQ ID NO: 1, at least 62, 65, 67 , 70, 72, 75, 77, 80, 82, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98% amino acid sequence identity, or at least 99% amino acid sequence identity The column identities are in this order of preference.

[0068] One embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention In this study, the alkylated amino acids were cysteine, methionine, serine, and threonine. consisting of tyrosine, lysine, arginine, valine, glutamic acid and aspartic acid The compound is one or more selected from the group consisting of:

[0069] All of these amino acids are well-known chemical compounds, and their (L-isomers) are non-interacting. The chemicals included are Cysteine: CAS Registry Number: 52-90-4, Methionine: CAS No.: 59-51-8; Serine: CAS No.: 56-45-1; Threonine: CAS No. : 80-68-2; Tyrosine: CAS No.: 60-18-4; Lysine: CAS No.: 56 -87-1; Arginine: CAS No.: 74-79-3; Valine: CAS No.: 72-1 8-4; glutamic acid: CAS number: 56-86-0; and aspartic acid: CAS number The number is 56-84-8.

[0070] Alkylation of the sulfhydryl group of the cysteine ​​side chain results in the formation of alkylene thioethers. The alkylene amine group replaces the hydrogen atom from the sulfhydryl group, resulting in a cysteine ​​that I replaced it.

[0071] Alkylation of the thioether group in the side chain of methionine gives methyl groups with tertiary thioethers. An alkyleneamine group replaced the hydrogen atom from the thioether group, resulting in onine.

[0072] Alkylation of the amino group on the side chain of arginine or lysine results in alkylation of alkyl groups attached to the amine side chain. This results in arginine or lysine bearing an alkyleneamine group.

[0073] Hydroxylated side chains of serine, threonine, tyrosine, aspartic acid, or glutamic acid Alkylation of the silyl group results in a cerium compound with an alkylene amine attached as an alkyl ether. The amino acid sequence provides threonine, tyrosine, glutamic acid, or aspartic acid.

[0074] Alkylation of the amino group of valine produces a valine having an alkylene amine attached to the amine group. Brings phosphorus.

[0075] In a preferred embodiment, the alkyleneamine attached by reaction with an aziridine is It is an ethyleneimine group.

[0076] A Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention In embodiments, the alkylated amino acid is an alkyl group of formula (2) [ka] In the formula, R1 is H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, bromine, alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl alkyl, alkenyl, alkynyl, alkylaryl, aryl; Each of arylalkyl and cycloalkyl is selected from carbonyl, hydroxyl, alkyl and and haloalkyl, Preferably, R1 is H, acetyl, or hydroxyethyl. is selected from the group consisting of:

[0077] R2' and R2'' are each independently selected from H and alkyl. R2' and R2'' are each independently H and C 1~6 Select from alkyl Preferably, R2' and R2'' are each independently H, methyl, ethyl Preferably, the alkyl group is selected from the group consisting of propyl, isopropyl, butyl, and isobutyl. Preferably, at least one of R2' and R2'' is H. Preferably, R2' and R2'' are H. 2'' is H.

[0078] R3' and R3'' are each independently selected from the group consisting of H and alkyl. Suitably, R3' and R3'' are each independently selected from H and C 1~6 Archi Preferably, R3' and R3'' are each independently selected from H, methyl, Preferably, the alkyl group is selected from the group consisting of butyl, ethyl, propyl, isopropyl, butyl and isobutyl. Preferably, at least one of R3' and R3'' is H. Preferably, R3' and R 3'' is H.

[0079] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof, Alkyl has 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 Preferably, the alkenyl group has from 1 to 6 carbon atoms, most preferably from 1 to 3 carbon atoms. Alkyl, alkynyl and cycloalkyl have 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms. carbon atoms, more preferably 2 to 6 carbon atoms, and most preferably 2 to 3 carbon atoms Suitably, aryl has 5 to 12 carbon atoms, preferably 5 to 10 carbon atoms. More preferably, it has 6 to 10 carbon atoms.

[0080] One embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention In the same manner, the alkylated amino acid is alkylated with an alkyl group of formula (2) In the formula, - R1 is H; -R2' is H; -R2' is ethyl; -R1 is acetyl; -R2' is methyl; - R1 is ethanol; -R2'isobutyl; -R2'' is H; -R3' is H; or -R3'' is H.

[0081] Preferred embodiments of the Cryptosporidium gp40 protein or immunogenic portions thereof according to the present invention include: In a preferred embodiment, the alkyl group of formula (2) is - R1 is H, R2' is H, R2'' is H, R3' is H, and and R3'' is H; -R1 is C(=O)CH3, R2' is H, R2'' is H, and R3' is and R3'' is H; -R1 is H, R2' is CH2CH3, R2'' is H, and R3' is H and R3'' is H; -R1 is H, R2' is CH3, R2'' is H, and R3' is H; and R3'' is H; -R1 is CH2CH2OH, R2' is H, R2'' is H, and R3' is and R3'' is H; and - R1 is H, R2' is C(CH3)3, R2'' is H, and R3' is H and R3'' is H; The group has one of the combinations of substituents from the group:

[0082] One embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention In the same manner, the alkylated amino acids are valine, glutamic acid, and aspartic acid. and one or more selected from the group consisting of:

[0083] Further analysis was performed using incubation with aziridine as described in the Examples. showed that these amino acids of gp40 are preferentially alkylated by .

[0084] As noted above, the Cryptosporidium gp40 protein according to the present invention is SEQ ID NO: 1 or a homolog thereof. The amino acids correspond to E88, E94 and D129 of SEQ ID NO:1.

[0085] Note: E and D are the one-letter IUPAC codes for glutamic acid and acetylcholine, respectively. The amino acid code for hydroxybenzoic acid is 88. For example, it refers to glutamic acid, which is found in the

[0086] In a preferred embodiment, the gp40 protein of the present invention comprises a sequence number incorporating SEQ ID NO:1. More preferably, gp40 comprises a portion of amino acid numbers 19 to 20 of SEQ ID NO: 3. 7; amino acid numbers 15 to 207 of SEQ ID NO: 3; 10: to 207; 5 to 207; 4 to 207 ; 3 to 207; 2 to 207; or 1 to 207, in this order of preference.

[0087] As described in the Examples, the One form of gp40 protein according to the present invention that is used is that comprising amino acid numbers 2 to 3 of SEQ ID NO: 3. Apparently, the partial signal sequence was not cleaved, but the N-terminal methionine residue was removed. The amino acid residue was cleaved, leaving the valine at position 2 (V2) exposed at the N-terminus of gp40. Interestingly, the valine was also alkylated by aziridine incubation. Ta.

[0088] Therefore, the Cryptosporidium gp40 protein or immunogen thereof according to the present invention and alkyl selected from valine, glutamic acid and aspartic acid In one embodiment of the modified amino acid, valine is selected from the amino acid number (aa.no.) of SEQ ID NO: 3. .) valine corresponding to the valine in SEQ ID NO: 2, and glutamic acid corresponding to amino acid number 1 in SEQ ID NO: 3. Glutamic acid corresponding to glutamic acid 06 or glutamic acid 112 and / or the aspartic acid is asparagine at amino acid number 147 of SEQ ID NO: 3. Aspartic acid is the counterpart of acetic acid.

[0089] In a further aspect, the present invention provides a method for the preparation of Cryptosporidium gp4, any of which is according to the present invention. The present invention relates to compositions comprising the .DELTA.I. protein or immunogenic portions thereof.

[0090] As described, the Cryptosporidium spp. according to the present invention comprises an alkylated amino acid. The gp40 protein and immunogenic portions thereof can be combined with aziridines as described herein. It can be prepared by incubation.

[0091] Therefore, in a further aspect of the present invention, the Cryptosporidium gp The 40 protein or immunogenic portion thereof may be a Cryptosporidium gp40 protein or or a composition containing an immunogenic portion thereof with aziridine. It can be obtained by

[0092] Similarly, in a further aspect, the present invention provides a Cryptosporidium gp4 strain according to the present invention. A method for the preparation of a Cryptosporidium spp. protein or immunogenic portion thereof, comprising: A composition containing the gp40 protein or an immunogenic portion thereof is mixed with aziridine. The present invention relates to a method comprising the step of incubating a

[0093] CAUTION: Aziridine must be stored, handled, and disposed of in a safe and compliant manner. Aziridines are toxic and mutagenic chemicals that must be avoided. It can be neutralized by incubation with, for example, sodium thiosulfate (Na2S2O3). This can be done.

[0094] The aziridine ring is a three-membered heterocyclic ring consisting of one amine group and two methylene groups. "Aziridine" is an organic chemical compound containing an aziridine ring and has the structural formula (1): [ka] It has.

[0095] R1 is H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, brosyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl alkyl, alkenyl, alkynyl, alkylaryl, aryl Each of alkyl and cycloalkyl is selected from the group consisting of carbonyl, hydroxyl, alkyl and halides. Preferably, R1 is substituted with a substituent selected from the group consisting of alkyl, aryl ... is selected from the group consisting of H, acetyl and hydroxyethyl.

[0096] R2' and R2'' are each independently selected from H and alkyl. R2' and R2'' are each independently H and C 1~6 Select from alkyl Preferably, R2' and R2'' are each independently H, methyl, ethyl Preferably, the alkyl group is selected from the group consisting of propyl, isopropyl, butyl, and isobutyl. Preferably, at least one of R2' and R2'' is H. Preferably, R2' and R2'' are H. 2'' is H.

[0097] R3' and R3'' are each independently selected from the group consisting of H and alkyl. Suitably, R3' and R3'' are each independently selected from H and C 1~6 Archi Preferably, R3' and R3'' are each independently selected from H, methyl, Preferably, the alkyl group is selected from the group consisting of butyl, ethyl, propyl, isopropyl, butyl and isobutyl. Preferably, at least one of R3' and R3'' is H. Preferably, R3' and R 3'' is H.

[0098] Suitably, alkyl has from 1 to 20 carbon atoms, preferably from 1 to 10 carbon atoms, more preferably More preferably, it has 1 to 6 carbon atoms, and most preferably 1 to 3 carbon atoms. Preferably, alkenyl, alkynyl and cycloalkyl have 2 to 20 carbon atoms, more preferably Preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, most preferably 2 to 3 Suitably, the aryl has 5 to 12 carbon atoms, preferably 5 to It has 10 carbon atoms, more preferably 6 to 10 carbon atoms.

[0099] Aziridines commonly used for microbial inactivation include ethyleneimine (R1=H) and and 1-acetyl-ethyleneimine (R1 = acetyl).

[0100] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for the preparation of the aziridine moiety, R is H or It is an aziridine of formula (1) which is

[0101] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for the preparation of the aziridine moiety, - R1 is H; -R2' is H; -R2' is ethyl; -R1 is acetyl; -R2' is methyl; - R1 is ethanol; -R2'isobutyl; -R2'' is H; -R3' is H; or -R3'' is H; It is an aziridine of formula (1).

[0102] The aziridine ring is highly reactive, reacting with one of the methylene groups, e.g., proteins. This allows the ring to open, resulting in alkylation of the sulfhydryl group. At alkaline pH, the amino acid of methionine is easily alkylated by aziridine. Oethers can also be alkylated with aziridines.

[0103] During the alkylation of proteins with aziridines according to the present invention, the ring-opening of the aziridines The resulting ring becomes attached as an alkyl group to one or more of the amino acids of the protein. For example, one or more amino acids may be alkylated with an alkyl group of formula (2) to form a compound of formula ( In 2), the dotted line represents the bond to an amino acid. [ka] R1 is H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, brosyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl alkyl, alkenyl, alkynyl, alkylaryl, aryl Each of alkyl and cycloalkyl is selected from the group consisting of carbonyl, hydroxyl, alkyl and halides. Preferably, R1 is substituted with a substituent selected from the group consisting of alkyl, aryl ... is selected from the group consisting of H, acetyl and hydroxyethyl.

[0104] R2' and R2'' are each independently selected from H and alkyl. R2' and R2'' are each independently H and C 1~6 Select from alkyl Preferably, R2' and R2'' are each independently H, methyl, ethyl Preferably, the alkyl group is selected from the group consisting of propyl, isopropyl, butyl, and isobutyl. Preferably, at least one of R2' and R2'' is H. Preferably, R2' and R2'' are H. 2'' is H.

