Cryptosporidiosis vaccine

Abstract

The present invention is based on the discovery that incubating Cryptosporidium gp40 protein with aziridine significantly increases the immunogenicity of the Cryptosporidium gp40 protein. When used as a vaccine, this allows for a reduced dose, thereby improving economic feasibility and safety. As a result, aziridine-treated gp40 can now be used as a safe and effective subunit vaccine for humans or non-human animals against cryptosporidiosis. Specifically, in the case of neonatal ruminants, vaccination via colostrum transfer has been shown to be highly effective in reducing the clinical signs of cryptosporidiosis, particularly diarrhea.

Description

[Technical field]

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

[0002] Cryptosporidium is a protozoan parasite of the phylum Apicomplexa. In mammals, Cryptosporidium resides primarily in the intestine and causes cryptosporidiosis, a disease characterized primarily by diarrhea accompanied by cramps, dehydration, and secondary infections. Symptoms can range from mild in immunocompetent humans or non-human animals to severe in more vulnerable hosts, such as young or immunocompromised animals. In human medicine, the disease is most prominent in malnourished children and AIDS patients. In veterinary medicine, disease in newborn ruminants: sheep, goats, and especially calves, is of major concern for animal welfare and economic reasons. The main pathogen is Cryptosporidium parvum, which occurs in zoonotic and human-infectious genotypes. Many other Cryptosporidium species, such as the human pathogen Cryptosporidium hominis (C. hominis), also cause cryptosporidiosis.

[0003] The parasitic stage released by infected hosts is the oocyst, which is highly infective and resistant to environmental conditions and many disinfectants. Thus, infection can spread rapidly through fecal material, for example by close contact or through fecal contamination of food or drinking water.

[0004] Several drugs have been approved for the therapeutic treatment of cryptosporidiosis. Nitazoxanide (a thiazolide) is indicated for use in immunocompetent humans, and Halocure® (halofuginone, MSD Animal Health) can be used in newborn calves. Ideally, an effective vaccine should also be developed, but this has proven extremely difficult as a result of the complex life cycle of this parasite and because in vitro culture of the parasite is not possible. As a result, there is currently no registered vaccine against cryptosporidiosis.

[0005] This is not for lack of attempts, as a wide variety of experimental vaccines against cryptosporidiosis have been tested over time, including live attenuated, inactivated, lysate and subunit vaccines. As subunit vaccines, many proteins from different parasite stages have been attempted. For a review, see Lemieux et al. (2018, Pathogens, vol. 7, DOI: 10.3390 / pathogens7010002).

[0006] One of many candidate antigens for a subunit vaccine is the 40 kDa mucin-type glycoprotein from Cryptosporidium gp40, which is present on the surface of motile parasite stages and is highly glycosylated.

[0007] It should be noted that the nomenclature of Cryptosporidium proteins is confusing, and gp40 (or its coding gene or gene product) is also called Cpgp40 / 15 (Cevallos et al., 2000, Inf. & Imm., vol. 68, p. 4108-4116); gp15 / 45 / 60 (Strong et al., 2000, Inf. & Imm., vol. 68, p. 4117-4134); or S60 (Winter et al., 2000, Funct. Integr. Genomics, vol. 1, p. 207-217). The Cp17 protein is also related (Priest et al., 2000 Mol. Biochem. Parasit., vol. 106, p. 261-271). The differences in molecular weights shown reflect variability in sequence and glycosylation levels. It should also be noted that the antigen designated "Cp15 / 60" is a different protein (Jenkins et al., 1993, Inf. and Imm., vol. 61, p. 2377-2382; GenBank Accession No. U22892). The same is true for the antigens designated "CP15" (GenBank Accession No. L34568) or "cp41" (WO 01 / 040439).

[0008] Expression of gp40 in recombinant expression systems and use for (passive) vaccination has been suggested for many years, for example in WO 93 / 024649, WO 01 / 040248, WO 01 / 077293 and US 2002 / 0081312. However, a functional vaccine is still not available.

[0009] In the case of neonatal ruminants, who are usually infected at a very early age, active vaccination would only confer protection after 3-4 weeks, which is too late in practice. However, a simple method of passive vaccination against cryptosporidiosis is possible by colostrum transfer, for example by feeding calves with colostrum from vaccinated cows. Such vaccination of neonatal calves against neonatal diarrhea, also called calf diarrhea, by colostrum transfer is described, for example, in WO 01 / 045735 and WO 2011 / 056175. This method of protection would also be consistent with the administration currently applied against other causes of neonatal calf diarrhea, such as by Escherichia coli, bovine coronavirus, bovine rotavirus and Clostridium. Passive vaccination of humans by feeding colostrum was also effective, see Lemieux et al., 2018 (supra).

[0010] Thus, many publications have described the isolation or expression of Cryptosporidium antigens and their study in diagnostic or antigenic studies. Some of the antigens have also been suggested to be used for active or passive vaccination of humans or animals. However, no commercial vaccine has been approved so far. Thus, there is a need in the art for a safe and effective vaccine against cryptosporidiosis.

[0011] Aziridines are organic chemical compounds that contain an aziridine ring. Due to their tendency to damage nucleic acids by alkylation reactions, causing cross-linking and strand breaks, these compounds are used in human medicine as anticancer drugs. In biotechnology, this property of aziridines is used for the inactivation of microorganisms such as bacteria and viruses.

[0012] Next to nucleic acids, aziridines can react with proteins by alkylation of nucleophilic sites on amino acids, for example, by reacting with sulfur-containing side chains such as sulfhydryl or thioether groups, with nitrogen-containing side chains such as amino groups, or with oxygen-containing side chains such as hydroxy groups.

[0013] These reactions may occur with one or more of the amino acids of the protein, although it is not completely understood which amino acid(s) reacts with the aziridine to become alkylated.

[0014] The aziridine most commonly used for virus inactivation is ethyleneimine (EI), mainly in the form of binary ethyleneimine (BEI), which has been reviewed by H. Bahnemann (1990, Vaccine, vol. 8, p. 299-303).

[0015] Although a hazardous chemical, the use of EI is preferred over conventional virus inactivation with formalin due to its more predictable, i.e., linear, kinetics, and because EI reacts preferentially with nucleic acids, it is less damaging to the immunogenicity of protein antigens than formalin (Blackburn & Besselaar, 1991, J. of Virol. Methods, vol. 33, p. 367-374; Hulskotte et al., 1997, Vaccine, vol. 15, p. 1839-1845).

[0016] There are some reports that describe the incubation of protein antigens with chemicals to increase antigenicity, but only for protein cross-linking reactions such as with formalin and / or heat treatment (Grovit-Ferbas et al., 2000, J. of Virol., vol. 74, p. 5802-5809). Other incubations of subunit proteins with inactivating agents have only been described for the detoxification of bacterial toxins with formalin. As for aziridine, incubation with subunit proteins has not been described outside the context of its use for the inactivation of microorganisms. Incubation with aziridine has not been described for Cryptosporidium gp40 protein.

[0017] Several papers have described negative effects on immunogenicity from the effect of aziridines on proteins during inactivation reactions and recommended caution, with Chen et al. (1999, J. of Exp. Med., vol. 189, p. 1757-1764) describing loss of antigenicity due to S-alkylation of cysteines in proteins. To reduce such reactivity, WO 98 / 51660 describes the development of polymers of EI that are even more specific for nucleic acids. WO 98 / 45415 also describes inactivation with EI at acidic pH levels to reduce adverse reactions with viral proteins. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] WO 01 / 040439 [Patent Document 2] International Publication No. 93 / 024649 [Patent Document 3] WO 01 / 040248 [Patent Document 4] WO 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 Document]

[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] It is an object of the present invention to overcome the shortcomings of the prior art and to address this need in the art by providing a safe and effective vaccine against cryptosporidiosis. [Means for solving the problem]

[0021] When we attempted to develop a cryptosporidiosis vaccine based on recombinantly expressed gp40 protein, early results were promising. Namely, gp40 could be expressed in Escherichia coli as described in the literature and used with standard adjuvants to vaccinate pregnant heifers. After parturition, colostrum was obtained, which contained high levels of gp40-specific antibodies. Feeding the calves with colostrum protected them from disease resulting from a severe Cryptosporidium parvum challenge infection. Unfortunately, the level of recombinant expression was not high and the expressed gp40 protein was poorly immunogenic, necessitating the application of relatively high antigen doses. Under these conditions, the production of a cryptosporidiosis vaccine based on recombinantly expressed gp40 was not considered feasible for commercialization.

[0022] A further problem was that the gp40 protein appeared to be highly reactive upon vaccination: when immunologically effective doses of gp40 were administered to cattle as a standard emulsion vaccine, there was good overall safety and no effect on pregnancy, but unacceptable levels of local vaccination reactions occurred, particularly using the subcutaneous route of administration, and these local reactions increased after booster vaccinations.

[0023] The use of lower antigen doses reduced local vaccination side reactions to tolerable levels, but unfortunately these doses did not induce sufficient antibody levels in the colostrum to provide effective passive vaccination of calves.

[0024] The inventors had no clue as to how to overcome these problems in order to develop an affordable, safe and effective vaccine against cryptosporidiosis.

[0025] Surprisingly, it has been found that the objects of the present invention can be met and one or more of the disadvantages of the prior art can be overcome by incubating gp40 protein with aziridine prior to its use as a vaccine. This incubation has been found to significantly increase the immunogenicity of gp40, resulting in higher titers of gp40-specific antibodies in the vaccinated mammalian target and, consequently, in the colostrum obtained from the vaccinated target, compared to immunization with the same amount of gp40 that has not been incubated with aziridine.

[0026] This discovery allows the antigen mass of gp40 used for vaccination to be lowered 10-20 fold compared to gp40 not incubated with aziridine, but still induce sufficient antibody levels in the colostrum to provide effective passive immune protection in the offspring against severe Cryptosporidium challenge infection. Next to providing an effective vaccine against cryptosporidiosis, the significant reduction in gp40 antigen mass per possible vaccine dose solved two further problems: it reduced the level of local reactions such that vaccination at effective doses became safe. Furthermore, it made cryptosporidiosis vaccines based on recombinantly expressed gp40 economically feasible.

[0027] It is known that gp40-specific antibodies can inhibit the entry of Cryptosporidium parasites into host cells, and as a result, if vaccination against cryptosporidiosis is achieved passively, for example by feeding colostrum from a vaccinated mammal, the increased levels of antibodies in the colostrum will result in increased local levels of antibodies in the gastrointestinal tract, providing protection against parasite invasion.

[0028] However, exactly how or why aziridine incubation increases the immunogenicity of gp40 is not known.

[0029] Although the inventors do not wish to be bound by any theory or model that may explain these findings, they expect that the chemical changes to the gp40 protein induced by incubation with aziridine, in particular the alkylation of one or more amino acids, will provoke a different response from the immune system of the vaccinated human or non-human animal target compared to the immune response to gp40 that was not alkylated by aziridine. Next to the observed increase in the level of anti-gp40 antibodies generated, there are likely also one or more other changes to the antibody profile generated, such as in the binding activity and / or specificity of the antibodies generated. Additionally, there may be a change in the type or level of the cellular immune response activated.

[0030] This was not at all obvious from any disclosure in the prior art. On top of the many publications reporting damage to immunogenic epitopes upon chemical inactivation, the positive effect on immunogenicity by aziridine incubation was completely unexpected. It goes without saying that an increase in immunogenicity of this magnitude is completely unexpected. Furthermore, no similar effect was observed when gp40 was incubated with formalin, Triton X-100, or after gamma irradiation.

[0031] Thus, in one aspect, the present invention relates to a Cryptosporidium gp40 protein or an immunogenic portion thereof, characterized in that said gp40 protein and said portions thereof comprise one or more alkylated amino acids.

[0032] A "protein" is a molecular chain of amino acids as defined herein. Polypeptides, peptides and oligopeptides, among others, are included within the definition of protein. A protein may be of natural or synthetic origin and may be a native or mature protein, a pre- or pro-protein, or a portion of a protein.

[0033] The term "Cryptosporidium" refers to a genus of parasites in the phylum Apicomplexa and subclass Coccidium. These microorganisms have features that characterize the taxonomic class, such as morphological, genomic and biochemical characteristics, as well as biological characteristics, such as physiological, immunological or pathological behavior. Many species of Cryptosporidium parasites are known. They can infect a wide variety of non-human animals as well as humans.