[0105] R3' and R3'' are each independently selected from the group consisting of H and alkyl. Suitably, R3' and R3'' are each independently selected from H and C 1~6 Archi Preferably, R3' and R3'' are each independently selected from H, methyl, Preferably, the alkyl group is selected from the group consisting of butyl, ethyl, propyl, isopropyl, butyl and isobutyl. Preferably, at least one of R3' and R3'' is H. Preferably, R3' and R 3'' is H.

[0106] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for the preparation of the aziridine moiety, - R1 is H, R2' is H, R2'' is H, R3' is H, and and R3'' is H; -R1 is C(=O)CH3, R2' is H, R2'' is H, and R3' is and R3'' is H; -R1 is H, R2' is CH2CH3, R2'' is H, and R3' is H and R3'' is H; -R1 is H, R2' is CH3, R2'' is H, and R3' is H; and R3'' is H; -R1 is CH2CH2OH, R2' is H, R2'' is H, and R3' is and R3'' is H; and - R1 is H, R2' is C(CH3)3, R2'' is H, and R3' is H and R3'' is H; is an aziridine of formula (1) having one of the combinations of substituents from the group

[0107] In a preferred embodiment of the aziridine for the present invention, one of the conditions selected from the following is satisfied: Applies. ethyleneimine preferably has CAS number 151-56-4, -2-ethyl-ethyleneimine preferably has CAS number 2549-67-9, -1-acetyl-ethyleneimine preferably has CAS number 460-07-1, -2-methyl-ethyleneimine preferably has CAS number 75-55-8, -1-ethyleneimine-ethanol preferably has CAS number 1072-52-2 the law of nature, 2-Isobutyl-ethyleneimine is preferably CAS number 3647-37-8. do.

[0108] As described, such incubation with aziridine inhibits the growth of cryptosporins. Polydum gp40 protein or immunogenic portions thereof, particularly cysteine, methionine , serine, threonine, tyrosine, lysine, arginine, valine, glutamic acid and amino acids This alkylation results in the alkylation of an amino acid selected from the group consisting of hydroxybenzoates, ... confers increased immunogenicity to such proteins or portions thereof.

[0109] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In the case of a method for preparing a soluble portion, "incubating" refers to the addition of an aqueous solution containing the indicated ingredients. Combining the compositions and allowing them to interact for a specific time and under specific conditions Suitable conditions for this incubation include the following: A condition that results in a detectable increase in the immunogenicity of a protein or portion thereof. All such incubations with the enzyme are well known to those skilled in the art and may be carried out in a timely manner if necessary. A wide variety of conditions and conditions are readily available for optimization and adaptation by routine methods. This can be done using parameters.

[0110] For example, the incubation can be carried out over a wide range of temperatures. Incubation with aziridine is preferably performed at temperatures above 0°C, more preferably between 1 and 55°C, and more preferably between 5 and 60°C. The temperature may be 50°C, 10-40°C, or 15-40°C, in that order of preference.

[0111] Similarly, incubation with aziridine can be carried out at a wide variety of pH values. However, alkylation of amino acids with aziridines occurs only at very acidic pH levels. It is most effective at pH values ​​that are not too acidic, as it can be less efficient. This would be incubation.

[0112] Thus, in one embodiment of the method, incubation with aziridine converts 4 to At a pH above 4.5, 5, 5.5, 6, 6.5, 7 or more preferably at ... or above pH 7.5, in that order of preference.

[0113] The upper limit of the pH value for incubation with aziridine in this method is It is easily determined in relation to other parameters of the incubation. Incubation is performed at a pH of less than 12, less than 10, or less than 9, in that order of preference. It is carried out in order.

[0114] As one skilled in the art will appreciate, aziridine is consumed during the incubation reaction; The concentration can only be reliably determined at the start of the incubation.

[0115] Therefore, the Cryptosporidium gp40 protein or Immunogenic portions thereof, as well as Cryptosporidium gp40 proteins or In one embodiment of the method for preparing the immunogenic portion, at the start of incubation The concentration of aziridine in this order of preference is at least 0.1 millimolar, more preferably Preferably at least 0.2, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 , 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 20 , 30, 40 or at least 50 millimolar.

[0116] The upper limit of the aziridine concentration can be easily determined. The aziridine concentration at the start of the reaction is, in order of preference, less than 1 molar, 0. It is less than 5 molar, or less than 0.1 molar.

[0117] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for preparing a functional portion, a composition comprising a protein or a fragment thereof is The duration of the aziridine incubation should be at least 10 minutes, in this order of preference. More preferably, at least 20, 30, 40, 50, 60 minutes, 1.5 hours, 2, 3, 4, 5, 6, 8, 10, 12, 15, 24, or at least 36 hours. The duration is overnight (i.e., 12-18 hours).

[0118] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for the preparation of the functional moiety, after incubation with aziridine, An addition step is carried out which results in the neutralization of any remaining aziridine.

[0119] In a preferred embodiment, the neutralization step is performed after the incubation reaction. Add the appropriate amount of thiosulfate to the mixture and allow it to stand for an appropriate length of time to complete the neutralization. This is done by incubation for a certain period of time. For example: 10-100 mM thiosulfate Neutralization by addition of sodium and incubation at 15-30°C for 15-90 minutes hmm.

[0120] In practice, the amount of thiosulfate used for neutralization is a slight excess. This acts as a guarantee: after the neutralization reaction, some thiosulfate If still remaining, it is certain that all of the aziridine has disappeared.

[0121] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for the preparation of the aziridine moiety, the aziridine is an ethyleneimine (R = H ), 2-ethyl-ethyleneimine (R2'=ethyl), 1-acetyl-ethyleneimine ( R1 = acetyl), 2-methyl-ethyleneimine (R2' = methyl), 1-ethyleneimine 2-isobutyl-ethyleneimine (R = ethanol) and 2-isobutyl-ethyleneimine (R = isobutyl).

[0122] In a preferred embodiment of the aziridine for the present invention, one of the conditions selected from the following is satisfied: Applies. ethyleneimine preferably has CAS number 151-56-4, -2-ethyl-ethyleneimine preferably has CAS number 2549-67-9, -1-acetyl-ethyleneimine preferably has CAS number 460-07-1, -2-methyl-ethyleneimine preferably has CAS number 75-55-8, -1-ethyleneimine-ethanol preferably has CAS number 1072-52-2 the law of nature, 2-Isobutyl-ethyleneimine is preferably CAS number 3647-37-8. do.

[0123] Because aziridine is such a dangerous chemical, the incubation time for this invention is In the present invention, it is preferably used in diluted form.

[0124] In the case of ethyleneimine, this results in the formation of the so-called "binary ethyleneimine" (BEI). This can be conveniently achieved by using alkaline conditions and e.g. For example, bromoethylamine hydrobromide (BEA) under gentle heating to about 37°C. The reaction product of the cyclization. Alkaline conditions are conveniently achieved by adding, for example, sodium hydroxide. This is all well known in the art and can be found, for example, in Bahne See Mann 1990 (ibid.).

[0125] Therefore, the Cryptosporidium gp40 protein or Immunogenic portions thereof, as well as Cryptosporidium gp40 proteins or In a preferred embodiment of the method for the preparation of the immunogenic portion thereof, the aziridine is ethyl or binary ethyleneimine.

[0126] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for the preparation of the aziridine moiety, prior to incubation with the aziridine An additional step is performed in which the BEI is produced.

[0127] Next, a Cryptosporidium gp40 protein or immunogenic portion thereof is A suitable amount of the produced BE as aziridine for incubation with the composition containing Use I.

[0128] Preferably, the BEI is produced from the reaction of BEA with a hydroxide.

[0129] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for preparation of an immunogenic portion thereof, the gp40 protein or an immunogenic portion thereof is The isolate is from Cryptosporidium parvum, more preferably genotype II It is from Cryptosporidium parvum.

[0130] For the purposes of the present invention, "from Cryptosporidium parvum" refers to a protein or directly by isolation of nucleic acids or by using other methods such as Cryptosporidium pallidum Indirectly based on sequence information from the gp40 protein, The partial amino acid sequence of the gp40 protein from the species Cryptosporidium parvum Indicates that it is based on

[0131] Therefore, the Cryptosporidium gp40 protein or immunogenic portion thereof of the present invention The component is preferably an isolated protein, and is preferably a Cryptosporidium gp40 protein of the present invention. The protein or immunogenic portion thereof is capable of binding to live Cryptosporidium in its natural context. This means that it is not in or on the parasite.

[0132] Cryptosporidium for use in incubation with aziridine of the present invention Compositions containing the cerebellar gp40 protein or immunogenic portions thereof can be produced in different ways. Cryptosporidium can be isolated from live infected host organisms. The protein or a portion thereof may also be isolated from a cell-free transcription system. It can be produced in

[0133] However, the most convenient and scalable approach to industrial production is to use recombinant expression systems. Cryptosporidium gp4 for use in the present invention by controlled in vitro expression The production of a desired protein or immunogenic portion thereof is achieved by such expression systems. Use a cell culture system of prokaryotic or eukaryotic cells genetically engineered to express the protein. Alternatively, the cells can be transfected with a gene that induces the cells to express a desired protein. Examples of recombinant expression systems include recombinant Chinese hamster ovary (CHO) cells, Escherichia coli, Bacillus subtilis (B acillus species or Staphylococcus carnosus cus carnosus) or yeast species, e.g., Saccharomyces cerevisiae Saccharomyces cerevisiae or Pichia pasteures ( Pichia pastores) and recombinant viruses for expression from host cells. Examples of the use of baculovirus-insect cell systems or adenovirus-mammalian cell systems are be.

[0134] Therefore, the Cryptosporidium gp40 protein or Immunogenic portions thereof, as well as Cryptosporidium gp40 proteins or In one embodiment of the method for the preparation of an immunogenic portion thereof, the gp40 protein or A composition comprising a portion of the compound is expressed by a recombinant expression system, preferably a baculovirus-insect cell Additional steps are taken to produce the product by the current system.

[0135] The baculovirus-insect cell expression system has been well known since the 1980s, and for reviews see ,Chambers et al.,2018,Curr.Protoc.Protei See Sci., vol. 91, pp. 5.4.1-5.4.6.

[0136] More preferred is the use of the sequence of SEQ ID NO:2 as a heterologous insert into a recombinant baculovirus. Expression of Cryptosporidium gp40 protein by expressing recombinant DNA sequences Produced using a baculovirus-insect cell expression system.

[0137] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for the preparation of a functional portion, the method comprises administering to the subject a therapeutically effective amount of ... The composition (before aziridine incubation) was expressed in a baculovirus-insect cell expression system. The composition may be produced from an insect cell culture, e.g., as a whole culture, or a portion of such a culture, for example the supernatant or cell pellet after centrifugation of an insect cell culture, Alternatively, it may be collected as the filtrate or retentate after filtration.

[0138] More preferably, the antibody comprises a Cryptosporidium gp40 protein or an immunogenic portion thereof. The composition containing the baculovirus-insect cell expression system is the supernatant or filtrate of the culture. After gravity settling of the culture, for example, by standing overnight or by centrifugation, The filtrate is what passes through the filter during filtration.

[0139] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for the preparation of a functional portion, the method comprises administering to the subject a therapeutically effective amount of ... The composition is produced by a recombinant expression system, and after production and harvesting said composition from the expression system: An additional step is carried out before the incubation with aziridine, which step comprises Preferably, the purification is by column chromatography. More preferably, The p40 protein or a portion thereof is prepared as a histidine derivative for metal affinity chromatography. a tag that facilitates purification of the composition by column chromatography, such as a ribonucleotide tag. include.

[0140] Incubation with aziridine is also an effective means for chemical inactivation of microorganisms. Therefore, the alkylated protein of the present invention or its alkylated derivatives The preparation of such immunogenic moieties can be advantageously combined with the inactivation of microorganisms, e.g., viruses or bacteria. It can be adjusted.

[0141] Therefore, the Cryptosporidium gp40 protein or Immunogenic portions thereof, as well as Cryptosporidium gp40 proteins or In one embodiment of the method for preparing the immunogenic portion thereof, Cryptosporidium gp4 Compositions comprising the protein or immunogenic portions thereof also contain microorganisms. For example, it may be the Cryptosporidium parvum parasite inactivated with aziridine, Dilysine inactivates the parasite but also causes alkylation of the gp40 protein. Preferably, the microorganism is a virus, e.g., as used in a recombinant expression system. Rus or bacteria.

[0142] Conditions for inactivating many microorganisms using aziridine are well known in the art. Any desired adaptation or optimization can be easily performed by routine methods. Cut.

[0143] Cryptosporidium gp40 protein obtainable by the present invention or its immunogenicity and the Cryptosporidium gp40 protein or immunogen thereof according to the present invention. In one embodiment of the method for the preparation of the functional moiety, the method comprises the steps of: One or more may apply. - incubation with aziridine above 0°C, more preferably between 1 and 55°C; The temperature range is 5-50°C, 10-40°C, or 15-40°C, in that order of preference. ru; - incubation with aziridine at a pH above 4, more preferably 4.5, At pH greater than 5, 5.5, 6, 6.5, 7, or at pH greater than 7.5, this preferred in the order of urgency; - Incubation with aziridine at a pH below 12, below 10, or below 9 , in this order of preference; The concentration of aziridine at the start of the incubation should be in this order of preference: At least 0.1 millimolar, more preferably at least 0.2, 0.3, 0.5, 1.0 ,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5,6,7,8,9,1 at 0, 11, 12, 13, 15, 20, 30, 40, or at least 50 millimolar concentrations be; The concentration of aziridine at the start of the incubation is, in this order of preference, 1 mol less than 0.5 molar, or less than 0.1 molar; - the duration of incubation of a composition containing a protein or a fragment thereof with aziridine The duration should be at least 10 minutes, more preferably at least 20 minutes, in this order of preference. 30, 40, 50, 60 minutes, 1.5 hours, 2, 3, 4, 5, 6, 8, 10, 12, 15, 24, or at least 36 hours; in one embodiment, the duration is overnight (i.e., 1 2 to 18 hours); -An additional step is carried out after incubation with aziridine, which removes any residual In a preferred embodiment, the neutralization is carried out by neutralizing the aziridine present in the reaction mixture with an appropriate amount of thiosulfonyl ether. This is done by adding sulfate to the incubation mixture; -Aziridine is ethyleneimine, 2-ethyl-ethyleneimine, 1-acetyl-ethyleneimine 1-Ethyleneimine-ethanol and 2-methyl-ethyleneimine isobutyl-ethyleneimine; For aziridines, one of the following conditions applies: o Ethyleneimine preferably has CAS number 151-56-4; o2-Ethyl-ethyleneimine preferably has CAS number 2549-67-9; o1-acetyl-ethyleneimine preferably has CAS number 460-07-1; o2-methyl-ethyleneimine preferably has CAS number 75-55-8; o1-Ethyleneimine-ethanol preferably has CAS number 1072-52-2. and o2-Isobutyl-ethyleneimine is preferably CAS number 3647-37-8 ru; the aziridine is an ethyleneimine or a binary ethyleneimine; - an additional step is carried out before the incubation with aziridine, in which BEI is generated; an appropriate amount of the generated BEI is then added to Cryptosporidium Incubation with a composition containing gp40 protein or an immunogenic portion thereof Preferably, BEI is used as an aziridine for the reaction with the hydroxide of BEA. Generated from; -gp40 protein or immunogenic portions thereof from Cryptosporidium parvum more preferably from genotype II Cryptosporidium parvum It is something; by recombinant expression systems, preferably by baculovirus-insect cell expression systems, Proteins or proteins to be incubated with aziridine in later steps of the process is preferably carried out in order to produce a composition containing a portion thereof; more preferably , by expressing the recombinant DNA sequence of SEQ ID NO: 2 as a heterologous insert, Production of Sporidioma gp40 protein using a baculovirus-insect cell expression system ; - produced by the baculovirus-insect cell expression system and extracted from insect cell cultures, e.g. Whole cultures include the supernatant or cell pellet after centrifugation of insect cell cultures, or the filtrate. or Cryptosporidium gp40 protein or its a composition comprising an immunogenic portion; - a composition comprising a Cryptosporidium gp40 protein or an immunogenic portion thereof, The supernatant or filtrate of a baculovirus-insect cell expression system culture; - Producing and harvesting compositions containing proteins or parts thereof by recombinant expression systems. An additional step is carried out after the step of preparing the composition, which step comprises purifying the composition; preferably, the purification by column chromatography; more preferably, Cryptosporidium g The p40 protein or immunogenic portions thereof may be used for metal affinity chromatography. and a histidine tag, such as a histidine tag, to facilitate purification of the composition by column chromatography. Contains tags that - a composition comprising a Cryptosporidium gp40 protein or an immunogenic portion thereof, It also includes microorganisms; preferably the microorganisms are viruses or bacteria.

[0144] Therefore, the Cryptosporidium gp40 protein or In a preferred embodiment of the immunogenic portion thereof, the aziridine is ethyleneimine or binary ethyleneimine; - Cryptosporidium gp40 is from Cryptosporidium parvum Ru, - a composition comprising a Cryptosporidium gp40 protein or an immunogenic portion thereof, is a supernatant or filtrate from a baculovirus-insect cell expression system culture, and - the supernatant or filtrate is purified by column chromatography; One or more or all of the features selected from the following may be applied:

[0145] Similarly, the present invention provides a method for the production of Cryptosporidium gp40 protein or its immunogenicity. In a preferred embodiment of the method for the preparation of a moiety, the aziridine is ethyleneimine or binary ethyleneimine; - Cryptosporidium gp40 is from Cryptosporidium parvum Ru, - a composition comprising a Cryptosporidium gp40 protein or an immunogenic portion thereof, is a supernatant or filtrate from a baculovirus-insect cell expression system culture, and - the supernatant or filtrate is purified by column chromatography; One or more or all of the features selected from the following may be applied:

[0146] As described, the present invention provides a safe and effective vaccine against cryptosporidiosis. The present invention provides an immunogen for the antibody containing one or more alkylated amino acids. Cryptosporidium gp40 or an immunogenic portion thereof has increased immunogenicity. Therefore, it is particularly useful in that respect.

[0147] Thus, in a further aspect, the present invention provides a method for treating human infections against cryptosporidiosis. or for use in a vaccine for the protection of non-human animal targets according to the invention. or a cryptosporin obtainable by the present invention or obtainable by a method according to the present invention. The present invention relates to the rhesus maize gp40 protein or immunogenic portions thereof.

[0148] Similarly, in a further aspect, the present invention provides a method for treating human or animal diseases against cryptosporidiosis. or a compound according to the invention for the manufacture of a vaccine for the protection of non-human animal targets. or obtainable by the method according to the invention. The present invention relates to the use of p40 proteins or immunogenic portions thereof.

[0149] A "vaccine" is generally defined as a composition containing an immunogen and a pharmaceutically acceptable carrier. Immunogens induce an immunological response in vaccinated targets, and this The response is effective in protecting against disease or against infection or its consequences. Protection is achieved by reducing the burden or duration of replication of the pathogen targeted by vaccination. This in turn relates to the shortening of the lesions, associated with diseases caused by pathogens. A reduction in the number, intensity, or severity of symptoms and clinical signs in vaccinated targets. and bring about.

[0150] Determining the efficacy of a vaccine according to the present invention is well within the skill of ordinary medical personnel. For example, by monitoring the immunological response after vaccination or after challenge infection. Target disease manifestations, e.g., by examining the appearance of subsequent clinical symptoms or mortality. by monitoring clinical scores, serological parameters, or re-isolation of the challenge pathogen These results are consistent with the vaccination-challenge response seen in sham-vaccinated animals. The relevant pathogens, their symptoms and diseases can be measured and characterized. Many methods of attachment are known in the art.

[0151] Cryptosporidium gp according to the invention for use in a vaccine according to the invention 40 protein or immunogenic portions thereof, and the use thereof for the preparation of a vaccine according to the present invention. Their use is effective in protecting against "cryptosporidium disease." A cluster of symptoms caused by infection with Cryptosporidium parasites in a susceptible target The symptoms mainly involve diarrhea, but some species of this parasite can also cause respiratory illness. For veterinary cryptosporidiosis symptoms, see The Merc Please refer to the "K Veterinary Manual" (cited above).

[0152] In the context of this invention, "protection against cryptosporidiosis..." means In reducing the severity and / or duration of diarrhea caused by parasitic gallstones The vaccine according to the present invention is effective in this respect, as demonstrated in the Examples section. It is valid.

[0153] Protection occurs in the vaccinated target itself. It also occurs in the immunized mammalian target. In some cases, protection can be indirectly provided by colostrum transfer. Milk is collected from pregnant mammals before and after birth, and the milk (or a product thereof) is used to The present invention relates to administering a therapeutic antibody (antibody that induces a leukemia) to a target human or non-human animal.

[0154] The result of this protection is a restoration of the overall health of the (passively) vaccinated target. In veterinary medicine, protection against cryptosporidiosis is achieved through (passive) vaccination. This results in increased economic performance in the treated animals, due to reduced mortality, Improved average daily weight gain, improved feed conversion ratio, improved milk production, improved reproductive output, and / or reduced healthcare costs.

[0155] In addition to reducing the symptoms of disease, especially diarrhea, protection is provided by reducing the risk of infection in the herd or flock environment and by reducing the risk of infection in the flock .... Reduced shedding of oocysts by vaccinated targets within a geographic area As a result, the protection of the present invention is It also results in a reduced prevalence of the Cryptosporidium parasite.

[0156] Cryptosporidium g according to the invention for use in a vaccine according to the invention of the p40 protein or immunogenic parts thereof and for the production of a vaccine according to the invention In a preferred embodiment of these uses, the vaccine is for the protection of ruminants. , more preferably for the protection of newborn calves by colostrum transfer.

[0157] In the context of the present invention, "ruminant" refers to any cud-chewing animal associated with veterinary or commercial farming operations. It relates to animals. Preferably, it refers to bovine, caprine, ovine or deer animals. More preferably Preferred are cattle, goats and sheep. Most preferred ruminant animals are bovine animals.

[0158] The "cattle" of the present invention includes taurine cattle (Bos taurus), zebu cattle, Cattle (Bos indicus), buffalo, bison, yak or Victoire. The cattle are dairy or beef cattle, or the parent stock of dairy or beef cattle breeds. It can be any type of thok.

[0159] To achieve protection against cryptosporidiosis as described, one or more Cryptosporidium gp40 protein according to the present invention containing a number of alkylated amino acids The protein or an immunogenic portion thereof is formulated and administered as a vaccine.

[0160] Thus, in a further aspect, the present invention provides a method for treating human infections against cryptosporidiosis. or a vaccine for a non-human animal target, according to the invention or prepared according to the invention Cryptosporidium gp40 proteins obtainable or obtainable by the method according to the present invention and a pharmaceutically acceptable carrier. do.

[0161] A "pharmaceutically acceptable carrier" is a substance that can be administered to a patient without causing any (serious) adverse effects. Such carriers may be aqueous solutions, e.g., water, It may be a buffer or a culture medium.

[0162] A preferred pharmaceutically acceptable carrier for the vaccine according to the present invention is insect cell culture medium. The medium may be saline, or a buffer such as PBS or 50 mM HEPES.

[0163] In addition, the pharmaceutically acceptable carrier may act as a bulking agent, stabilizer, preservative, or adjuvant. Further additives and excipients may be included. Details and examples are well known, e.g. For example, “Remington: the science and practice of pharmacy” (2000, Lippincott, USA, ISBN: 6833 06472) and "Veterinary vaccinology" (P.Past oret et al. ed., 1997, Elsevier, Amsterdam, I SBN 0444819681).

[0164] Furthermore, the vaccine according to the present invention may contain an adjuvant. In the case of immunizations, adjuvants further increase the target immune response to the subunit antigen. .

[0165] Therefore, in one aspect, the vaccine according to the invention comprises an adjuvant. It is a sign.