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

[0035] As known in the art, the classification of a microorganism in a particular taxon is based on a combination of its characteristics. Thus, the present invention also includes other species of Cryptosporidium classified in this genus. Similarly, it refers to parasites that are subclassified in some way from this genus, such as, for example, as subspecies, strains, isolates, genotypes, variants, subtypes or subgroups.

[0036] Furthermore, while a particular parasite of the invention may currently be assigned to such a species or genus, it will be apparent to one of skill in the art that such assignments are taxonomic classifications that may change over time, as new insights may result in reclassification into new or different taxa. However, such reclassified parasites remain within the scope of the invention, as this does not change the parasite itself or its antigenic repertoire, but only its scientific name or classification.

[0037] Samples of Cryptosporidium parasites for use in the present invention can be obtained from various sources, for example, from humans or as wild isolates from wild or farmed non-human animals, or from various laboratories, (depository) institutions, or (veterinary) universities. Also, much of the genetic information about Cryptosporidium and gp40 is digitally available in public sequence databases, such as NCBL's GenBank and EMBL's EBI. A public database that specializes in sequences from Cryptosporidium is CryptoDB, which is available online at cryptodb.org.

[0038] In the present invention, "gp40" is a protein comprising the amino acid sequence of SEQ ID NO: 1 or a homologue thereof. The same sequence is also shown in FIG. 1 in single letter IUPAC code. This particular sequence corresponds to the core sequence of gp40 protein from amino acid number 31 to number 220 from GenBank Accession No. AAF78345.1 (Strong et al., 2000, supra). This GenBank entry shows the amino acid sequence of the 60 kDa precursor protein from Cryptosporidium parvum Iowa strain, a U.S. bovine isolate and of genotype II.

[0039] In nature, gp40 is expressed as a precursor glycoprotein, with the N-terminal 2 / 3 of the precursor being gp40 and the C-terminal 1 / 3 being gp15. The precursor has an N-terminal signal sequence and a polyserine chain (both in the gp40 portion) and a C-terminal GPI anchor (in gp15). After cleavage of the precursor, the two proteins interact and attach to the apical surface region of the merozoite and sporozoite stages of Cryptosporidium, where they are involved in attachment and invasion of host cells. In nature, gp40 is heavily glycosylated, primarily with O-linked GalNac structures on threonine and serine amino acids.

[0040] The gene encoding the precursor protein of gp40 from Cryptosporidium parvum is listed in the CryptoDB database (supra) under gene number cgd6_1080.

[0041] A "homolog" of the Cryptosporidium gp40 polyprotein of the present invention is a protein comprising an amino acid sequence that has at least 60% amino acid sequence identity with SEQ ID NO: 1 when aligned to the entire length of SEQ ID NO: 1. Sequence alignments should be performed using the "blastp" algorithm with standard parameters from the NCBI BLAST™ suite of alignment software available online at blast.ncbi.nlm.nih.gov.

[0042] This spread in sequence identity levels is required to encompass the natural variation in the amino acid sequence of the Cryptosporidium gp40 protein of the present invention, which is known to be highly heterogeneous (see also Strong et al., 2000, supra). For example, even among gp40 proteins from Cryptosporidium parvum genotype II isolates, there is a variation in amino acid sequence identity levels of up to 23% across the sequence of SEQ ID NO:1. This results from some scattered amino acid sequence variation on the one hand, and from variation in the length of the polyserine tether on the other hand. This polyserine tether in Cryptosporidium gp40 can have from 6 to 25 consecutive serines, all of which are functional gp40 proteins. See, for example, the Cryptosporidium parvum gp40 genotype II homologs in GenBank accession numbers AOA32955.1 (6 serines) and ACR78128.1 (25 serines). When aligned to the entire length of SEQ ID NO:1 using the blastp program, they show 77 and 96% amino acid sequence identity, respectively.

[0043] Further variation in the gp40 amino acid sequence occurs in Cryptosporidium parvum genotype I isolates. As noted in Table 1 of Strong et al. (2000, supra), the amino acid identity between the gp15 / 45 / 60 precursor proteins from type I and type II isolates can be as low as 67%, yet still be functional gp40 proteins.

[0044] On the topic of Cryptosporidium parvum gp40 diversity, see also Leav et al., 2002, Inf. and Imm., vol. 70, p. 3881-3890; and Wu et al., 2003, Appl. Environ. Microbiol., vol. 69, p. 4720-4726.

[0045] Further levels of variability in the gp40 amino acid sequence come from gp40 homologs from closely related species such as Cryptosporidium hominis, which share amino acid sequence identity with SEQ ID NO:1 down to approximately 60%.

[0046] However, all of these homologous gp40 proteins can be used in the present invention.

[0047] To be capable of being alkylated by incubation with aziridine in the present invention, a homologue of Cryptosporidium gp40 has at least one amino acid selected from cysteine, methionine, serine, threonine, tyrosine, lysine, arginine, valine, glutamic acid and aspartic acid.

[0048] As used herein, the term "comprise" (and variations such as "comprising," "comprises," and "comprised") refers to all elements encompassed by or contained within a section, paragraph, claim, or the like of the text in which the term is used, and any possible combinations contemplated for the present invention, even if such element or combination is not explicitly recited, and does not refer to the exclusion of any such element or combinations.

[0049] Thus, any such section, paragraph, claim, etc. of the text may also relate to one or more aspects in which the term "comprising" (or variations thereof) is replaced with terms such as "consist of," "consisting of," or "consist essentially of."

[0050] An "alkylated amino acid" according to the present invention is an amino acid in which a sulfur, nitrogen or oxygen atom has been alkylated by reaction with an aziridine, so that the amino acid has an additional alkyl group compared to the natural structural formula of the amino acid. Alkylated amino acids relate, for example, to cysteine ​​amino acids whose side chain sulfhydryl group has been alkylated to an alkylene thioether, or methionine amino acids whose side chain thioether has been alkylated to a tertiary thioether; lysine or arginine whose side chain amino group has been alkylated to an alkylamine group; or serine, threonine, tyrosine, glutamic acid or aspartic acid whose side chain hydroxy group has been alkylated to an alkyl ether.

[0051] The alkylation reaction that occurs upon incubation with aziridine depends on the availability of such amino acids for reaction. For example, if two cysteines are connected by a sulfur bridge, the two cysteines are under non-reducing conditions and are not available for alkylation. On the other hand, an amino acid in which one of its groups is normally unreactive can be alkylated on the amino or carboxy group of such a group if such a group is exposed, for example, at the N-terminus or C-terminus of a protein fragment. For example, if a gp40 protein or a portion thereof begins with a valine, the amino group exposed at the N-terminus of that valine can be alkylated upon reaction with aziridine.

[0052] As will be appreciated by those skilled in the art, portions of Cryptosporidium gp40 proteins that contain one or more alkylated amino acids according to the invention may equally be used to induce increased levels of gp40-specific antibodies in immunized targets. The portion of the gp40 protein must be an "immunogenic portion" of the gp40 protein in the sense that it must be capable of inducing gp40-specific antibodies that can be used to protect against cryptosporidiosis. This is provided, inter alia, by portions having one or more alkylated amino acids, as described herein.

[0053] One of skill in the art can readily determine whether a portion of the gp40 protein or a homolog thereof is such an immunogenic portion by immunizing a target and testing whether gp40-specific antibodies are produced.

[0054] To ensure immunogenic efficacy for the present invention, an immunogenic portion of a Cryptosporidium gp40 protein comprising one or more alkylated amino acids has at least 50 contiguous amino acids from SEQ ID NO:1 and includes at least one alkylated amino acid selected from a glutamic acid corresponding to the glutamic acid at amino acid number (aa.no.) 88 or 94 of SEQ ID NO:1, and an aspartic acid corresponding to the aspartic acid at amino acid number 129 of SEQ ID NO:1.

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

[0056] Each of the Cryptosporidium gp40 proteins or their immunogenic parts according to the present invention can be modified in different ways, as is well known in the art. Examples are glycosylation, lipidation or pegylation. Also, further parts of the protein can be added, such as carriers, haptens, signal sequences, anchor sequences, markers or tags. Any of these modifications can be useful in the expression, detection and / or purification of the protein. Also, such modifications can be useful to adapt or even increase immunogenicity.

[0057] For example, the Examples provided herein below describe the use of a Cryptosporidium gp40 protein containing one or more alkylated amino acids with a C-terminal 6x-histidine tag, which had no effect on immunogenicity but allowed for purification of the gp40 protein by metal affinity chromatography, for example, when the protein was in the harvest of a recombinant expression system.

[0058] The N-terminal coding sequence of the gp40 protein used in the vaccination experiments described in the Examples was extended with a portion of the native signal sequence, An N-terminal methionine was encoded to initiate its transcription, which improved expression levels in the baculovirus-insect cell expression system without inducing secretion from insect cells.

[0059] The amino acid sequence of the Cryptosporidium gp40 protein used in the examples is shown in SEQ ID NO:3. This protein was expressed from a recombinant gene that contains the entire amino acid sequence of SEQ ID NO:1 with additional N-terminal and C-terminal sequences as indicated, providing a His tag, an additional methionine, and a partial signal sequence. Additionally, this gene encoding gp40 for the present invention was codon-optimized for the codon preferences of the baculovirus AcMNPV polyhedrin gene. The encoding nucleotide sequence is provided in SEQ ID NO:2.

[0060] Alkylation of Cryptosporidium gp40 protein or its immunogenic portion with aziridine results in an increase in the immunogenicity of the protein of the portion. The term "immunogenicity" 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 humoral and / or cellular type, and protection can be achieved in various ways, as described below. Immunogenicity in the context of vaccination is also referred to as "efficacy."

[0061] The increase in immunogenicity of a protein can be easily determined by those skilled in the art by performing comparative immunization experiments. In the present invention, this can mean comparing the immune response in a non-human animal immunized with a Cryptosporidium gp40 protein comprising one or more alkylated amino acids according to the present invention to the immune response of an animal immunized with gp40 that does not comprise one or more alkylated amino acids, where all other parameters and conditions are substantially identical. In accordance with the present invention, the increased immunogenicity results in a higher amount of gp40-specific antibodies being generated in the serum of the target by the Cryptosporidium gp40 protein comprising one or more alkylated amino acids compared to a similar dose of gp40 that did not comprise one or more alkylated amino acids. The level and specificity of the serological response can be easily measured by any suitable serological diagnostic technique, such as ELISA, IFT or AlphaLisa. For optimal comparison of the effect of the present invention, gp40 that did not comprise one or more alkylated amino acids should be mock-incubated, i.e. incubated under the same conditions as the aziridine-incubation, except that the aziridine is not present.

[0062] For example, following a single immunization of cattle with 10 μg of alkylated or mock treated gp40 in a standard oil adjuvant, Cryptosporidium gp40 protein containing one or more alkylated amino acids according to the present invention induced titers of gp40-specific antibodies that were 2-4 Log2 units higher compared to titers generated by gp40 not containing one or more alkylated amino acids when measured by standard antibody ELISA using coated gp40, representing up to a 16-fold increase in gp40-specific antibody titers resulting from alkylation with aziridine.

[0063] In the present invention, a gp40-specific antibody is an antibody that recognizes and binds to gp40 in a "specific" manner. In the art, this means that binding and recognition are correlated with the concentration of antibody and antigen. As a result, in the case of specific binding, dilution of either antigen or antibody should show a gradual decrease in binding levels, as detectable in standard serological assays. [Brief description 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. [Diagram 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 given in Examples 3 and 4. The test groups were: rgp40His = vaccine with non-alkylated 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 Rotavec Corona vaccine and vaccine with non-alkylated Cryptosporidium gp40 protein. [Diagram 3] Results of a vaccination-challenge study in calves with regard to diarrhea severity. The horizontal axis shows the number of days after challenge. The vertical axis shows the mean daily diarrhea scores for the rgp40His and rgp40His+BEI vaccine groups. 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 in SEQ ID NO:3) on the horizontal axis and the number of times that 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. [Diagram 5] Health scoring results of vaccination-challenge experiment using very low dose of gp40 protein to generate colostrum with large amount of antibodies. Horizontal axis is days after challenge. Vertical axis represents health score according to Wisconsin-Madison scale. Details are described in Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 present invention, a homologue is a protein comprising an amino acid sequence having at least 60% amino acid sequence identity with SEQ ID NO:1 when aligned against the entire length of SEQ ID NO:1 as defined herein.

[0067] More preferably, homologues of Cryptosporidium gp40 protein for purposes of the present invention have 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, in this order of preference, to SEQ ID NO:1 when aligned against the entire length of SEQ ID NO:1.

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

[0069] All of these amino acids are well known chemical compounds, and the non-incorporated chemical forms of these (their L-isomers) are: Cysteine: CAS Number: 52-90-4 Methionine: CAS Number: 59-51-8; Serine: CAS Number: 56-45-1; Threonine: CAS Number: 80-68-2; Tyrosine: CAS Number: 60-18-4; Lysine: CAS Number: 56-87-1; Arginine: CAS Number: 74-79-3; Valine: CAS Number: 72-18-4; Glutamic acid: CAS Number: 56-86-0; and Aspartic acid: CAS Number: 56-84-8.

[0070] Alkylation of the side chain sulfhydryl group of cysteine ​​resulted in a cysteine ​​bearing an alkylene thioether, in which an alkylene amine group replaced the hydrogen atom from the sulfhydryl group.

[0071] Alkylation of the side chain thioether group of methionine resulted in a methionine bearing a tertiary thioether, with an alkyleneamine group replacing the hydrogen atom from the thioether group.

[0072] Alkylation of the side chain amino group of arginine or lysine results in an arginine or lysine having an alkyleneamine group attached to the amine side chain.

[0073] Alkylation of the side chain hydroxy group of serine, threonine, tyrosine, aspartic acid, or glutamic acid results in the serine, threonine, tyrosine, glutamic acid, or aspartic acid having an alkylene amine attached as an alkyl ether.

[0074] Alkylation of the amino group of valine results in a valine having an alkyleneamine attached to the amine group.

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

[0076] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the alkylated amino acid is an alkyl group of formula (2) [ka] wherein R1 is selected from the group consisting of H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, brosyl, alkenyl, alkynyl, alkylaryl, arylalkyl, and cycloalkyl, each of which may be substituted with a substituent selected from the group consisting of carbonyl, hydroxyl, alkyl, and haloalkyl; Preferably, R1 is selected from the group consisting of H, acetyl and hydroxyethyl.

[0077] R2' and R2'' are each independently selected from H and alkyl. Suitably, R2' and R2'' are each independently selected from H and C. 1~6 Preferably, R2' and R2'' are each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, and isobutyl. Preferably, at least one of R2' and R2'' is H. Preferably, R2' and R2'' are 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 the group consisting of H and C. 1~6 Preferably, R3' and R3" are each independently selected from H, methyl, ethyl, propyl, isopropyl, butyl, and isobutyl. Preferably, at least one of R3' and R3" is H. Preferably, R3' and R3" are H.

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

[0080] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the alkylated amino acid is alkylated with an alkyl group of formula (2), wherein - 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] In a preferred embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the alkyl group of formula (2) is - R1 is H, R2' is H, R2'' is H, R3' is H, and R3'' is H; -R1 is C(=O)CH3, R2' is H, R2'' is H, R3' is H, and R3'' is H; - R1 is H and R2' is CH 2 CH 3 where R2″ is H, R3′ is H, and R3″ is H; - R1 is H and R2' is CH 3where R2″ is H, R3′ is H, and R3″ is H; -R1 is CH 2 CH 2 OH, R2' is H, R2'' is H, R3' is H, and R3'' is H; and -R1 is H and R2' is C(CH 3 ) 3 where R2″ is H, R3′ is H, and R3″ is H; has one of the combinations of substituents from the group:

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

[0083] As described in the Examples, further analysis showed that these amino acids of gp40 were preferentially alkylated by incubation with aziridine.

[0084] As noted above, the Cryptosporidium gp40 protein according to the present invention comprises the amino acid sequence of SEQ ID NO: 1 or a homologue thereof. Thus, the alkylated amino acids preferably included are those corresponding to E88, E94 and D129 of SEQ ID NO: 1.

[0085] Note: E and D are the amino acid codes for glutamic acid and aspartic acid, respectively, in the one-letter IUPAC code. Thus, "E88" refers to glutamic acid at amino acid number 88, etc.

[0086] In a preferred embodiment, the gp40 protein of the present invention comprises a portion of SEQ ID NO:3 that incorporates SEQ ID NO:1. More preferably, gp40 comprises amino acid numbers 19 to 207 of SEQ ID NO:3; 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 that order of preference.

[0087] As described in the Examples, one form of gp40 protein according to the invention found after recombinant expression and aziridine incubation contained amino acids 2-207 of SEQ ID NO:3. Apparently, the partial signal sequence was not cleaved, but the N-terminal methionine was cleaved, leaving a valine at position 2 (V2) exposed at the N-terminus of gp40. Interestingly, that valine was also alkylated by aziridine incubation.

[0088] Thus, in one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention and of the alkylated amino acids selected from valine, glutamic acid and aspartic acid, the valine is a valine corresponding to the valine at amino acid number (aa.no.) 2 of SEQ ID NO: 3, the glutamic acid is a glutamic acid corresponding to the glutamic acid at amino acid number 106 or the glutamic acid at amino acid number 112 of SEQ ID NO: 3, and / or the aspartic acid is an aspartic acid corresponding to the aspartic acid at amino acid number 147 of SEQ ID NO: 3.

[0089] In a further aspect, the present invention relates to compositions comprising a Cryptosporidium gp40 protein or an immunogenic portion thereof, any of which is according to the present invention.

[0090] As described, Cryptosporidium gp40 proteins and immunogenic portions thereof according to the invention that contain alkylated amino acids can be prepared by incubation with an aziridine as described herein.

[0091] Thus, in a further aspect of the invention, the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention is obtainable by incubating a composition comprising the Cryptosporidium gp40 protein or immunogenic portion thereof with aziridine.

[0092] Similarly, in a further aspect, the present invention relates to a method for the preparation of a Cryptosporidium gp40 protein or an immunogenic portion thereof according to the present invention, comprising the step of incubating a composition comprising a Cryptosporidium gp40 protein or an immunogenic portion thereof with aziridine.

[0093] CAUTION: Aziridine is a toxic and mutagenic chemical that must be stored, handled, and disposed of in a safe and regulatory compliant manner. Aziridine can be prepared by the conversion of thiosulfates, e.g., sodium thiosulfate (Na 2 S 2 O 3 ) can be neutralized by incubation with

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

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

[0096] R2' and R2'' are each independently selected from H and alkyl. Suitably, R2' and R2'' are each independently selected from H and C. 1~6Preferably, R2' and R2'' are each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, and isobutyl. Preferably, at least one of R2' and R2'' is H. Preferably, R2' and R2'' are 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 the group consisting of H and C. 1~6 Preferably, R3' and R3" are each independently selected from H, methyl, ethyl, propyl, isopropyl, butyl, and isobutyl. Preferably, at least one of R3' and R3" is H. Preferably, R3' and R3" are H.

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

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

[0100] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable by the present invention, as well as the method for preparing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention, the aziridine is of formula (1) in which R1 is H or acetyl.

[0101] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable according to the invention, as well as the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the aziridine is - 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 and can be opened by reaction of one of the methylenes, e.g. with proteins, resulting in alkylation of the sulfhydryl group. The amino acid cysteine ​​is readily alkylated by aziridine. At alkaline pH, the thioether of methionine can also be alkylated by aziridine.

[0103] Upon alkylation of a protein with an aziridine for the purposes of the present invention, the opened ring of the aziridine becomes attached as an alkyl group to one or more of the amino acids of the protein. For example, one or more amino acids are alkylated with an alkyl group of formula (2), where the dotted line represents the bond to the amino acid. [ka] R1 is selected from the group consisting of H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, brosyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl, each of which may be substituted with a substituent selected from the group consisting of carbonyl, hydroxyl, alkyl and haloalkyl. Preferably, R1 is selected from the group consisting of H, acetyl and hydroxyethyl.

[0104] R2' and R2'' are each independently selected from H and alkyl. Suitably, R2' and R2'' are each independently selected from H and C. 1~6 Preferably, R2' and R2'' are each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, and isobutyl. Preferably, at least one of R2' and R2'' is H. Preferably, R2' and R2'' are 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 the group consisting of H and C. 1~6 Preferably, R3' and R3" are each independently selected from H, methyl, ethyl, propyl, isopropyl, butyl, and isobutyl. Preferably, at least one of R3' and R3" is H. Preferably, R3' and R3" are H.

[0106] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable according to the invention, as well as the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the aziridine is - R1 is H, R2' is H, R2'' is H, R3' is H, and R3'' is H; -R1 is C(=O)CH3, R2' is H, R2'' is H, R3' is H, and R3'' is H; - R1 is H and R2' is CH 2 CH 3 where R2″ is H, R3′ is H, and R3″ is H; - R1 is H and R2' is CH 3 where R2″ is H, R3′ is H, and R3″ is H; -R1 is CH 2 CH 2 OH, R2' is H, R2'' is H, R3' is H, and R3'' is H; and -R1 is H and R2' is C(CH 3 ) 3 where R2″ is H, R3′ is H, and R3″ is H; The aziridines of formula (1) have one of the combinations of substituents from the group:

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

[0108] As described, such incubation with aziridine results in alkylation of the Cryptosporidium gp40 protein or immunogenic portion thereof, particularly of amino acids selected from cysteine, methionine, serine, threonine, tyrosine, lysine, arginine, valine, glutamic acid and aspartic acid, which then results in increased immunogenicity of such protein or portion thereof.

[0109] In the case of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable according to the invention, as well as the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, "incubating" includes the general procedure of combining an aqueous composition containing the indicated components and allowing them to interact for a certain time and under certain conditions. Suitable conditions for this incubation are those that result in a detectable increase in the immunogenicity of the gp40 protein or portion thereof. Such incubation with aziridine can be carried out using a wide variety of conditions and parameters that are all well known to those skilled in the art and readily available for optimization and adaptation by routine methods, if necessary.

[0110] For example, the incubation can be carried out over a wide range of temperatures. Preferably, the incubation with aziridine is carried out at a temperature above 0° C., more preferably at a temperature between 1-55° C., 5-50° 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, most effective will be incubation at a less acidic pH value, as alkylation of amino acids with aziridine can be less efficient at very acidic pH levels.

[0112] Thus, in one embodiment of the method, incubation with the aziridine is carried out at a pH greater than 4, more preferably greater than 4.5, 5, 5.5, 6, 6.5, 7, or greater than 7.5, in that order of preference.

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

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

[0115] Thus, in one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable by the present invention, as well as the method for the preparation of a Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention, the concentration of aziridine at the start of incubation is, 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, 10, 11, 12, 13, 15, 20, 30, 40 or at least 50 millimolar.

[0116] The upper limit of the aziridine concentration can be readily determined. In one embodiment, the concentration of aziridine at the start of the incubation is less than 1 molar, less than 0.5 molar, or less than 0.1 molar, in that order of preference.

[0117] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable by the present invention, as well as the method for the preparation of a Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention, the period of incubation of the composition comprising the protein or fragment thereof with aziridine is, in this order of preference, at least 10 minutes, 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. In one embodiment, the period is overnight (i.e., 12-18 hours).

[0118] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable according to the invention, as well as the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, an additional step is carried out after incubation with aziridine, which step results in neutralization of any remaining aziridine.

[0119] In a preferred embodiment, the neutralization step is performed by adding an appropriate amount of thiosulfate to the incubation mixture after the incubation reaction, and incubating for an appropriate length of time to complete the neutralization, for example: neutralization by adding 10-100 mM sodium thiosulfate, and incubating at 15-30° C. for 15-90 minutes.

[0120] In practice, the amount of thiosulfate used for neutralization is a slight overdose, which acts as a guarantee: if some thiosulfate remains after the neutralization reaction, it is certain that all of the aziridine has disappeared.

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

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

[0123] Because aziridine is such a hazardous chemical, it is preferably used in a diluted form in incubations for the present invention.

[0124] In the case of ethyleneimines, this can be conveniently achieved by using so-called "binary ethyleneimines" (BEIs). BEIs are the reaction product of the cyclization of bromoethylamine-hydrobromide (BEA) under alkaline conditions and gentle heating, e.g. to about 37°C. The alkaline conditions can be conveniently provided by the addition of, e.g., sodium hydroxide. All this is well known in the art, see, e.g., Bahnemann 1990, supra.

[0125] Thus, in a preferred embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable according to the invention, as well as the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the aziridine is an ethyleneimine or is a binary ethyleneimine.

[0126] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable by the present invention, as well as the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention, an additional step is carried out prior to incubation with aziridine, in which BEI is produced.

[0127] An appropriate amount of the produced BEI is then used as the aziridine for incubation with a composition containing Cryptosporidium gp40 protein or an immunogenic portion thereof.