[0166] "Adjuvants" are well-known vaccine components that nonspecifically stimulate the target immune response. Many different adjuvants are known in the art. Examples of adjuvants include complete or Incomplete Freund's adjuvant, vitamin E or α-tocopherol, non-ionic bleach Lock polymers and polyamines, such as dextran sulfate, Carbopol™ , pyran, saponin, such as Quil A (trademark) or Q-vac (trademark). Ponin and vaccine components may be combined in ISCOM™.

[0167] In addition, peptides such as muramyl dipeptide, dimethylglycine, and tuftsin, mineral oil, For example, Bayol™, Drakeol™, Klearol™ or M arcol™, Montanide™ or light mineral (paraffin) oil; non-mineral oils such as arylene, squalane, etc.; vegetable oils or their derivatives, e.g., ethyl oleate ISA (Seppic) or Di luvacForte™ and Xsolve™ (both MSD Animal Combination products such as Al Health can also be used to advantage. An alternative is the SVEA adjuvant ( The use of squalane and vitamin E acetate.

[0168] A handbook on adjuvants and their use and effects is available in the Vaccination e adjuvants''(Methods in molecular medicine ine, vol. 42, D. O'Hagan ed., 2000, Humana pre ss,NJ,ISBN:0896037355).

[0169] In one embodiment of the vaccine according to the invention, the adjuvant is a mixture of an aluminum salt and an oil. The oil may be a mineral oil or a non-mineral oil, preferably one or more selected from the group consisting of: The oil is a mineral oil. The aluminum salt is preferably aluminum hydroxide.

[0170] A mineral oil adjuvant commonly used in veterinary vaccines is Drakeol (registered trademark). Mark) 6VR (Penreco), Marcol (registered trademark) 52 (Exxon Mobi le) and light (or white) such as Klearol® (Sonneborn) (color) liquid paraffin oil. Alternatively, Montanide from Seppic, France and pre-mixed mineral oil / emulsifier mixtures such as ®range.

[0171] Common non-mineral oil adjuvants are squalene and squalane (shark liver oil), olein The oil phase contains emulsifiers and stabilizers such as ethyl acetate and tocopherol (vitamin E). The composition may contain excipients such as:

[0172] A common emulsifier for vaccines is sorbitan monooleate (Span® 8 0) and polyoxyethylene-sorbitan monooleate (polysorbate 80 or Tween® 80. Common emulsion stabilizers are benzyl alcohol, Cholesterol and triethanolamine.

[0173] A commonly used aluminum salt is aluminum hydroxide, e.g., Alhydrog el(trademark)(Brennetag Biosector), Rehydragel(trademark) ) (Reheis) and Rehsorptar™ (Armour Pharma (e.g., eutical).

[0174] Vaccines containing oil-based adjuvants should be formulated as emulsions of aqueous and oil phases. Preferably, the emulsion is water-in-oil (w / o), oil-in-water (o / w), or water-in-water. Species selected from water-in-oil (w / o / w) and double oil emulsions (o / w / o) It is of the same kind.

[0175] More preferably, it is adjuvanted with oil and formulated as a water-in-oil emulsion. The vaccine according to the present invention is

[0176] Thus, in one embodiment of the vaccine according to the invention, the vaccine is a water-in-oil emulsion. It is formulated as a drug.

[0177] In a preferred embodiment of the vaccine according to the invention comprising an adjuvant, the adjuvant is an oil and preferably the oil is a light paraffin oil.

[0178] More preferably, the adjuvant also comprises an aluminium salt, which is preferably is aluminum hydroxide.

[0179] In a preferred embodiment of the vaccine according to the invention, said vaccine is administered to a pregnant mammal. Preferably, the pregnant mammal is a ruminant, More preferably, it is a bovine animal.

[0180] This allows for the collection of colostrum from the mammal before or after birth, The milk then provides antibodies to humans or non-human animals through the provision of colostrum or antibodies from the colostrum. It can be used to provide passive protection against liptosporidiosis.

[0181] The vaccine according to the invention can be administered to human or non-human targets by different application routes. This can be done.

[0182] In one embodiment, the vaccine according to the invention is administered by the parenteral route, i.e. through the skin. The preferred route of administration is, for example, intramuscular, intraperitoneal, intradermal, submucosal or subcutaneous. is by intradermal, intramuscular or subcutaneous route.

[0183] The volume per dose of the vaccine according to the invention depends on the characteristics of the specific vaccine to be applied, the target The route of administration can be selected according to the intended characteristics and the intended route of administration. For adult cattle, the preferred dose is 0.01-10 ml per dose. The recommended volume is 0.5 ml for the subcutaneous route and 1-2 ml for the intramuscular route.

[0184] The beneficial effect of alkylation with aziridine on the present invention is that it provides a good level of protection. A gp40 protein that is economical but does not contain one or more alkylated amino acids or a portion thereof, a smaller amount of Cryptosporidium gp40 protein according to the present invention The need for the protein or immunogenic portion thereof to be used in the vaccine dose. The choice of the amount of gp40 protein or portion thereof per dose depends on the characteristics of the vaccine and the target. This can be done by a person skilled in the art based on the above.

[0185] Thus, in one aspect, the vaccine according to the invention contains 0. 0.1 to 50 μg of the Cryptosporidium gp40 protein according to the present invention or its immunogen Preferably, the vaccine contains, in this order of preference, 0.0 per dose. 5 to 20 μg, 0.1 to 10 μg, or 0.1 to 5 μg of the cryptosporin according to the present invention The antibody comprises the cerebrospinal fluid gp40 protein or an immunogenic portion thereof.

[0186] Most preferably, the vaccine is administered at a dose of 0.5-2 μg per adult cow. The present invention comprises a Cryptosporidium gp40 protein or an immunogenic portion thereof.

[0187] In one embodiment of the vaccine according to the invention, the vaccine is intended for vaccination of cattle. In some cases, the vaccine contains less than 10 μg of Cryptosporidium according to the present invention per dose. gp40 protein or an immunogenic portion thereof. The vaccines by Maki contain less than 9, 8, 7, 6, 5, 4, 3 μg, or 2 μg per dose. less than 100 mg of Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention include.

[0188] Most preferably, the vaccine is administered at a dose of 1 μg of the present invention per adult cow. The present invention includes a Cryptosporidium gp40 protein or an immunogenic portion thereof.

[0189] Cryptosporidium gp40 protein or its immunization according to the present invention per dose The amount of virulent moiety can be assayed in the ready-to-use vaccine emulsion. This can be done by using standard biochemical testing procedures, e.g., by breaking down emulsions. This can be done by examining the aqueous phase using SDS-PAGE. The amount of protein can be determined by comparison with a known amount of a standard protein, e.g., albumin. For example, the Protein Protocols Handbook ok,2nd edition,September 2002,ed.JMWal ker,Humana Press Inc.,Totowa,NJ;Chapter Please refer to 29, pp. 237-242.

[0190] Preferably, the Cryptosporidium gp40 protein or immunogen thereof according to the present invention The amount of the active moiety is determined by antigen mass ELISA as described below. Protein quantification is performed using liquid chromatography such as HPLC. This can be done by mass spectrometry or by immunoassay.

[0191] Preferably, Cryptosporidium gp40 protein or The amount of the immunogenic portion thereof may be adjusted before mixing with the aluminum salt and / or after emulsification with the oil phase. Before the vaccine is administered, it is determined in the aqueous phase of the vaccine according to the invention.

[0192] The administration regime of the vaccine according to the invention depends on the type of protection intended, i.e. vaccination. Vaccination to provide active protection for the target itself, or passive vaccination The animals are selected based on vaccination to produce colostrum for the animals.

[0193] Active vaccination of a target can mobilize humoral and cellular immune responses, advantageously Active vaccination of the target can be timed to suit the needs of the patient. Regardless of the age, weight, sex, immunological status and other parameters of the target Active vaccination can be given as a single dose followed by, e.g. For example, a booster vaccination about two weeks later, followed by annual revaccination. can be done.

[0194] However, passive vaccination via colostrum transfer is almost exclusively antibody mediated. Therefore, in order to obtain high levels of gp40-specific antibodies in colostrum, Vaccinations for milk production should be timed with respect to the expected date of calving. For different species of animals, the gestation period is known and therefore this vaccination The timing of the insemination of milk can be selected and optimized to produce colostrum in cows. Pregnant cows are preferably administered two doses of the vaccine according to the invention, i.e., two doses at the expected time. Booster vaccinations in the period 1 to 4 weeks before parturition and 1 to 4 weeks after booster vaccinations A primary vaccination is administered 12 weeks prior. Preferably, a booster vaccination is administered. is given 2-4 weeks before expected delivery, with the prime given 2-6 weeks after the booster vaccination. If the cow has already received a prime and booster vaccination for a previous pregnancy, If you have had the vaccine, a single dose is sufficient for subsequent pregnancies. Possibly, 1 to 12 weeks before expected delivery, preferably 2 to 8 weeks before expected delivery. is given for a period.

[0195] Preferably, the method, timing and volume of administration of the vaccine according to the present invention are adapted to target To reduce stress and labor costs, the target human or non-human animal needs These other vaccines will be integrated into the existing vaccination schedule. The operations can be simultaneous, concurrent or sequential. They can be administered in any suitable form and in a manner compatible with their approved uses.

[0196] In a preferred embodiment, the vaccine according to the invention is administered to pregnant cows and the colostrum In combination with other vaccines to protect newborn calves against metastatic neonatal diarrhea Examples of such other vaccines are: Guardian® (Merck) is a water-in-oil emulsion containing 1,000 mg of glycerol and is administered subcutaneously. (K Animal Health) and both mineral oil and aluminum salts as adjuvants. Rotavec® Co is a water-in-oil emulsion containing rona (MSD Animal Health). The preferred combination method is This is "associated non-mixed" use.

[0197] Depending on the target animal, the target disease, the route of administration, etc., the vaccine or It may be desirable to adapt the administration parameters. This is well within the skill of the art. This is within the scope of the vaccine and generally involves fine-tuning the efficacy of the vaccine. This can be done by adapting the amount, quantity, frequency, route, excipients, etc.

[0198] The vaccine of the present invention may contain other additives such as stabilizers, carriers, adjuvants, diluents, emulsions, etc. It goes without saying that it is within the scope of the present invention to mix such compounds. The agent is "Remington" and "Veterinary Vaccinology (all cited above) and other well-known handbooks.

[0199] In the case of a vaccine according to the invention, further combination with additional immunoactive components may be carried out. This may be advantageous to boost immune protection already provided or to enhance immune protection already provided. These findings may help extend the immune defenses gained from these antibodies to other pathogens.

[0200] Thus, in one embodiment, the vaccine according to the invention comprises at least one additional immunization Contains active ingredients.

[0201] Such "additional immunoactive components" may be antigens, immunopotentiators, adjuvants or immunosuppressants. It may be an immunomodulator, a cytokine, another vaccine or any combination thereof. This provides advantages in terms of cost, efficiency, and target protection. A vaccine may itself be added to another vaccine.

[0202] General techniques and procedures applied to the manufacture of vaccines under well-known standards for pharmaceutical manufacturing. and considerations, such as government mandates and regulations (Pharmacopoeias, 9CFR) and well-known hand Books (Veterinary vaccinology and Remingt Generally, such vaccines are prepared sterilely. and prepared using pharmaceutical-grade excipients.

[0203] Such preparations are subjected to microbiological testing for sterility and the absence of adventitious agents. The studies may include in vivo or in vitro testing to confirm efficacy and safety. The vaccine is released for sale after completion of tests for quality, quantity, sterility, safety and efficacy. All of these are well known to those skilled in the art.

[0204] Therefore, in a further aspect, the present invention relates to a method for producing a vaccine according to the present invention. A method according to the invention or obtainable by the invention or a method according to the invention The Cryptosporidium gp40 protein or an immunogenic portion thereof obtainable by the method of The present invention relates to a method comprising the step of formulating the extract into cutin.

[0205] Such formulations may be used in combination with pharmaceutically acceptable carriers of the gp40 protein or immunogenic portions thereof. Such formulations may also include simple mixing with a carrier that may be used. Such formulations may also include emulsification. admixture with an adjuvant and / or with at least one additional immunoactive component This may include cases where

[0206] The variant of the vaccine against cryptosporidiosis according to the invention is The colostrum is produced by pregnant mammals immunized with clostridium erythropoietin. Highly effective for passive vaccination of human or non-human animal targets against liposporidiosis Colostrum can be given to or drunk by the target, and the crypts providing intestinal, mucosal and / or systemic immune defense against sporidiosis; or Antibodies derived from such colostrum can be administered.

[0207] Colostrum is used to vaccinate pregnant mammals with the vaccine according to the invention before birth. It can be generated by

[0208] Thus, in a further aspect, the present invention provides a method for treating a cancer cell comprising administering to a patient a cancer cell according to the present invention or obtained by the present invention. Cryptosporidium gp40 antigen obtainable or obtainable by the method according to the present invention. 1. A method for producing colostrum containing antibodies against a protein or immunogenic portion thereof, comprising: a. vaccinating a pregnant mammal at least once with a vaccine according to the present invention The process and b. collecting colostrum from the mammary gland of said mammal; The present invention relates to a method, including:

[0209] As is well known, "colostrum" is the milk secreted by the mammary glands of mammals before and after birth.

[0210] Preferably, the colostrum of the present invention is obtained from the mother's colostrum 1 day before to 4 days after birth, more preferably from the mother's colostrum 1 hour after birth. This is milk secreted over a period of ~72 hours.

[0211] More preferably, the colostrum according to the present invention is produced by a ruminant animal, and even more preferably by the It is secreted by a bovine animal as defined herein. Even more preferably, Colostrum is collected from the cow after calving at the first, second, third and fourth milkings. can be.

[0212] In one embodiment of the method for producing colostrum according to the invention, the target is a ruminant, preferably The target is a cow as defined herein.

[0213] The colostrum according to the present invention is capable of inhibiting the Cryptosporidium gp40 protein according to the present invention. or an immunogenic portion thereof, and these antibodies may be vaccinated with gp40 protein or a portion thereof that did not contain the modified amino acid higher concentrations and / or binding activity compared to antibodies in colostrum from mammals Or the specificity is different.

[0214] Vaccination of pregnant mammals is preferably carried out with a vaccine as described herein. This is done according to the vaccination schedule.

[0215] Collection of colostrum is preferably performed between 1 day before birth and 4 days after birth, as described herein. More preferably, the procedure is carried out for at least three days starting on the day of delivery.

[0216] In a further aspect, the present invention provides a method for treating cryptosporidiosis in humans or non-humans. Obtainable by the method for producing colostrum according to the invention for use in the protection of animal targets Regarding colostrum.

[0217] In a preferred embodiment of the colostrum for use according to the invention, protection is provided by the colostrum or by the colostrum-derived This is achieved by delivering the target of the incoming antibody.

[0218] As described, the present invention provides active and passive vaccines against cryptosporidiosis. Regarding both the kuching vaccination plan.

[0219] In a further aspect, the present invention provides a method for treating cryptosporidiosis in humans or non-humans. A method for the protection of an animal target, comprising administering a vaccine according to the invention to said target at least once. The method includes administering the compound once.

[0220] In a further aspect, the present invention provides a method for treating cryptosporidiosis in humans or non-humans. A method for the protection of animal targets, comprising the step of: providing the colostrum according to the present invention to said target.

[0221] The method of protection using colostrum for the present invention also comprises the steps of: The use of a preparation of gp40-specific antibodies purified from colostrum or from colostrum according to the invention It relates to passive vaccination of targets.

[0222] When passive protection methods concern cattle, there are different animal protection methods that are applied to cattle for different purposes. A further distinction can be made regarding the method of birth: dairy calves are generally These calves are separated from their mothers on the day of birth and should be fed colostrum. Vaccination with colostrum is achieved by active collection of colostrum and administering it to the young during the first week of life. Preferably, dairy calves are fed a diet of: For at least 3 days, more preferably at least 4 days, or at least 5 days, The first dose of colostrum should be given within 8 hours of birth, preferably at 6 hours of age. It should be administered to calves within 2 weeks of birth or within 4 hours of birth.

[0223] Colostrum feeding can be done once or twice a day, preferably feeding is done once a day.

[0224] In beef cattle, the concentration of antibodies in colostrum is higher than in dairy cattle because they produce less milk. Furthermore, beef calves are usually left with their mothers after birth. The provision of colostrum for this protection method is achieved by allowing the calf to ingest the colostrum by suckling. Preferably, beef calves are fed in this order of preference for at least 3 days. and administering the vaccines described herein for at least 4 days, or more preferably for at least 5 days. The calf is allowed to suckle colostrum from the cow that has been vaccinated. should be given within 8 hours of birth, preferably within 6 hours or within 4 hours of birth. is.

[0225] In one embodiment of both methods for protection according to the invention, the target is a ruminant, preferably The target is a cow as defined herein.

[0226] The invention will now be further illustrated by the following non-limiting examples. [Example]

[0227] [Example 1] Cryptosporidiosis vaccine production 1.1. Recombinant Protein Constructs Cryptosporidium parvum, Iowa strain, genotype II derived from SEQ ID NO: 1 Starting from the core amino acid sequence, recombinant gp4 for use in vaccination studies The protein was constructed to enable purification by nickel column chromatography. To achieve this, we extended this sequence with a C-terminal 6x histidine tag.

[0228] In the case of the Cryptosporidium gp40 protein, cleavage of the N-terminal signal sequence In an expression system where the fragment is amino acid number 20, the fragment is expressed as native gp40 starting at amino acid number 31. It is known that the nucleotide sequence is different from that of proteins. O'Connor et al. (2007, Mo (I. Bioch. Parasit., vol. 152, pp. 149-158) In the present invention, amino acid 14 from GenBank entry AAF78345.1 is The N-terminal gp40 signal sequence was partially restored by using ~30.

[0229] Additionally, an N-terminal methionine was added to initiate transcription. It was efficiently expressed in the baculovirus-insect cell expression system, but the secretion was not As a result, expression is cytoplasmic and is not tolerated in most insects. gp40 could be harvested from the culture supernatant at the end of the culture period when the cells were lysed.

[0230] Accuracy of Cryptosporidium gp40 protein used in vaccination trials The amino acid sequence of the AcMNPV baculovirus gene is shown in SEQ ID NO: 3. It was expressed from recombinant DNA that was codon-optimized based on the codon preference of the rehedrin gene. The recombinant DNA sequence used was that of SEQ ID NO: 2. Polyhedrin promoter A baculovirus vector based on the pVL1393 plasmid drives expression from Insert this recombinant gene as a BamH1-EcoR1 fragment into a transfer vector. Homologous recombination with linearized AcMNPV genomic DNA was performed using standard procedures. Stably transfected recombinant baculoviruses were generated by the following procedure. A recombinant vector expressing the recombinant Cryptosporidium gp40 protein (rgp40His) was used. The curovirus was isolated, plaque purified, and amplified.

[0231] Expression of Rgp40His was confirmed by SDS-Page of insect cell culture supernatant. SDS-Page showed the expressed protein as a band of approximately 32 kDa. Western using reclonal anti-gp40 antiserum and anti-His monoclonal antibody Further testing was performed by blot and immunofluorescence assays. After several tests for the stability and sterility of the recombinant baculovirus, It was designated as the master seed virus.

[0232] 1.2. Expression and Harvesting Rgp40His was produced from Sf9 insect cells infected with recombinant baculovirus seeds. Cells were cultured in commercially available animal compound-free insect cell culture medium SF900II™. Small-scale cultures of 0.5 to 2 liters were performed in the laboratory. Large-scale production of the culture was carried out in a production preparation facility. Generally, the following schedule was followed: Applied: Clean Sf9 cells were produced in increasing volumes of culture. Once cells were produced, they were concentrated and replated in fresh medium at 1.6 x 10^6 cells / ml. These were infected at a multiplicity of infection of 0.002 and incubated at 28°C for 5 days. At the end of the culture period, the supernatant was collected, which was collected by centrifugation at small scale and by deep cell culture at large scale. When expressed at small scale, the expression was followed by clarification by filtration. 1 hour incubation at room temperature with on® X-100, followed by The recombinant baculovirus was inactivated using gamma irradiation by If so, all downstream processing must be carried out in a contained facility using closed connections. The majority of the recombinant baculovirus is removed by different filtration and purification steps. Any remaining virus was then destroyed during aziridine treatment.

[0233] For purification, the collected rgp40His was applied to a Ni-Sepharose column. The column was then washed and rgp40His was eluted with imidazole. Imidazole was removed by diafiltration against 50 mM HEPES buffer at pH 7.5. Next, rgp40His was sterile filtered through a 0.2 μm membrane filter and aziridin was added. BEI was incubated with equal volumes of 1.09M BEA and 1.91M BEA. This was prepared by combining 545 mM BEI with 100 mM NaOH. A stock solution was then prepared. B was then added to the composition containing rgp40His to make a 33 mM solution. The mixture with BEI was incubated at room temperature for 24 hours. Sodium chloride was added to the solution at 33 mM. This was incubated at room temperature for one hour, and the pH was measured. The result was 7.1.

[0234] For comparison, buffer was added instead of aziridine for mock incubations. Cryptosporidium erythropoeias containing no one or more alkylated amino acids, except that Zyme gp40 was produced in the same manner.

[0235] 1.3. Determination of antigen mass First, a dilution range of known amounts of a standard protein was analyzed by SDS-PAGE. The amount of p40His protein was measured. The stained gel was then analyzed by densitometry. This test was later developed to be more accurate, i.e. specific, robust, and Assay performed by competitive antigen mass ELISA, fully validated for robustness, linearity, and precision. Briefly, antigen mass ELISA was performed as follows: Microtitration plates at 50 ng / well in ELISA buffer and overnight at 4 °C. wells were coated with purified rgp40His (unalkylated). The next day, the plates were washed and post-coated with casein-containing buffer for 1 hour at 37°C. On a separate microtiter plate, unknown concentrations of Test samples for rgp40His were placed in ELISA buffer (containing 0.05% polysorbate 80). A fixed amount of anti-gp40 mouse monoclonal antibody was added. This mixture was pre-incubated at 37°C for 1 hour, and then applied to the coated plates. The plates were then transferred to a tray and incubated in ELISA buffer at 37°C for 1 hour. The plate was washed and incubated in ELISA buffer at 37°C for 1 hour to detect peroxidase activity. Plates were incubated with conjugated goat anti-mouse IgG. The amount of bound gp40 monoclonal antibody was detected by tetramethyl-glucose precipitation at room temperature for 15 minutes. Peroxidase is visualized by enzymatic conversion of benzidine and this reaction is then quenched using sulfuric acid. The optical density of the yellow color in each well was measured at 450 nm. - The amount of conjugate is inversely related to the amount of antigen in the test sample. For three sample points, a software program using the Logit-Log algorithm was used. This was treated by gram. Samples were also measured at least in duplicate and the test Appropriate positive and negative controls were also used. Test values ​​were then calculated based on the standardized reference sample values. The antigen mass value of gp40 in the sample was calculated in micrograms / mL.

[0236] 1.4. Vaccine formulation It is then combined with suitable adjuvants and emulsifiers, all using well-known methods and materials. By combining column-purified and aziridine-incubated or pseudotyped The incubated rgp40His was used for further use as an emulsion vaccine. Briefly, sterile saline (0.85% w / v sodium chloride) rgp40His was added to the required concentration. In parallel, ISA ( The oil phase was prepared with Seppic 70VG (Seppic, France). Water-in-oil homogenizer using an Iverson™ or Dispax™ homogenizer Emulsify to an emulsion. Monitor the temperature increase during homogenization and keep it below 50°C. The resulting emulsion was stored at 4°C until filled into suitable containers. The resulting product undergoes various tests for stability and sterility before shipping.

[0237] Further adjuvants, depending on the intended geographic market or combination with other antigens, Specifically, batches of emulsions containing aluminum hydroxide were also prepared, in which case: First, the aqueous rgp40His preparation was diluted with 3% w / v Alhydrogel ( Combine with a heat-sterilized suspension of Brenntag® and mix at room temperature with stirring. The sample was then absorbed into aluminum for 30-60 minutes. Then, the antigen and aluminum were mixed as described above. This mixture of minium was emulsified in an oil phase.