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

[0129] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable by the present invention, as well as the method for preparing a Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention, the gp40 protein or immunogenic portion thereof is from Cryptosporidium parvum, more preferably from genotype II Cryptosporidium parvum.

[0130] For purposes of the present invention, "from Cryptosporidium parvum" indicates that the amino acid sequence of the gp40 protein or a portion thereof is based on the gp40 protein from the species Cryptosporidium parvum, either directly, by isolation of the protein or encoding nucleic acid, or indirectly, for example, based on sequence information from such a Cryptosporidium parvum gp40 protein.

[0131] Thus, the Cryptosporidium gp40 protein or immunogenic portion thereof of the present invention is preferably an isolated protein, meaning that the Cryptosporidium gp40 protein or immunogenic portion thereof of the present invention is not in its natural context, in or on a living Cryptosporidium parasite.

[0132] Compositions comprising the Cryptosporidium gp40 protein or immunogenic portions thereof for use in incubation with aziridine of the present invention can be produced in different ways, including isolation from Cryptosporidium parasites obtained from live infected host organisms, or the protein or portions thereof can be produced in cell-free transcription systems.

[0133] However, most convenient and scalable to industrial production levels is the production of Cryptosporidium gp40 proteins or immunogenic portions thereof for use in the present invention by in vitro expression using a recombinant expression system. Such expression systems can use cell culture systems of prokaryotic or eukaryotic cells genetically engineered to express the desired protein. Alternatively, the cells can be a host for a recombinant microorganism, such as a virus, which induces the cells to express the desired protein. Examples of recombinant expression systems are genetically engineered Chinese hamster ovary (CHO) cells, bacteria such as Escherichia coli, Bacillus species or Staphylococcus carnosus, or yeast species, such as Saccharomyces cerevisiae or Pichia pastores. Examples of the use of recombinant viruses for expression from host cells are the baculovirus-insect cell system or the adenovirus-mammalian cell system.

[0134] Thus, in one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable by the present invention, as well as in one embodiment of the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention, an additional step is carried out to produce a composition comprising the gp40 protein or portion thereof by a recombinant expression system, preferably by a baculovirus-insect cell expression system.

[0135] The baculovirus-insect cell expression system has been well known since the 1980s, for a review see Chambers et al., 2018, Curr. Protoc. Protein Sci., vol. 91, p. 5.4.1-5.4.6.

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

[0137] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable according to the invention, as well as the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, a composition comprising the gp40 protein or portion thereof (prior to aziridine incubation) is produced by a baculovirus-insect cell expression system, said composition being harvested from an insect cell culture, e.g. as a whole culture or a portion of such a culture, e.g. as the supernatant or cell pellet after centrifugation of an insect cell culture, or as the filtrate or retentate after filtration.

[0138] More preferably, the composition comprising Cryptosporidium gp40 protein or an immunogenic portion thereof is the supernatant or filtrate of a baculovirus-insect cell expression system culture. The supernatant can be obtained after gravity settling of the culture, e.g., by standing overnight, or by centrifugation. The filtrate is that which passes through the filter during filtration.

[0139] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable according to the invention, and of the method for the preparation of a Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, a composition comprising the gp40 protein or portion thereof is produced by a recombinant expression system, and after production and harvesting of said composition from the expression system, an additional step is performed prior to incubation with aziridine, which step comprises purification of said composition. Preferably, purification is by column chromatography. More preferably, the gp40 protein or portion thereof comprises a tag that facilitates purification of said composition by column chromatography, such as a histidine tag for metal affinity chromatography.

[0140] Since incubation with aziridine is also an effective means for the chemical inactivation of microorganisms, the preparation of the alkylated proteins or alkylated immunogenic portions thereof of the invention can therefore be advantageously combined with the inactivation of microorganisms, such as viruses or bacteria.

[0141] Thus, in one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable according to the invention, as well as the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the composition comprising the Cryptosporidium gp40 protein or immunogenic portion thereof also contains a microorganism. The microorganism may for example be a Cryptosporidium parvum parasite inactivated with aziridine, which inactivates the parasite but also causes the gp40 protein to be alkylated. Preferably, the microorganism is a virus or a bacterium, for example as used in a recombinant expression system.

[0142] Conditions for inactivating many microorganisms using aziridines are well known in the art. Any desired adaptation or optimization can be readily carried out by routine methods.

[0143] In one embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtainable according to the invention, as well as the method for the preparation of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, one or more of the conditions selected from the group consisting of: - the incubation with aziridine is carried out at a temperature above 0°C, more preferably at 1-55°C, 5-50°C, 10-40°C or 15-40°C, in this order of preference; the incubation with the aziridine is carried out at a pH above 4, more preferably at a pH above 4.5, 5, 5.5, 6, 6.5, 7 or at a pH above 7.5, in this order of preference; - the incubation with aziridine is carried out at a pH of less than 12, less than 10, or less than 9, in that order of preference; the concentration of aziridine at the start of the incubation is, 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, 10, 11, 12, 13, 15, 20, 30, 40, or at least 50 millimolar; the concentration of aziridine at the start of the incubation is, in this order of preference, less than 1 molar, less than 0.5 molar, or less than 0.1 molar; - the duration of incubation of the composition comprising the protein or fragment thereof and the aziridine is, in this order of preference, at least 10 minutes, 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; in one embodiment the duration is overnight (i.e. 12-18 hours); - after incubation with aziridine, an additional step is carried out which results in neutralization of any remaining aziridine; in a preferred embodiment, neutralization is carried out by addition of an appropriate amount of thiosulfate to the incubation mixture; -aziridine is selected from ethyleneimine, 2-ethyl-ethyleneimine, 1-acetyl-ethyleneimine, 2-methyl-ethyleneimine, 1-ethyleneimine-ethanol and 2-isobutyl-ethyleneimine; For aziridines, one of the following conditions applies: o Ethyleneimine preferably has the CAS number 151-56-4; o2-Ethyl-ethyleneimine preferably has the CAS number 2549-67-9; o1-acetyl-ethyleneimine preferably has the 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 preferably has CAS number 3647-37-8; the aziridine is an ethyleneimine or a binary ethyleneimine; - an additional step is performed prior to incubation with the aziridine, in which the BEI is generated; an appropriate amount of the generated BEI is then used as the aziridine for incubation with a composition comprising a Cryptosporidium gp40 protein or an immunogenic portion thereof; preferably, the BEI is generated from reaction of the BEA with a hydroxide; - the gp40 protein or an immunogenic portion thereof is from Cryptosporidium parvum; more preferably, from genotype II Cryptosporidium parvum; - an additional step is carried out to produce a composition comprising the protein or a part thereof, which is to be incubated with aziridine in a later step of the method, by a recombinant expression system, preferably a baculovirus-insect cell expression system; more preferred is the baculovirus-insect cell expression system production of Cryptosporidium gp40 protein by expressing the recombinant DNA sequence of SEQ ID NO:2 as a heterologous insert; - a composition comprising a Cryptosporidium gp40 protein or an immunogenic portion thereof produced by a baculovirus-insect cell expression system and harvested from an insect cell culture, e.g. as a whole culture, as a supernatant or cell pellet after centrifugation of the insect cell culture, or as a filtrate or retentate; - the composition comprising Cryptosporidium gp40 protein or an immunogenic portion thereof is a supernatant or filtrate of a baculovirus-insect cell expression system culture; - an additional step is performed after the recombinant expression system produces and harvests a composition comprising the protein or a portion thereof, which step includes purifying said composition; preferably, the purification is by column chromatography; more preferably, the Cryptosporidium gp40 protein or an immunogenic portion thereof includes a tag that facilitates purification of said composition by column chromatography, such as a histidine tag for metal affinity chromatography; and The composition comprising the Cryptosporidium gp40 protein or an immunogenic portion thereof also contains a microorganism; preferably, the microorganism is a virus or a bacterium.

[0144] Thus, in a preferred embodiment of the Cryptosporidium gp40 protein or immunogenic part thereof obtainable according to the invention, the aziridine is an ethyleneimine or a binary ethyleneimine; - the Cryptosporidium gp40 is from Cryptosporidium parvum; - the composition comprising the 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] Likewise, in a preferred embodiment of the method for the preparation of Cryptosporidium gp40 protein or an immunogenic part thereof according to the invention, the aziridine is an ethyleneimine or a binary ethyleneimine; - the Cryptosporidium gp40 is from Cryptosporidium parvum; - the composition comprising the 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 immunogens for a safe and effective vaccine against cryptosporidiosis. Cryptosporidium gp40 or immunogenic portions thereof according to the present invention that contain one or more alkylated amino acids are particularly useful in that regard since they have increased immunogenicity.

[0147] Thus, in a further aspect, the present invention relates to a Cryptosporidium gp40 protein or an immunogenic part thereof according to the present invention, or obtainable by the present invention, or obtainable by a method according to the present invention, for use in a vaccine for the protection of human or non-human animal targets against cryptosporidiosis.

[0148] Similarly, in a further aspect, the present invention relates to the use of a Cryptosporidium gp40 protein or an immunogenic part thereof according to the present invention, or obtainable by the present invention, or obtainable by a method according to the present invention, for the manufacture of a vaccine for the protection of human or non-human animal targets against cryptosporidiosis.

[0149] It is well known that a "vaccine" is a composition that comprises an immunogen and a pharmaceutically acceptable carrier. The immunogen induces an immunological response in the vaccinated target, and this response is effective in protecting against disease or against infection or its consequences. Protection refers to reducing the load or shortening the duration of replication of the pathogen against which vaccination is aimed. This then leads to a reduction in the number, intensity or severity of the lesions, associated symptoms and clinical signs of the disease caused by the pathogen in the vaccinated target.

[0150] Determination of the effectiveness of a vaccine according to the invention is well within the skill of ordinary medical personnel and can be carried out, for example, by monitoring the immunological response after vaccination, or by examining the appearance of clinical symptoms or mortality after challenge infection, for example by monitoring the target disease signs, clinical scores, serological parameters, or by re-isolation of the challenge pathogen, and comparing these results to the vaccination-challenge response seen in sham-vaccinated animals. Many methods of measuring and characterizing the relevant pathogens, their symptoms and diseases are known in the art.

[0151] The Cryptosporidium gp40 protein or immunogenic parts thereof according to the invention for use in a vaccine according to the invention and their use for the manufacture of a vaccine according to the invention are effective in protecting against "cryptosporidium disease". This term refers to the collection of symptoms caused by infection of a susceptible target with the Cryptosporidium parasite. Primarily such symptoms relate to diarrhea, although some species of this parasite can also induce respiratory disease. For symptoms of veterinary cryptosporidiosis, see "The Merck veterinary manual" (supra).

[0152] In the context of the present invention, "protection against cryptosporidiosis..." relates to the reduction in the severity and / or duration of diarrhea caused by the Cryptosporidium parasite. The vaccine according to the present invention is effective in this respect, as demonstrated in the Examples section.

[0153] Protection occurs in the vaccinated target itself. Also, when immunizing mammalian targets, protection can be generated indirectly by colostrum transfer, which involves collecting breast milk from a vaccinated pregnant mammal before or after birth and feeding the breast milk (or antibodies derived therefrom) to the target human or non-human animal.

[0154] The result of this protection is the restoration of the overall health of the (passively) vaccinated target. In the veterinary field, protection against cryptosporidiosis leads to an increase in economic performance in (passively) vaccinated animals, reflected in one or more of the following parameters: reduced mortality, improved average daily weight gain, improved feed conversion ratio, improved milk production, improved reproductive output, and / or reduced medical costs.

[0155] In addition to reducing the symptoms of the disease, particularly diarrhea, the protection relates to a reduced likelihood of infection with the Cryptosporidium parasite due to reduced shedding of oocysts by vaccinated targets into the environment and geographical area of ​​the herd or flock. As a result, the protection of the present invention also results in a reduced prevalence of the Cryptosporidium parasite.

[0156] In a preferred embodiment of the Cryptosporidium gp40 protein or an immunogenic part thereof according to the invention for use in a vaccine according to the invention, and of their use for the manufacture of a vaccine according to the invention, 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" relates to any ruminant animal associated with veterinary or commercial breeding operations. Preferably, it refers to bovine, caprine, ovine or cervid animals. More preferred are bovine, caprine and ovine animals. The most preferred ruminant animals are bovine animals.

[0158] A "cattle" according to the present invention is a taurine cattle (Bos taurus), a zebu cattle (Bos indicus), a buffalo, a bison, a yak or a vicente. The cattle can be any type of dairy or beef cattle, or a parent stock of a dairy or beef cattle breed.

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

[0160] Thus, in a further aspect, the present invention relates to a vaccine for a human or non-human animal target against cryptosporidiosis, comprising a Cryptosporidium gp40 protein or an immunogenic part thereof according to the present invention or obtainable by the present invention or obtainable by a method according to the present invention, and a pharma- ceutically acceptable carrier.

[0161] A "pharmaceutically acceptable carrier" aids in the manufacture, administration and / or storage of a vaccine without causing (serious) adverse effects. Such a carrier may be an aqueous solution, such as water, a buffer solution or a culture medium.

[0162] Preferred pharma- ceutically acceptable carriers for vaccines according to the invention are insect cell culture media or buffers such as saline, PBS or 50 mM HEPES.

[0163] In addition, the pharma- ceutically acceptable carrier may contain further additives and excipients, such as bulking agents, stabilizers, preservatives or adjuvants. Details and examples are well known and can be found in handbooks such as "Remington: the science and practice of pharmacy" (2000, Lippincott, USA, ISBN: 683306472) and "Veterinary vaccinology" (P.Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681).

[0164] Furthermore, the vaccine according to the invention may comprise an adjuvant, which, particularly in the case of subunit vaccines, further increases the target immune response against the subunit antigen.

[0165] Thus, in one aspect, the vaccine according to the invention is characterised in that it comprises an adjuvant.

[0166] An "adjuvant" is a well-known vaccine component that non-specifically stimulates the target immune response. Many different adjuvants are known in the art. Examples of adjuvants are complete or incomplete Freund's adjuvant, vitamin E or α-tocopherol, non-ionic block polymers and polyamines such as dextran sulfate, Carbopol™, pyran, saponins such as Quil A™ or Q-vac™. Saponins and vaccine components can be combined in ISCOM™.

[0167] In addition, peptides such as muramyl dipeptide, dimethylglycine, tuftsin, mineral oils such as Bayol™, Drakeol™, Klearol™ or Marcol™, Montanide™ or light mineral (paraffin) oils; non-mineral oils such as squalene, squalane; vegetable oils or their derivatives such as ethyl oleate are often used as adjuvants. Combination products such as ISA™ (Seppic) or DiluvacForte™ and Xsolve™ (both MSD Animal Health) can also be used advantageously. A further option is the use of the SVEA adjuvant (comprising squalane and vitamin E-acetate) disclosed in WO 2018 / 115435.

[0168] A handbook on adjuvants and their use and effects is "Vaccine adjuvants" (Methods in molecular medicine, vol. 42, D. O'Hagan ed., 2000, Humana press, NJ, ISBN: 0896037355).

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

[0170] Commonly used mineral oil adjuvants in veterinary vaccines are light (or white) liquid paraffin oils such as Drakeol® 6VR (Penreco), Marcol® 52 (Exxon Mobile) and Klearol® (Sonneborn), or premixed mineral oil / emulsifier mixtures such as Seppic's Montanide® range from France.

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

[0172] Common emulsifiers for vaccines are sorbitan monooleate (Span® 80) and polyoxyethylene-sorbitan monooleate (Polysorbate 80 or Tween® 80). Common emulsion-stabilizers are benzyl alcohol and triethanolamine.

[0173] A commonly used aluminum salt is aluminum hydroxide, for example Alhydrogel™ (Brenntag Biosector), Rehydragel™ (Reheis), and Rehsorptar™ (Armour Pharmaceutical).

[0174] Vaccines containing oil-based adjuvants may be formulated as an emulsion of aqueous and oil phases, preferably the emulsion is of a type selected from water-in-oil (w / o), oil-in-water (o / w), water-in-oil-in-water (w / o / w) and double oil emulsions (o / w / o).

[0175] More preferred are vaccines according to the invention that are oil adjuvanted and formulated as water-in-oil emulsions.

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

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

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

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

[0180] This allows for the collection of colostrum from said mammal before or after birth, which can then be used to provide passive protection against cryptosporidiosis to humans or non-human animals by the provision of antibodies from the colostrum or colostrum.

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

[0182] In one embodiment, the vaccine according to the invention is administered by parenteral route, i.e. through the skin, for example intramuscularly, intraperitoneally, intradermally, submucosally or subcutaneously. Preferred routes of administration are by intradermal, intramuscular or subcutaneous route.

[0183] The volume per dose of the vaccine according to the invention can be selected according to the characteristics of the specific vaccine applied, the characteristics of the target and the intended application route. Parenteral injection is generally performed at 0.01-10 ml / target dose. For adult cattle, the preferred volume per dose is 0.5 ml for the subcutaneous route and 1-2 ml for the intramuscular route.

[0184] An advantageous effect of alkylation with aziridine for the present invention is that a smaller amount of Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention needs to be used in a vaccine dose compared to a gp40 protein or portion thereof that does not contain one or more alkylated amino acids while still obtaining a good level of protection. The selection of the amount of gp40 protein or portion thereof per dose can be made by one skilled in the art based on the characteristics of the vaccine and the target.

[0185] Thus, in one embodiment, the vaccine according to the invention comprises 0.01-50 μg of Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention per vaccine dose. Preferably, the vaccine comprises, in this order of preference, 0.05-20 μg, 0.1-10 μg, or 0.1-5 μg of Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention per dose.

[0186] Most preferably, the vaccine comprises 0.5-2 μg of Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention per dose per adult cattle.

[0187] In one embodiment of the vaccine according to the invention, when the vaccine is intended for vaccination of cattle, the vaccine comprises less than 10 μg of Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention per dose. Preferably, the vaccine according to the invention intended for cattle comprises less than 9, 8, 7, 6, 5, 4, 3 μg, or less than 2 μg of Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention per dose.

[0188] Most preferably, the vaccine comprises 1 μg of Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention per dose per adult cattle.

[0189] The amount of Cryptosporidium gp40 protein or its immunogenic part per dose according to the present invention can be analyzed in the ready-to-use vaccine emulsion. This can be done by using standard biochemical testing procedures, for example by breaking the emulsion and examining the aqueous phase using SDS-PAGE. The amount of protein is then determined by comparing with a known amount of a standard protein, for example albumin. See, for example, The Protein Protocols Handbook, 2nd edition, September 2002, ed. JM Walker, Humana Press Inc., Totowa, NJ; Chapter 29, p.237-242.

[0190] Preferably, the amount of Cryptosporidium gp40 protein or immunogenic portion thereof according to the present invention is determined by antigen mass ELISA as described below. Alternatively, protein quantification can be performed by using liquid chromatography such as HPLC or by mass spectrometry.

[0191] Preferably, the amount of Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention per dose is determined in the aqueous phase of a vaccine according to the invention before mixing with the aluminium salt and / or emulsifying with the oil phase.

[0192] The administration regimen of the vaccine according to the invention is selected based on the type of protection intended, i.e. vaccination to provide active protection of the vaccinated target itself, or vaccination to generate colostrum for passive vaccination.

[0193] Active vaccination of target can mobilize humoral and cellular immune response, and can be timed as desired.Active vaccination of target can be performed regardless of the age, weight, sex, immunological status and other parameters of the target to be vaccinated.Active vaccination can be given as a single dose, followed by a booster vaccination, for example, about two weeks later, and then annual revaccination.

[0194] However, passive vaccination via colostrum transfer is almost exclusively mediated by antibodies. Therefore, in order to obtain high levels of gp40-specific antibodies in the colostrum, vaccination for the production of such colostrum must be timed with respect to the expected date of calving. For different species of mammals, the gestation period is known and therefore the timing of this vaccination can be selected and optimized. To produce colostrum in cows, pregnant cows are preferably vaccinated twice with the vaccine according to the invention, namely a booster vaccination in the period 1-4 weeks before expected calving and a prime vaccination 1-12 weeks before the booster vaccination. Preferably, a booster vaccination is given 2-4 weeks before expected calving and a prime vaccination 2-6 weeks before the booster vaccination. If the cow has already received a prime and booster vaccination for a previous pregnancy, it may be sufficient to administer only a single vaccination for the subsequent pregnancy, given in the period 1-12 weeks before expected calving, preferably 2-8 weeks before expected calving.

[0195] Preferably, the method, timing and volume of administration of the vaccine according to the invention is integrated into existing vaccination schedules of other vaccines that the target human or non-human animal may require, to reduce stress on the target and reduce labor costs. These other vaccines can be administered in a simultaneous, concurrent or sequential manner, and in a manner compatible with their approved use.

[0196] In a preferred embodiment, the vaccine according to the invention is administered to pregnant cows in combination with other vaccines aimed at protecting newborn calves against neonatal diarrhea due to colostrum transfer. Examples of such other vaccines are: Guardian® (Merck Animal Health), a W / O emulsion with mineral oil adjuvant, administered subcutaneously, and Rotavec® Corona (MSD Animal Health), a W / O emulsion with both mineral oil and aluminum salts as adjuvants, administered intramuscularly. A preferred method of combination is the so-called "associated non-mixed" use.

[0197] Depending on the target animal, the targeted disease, the route of administration, etc., it may be desirable to adapt the vaccine according to the invention or elements of its administration. This is well within the capabilities of the skilled artisan and generally involves fine-tuning the efficacy of the vaccine. This can be done, for example, by adapting the vaccine dose, amount, frequency, route, excipients, etc.

[0198] It is, of course, within the scope of the present invention to incorporate other compounds into the vaccines according to the present invention, such as stabilizers, carriers, adjuvants, diluents, emulsions, etc. Such additives are described in well-known handbooks such as "Remington" and "Veterinary Vaccinology" (both cited above).

[0199] In the case of the vaccines according to the invention, it may be advantageous to carry out further combinations with additional immunoactive components, which may serve to boost the immune protection already conferred or to extend the immune protection already conferred to other pathogens.

[0200] Thus, in one embodiment, the vaccine according to the invention comprises at least one additional immunoactive component.

[0201] Such an "additional immunoactive component" may be an antigen, an immune enhancer, an adjuvant or immune modulator, a cytokine, another vaccine or any combination of these. This provides advantages in terms of cost, efficiency and target protection. Alternatively, the vaccine according to the invention may itself be added to another vaccine.

[0202] General techniques and considerations applicable to the manufacture of vaccines under well-known standards for pharmaceutical manufacturing are described, for example, in government directives and regulations (pharmacopoeias, 9 CFR) and well-known handbooks (Veterinary vaccinology and Remington, both supra). Generally, such vaccines are prepared sterile and using pharmaceutical quality grade excipients.

[0203] Such preparations may incorporate microbiological tests for sterility and absence of adventitious agents, and may include in vivo or in vitro tests to ascertain efficacy and safety. After completion of tests for quality, quantity, sterility, safety and efficacy, the vaccine may be offered for sale, all of which are well known to those skilled in the art.

[0204] Thus, in a further aspect, the present invention relates to a method for the manufacture of a vaccine according to the invention, comprising the step of formulating a Cryptosporidium gp40 protein or an immunogenic part thereof according to the invention or obtainable by the invention or obtainable by a method according to the invention into a vaccine.

[0205] Such formulations may include simple mixing of the gp40 protein or immunogenic portion thereof with a pharma- ceutically acceptable carrier, or may include mixing with an adjuvant as described, including emulsification, and / or mixing with at least one additional immunoactive ingredient.

[0206] A variant of the vaccine against cryptosporidiosis according to the present invention is colostrum produced by a pregnant mammal immunized with the vaccine according to the present invention. This colostrum is highly effective for passive vaccination of human or non-human animal targets against cryptosporidiosis. The colostrum can be fed to or drunk by the target, providing intestinal, mucosal and / or systemic immune protection against cryptosporidiosis. Alternatively, antibodies derived from such colostrum can be administered.

[0207] Colostrum can be produced by vaccinating a pregnant mammal with a vaccine according to the invention prior to parturition.

[0208] Thus, in a further aspect, the present invention relates to a method for producing colostrum comprising antibodies against a Cryptosporidium gp40 protein or an immunogenic part thereof according to the invention or obtainable by the invention or obtainable by a method according to the invention, comprising: a. vaccinating a pregnant mammal at least once with a vaccine according to the invention; b. collecting colostrum from the mammary gland of said mammal; The present invention relates to a method comprising the steps of:

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

[0210] Preferably, the colostrum of the present invention is milk secreted in the period from 1 day before to 4 days after parturition, more preferably from 1 hour to 72 hours after parturition.

[0211] More preferably, the colostrum according to the invention is secreted by a ruminant animal, even more preferably by a bovine animal as defined herein. Even more preferably, the colostrum according to the invention is collected from said cow after calving by the first, second, third and fourth milking.