[0238] Long-term stability studies for a 1- and 2-year shelf life at 4°C are underway. Intermediate and rapid stability test results for 15 months at 4°C and 3 weeks at 30 or 37°C The quality of the emulsion remained good throughout, and the antigenicity found was The average change in amount was within 10% of the starting composition, which indicates long-term stability under refrigerated conditions. is a good indicator of

[0239] [Example 2] Preparation for vaccination-challenge testing To evaluate the efficacy of various compositions tested as cryptosporidiosis vaccines To develop and optimize models for effective vaccination-challenge trials that can Over the years, several studies have been carried out in laboratory animals. The following settings have been used to assess in vivo efficacy: It turns out that this is best illustrated by: The vaccine was prepared as described above: Cryptosporidiosis gp40 protein was added to the vaccine. The cells were expressed in a human ovarian virus-insect cell expression system, harvested at the end of the culture, and analyzed by metal affinity chromatography. Purified by column chromatography (or not) and mixed with aziridine in ink. Then, either oil or oil and aluminum salt was added. The protein is formulated with an adjuvant and emulsified as a W / O emulsion. did.

[0240] Animal Models For passive vaccination studies, target animals were Holstein-Friesian cattle from approximately 1 year of age. Healthy dairy cows of the Anne breed, both heifers and cows that have given birth more than once, Cows were selected from the third trimester of pregnancy and had background levels of gp40-specific antibodies. The serum was tested for the presence of HIV only. No acclimation was required. Feed and water were provided according to standard cattle management practices. Animals were marked with a unique organism number via ear tag.

[0241] 2.2.Vaccination Aziridine-treated or sham-treated mice were treated with 10 mg of rgp40His per dose. The vaccine was administered in a volume of 2 ml, emulsified as W / O in mineral oil, and administered intramuscularly in the neck. The timing was a booster vaccination 3 weeks before the expected delivery date and an initial vaccination 6 weeks before the booster vaccination. Cows were also exposed to pathogens other than Cryptosporidium parvum. To prevent neonatal diarrhea in cattle, Rotavec® C was administered 4 weeks before the expected delivery date. Patients were vaccinated with the orona vaccine. Serum samples were collected at specific time points to measure antibody Tested by ELISA.

[0242] After calving, colostrum was collected as the first, second, and third milkings, with an average of 10 ... Four to five liters of colostrum were obtained. The milk was pooled and aliquoted into 250 ml jars, then placed in a water bath at 56°C for 30-45 minutes. The mixture was pasteurized using a 200 ml aliquot. Aliquots were then divided and kept frozen until use. Use a water bath set to 43°C, checking the water temperature regularly, or warm it by hand. Thawing was controlled by using tap water containing 100% ethanol. When fed to calves, the colostrum was (i.e., about 40°C).

[0243] Newborn calves were collected at birth, housed individually, and tested for anti-gp40 antibodies from one of the vaccine groups. A 3-liter bolus of colostrum was given within 4 hours of birth, followed by a 2-hour feeding. , a challenge dose of live Cryptosporidium parvum oocysts was given to calves.

[0244] Group sizes were 5–10 calves, and unprotected challenge groups were included in all experiments. This group was given sham colostrum. The use of unprotected non-induced indicators was not found to be beneficial. Calves included in the study were at least 3 months old at birth and were excluded from further studies. The weight was 0kg.

[0245] Calves were housed in single animal boxes in an isolated facility. Caregivers changed gloves and footwear before treating each animal.

[0246] Each box for one animal consisted of two compartments: one containing wood chips and one It consisted of a metal grid on a horizontal smooth surface plate above the waste collection channel. Calves were raised on wood chips for the first three days of life. At the end of the third day, the calves were placed on a second The smooth surface plate was used to assess stool consistency and determine diarrhea scores. The following scoring was performed on the fecal material produced during each scoring time point: To ensure consistent results, the plates were washed after each scoring time point.

[0247] Calves were fed twice daily; colostrum was first fed to the calves, followed by colostrum (first feeding). or from the second feeding onwards, a daily diet consisting of colostrum and milk replacer according to the manufacturer's specifications. I got it.

[0248] 2.3. Trigger 1 x 10^6 live Cryptosporidium parvum, Iowa strain per calf The challenge inoculum was prepared immediately prior to administration using 50 ml of milk replacer. This was orally administered to calves. The calves were then given either gp40- or anticoagulant in milk replacer. Colostrum feedings were given twice daily, totalling 2-2.5 liters of milk per day. 0.25-0.5 liters of colostrum for several days.

[0249] The challenge dose was chosen to produce repeatable and consistent diarrhea, which was protective. The effects were severe (but transient) in all unchallenged controls.

[0250] Live oocysts were obtained from Waterborne Inc., New Orleans, US. A. The oocysts were freshly released within 2 months before the start of the animal experiment. The survival rate of the parasites after excystation (test criteria are 50-100%), plus H The infectivity of the CT-8 cell layer was found to be sufficient both before the start of the experiment and at the end of the experiment. It was.

[0251] After induction, calves were monitored for 14 days, with the decline typically occurring between 4 and 11 days. Diarrhea developed and peaked in severity between days 5 and 10. Antibody levels were measured in the colostrum and serum of the calves on day 1.

[0252] 2.4. Scoring effectiveness To best quantify and evaluate the effect of vaccination against challenge infection, time is needed. Several methods were tested for quantity and consistency using only male calves. Faeces were collected in bags from all calves for analysis. However, this proved to be inaccurate due to loss of material. There are a number of observational scoring parameters, particularly general health, hydration, and buttock appearance. The diarrhea score was recorded twice a day. Ring parameters are based on S. McGuirk, 2008 (Vet. Clin. North Am. Food Anim. Pract., vol. 24, pp. 139-153) The parameters used for observation were based on the Wisconsin-Madison scale. The data are shown in Table 1. With some experience, animal caretakers can determine scores consistently. came.

[0253] If diarrhea becomes pronounced, a test for Cryptosporidium in the feces should be administered. is a commercially available test kit that uses liquid chromatography for specific test strips: BIO K 306(trademark)-Rainbow Calf Scours(Bio-X Dia The experiment was carried out using a chemosensor (Gnostics, Belgium).

[0254] To determine the reduction in shedding, oocysts were counted in fecal samples. The counts were as follows: a 50 ml sample was taken from the collected feces and used until The samples were serially diluted and placed on a diagnostic microscope slide and stored at 2-8°C. The slides were then stained red according to the Ziehl-Neelsen (acid-fast) method. The stained oocysts were counted by light microscopy. Results were expressed as oocysts per gram of feces. Expressed as cysts.

[0255] [Table 1]

[0256] [Example 3] Results of vaccination-challenge experiments 3.1. Recombinant expression of gp40: Optimized baculovirus-insect cell expression system: partial signal sequence and coding sequence This adaptation allows for greater protein production than previously used E. coli expression systems. However, crude gp40 harvest from insect cell cultures, i.e., only centrifuged and inactivated with Triton X-100 and gamma irradiation. When vaccinated with HIV, immunogenicity to the standard 20 μg dose was similarly inactivated. The immunogenicity of a similar dose of column-purified gp40 was not as good as that of a similar dose of column-purified gp40. Therefore, His-tag fusion constructs and nickel column purification were used as standards.

[0257] 3.2. Preparation of vaccine formulations Both the oil and oil plus aluminum adjuvants used had no effect on the growth of the folic acid in pregnant cows. As a result, effective antibody titers were induced in the colostrum of cows. For cattle that have never been vaccinated with flu vaccine, a single vaccination is not sufficient. and the presence of antibodies in colostrum that were protective against severe challenge as applied in these studies. A prime-boost vaccination regimen was required to induce levels of Naive cattle produced 9-12 Log2 gp40-specific antibody antiserum in antibody ELISA. Therefore, titers up to 10 Log2 were observed at background levels. It was thought that one or more alkylated amino acids were present in the W / O emulsion vaccine. After vaccination with a 10 microgram dose of amino acid-free purified gp40 This titer increased to 12-13 Log2 after the first vaccination and 15-1 after the booster vaccination. It increased to 7 Log2.

[0258] Colostrum containing gp40-specific antibody titers of approximately 20 Log2 is collected from these cows. As a result, purified RGP that was not incubated with aziridine A vaccine dose of 10 μg of 40His was used in the primary and booster experiments. When applied in combination with the vec Corona vaccine, it is effective against gp40 vaccination Vaccination against rotavirus, coronavirus or Escherichia coli No impact on efficacy was measured, see also Figure 2. These vaccines were effectively combined It was concluded that they could be combined.

[0259] Since there was no difference in colostrum intake between once a day and twice a day, we decided to continue with once a day. A total of 5 days of colostrum intake was also found to be sufficient.

[0260] 3.3. Rainbow diarrhea test All animals that received the challenge inoculum and had a diarrhea score of 2 or 3 were cryptically Tested positive for the presence of sporidium.

[0261] 3.4. Side effects Vaccination of cattle with an emulsified rgp40His vaccine is generally safe. There were no systemic effects or effects on pregnancy, but vaccination It induces local reactions at the site of the injury, which are palpable and show signs of inflammation at necropsy. A similar vaccine in emulsion, Rotavec Corona vaccine, This was not related to the presence of gp40 protein, as it did not induce such a local reaction. It became clear that this was the case.

[0262] The only way to reduce these reactions was to reduce the dose of gp40, but this was not effective. The reduction in dosage while maintaining efficacy can be achieved by administering one or more alkylated amino acids of the present invention. This was only possible using the Cryptosporidium gp40 protein, which contains amino acids. Ta.

[0263] [Example 4] Effect of aziridine incubation in vaccination-challenge experiments 4.1.Effect on antibody titers Vaccination of groups of five pregnant cows each as described in Example 3 above In the experiment, 10 in W / O emulsion with oil + aluminum adjuvant was used. Two doses of 1 μg of rgp40His were given to cows. The same formulation was used with or without incubation of the gin. The group that received only the vec Corona vaccine served as a negative control. The group includes vaccines with gp40 that do not contain one or more alkylated amino acids. He received both the blood test and the Rotavec Corona vaccine. Serum antibody titers were measured weekly and determined by antibody ELISA. Titers below 10 log2 were set at 10. Antibodies over time in all gp40-vaccinated animals were measured and considered background. The seroconversion profiles were similar. Only the Rotavec Corona vaccine showed a significant difference in gp The combined vaccine titer results showed that the two vaccine types This shows that there is no interference between the groups.

[0264] A clone containing the same amount of gp40 antigen but containing one or more alkylated amino acids Vaccines containing the R. typhimurium gp40 protein are non-alkylated gp40 The antibody titer induced in cattle was significantly higher than that of the 40 vaccine. The IgG4-related ... The titer of colostrum obtained from the immunized gp40 vaccine was very high at 24 Log2. It became clear that:

[0265] 4.2. Effect on emissions For shedding of oocysts by calves after challenge as a result of passive vaccination The effect was moderate but significant. Typically, between days 4 and 14, Vaccination from 10^9 oocysts in unvaccinated, induced calves 10^8 in the calf and 10^8 in the total number of oocysts per 100 grams of feces. However, due to the shorter duration of diarrhea seen in calves, Consequently, fecal output was much lower: 2 days in calves vaccinated with BEI-treated gp40. The overall reduction in oocyst shedding with inoculation was certainly relevant.

[0266] 4.3. Safety implications The gp40 protein combined with mineral oil adjuvant was found to be highly reactive. This was demonstrated when subcutaneous vaccination and prime-boost regimens were applied. This effect was most evident after aziridine incubation of gp40 protein. The condition did not worsen with treatment.

[0267] 10 μg of gp40 (alkylated or alkylated) with mineral oil adjuvant and adult Holstein cows were administered the first and second doses of 100 mg ... Vaccines given as booster vaccinations should be up to 10cm in diameter and 2cm thick. However, apart from appearing severe, the vaccine The chin-vaccinated cows showed no signs of suffering from this local reaction: No fever was observed and there was no effect on appetite or pregnancy.