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

[0213] Colostrum according to the present invention contains antibodies against the Cryptosporidium gp40 protein or immunogenic portions thereof according to the present invention, which antibodies have a higher concentration and / or different binding activity or specificity compared to antibodies in colostrum from a mammal vaccinated with a gp40 protein or portion thereof that did not contain one or more alkylated amino acids.

[0214] Vaccination of the pregnant mammal is preferably carried out according to the vaccination schedule described herein.

[0215] Collection of colostrum, as described herein, preferably occurs for at least 3 days starting from 1 day before calving to 4 days after calving, more preferably on the day of calving.

[0216] In a further aspect, the present invention relates to colostrum obtainable by the method for producing colostrum according to the invention, for use in the protection of human or non-human animal targets against cryptosporidiosis.

[0217] In a preferred embodiment of colostrum for use according to the invention, protection is achieved by providing colostrum or colostrum-derived antibodies to target.

[0218] As described, the present invention relates to both active and passive vaccination regimens against cryptosporidiosis.

[0219] In a further aspect, the present invention relates to a method for the protection of a human or non-human animal target against cryptosporidiosis, comprising at least one administration to said target of a vaccine according to the invention.

[0220] In a further aspect, the present invention relates to a method for the protection of a human or non-human animal target against cryptosporidiosis, comprising feeding said target with colostrum obtainable by a method according to the invention or with colostrum according to the invention.

[0221] The method of protection using colostrum for the purposes of the present invention also relates to passive vaccination of targets using a preparation of gp40 specific antibodies obtained from colostrum obtainable by the method according to the invention or purified from colostrum according to the invention.

[0222] When the method of passive protection concerns cattle, a further distinction can be made between different husbandry methods applied to cattle for different purposes. That is, for dairy calves, which are generally separated from their mothers on the day of birth, vaccination by administering colostrum is carried out by active collection of colostrum and by feeding it to the calf during the first week of life. Preferably, dairy calves are fed with colostrum according to the invention for at least 3 days, more preferably at least 4 days or at least 5 days, in this order of preference. The first dose of colostrum should be administered to the calf within 8 hours of birth, preferably within 6 hours 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 the amount of milk produced is less. Also, beef cattle calves are usually left with their mothers after birth. In that case, the supply of colostrum for the protection method according to the present invention is carried out by suckling the calf. Preferably, the beef cattle calves are allowed to suckle colostrum from the vaccinated cattle described herein for at least 3 days, more preferably at least 4 days or at least 5 days, in this order of preference. The calf should be given access to suckle colostrum within 8 hours of birth, preferably within 6 hours of birth or within 4 hours of birth.

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

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

[0227] [Example 1] Creation of a cryptosporidiosis vaccine 1.1. Recombinant Protein Constructs A recombinant gp40 protein for use in vaccination studies was constructed starting from the core amino acid sequence of SEQ ID NO: 1 derived from Cryptosporidium parvum, Iowa strain, genotype II. This sequence was extended with a C-terminal 6x histidine tag to allow purification by nickel column chromatography.

[0228] In the case of Cryptosporidium gp40 protein, it is known that the truncation of the N-terminal signal sequence is different in the expression system where the truncation is at amino acid number 20 compared to the native gp40 protein starting at amino acid number 31. See O'Connor et al. (2007, Mol. Bioch. Parasit., vol. 152, p. 149-158). In the present invention, the N-terminal gp40 signal sequence was partially restored by using amino acids 14-30 from GenBank entry AAF78345.1.

[0229] In addition, an N-terminal methionine was added to initiate transcription. This construct could be efficiently expressed in a recombinant baculovirus-insect cell expression system but did not allow secretion or glycosylation. As a result, expression was cytoplasmic and gp40 could be harvested from the culture supernatant at the end of the culture period when most of the insect cells had lysed.

[0230] The exact amino acid sequence of the Cryptosporidium gp40 protein used in the vaccination experiment is that of SEQ ID NO: 3. It was expressed from a recombinant DNA that was codon-optimized based on the codon preference of the AcMNPV baculovirus polyhedrin gene. The recombinant DNA sequence used was that of SEQ ID NO: 2. The recombinant gene was inserted as a BamH1-EcoR1 fragment into a baculovirus transfer vector based on the pVL1393 plasmid, driving expression from the polyhedrin promoter. Stably transfected recombinant baculoviruses were generated by homologous recombination with linearized AcMNPV genomic DNA using standard procedures. Recombinant baculoviruses expressing His-tagged recombinant Cryptosporidium gp40 protein (rgp40His) were isolated, plaque-purified and amplified.

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

[0232] 1.2. Expression and Harvesting Rgp40His was produced from Sf9 insect cells infected with a recombinant baculovirus seed. Cells were cultured in the 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 runs of up to 500 L cultures were performed in a production preparation facility. In general, the following schedule was applied: clean Sf9 cells were produced in increasing volumes of culture. Once enough clean cells were produced, the cells were concentrated and reseeded in fresh medium at 1.6x10^6 cells / ml. These were infected with a multiplicity of infection of 0.002 and incubated at 28°C for 5 days. At the end of the culture, the supernatant was harvested. This was done by centrifugation on a small scale and by clarification by depth filtration on a large scale. When expressed on a small scale, the recombinant baculovirus was then inactivated using 0.1% Triton® X-100 incubation at room temperature for 1 hour followed by gamma irradiation by a commercial company. When produced on a large scale, all downstream processing was performed in a contained facility using closed connections, and the majority of the recombinant baculovirus was 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. The imidazole was then removed by diafiltration against 50 mM HEPES buffer at pH 7.5. The rgp40His was then sterile filtered through a 0.2 μm membrane filter and incubated with aziridine. BEI was prepared by combining equal volumes of 1.09 M BEA and 1.91 M NaOH. This produced a stock solution of 545 mM BEI. BEI was then added to the composition containing rgp40His to make it 33 mM. The mixture with BEI was incubated at room temperature for 24 hours. Sodium thiosulfate was then added at 33 mM. This was incubated at room temperature for one hour and the pH was measured to be 7.1.

[0234] For comparative experiments, Cryptosporidium gp40 was produced in the same manner but without one or more alkylated amino acids, except that buffer was added instead of aziridine for mock incubations.

[0235] 1.3. Determination of antigen mass First, the amount of rgp40His protein was measured by SDS-PAGE along a dilution range of known amounts of standard protein. The bands were then quantified using densitometry of stained gels. This test was later replaced by a more accurate test, namely the competitive antigen mass ELISA, which was fully validated for specificity, robustness, linearity and precision. Briefly, the antigen mass ELISA was performed as follows: wells of a microtitration plate were coated with purified rgp40His (not alkylated) at 50 ng / well in coating buffer and overnight at 4°C. The next day, the plate was washed and post-coated with casein-containing buffer for 1 hour at 37°C and then washed again. On a separate microtiter plate, test samples of unknown concentrations of rgp40His were serially diluted in ELISA buffer (containing 0.05% polysorbate 80) and a fixed amount of anti-gp40 mouse monoclonal antibody was added. This was pre-incubated at 37°C for 1 hour, then the mixture was transferred to the coated plate and incubated in ELISA buffer at 37°C for 1 hour. The plate was then washed and the amount of gp40 monoclonal antibody bound to the plate was detected by incubation with peroxidase-conjugated goat anti-mouse IgG in Elisa buffer at 37°C for 1 hour. Peroxidase was visualized by enzymatic conversion of tetramethylbenzidine for 15 minutes at room temperature, and the reaction was stopped using sulfuric acid. The optical density of the yellow color in each well was measured at 450 nm. The amount of peroxidase-conjugate is inversely related to the amount of antigen in the test sample. For at least three sample points from the dilution series, this was processed by a software program using a Logit-Log algorithm. Samples were also measured at least in duplicate, and appropriate positive and negative controls were also used in the test. The value of the antigen mass of gp40 in the test sample was then calculated in micrograms / mL based on the standardized reference sample value.

[0236] 1.4. Vaccine formulation The column purified, aziridine-incubated or mock-incubated rgp40His was then formulated as an emulsion vaccine for further use by combining with appropriate adjuvants and emulsification, all using well-known methods and materials. Briefly, rgp40His was spiked in sterile saline (0.85% w / v sodium chloride) to the required concentration. In parallel, the oil phase was prepared with ISA™ 70VG (Seppic, France) by sterile filtration. Both phases were combined and emulsified into a water-in-oil emulsion using a Silverson™ or Dispax™ homogenizer. The gradual increase in temperature during homogenization was monitored and maintained below 50°C. The resulting emulsion was stored at 4°C until filling into appropriate containers. The filled product was subjected to various tests for stability and sterility before shipping.

[0237] Depending on the intended geographic market or combination with other antigens, batches of emulsions were also prepared containing an additional adjuvant, specifically aluminium hydroxide. In this case, the aqueous rgp40His preparation was first combined with a heat-sterilized suspension of 3% w / v Alhydrogel® (Brenntag) in saline and allowed to absorb into the aluminium for 30-60 minutes at room temperature with stirring. This mixture of antigen and aluminium was then emulsified with an oil phase as described above.

[0238] Long-term stability studies for 1 and 2 year shelf life at 4°C are ongoing. However, results from intermediate and rapid stability studies for 15 months at 4°C and 3 weeks at 30 or 37°C are instructive. Emulsion quality remained good throughout, with the average change in antigen mass found being within 10% of the starting composition. This is a good indication of long-term stability under refrigerated conditions.

[0239] [Example 2] Vaccination-challenge test preparation Over the years, several studies in laboratory animals have been carried out to develop and optimize a model for an effective vaccination-challenge study capable of evaluating the efficacy of various compositions tested as cryptosporidiosis vaccines. It has emerged that the following setup best represents in vivo efficacy: The vaccine was prepared as described above: cryptosporidiosis gp40 protein was expressed in a baculovirus-insect cell expression system, harvested at the end of the culture, purified (or not) by metal affinity chromatography, incubated (or not) with aziridine, and then the protein was formulated with an adjuvant, either oil or oil and aluminum salt, and emulsified as a W / O emulsion.

[0240] 2.1.Animal models For passive vaccination studies, target animals were healthy Holstein-Friesian dairy cows from about 1 year of age, selected from both heifers and cows that had parous more than once. The cows were in the third trimester of pregnancy and their sera were tested for only background levels of gp40-specific antibodies. The cows were treated on the farm where they were kept, so no acclimation was required. Feed and water were provided according to standard cattle management practices. The animals were marked with a unique organism number via ear tag.

[0241] 2.2. Vaccination Vaccines containing 10 mg rgp40His / dose, either aziridine-treated or sham-treated, emulsified W / O in mineral oil, were administered in a 2 ml volume and given intramuscularly in the neck. Timing was a prime 6 weeks before booster and a booster 3 weeks before expected calving. Cows were also vaccinated with Rotavec Corona vaccine 4 weeks before expected calving to prevent neonatal diarrhea in calves from pathogens other than Cryptosporidium parvum. Serum samples were taken at specific time points and tested by antibody ELISA.

[0242] After calving, colostrum was collected as the first, second and third milkings, yielding an average of 4-5 litres of colostrum per cow per milking. Colostrum was pooled for each vaccination study group and each milking time point, aliquoted into 250 ml jars and pasteurised using a water bath at 56°C for 30-45 min. Aliquots were then divided and kept frozen until use. Thawing was controlled by using a water bath set at 43°C, with regular checking of the water temperature, or by using hand-warmed tap water. When fed to the calves, the colostrum was hand-warmed (i.e., approximately 40°C).

[0243] Neonatal calves were collected at birth, housed individually, and fed a 3-liter bolus of anti-gp40 colostrum from one of the vaccine groups within 4 hours of birth, then fed a challenge dose of live Cryptosporidium parvum oocysts 2 hours after the first feeding.

[0244] Group sizes were 5-10 calves and an unprotected induced group was included in all experiments; this group was fed sham colostrum. The use of unprotected uninduced indicators was not found to be beneficial and was excluded in subsequent experiments. Calves included in the studies weighed at least 30 kg at birth.

[0245] Calves were kept in single animal boxes in an isolated facility. Contact between calves was not possible. Breeders changed gloves and footwear before treating each animal.

[0246] Each box for one animal consisted of two compartments: one containing wood shavings and one consisting of a metal grid on a horizontal smooth surface plate above the waste collection channel. Calves were kept on the wood shavings for the first three days of life. At the end of the third day, calves were transferred to the second compartment. The smooth surface plate allowed for the assessment of stool consistency and the determination of a diarrhea score. This plate was washed after each scoring time point so that the next scoring could be performed on the fecal material produced between each scoring time point.

[0247] Calves were fed twice daily; colostrum was first fed to the calves, after which they were fed daily with colostrum (first feeding) or colostrum and milk replacer according to the manufacturer's specifications from the second feeding onwards.

[0248] 2.3. Trigger A challenge inoculum was prepared immediately prior to administration using 1 x 10^6 live Cryptosporidium parvum, Iowa strain oocysts per calf. Calves were orally administered this in 50 ml of milk replacer. Calves were then fed two feedings of (gp40- or control vaccine) colostrum in milk replacer per day: 0.25-0.5 liters of colostrum in a total of 2-2.5 liters of milk per day for several days.

[0249] This challenge dose was selected to induce repeatable and consistent diarrhea, which was severe (but transient) in all of the unprotected challenge controls.

[0250] Live oocysts were purchased fresh from Waterborne Inc. New Orleans, USA. Oocysts were freshly released no more than 2 months prior to the start of the animal experiments. Parasite viability upon excystation (test criteria are 50-100%), as well as infectivity of HCT-8 cell layers, were found to be sufficient both before the start of the experiment and at the end of the experiment.

[0251] After challenge, calves were monitored for 14 days, with diarrhea typically developing between days 4 and 11 and peaking in severity between days 5 and 10. Antibody levels were measured in colostrum and calf serum 1 day before feeding and 3 days after feeding.

[0252] 2.4. Scoring of Efficacy Several methods were tested over time to best quantify and evaluate the effectiveness of vaccination against challenge infection. Initially, only male calves were used and feces was collected in bags from all calves to analyze for volume and consistency. This proved to be inaccurate as the calves would lie down when sick and material would be lost. It was found that the most clinically relevant was to record a number of observational scoring parameters twice daily, specifically scores for general health, dehydration, rump appearance and diarrhea. The diarrhea scoring parameters used were based on the Wisconsin-Madison scale described in S. McGuirk, 2008 (Vet. Clin. North Am. Food Anim. Pract., vol. 24, p. 139-153). The parameters used for observation are shown in Table 1. With some experience, the animal breeders were able to determine the scores consistently.

[0253] Once diarrhea became evident, testing for Cryptosporidium in the faeces was applied, performed using a commercial test kit using liquid chromatography on a specific specimen: BIO K 306™ - Rainbow Calf Scours (Bio-X Diagnostics, Belgium).

[0254] To determine the reduction in shedding, an enumeration of oocysts in fecal samples was performed. The enumeration of oocysts was as follows: a 50 ml sample was taken from the collected feces and stored at 2–8 °C until use. The samples were serially diluted, the samples were placed on diagnostic microscope slides and air dried for 1 h. The slides were stained according to the Ziehl-Neelsen (acid-fast) method and the red-stained oocysts were counted by light microscopy. The results were expressed as oocysts per gram of feces.

[0255] [Table 1]

[0256] [Example 3] Results of vaccination-challenge experiments 3.1. Recombinant expression of gp40: The optimized baculovirus-insect cell expression system, with partial signal sequences and codon usage adaptation, was found to give more protein than the previously used Escherichia coli expression system. However, when vaccinated with unpurified gp40 harvest from insect cell cultures, i.e. only centrifuged and inactivated with Triton X-100 and gamma irradiation, the immunogenicity for a standard 20 μg dose was not as good as that for a similar dose of similarly inactivated and column-purified gp40. Therefore, the His-tag fusion construct and nickel column purification were used as a standard.

[0257] 3.2. Preparation of vaccine formulations Both the oil and oil + aluminum adjuvants used induced effective antibody titers in pregnant cows and, consequently, in the colostrum of the cows. However, for cows not previously vaccinated with gp40, a single vaccination was insufficient and a prime-boost vaccination regimen was necessary to induce levels of antibody in the colostrum that were protective against severe challenge as applied in these studies. In general, naive cows had antiserum titers of gp40-specific antibodies of 9-12 Log2 in antibody ELISA. Thus, titers up to 10 Log2 were considered to be background levels. After vaccination with a 10 microgram dose of purified gp40 without one or more alkylated amino acids in a water-in-oil emulsion vaccine, this titer increased to 12-13 Log2 after the prime vaccination and to 15-17 Log2 after the boost vaccination.

[0258] From these cows it was possible to collect colostrum containing gp40-specific antibody titers of approximately 20 Log2. As a result, a vaccine dose of 10 μg of purified rgp40His, not incubated with aziridine, was used in the primary and boost experiments. No effect of gp40 vaccination on the efficacy of vaccination against rotavirus, coronavirus or Escherichia coli was measured when applied in combination with the Rotavec Corona vaccine, see also Figure 2. It was concluded that these vaccines can be effectively combined.

[0259] Since there was no difference between once-daily or twice-daily colostrum intake, once-daily was applied from then on. Colostrum intake for a total of 5 days 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 tested positive for the presence of Cryptosporidium.

[0261] 3.4. Side effects Vaccination of cattle with an emulsified vaccine of rgp40His was generally safe, i.e. no systemic effects or effects on pregnancy were observed, but it did induce local reactions at the vaccination site that were palpable and showed signs of inflammation at necropsy, which was apparently related to the presence of the gp40 protein, as a similar vaccine in emulsion, the Rotavec Corona vaccine, did not induce such local reactions.

[0262] The only way to reduce these reactions was to reduce the dose of gp40, but reducing the dose while maintaining efficacy was only possible when using Cryptosporidium gp40 proteins containing one or more alkylated amino acids of the present invention.

[0263] [Example 4] Effect of aziridine incubation in vaccination-challenge experiments 4.1.Effect on antibody titers In a vaccination study of groups of five pregnant cows each, as described in Example 3 above, cows were given two doses of 10 μg rgp40His in a W / O emulsion adjuvanted with oil + aluminum. gp40 was column purified and incubated with or without aziridine. A group that received only the Rotavec Corona vaccine with the same formulation served as a negative control. An additional group was vaccinated both with gp40 without one or more alkylated amino acids and with the Rotavec Corona vaccine. Serum antibody titers were measured weekly and determined by antibody ELISA. Titers below 10 log2 were set to 10 and considered as background. The seroconversion profiles over time of all gp40-vaccinated animals were similar. The Rotavec Corona vaccine alone did not induce antibodies against gp40. The titer results of the combined vaccines indicate that there is no interference between the two vaccine types.

[0264] Vaccines containing the same amount of gp40 antigen but with Cryptosporidium gp40 protein containing one or more alkylated amino acids induced significantly higher antibody titers in cattle than the non-alkylated gp40 vaccine; 3-4 Log2 higher after the primary vaccination and 2 Log2 higher after the booster vaccination. This is shown in Figure 2. The titers of the resulting colostrum from the alkylated gp40 vaccination were found to be as high as 24 Log2.

[0265] 4.2.Effect on emissions The effect on oocyst shedding by calves after challenge as a result of passive vaccination was modest but significant. Typically, a 10-fold reduction in the total amount of oocysts per 100 grams of feces was observed between days 4 and 14, from 10^9 oocysts in unvaccinated challenged calves to 10^8 in vaccinated calves. However, fecal load was also much lower due to the shorter duration of diarrhea seen in calves: 6 days in unvaccinated calves vs. 2 days in calves vaccinated with BEI-treated gp40. Thus, the overall reduction in oocyst shedding due to vaccination was indeed relevant.

[0266] 4.3. Impact on Safety The gp40 protein combined with mineral oil adjuvant proved to be highly reactive, most evident when vaccinated by the subcutaneous route and when administered in a prime-boost regimen, an effect that was not exacerbated by aziridine incubation of the gp40 protein.

[0267] Vaccines with a dose of 10 μg gp40 (alkylated or non-alkylated) with mineral oil adjuvant given by the subcutaneous route as primary and booster vaccinations of adult Holstein cows induced swelling at the inoculation site up to 10 cm in diameter and up to 2 cm thick. However, apart from appearing severe, the vaccinated cows did not show any signs of suffering from this local reaction: no fever was observed and there was no effect on appetite or pregnancy.

[0268] Nonetheless, such swelling, even if asymptomatic and transient, has been determined to be undesirable.

[0269] Fortunately, the invention described herein allowed the dose of gp40 protein to be reduced to a safe level while maintaining good immunological efficacy: when the amount of protein per dose was adapted to 1 μg of alkylated gp40 protein, swelling after two subcutaneously administered doses was typically less than 5 cm in diameter and 1 cm in height, mostly only 2 cm in diameter. This was considered acceptable. Importantly, this 10-fold reduction in the amount of protein did not affect the efficacy of vaccination in that sufficiently high titers of gp40 antibodies could still be obtained in the colostrum.

[0270] 4.4.Effects on diarrhea From the vaccination-challenge experiment, diarrhea severity was scored based on the parameters shown in the last column of Table 1. Results for the two most relevant groups of calves are shown in Figure 3. The calves were fed colostrum from dams vaccinated with the BEI-treated gp40 vaccine or from dams vaccinated with the untreated gp40 vaccine. Challenge was given on day 1 after the first bolus of colostrum as described. The mean daily diarrhea score per group was determined and plotted over the duration of the 14-day experiment.

[0271] As can be clearly seen from Figure 3, diarrhea was much less severe and much shorter in the group fed colostrum from BEI-gp40 vaccinated cows. The difference is striking and highly relevant: the rgp40His+BEI vaccine group only had significant diarrhea (score above 1.0) for 2 days (days 7 and 8). However, the rgp40His vaccine group (gp40 mock-treated with aziridine) had significant diarrhea for 6 days (days 6-11). This also resulted in much less severe diarrhea in the rgp40His+BEI vaccine group: the maximum score was 1.2 compared to a maximum score of 2.8 in the mock BEI-treated vaccine group.

[0272] in conclusion: A truly effective cryptosporidiosis vaccine has now been discovered that is capable of significantly reducing both the severity and duration of diarrhea caused by a severe challenge infection with the Cryptosporidium parasite.

[0273] Also, the model of passive vaccination of newborn calves by feeding them with colostrum from vaccinated dams has proven to be very effective.

[0274] [Example 5] Ongoing research 5.1. Dose setting Ongoing dose-ranging studies are testing vaccine doses of 0.1-0.2-0.5-1-2.5 and 5 μg of alkylated gp40 protein according to the invention.

[0275] Initial results indicate that 1 μg of alkylated gp40 protein per dose in a prime-boost regimen induces sufficient levels of gp40-specific antibodies in pregnant cows. Thus far, two 1 μg doses given by subcutaneous route with mineral oil adjuvant induced titers of 19 Log2 Elisa units 2 weeks after the prime vaccination, which dropped to 18 at the 4th week of vaccination when the booster vaccination was given. The booster vaccination then increased the titer again to 22 at the 6th week after vaccination, which dropped slightly to a titer of 21 Log2 at the 8th week after the prime vaccination. At parturition, colostrum antibody values ​​were found to be 1-2 Log2 higher than serum Ab levels.

[0276] Considering that colostrum titers of 17-18 Log2 are sufficient to protect calves, an optimal vaccination protocol would aim to reach titers of 15-16 Log2 in the dams of vaccinated cows. As a result, even a vaccine dose of 0.5 μg of alkylated gp40 protein according to the present invention would be sufficient to reach such titer levels in the dams using a prime-boost regimen.

[0277] Alternatively, it is being investigated whether such protective titer levels in colostrum could be achieved by giving only a single vaccination to pregnant ruminants using 0.5 or 1 μg of alkylated gp40 protein per dose.

[0278] [Example 6] mass spectrometry To determine which amino acids were alkylated by aziridine incubation, we performed LC-MS / MS analysis of rgp40His incubated and not incubated with BEI. The proteins were digested in parallel with the different enzymes and the detected peptides were analyzed for the presence of a 42 Da adduct corresponding to alkylation with an ethyleneimine molecule.

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

[0280] 6.1.2 Intact mass analysis of gp40 Protein amounts were determined by BCA (Pierce) using the manufacturer's protocol. Approximately 6-8 μg of rgp40His samples were analyzed by liquid chromatography and mass spectrometry (LC-MS) using an Agilent 1290 LC and an Agilent 6545 QTOF MS. MassPrep™ Online Desalting cartridges (Waters) were used at 60 °C and a flow rate of 0.4 ml / min. LC buffer A consisted of 0.1% v / v formic acid in water and buffer B consisted of 0.1% formic acid in acetonitrile. The gradient was 5% to 80% buffer B in 5 min with a 1 min hold at 80% buffer B. Equilibration was performed at 5% buffer B with a total run time of 8 min. The column eluate was electrosprayed into the MS using a Dual AJS ESI source (typically 4 kV). MS scans were recorded between 100 and 3200 m / z at 1 spectrum / sec.