[0268] Nevertheless, such swelling, even if asymptomatic and transient, is undesirable. It was decided that

[0269] Fortunately, the invention described herein provides a method for the treatment of g This allowed the dose of p40 protein to be reduced to a safe level: The amount of protein was adjusted to 1 μg of alkylated gp40 protein, and the s.c. After two doses given, the swelling was typically less than 5 cm in diameter and 1 cm in height, mostly It was only 2 cm in diameter, which was considered acceptable. This 10-fold reduction in the amount of gp40 antibody still allows for a sufficiently high titer of gp40 antibody to be obtained in colostrum. This did not affect the efficacy of vaccination in terms of the possibility of vaccination.

[0270] 4.4.Effects on diarrhea From the vaccination-challenge experiment, the incidence of diarrhea was estimated based on the parameters shown in the last column of Table 1. The severity was scored and the results for the two most relevant groups of calves are shown in Figure 3. The calves were vaccinated with either BEI-treated gp40 vaccine or untreated gp40 vaccine. They were fed colostrum from vaccinated mothers. As described, the first bolus of colostrum After the test, challenge was given on day 1. The mean daily diarrhea score per group was determined and the results were analyzed over the 14-day period. The data were plotted over the duration of the experiment.

[0271] As can be clearly seen in Figure 3, colostrum from BEI-gp40 vaccinated cows The diarrhea was much less severe and much shorter in the group given cereals containing cereals containing gluten. The difference was striking. and highly correlated: the rgp40His+BEI vaccine group had significantly more diarrhea (1.0 However, only two days (days 7 and 8) had a score above r The gp40His vaccine group (gp40 mock-treated with aziridine) was administered for 6 days (6-11 On day 1, the patient had severe diarrhea. This resulted in a lower incidence of diarrhea in the rgp40His+BEI vaccine group. Diarrhea was also much less severe: compared with a maximum score of 2.8 in the sham BEI-treated vaccine group , the maximum score was 1.2.

[0272] in conclusion: Severity of diarrhea caused by severe challenge infection with Cryptosporidium parasites A truly effective cryptosporidium inhibitor that can significantly reduce both the severity and duration of infection. A vaccine for flu has been discovered.

[0273] Passive vaccination of newborn calves through colostrum supply from vaccinated dams is also possible. The species model proved to be very effective.

[0274] [Example 5] Ongoing research 5.1. Dose setting Ongoing dose-ranging studies are evaluating the efficacy and safety of 0.1-0.2-0.5-1-2.5 and 5 μg doses of this compound. Vaccine doses of alkylated gp40 protein by Ming are being tested.

[0275] Initial results show that in a prime-boost regimen, The p40 protein induces sufficient levels of gp40-specific antibodies in pregnant cows. So far, the efficacy of 1 μg subcutaneous administration with mineral oil adjuvant has been demonstrated. When given twice by the IV route, the 19Log2Elisa single dose was A booster vaccination was given, and by the fourth week after vaccination, a titer of The titer then decreased to 18. A booster shot then increased the titer again to 22 at 6 weeks post-vaccination. This decreased slightly to a titer of 21 Log2 by week 8 after the primary vaccination. At the time of parturition, colostrum antibody levels were found to be 1-2 Log2 higher than serum Ab levels. This is what happened.

[0276] Considering that a colostrum titer of 17-18 Log2 is sufficient to protect the calf, The optimal vaccination protocol is 15-16 L in the dams of vaccinated cows. The goal is to reach a titer of og2. As a result, 0.5 μg of the alkyl group according to the present invention Even vaccine doses of modified gp40 protein were not associated with increased risk of HIV infection in mothers using prime-boost regimens. would be sufficient to reach such titer levels.

[0277] Alternatively, such protective titer levels in colostrum may be achieved at 0.5 or 1 μg of alpha-glucan per dose. A single vaccination of pregnant ruminants using methylated gp40 protein It is being investigated whether this can be achieved by providing seeds.

[0278] [Example 6] mass spectrometry Determine which amino acids are alkylated by aziridine incubation For this purpose, rgp40H was incubated with and not incubated with BEI. LC-MS / MS analysis of is was performed. Proteins were digested in parallel with different enzymes. , which has a 42 Da adduct corresponding to alkylation with an ethyleneimine molecule. , and the detected peptides were analyzed.

[0279] 6.1 Materials and Methods 6.1.1 Protein samples tested Two batches of rgp40His with different initial concentrations of protein: by BCA , 4.1 mg / ml of Batch 01K and 1.0 mg / ml of Batch 12G were used. Both of these batches were incubated with BEI as described. A batch of rgp40His protein that was not incubated with gin, batch 2DJ was used at 0.62 mg / ml and this batch was washed to remove any remaining baculovirus. The extract was nanofiltered.

[0280] 6.1.2 Intact mass analysis of gp40 Protein quantity was determined by BCA (Pierce) using the manufacturer's protocol. The Agilent 1290LC and Agilent 6545QTOFMS were used. Approximately 6–8 μg of r was identified by liquid chromatography and mass spectrometry (LC-MS). The gp40His sample was analyzed using MassPrep™ Online Desalt. A LC cartridge (Waters) was used at 60°C and a flow rate of 0.4 ml / min. Buffer A consisted of 0.1% v / v formic acid in water, and buffer B consisted of 0.1% v / v formic acid in acetonitrile. % formic acid. The gradient was 5% to 80% buffer B in 5 min, followed by 80% buffer B for 1 min. Equilibration was performed with 5% buffer B, and the total run time was 8 minutes. Dual AJS An ESI source (typically 4 kV) is used to electrospray the column eluate into the MS. MS scans were recorded between 100 and 3200 m / z at 1 spectrum / sec. was done.

[0281] 6.1.3 Peptide mapping of gp40 Enzyme:trypsin, chymotrypsin, or Glu at a 1:50 enzyme:protein ratio C (Staphylococcus aureus) ) Protease V8) digestion was performed to perform peptide mapping of rgp40His. After incubation for 1 hour, the enzymes were added again at the same ratio to complete the digestion. Run overnight. Sep-Pak™ tC18, 1 cc, 50 mg sorbent cartridge Sample cleanup was performed with 0.1% in 90% acetonitrile (Waters). The peptide mixture was eluted with v / v formic acid. The solvent was removed in a Speedvac™ at room temperature. The peptide sample mixture was reconstituted in 0.1% formic acid.

[0282] Approximately 30 μg of the enzymatic digest was analyzed by LC-MS as described. nt AdvanceBio(TM) Peptide Map, C18, 1.2×150 The peptide mixture was separated using a 2.7 mm column at 60°C and 0.4 mL / min. LC buffers A and B were as described. The gradient used was 2% to 4% in 110 min. 5% Buffer B, followed by 45% to 95% Buffer B for 5 min, and 95% Buffer B for 10 min Equilibration was performed with 2% Buffer B, and the total run time was 145 minutes. A JS ESI source (typically 4 kV) was used to electrify the column eluate into the MS. MS scans were performed at 4 spectra / s between 100 and 1700 m / z. MS / MS scans were recorded between 150 and 3200 m / z at 2 spectra / s. per cycle for MS / MS with an abundance threshold of 10,000 counts. Up to five precursor ions were selected. The precursor ions were selected after five iterations for one minute. The selection range was set to medium (4 amu).

[0283] 6.1.4 MS data analysis Intact analysis MassHunter(TM) Qualitative Analysis B.07 0.00 software was used to deconvolute the LC peaks containing the protein fragments. The deconvolution algorithm used was "maximum entropy." The range was 16,000-23,000 daltons, and the m / z range was 800-2000. Baseline subtraction was turned on and the baseline factor was set to 7.00. The isotope width was set to "auto".

[0284] Peptide mapping MassHunter software was used to measure the mass of the Agilent QTOF MS / M The S data files were converted into MFG files. A compound list was created using the "Search for Compounds by / MS" function. Enable "Extract MS / MS" by extracting average MS / MS spectra for energy Other than that, the default settings were applied. Set the "Conformational Model" to peptide and the "Restricted Assigned Charge States" to a maximum of 7. The compound list was exported to MGF using the settings. No. 3 Database for Cryptosporidium parvum gp40 recombinant protein , MFG files were searched. Enzyme restriction was set to none.

[0285] Ethyleneimine (43.04 Da, C2H5N) is variable for all amino acids The MS tolerance was set to 25 ppm and the MS / MS tolerance was set to 0.1 The peptide charge was set to 2+, 3+ and 4+.

[0286] Using the same settings as above, a host cell protein database (Sf9, Spodoptera Spodoptera frugiperda, Uniprot (201909127, 26,506 entries) .

[0287] 6.2 Results In gp40 incubated with BEI, the protein reacted with β-glucan at a rapidly decreasing frequency. 1 / 4 contain no EI adducts, 1 / 4 have one adduct, and the rest have two or more adducts had.

[0288] Intact mass analysis showed no evidence of glycosylation or other post-translational modifications. Additionally, no significant database hits were found for Sf9 host cell proteins.

[0289] Protein fragments can be isolated using trypsin, chymotrypsin, or GluC digestion. A multiple digestion approach was applied to analyze these. Together, these revealed overlapping peptides. A complete set of EI adducts was generated with good sequence coverage and high confidence in the assignment of EI adducts. The top four modification sites were subjected to additional manual analysis.

[0290] PepMap results showed complete sequence coverage of gp40, but not the His-tag tail. The sequence coverage of the nucleotide sequence ... This is highly unlikely. The coverage of various peptide fragments is shown in Table 2.

[0291] In Table 2, the 1 (*) of all EI-alkylated peptides in the untreated protein sample ) results in a false positive score.

[0292] [Table 2]

[0293] PepMap analysis allowed for site-specific identification of EI adducts. Because of prior knowledge of the EI modification site, the identification of the EI modification site was performed using automated database searches. Each EI adduct increases the size by 42 Da, but The resolution of the method was ±1 Da, so a weight increase of 41–43 Da was analyzed. The S analysis software selects for multiple EI adducts on a single amino acid. Ta.

[0294] The combined data from both batches incubated with aziridine showed that gp40 amine Acids: 2V, 106E, 112E and 147D (numbered as in SEQ ID NO: 3) The results are shown in Figure 4. Amino acids were also found to be alkylated, but (much) less frequently: cysteine threonine, tyrosine, methionine, serine, threonine, tyrosine, lysine and arginine. Interestingly, aspartic acid (D) and glutamic acid (E) are the major modification sites. The frequency of other amino acids was low.

[0295] The Cryptosporidium gp40 protein used in these studies is SEQ ID NO: The recombinantly expressed gp40-His had the amino acid sequence of No. 3. The major protein detected in all samples was a 20.9 kDa base protein. The rgp40His gene lacks the initiation methionine. This freed the valine at the 2-position for alkylation.

[0296] [Example 7] Vaccination-challenge study with low-dose vaccine As shown in Example 5 above, the present invention, which includes extremely low doses of BEI-gp40, A dose-finding experiment was conducted using a vaccine containing the active vaccine administered to pregnant heifers. The calves were passively vaccinated and challenged. Specifically, the experiment was carried out as follows: First, the pregnant women were given a 2-month-old baby. Large-scale vaccination is achieved by vaccinating cows and collecting colostrum from the first and second milkings. The colostrum obtained contained a large amount of antibody. The amount of protein was 0.4 or 1.5 μg. Oil (ISA70) was used as an adjuvant. ) + aluminum (Alhydrogel) to the above water-in-oil emulsion and The vaccine was formulated as follows: the first and second doses were administered 7 and 3 weeks before the expected delivery date, respectively. The vaccine was administered by the subcutaneous route as a booster. Serum and colostrum samples were tested for specific IgG antibody responses to gp40 using .

[0297] The newborn calves were then fed this colostrum for 5 days and then infected with Cryptosporidium parvum. After induction, the cells were incubated in a Wisconsin-derived medium as described by McGuirk (supra). Diarrhea scores and other clinical scores were determined according to the Sinchin-Madison scale. Reported as a health score.

[0298] 7.1 Generation of serum containing large amounts of antibodies using low-dose vaccines Pregnant heifers (Holstein-Friesian cows) were used in two groups: Group 1: 11 animals received 1.5 μg gp40 per animal dose in a 2 ml volume / dose. The BEI-rgp40his subunit vaccine containing an adjuvant was administered subcutaneously for 2 days. The vaccinations were administered approximately 7 weeks and 3 weeks before the expected delivery date. In group 2, 12 animals were not vaccinated due to control colostrum production. All animals were also treated with Rotavac® Coro according to the manufacturer's instructions. He was vaccinated with the na(MSD AH) vaccine.

[0299] From each animal in Group 1 immediately before (on the same day as) each vaccination and one week after the second vaccination after vaccination with the Rotavec Corona vaccine A single blood sample was taken from Group 2 animals one week prior.

[0300] The mean anti-gp40 Elisa titers per group were calculated from blood samples from vaccinated cows. The results of the Qing studies are shown in Table 3 along with their standard deviations.

[0301] [Table 3]

[0302] At the start of the study, mean IgG antibody titers to gp40 were similar in both groups, with background It was round level.

[0303] As can be seen from Table 3, the results showed that in Group 1 after the first and second vaccinations However, both titers were significantly increased in the serum IgG titers against gp40 in the colostrum. Protective levels of antibodies were shown to be produced.

[0304] After calving, the first two milkings were collected from each cow, followed by passive vaccination / challenge experiments. The first milking was collected within 6 hours after parturition, and the second milking was stored for further use. On average, approximately 5 liters of milk was collected per cow per milking. Colostrum was collected. The results of colostrum IgG ELISA were reported as the mean per group with standard deviation. Both are shown in Table 4.

[0305] [Table 4]

[0306] Colostrum titers indicated that protective levels of anti-gp40 colostrum antibodies were produced in the colostrum from vaccinated groups. This indicates that the titers in colostrum from unvaccinated control animals were higher than those in the control animals. was significantly higher than that of the control group (p value < 0.001).

[0307] 7.2 Passive vaccination-challenge studies using highly antibody-rich colostrum In subsequent experiments, the large amounts of antibodies generated as described in Section 7.1 above were The colostrum containing the IgG was used for passive vaccination of newborn calves. After 5 days of feeding, the Cryptosporidium parvum parasitism was observed in the colostrum-fed chicks. It was then possible to test protection against challenge with the organism. calves were born on different days, so the test schedule was run from different starting dates. Calves were Holstein-Friesian calves weighing at least 34 kg at the start of the experiment. The duration of the test was less than 4 hours and no colostrum had been given prior to the test.

[0308] All calves received 3 liters of colostrum and a combination of colostrum and milk replacer within the first 4 hours of birth. The colostrum was given once a day for 5 consecutive days (Groups 1 and 2). After a period of time, all calves were inoculated with 10^4 Cryptosporidium parvum oocysts. Each calf was assessed twice daily for 14 days for stool consistency and health score.

[0309] The groups were divided as follows: 1. n = 8, colostrum from cows vaccinated with 1.5 μg BEI-gp40 vaccine; 5 days 2. n=8, colostrum from unvaccinated cows, 5 days

[0310] To confirm the uptake of anti-gp40 antibodies, blood samples were taken from the calves at the start of the experiment and on day 3. A fluid sample was taken.

[0311] Health scores according to Table 1 above, including scoring of fecal consistency twice daily (morning and afternoon), were administered. Daily health checks of each calf were performed for 14 days after challenge to determine CORE.

[0312] At least one test confirmed positive for Cryptosporidium parvum Until further notice, the commercially available Rainbow Calf Scours 4™ test (BIO- K288) for the presence of Cryptosporidium parvum or other enteric pathogens Fecal samples from all animals with diarrhea scores of 2 or 3 at any given time point were collected. The quality was tested once a day.

[0313] Serological results showed the following serum IgG titers to gp40: on day 1, all The titer of 100 mg / kg of 10 ... The mean serum titers were group 1: 20.6±0.4 and group 2: 10.9±0.7.

[0314] Rainbow test scores indicate that all diarrhea is due to Cryptosporidium parvum infection. Group 1 (cows vaccinated with 1.5 μg BEI-gp40) Most calves fed colostrum from a cow (5 days old) had Cryptosporidium in their feces on the 8th day after challenge. parvum, as well as Group 2 (colostrum from unvaccinated cows, 5 days). Most calves showed Cryptosporidium parvum in the feces 6 days after challenge.

[0315] The health score results are shown in Figure 5. These scores include a fecal consistency score and a Wisco The results were determined according to the Sinchin-Madison scale (supra) as outlined in Table 1 above. Apparently, calves fed colostrum from cows vaccinated with gp40 Calves fed colostrum without anti-gp40 antibodies did not become as ill as those fed colostrum without anti-gp40 antibodies. much better able to cope with a severe challenge infection with the Tosporidium parvum parasite. came.

[0316] 7.3 Conclusion at a dose of 1.5 μg of alkylated gp40 protein per animal dose of vaccine Effective passive protection against even severe Cryptosporidium parvum challenge infection We conclude that it is possible to induce in colostrum a level of anti-gp40 antibodies that can be used to treat breast cancer. It can be extracted.

Claims

1. Cryptosporidium gp40 protein or an immunogenic portion thereof, 40 proteins and portions thereof containing one or more alkylated amino acids A Cryptosporidium gp40 protein or an immunogenic portion thereof, characterized in that:

2. The alkylated amino acid is an alkyl group of formula (2) 【Chemical 1】 In the formula, R1 is H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, bromine, alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl and wherein the alkyl, alkenyl, alkynyl, alkylaryl, Each of arylalkyl and cycloalkyl is selected from the group consisting of carbonyl, hydroxyl, alkyl, and cycloalkyl. optionally substituted with a substituent selected from the group consisting of alkyl, aryl, and haloalkyl; R2' and R2'' are each independently selected from H and alkyl; R3' and R3'' are each independently selected from the group consisting of H and alkyl. can be; The gp40 protein or immunogenic portion thereof according to claim 1, wherein the gp40 protein or immunogenic portion thereof is alkylated with Minutes.

3. The alkyl group of formula (2) is - R1 is H, R2' is H, R2'' is H, R3' is H, and and R3″ is H; -R1 is C(=O)CH3, R2' is H, R2'' is H, and R3' is and R3'' is H; - R1 is H, R2' is CH2CH3, R2'' is H, and R3' is H and R3″ is H; - R1 is H, R2' is CH3, R2'' is H, and R3' is H; and R3″ is H; - R1 is CH2CH2OH, R2' is H, R2'' is H, and R3' is and R3'' is H; and - R1 is H, R2' is C(CH3)3, R2'' is H, and R3' is H and R3″ is H; 3. The gp40 protein according to claim 1 or 2, having one of the combinations of substituents from the group Proteins or immunogenic portions thereof.

4. The alkylated amino acid is cysteine, methionine, serine, threonine, thiamine, thiamine, thiamine-1, thiamine-2, thiamine-3, thiamine-4, thiamine-5, thiamine-6, thiamine-7, thiamine-8, thiamine-9, thiamine-10, thiamine-11, thiamine-12, thiamine- from the group consisting of lysine, lysine, arginine, valine, glutamic acid and aspartic acid The gp40 protein according to any one of claims 1 to 3, which is one or more selected from the group consisting of: A protein or immunogenic portion thereof.

5. The alkylated amino acids consist of valine, glutamic acid, and aspartic acid. The gp40 according to any one of claims 1 to 4, which is one or more selected from the group consisting of: A protein or immunogenic portion thereof.

6. The valine corresponds to the valine at amino acid number (aa.no.) 2 of SEQ ID NO:

3. and the glutamic acid is the glutamic acid at amino acid number 106 of SEQ ID NO: 3 or is a glutamic acid corresponding to the glutamic acid at amino acid number 112, and / or The aspartic acid corresponds to the aspartic acid at amino acid number 147 in SEQ ID NO:

3.

6. The gp40 protein or immunogenic portion thereof of claim 5, which is paragic acid.

7. A composition comprising a Cryptosporidium gp40 protein or an immunogenic portion thereof is administered to an adult.

7. The method according to claim 1, wherein the lysine is soluble in water and the lysine is soluble in water. Item 1. Cryptosporidium gp40 protein according to item 1. or immunogenic portions thereof.

8. the aziridine is an ethyleneimine or a binary ethyleneimine 、 - the Cryptosporidium gp40 is Cryptosporidium parvum (Cryp tosporidium parvum), - a composition comprising said Cryptosporidium gp40 protein or an immunogenic portion thereof is the supernatant or filtrate from a baculovirus-insect cell expression system culture, and - the supernatant or filtrate is purified by column chromatography; The clip according to claim 7, wherein one or more or all of the features selected from Ptosporidium gp40 protein or immunogenic portion thereof.

9. A Cryptosporidium gp40 protein according to any one of claims 1 to 6, or A method for the preparation of an immunogenic portion thereof, comprising: incubating a composition comprising the protein or an immunogenic portion thereof with aziridine. Including, a method.

10. the aziridine is an ethyleneimine or a binary ethyleneimine 、 - the Cryptosporidium gp40 is Cryptosporidium parvum (Cryp tosporidium parvum), - a composition comprising said Cryptosporidium gp40 protein or an immunogenic portion thereof is the supernatant or filtrate from a baculovirus-insect cell expression system culture, and - the supernatant or filtrate is purified by column chromatography; 10. The method according to claim 9, wherein one or more or all of the features selected from 。

11. To vaccines for the protection of human or non-human animal targets against cryptosporidiosis The method according to any one of claims 1 to 6 or claim 7 or 8 for use in or obtainable by the method of claim 9 or 10. Lysium gp40 protein or immunogenic portion thereof.

12. Vaccines for the protection of human or non-human animal targets against cryptosporidiosis A method according to any one of claims 1 to 6 or according to claim 7 or 8 for the manufacture or obtainable by the method of claim 9 or 10. Use of the gp40 protein or immunogenic portions thereof.

13. A vaccine for human or non-human animal targets against cryptosporidiosis. The vaccine is a vaccine according to any one of claims 1 to 6, or according to claim 7 or 8. Cryptosporidium erythropoeisis according to claim 9 or obtainable by the method of claim 10. The present invention relates to a method for producing a medicament for the treatment of rhesus malabsorption, comprising administering to a subject a therapeutically effective amount of ... a medicament for the treatment of rhesus malabsorption, a medicament for the treatment of rhesus malabsorption, a medicament for the treatment of Hmm, vaccines.

14. 14. The vaccine of claim 13, wherein the vaccine comprises an adjuvant.

15. 10. The vaccine according to claim 9, characterized in that it comprises at least one additional immunoactive component.

15. The vaccine according to 13 or 14.

16. The method according to any one of claims 1 to 6, or according to claim 7 or 8, or A Cryptosporidium gp40 protein obtainable by the method according to claim 9 or 10.

16. The method of claim 13, further comprising formulating the protein or an immunogenic portion thereof into a vaccine. A method for the manufacture of a vaccine according to any one of the preceding claims.

17. The method according to any one of claims 1 to 6, or according to claim 7 or 8, or A Cryptosporidium gp40 protein obtainable by the method according to claim 9 or 10.

1. A method for producing colostrum containing antibodies against a protein or immunogenic portion thereof, comprising: a. administering the vaccine according to any one of claims 13 to 15 to a pregnant mammal administering at least one vaccination; b. collecting colostrum from the mammary gland of said mammal; A method comprising:

18. 18. The method of claim 17, wherein the target is a ruminant, preferably the target is a cow. method.

19. For use in the protection of human or non-human animal targets against cryptosporidiosis 19. Colostrum obtainable by the method according to claim 17 or 18.

20. A method for the protection of human or non-human animal targets against cryptosporidiosis. and administering at least one dose of the vaccine according to any one of claims 13 to 15 to said target. A method, comprising:

21. A method for the protection of human or non-human animal targets against cryptosporidiosis. and obtainable by the method of claim 17 or 18 or as claimed in claim 19. providing colostrum to said target.

22. 22. The method of claim 20 or 21, wherein the target is a ruminant, preferably the target is a cow. The method described below.

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