[0281] 6.1.3 Peptide mapping of gp40 Peptide mapping of rgp40His was performed by digestion with enzymes: trypsin, chymotrypsin or GluC (Staphylococcus aureus protease V8) at 1:50 enzyme:protein ratio with 1 hour incubation. Afterwards, enzyme was added again at the same ratio and digestion continued overnight. Sample cleanup was performed with Sep-Pak™ tC18, 1 cc, 50 mg sorbent cartridges (Waters). Peptide mixtures were eluted with 0.1% v / v formic acid in 90% acetonitrile. Solvent was evaporated in a Speedvac™ at room temperature. Peptide sample mixtures were reconstituted in 0.1% formic acid.

[0282] Approximately 30 μg of enzymatic digests were analyzed by LC-MS as described. Peptide mixtures were separated at 60 °C, 0.4 mL / min using an Agilent AdvanceBio™ Peptide Map, C18, 1.2 × 150 mm, 2.7 micron. LC buffers A and B were as described. The gradient used was 2% to 45% buffer B in 110 min, followed by 45% to 95% buffer B in 5 min, with a 10 min hold at 95% buffer B. Equilibration was performed at 2% buffer B, with a total run time of 145 min. The column eluate was electrosprayed into the MS using a Dual AJS ESI source (typically 4 kV). MS scans were recorded between 100 and 1700 m / z at 4 spectra / sec. MS / MS scans were recorded between 150 and 3200 m / z at 2 spectra / sec. Up to five precursor ions were selected per cycle for MS / MS with an abundance threshold of 10000 counts. Precursor ions were excluded after five iterations for 1 min. The selection width was set to medium (4 amu).

[0283] 6.1.4 MS data analysis Intact Analysis Deconvolution of LC peaks containing protein fragments was performed using MassHunter™ Qualitative Analysis B.07.00 software. The deconvolution algorithm was "maximum entropy." The mass 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. Adduct was set to "proton" and isotope width was set to "auto."

[0284] Peptide mapping Agilent QTOF MS / MS data files were converted to MFG files using MassHunter software. For each data file, a compound list was created using the "Find compounds by automated MS / MS" function. "Extract MS / MS" was enabled by extraction of average MS / MS spectra for all collision energies. Default settings were otherwise applied. Compound lists were exported to MGF with "Remove isotopes and calculate" enabled, "Isotope model" set to peptide, and "Restricted assigned charge states" set to a maximum of 7. MFG files were searched against the Cryptosporidium parvum gp40 recombinant protein in Mascot™ SEQ ID NO:3 database. Enzyme restrictions were set to none.

[0285] Ethyleneimine (43.04 Da, C 2 H 5 N) was set as a variable modification for all amino acids. MS tolerance was set to 25 ppm and MS / MS tolerance was set to 0.1 Da. Peptide charge was set to 2+, 3+ and 4+.

[0286] Using identical settings as above, the MGF files were also searched against a host cell protein database (Sf9, Spodoptera frugiperda, Uniprot 201909127, 26,506 entries).

[0287] 6.2 Results For gp40 incubated with BEI, approximately one-quarter of the proteins contained no EI adducts, one-quarter had one adduct, and the remainder had two or more adducts, with rapidly decreasing frequencies.

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

[0289] A multiple digestion approach was applied to analyze protein fragments using digestion with trypsin, chymotrypsin or GluC. Together, these generated a complete set of overlapping peptides with good sequence coverage and high confidence in the assignment of EI adducts. Additional manual analysis was performed on the top four modification sites.

[0290] The PepMap results showed complete sequence coverage of gp40, but limited sequence coverage of the His-tag tail. However, it is highly unlikely that the EI adduct is located at a histidine. The coverage of the various peptide fragments is shown in Table 2.

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

[0292] [Table 2]

[0293] PepMap analysis allowed site-specific identification of EI adducts. Since there was prior knowledge about the adducts to be detected, identification of the EI modification site could be performed using an automated database search: each EI adduct increases the size by 42 Da, but since the resolution of the applied method had an accuracy of ±1 Da, weight gains of 41–43 Da were analyzed. Selection for multiple EI adducts on one amino acid was performed by the MS analysis software.

[0294] From the combined data of both batches incubated with aziridine, gp40 amino acids: 2V, 106E, 112E and 147D (numbered as in SEQ ID NO:3) appeared to be the major sites of alkylation. The results are shown in Figure 4. Several other amino acids were also found to be alkylated, but at a (much) lower frequency: cysteine, methionine, serine, threonine, tyrosine, lysine and arginine. Thus, interestingly, aspartic acid (D) and glutamic acid (E) appeared to be the major sites of modification, with other amino acids less frequent.

[0295] The Cryptosporidium gp40 protein used in these studies was recombinantly expressed gp40-His having the amino acid sequence of SEQ ID NO: 3. However, the major protein detected in all samples was rgp40His, which lacks the initiating methionine, with a base molecular weight of 20.9 kDa. Loss of the N-terminal methionine frees up the valine at position 2 for alkylation.

[0296] [Example 7] Vaccination-challenge study with low-dose vaccine A titration experiment was conducted using a vaccine according to the present invention with a very low dose of BEI-gp40, as shown in Example 5 above. Pregnant heifers were actively vaccinated and calves were passively vaccinated and challenged. The setup and performance were essentially as described above. Specifically, the experiment was conducted as follows: First, pregnant heifers were vaccinated and colostrum with large amounts of antibodies was obtained by collecting colostrum from the first and second milkings. The amount of alkylated gp40 protein used per animal dose was 0.4 or 1.5 μg. The vaccine was formulated as a water-in-oil emulsion as described above, using oil (ISA70) + aluminum (Alhydrogel) as adjuvant. The vaccine was administered by subcutaneous route as a primary and booster vaccination 7 and 3 weeks before expected calving, respectively. Serum and colostrum samples were tested for specific IgG antibody responses to gp40 using ELISA, as described above.

[0297] The newborn calves were then fed this colostrum for 5 days and then challenged with the Cryptosporidium parvum parasite. After challenge, diarrhea scores and other clinical scores were determined according to the Wisconsin-Madison scale as described by McGuirk (supra) and reported as health scores.

[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 were vaccinated twice by subcutaneous route with the adjuvanted BEI-rgp40his subunit vaccine containing 1.5 μg gp40 per animal dose in 2 ml volume / dose. Vaccinations were performed approximately 7 and 3 weeks before expected calving. In group 2, 12 animals were not vaccinated due to control colostrum production. All animals were also vaccinated with the Rotavac® Corona (MSD AH) vaccine according to the manufacturer's instructions.

[0299] Blood samples were taken from each animal in Group 1 immediately before (on the same day) each vaccination and one week after the second vaccination, and from animals in Group 2 once one week before vaccination with the Rotavec® Corona vaccine.

[0300] Serological results from vaccinated cows are shown in Table 3 as mean anti-gp40 Elisa titers per group 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 and at background levels.

[0303] As can be seen from Table 3, the results show a significant increase in serum IgG titers to gp40 in Group 1 after the first and second vaccinations. However, both titers indicated that protective levels of colostral antibodies were generated.

[0304] After calving, the first two milkings were collected from each cow and saved for use in subsequent passive vaccination / challenge experiments. The first milking was collected within 6 hours after calving and the second milking was collected within 20 hours after calving. On average, approximately 5 liters of colostrum was collected per cow per milking. Colostrum IgG Elisa results are shown in Table 4 as the mean per group with standard deviation.

[0305] [Table 4]

[0306] Colostrum titers showed that protective levels of anti-gp40 colostrum antibodies were produced in the colostrum from the vaccinated group, which were significantly higher than the titers in colostrum from unvaccinated control animals (p value < 0.001).

[0307] 7.2 Passive vaccination-challenge studies using colostrum containing large amounts of antibodies In subsequent experiments, colostrum with large amounts of antibodies generated as described in section 7.1 above was used for passive vaccination of newborn calves. This made it possible to test the protection conferred by the colostrum against challenge with the Cryptosporidium parvum parasite after feeding it for 5 days. A group of 8 newborn calves was used; the calves were born on different days and therefore progressed through the test schedule from different starting dates. The calves were at least 34 kg Holstein-Friesian, not more than 4 hours old at the start of the experiment, and had not been fed colostrum prior to the test.

[0308] All calves were fed 3 liters of colostrum or a combination of colostrum and milk replacer once daily for 5 consecutive days within 4 hours of birth (groups 1 and 2). All calves were orally challenged with 10^4 Cryptosporidium parvum oocysts 2-4 hours after the first feeding of colostrum. Fecal consistency and health scores of each calf were assessed twice daily for 14 days.

[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 uptake of anti-gp40 antibodies, blood samples were taken from the calves at the start and on day 3 of the experiment.

[0311] Daily health checks of each calf were performed for 14 days after induction to determine a health score according to Table 1 above, which included scoring fecal consistency twice daily (morning and afternoon).

[0312] Fecal material from all animals with diarrhea scores of 2 or 3 at any given time point was tested once daily for the presence of Cryptosporidium parvum or other enteric pathogens using the commercially available Rainbow Calf Scours 4™ test (BIO-K288) until at least one test was confirmed positive for Cryptosporidium parvum.

[0313] Serological results showed the following serum IgG titers against gp40: all titers were at the baseline level of 8.8 Log2 on day 1. On day 3 of colostrum feeding, the mean titers in calf serum were Group 1: 20.6±0.4 and Group 2: 10.9±0.7.

[0314] Rainbow test scores indicated that all diarrhea was due to Cryptosporidium parvum infection. Most calves in group 1 (colostrum from cows vaccinated with 1.5 μg BEI-gp40, 5 days) showed Cryptosporidium parvum in the feces 8 days after challenge, and most calves in group 2 (colostrum from non-vaccinated cows, 5 days) showed Cryptosporidium parvum in the feces 6 days after challenge.

[0315] The health score results are shown in Figure 5. These scores include fecal consistency scores and were determined according to the Wisconsin-Madison scale (ibid.) as outlined in Table 1 above. As can be seen, calves fed colostrum from cows vaccinated with gp40 did not become as sick as calves fed colostrum without anti-gp40 antibodies and were therefore much better able to cope with a vigorous challenge with the Cryptosporidium parvum parasite.

[0316] 7.3 Conclusion The conclusion can be drawn that even a dose of 1.5 μg of alkylated gp40 protein per animal dose of vaccine is capable of inducing levels of anti-gp40 antibodies in colostrum that can effectively passively protect against a vigorous Cryptosporidium parvum challenge infection.

Claims

1. 1. A Cryptosporidium gp40 protein, characterized in that said gp40 protein comprises amino acids alkylated with one or more binary ethyleneimines.

2. The alkylated amino acid is an alkyl group of formula (2) 【Chemistry 1】 wherein R1 is selected from the group consisting of H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, brosyl, alkenyl, alkynyl, alkylaryl, arylalkyl, and cycloalkyl, each of said alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl, and cycloalkyl being optionally substituted with a substituent selected from the group consisting of carbonyl, hydroxyl, alkyl, 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; The gp40 protein of claim 1 , wherein the gp40 protein is alkylated with

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

4. The gp40 protein according to any one of claims 1 to 3, wherein the alkylated amino acid is one or more selected from the group consisting of cysteine, methionine, serine, threonine, tyrosine, lysine, arginine, valine, glutamic acid and aspartic acid.

5. The gp40 protein according to any one of claims 1 to 4, wherein the alkylated amino acids are one or more selected from the group consisting of valine, glutamic acid and aspartic acid.

6. 6. The gp40 protein of claim 5, wherein the valine corresponds to the valine at amino acid number (aa.no.) 2 of SEQ ID NO:3, the glutamic acid corresponds to the glutamic acid at amino acid number 106 or the glutamic acid at amino acid number 112 of SEQ ID NO:3, and / or the aspartic acid corresponds to the aspartic acid at amino acid number 147 of SEQ ID NO:

3.

7. The method of claim 1, wherein the Cryptosporidium gp40 is from Cryptosporidium parvum. - the composition comprising the Cryptosporidium gp40 protein is a supernatant or filtrate from a baculovirus-insect cell expression system culture; and - the supernatant or filtrate is purified by column chromatography; 2. The Cryptosporidium gp40 protein of claim 1 , wherein one or more or all of the features selected from the following are applied:

8. A method for the preparation of a Cryptosporidium gp40 protein according to any one of claims 1 to 6, comprising incubating a composition comprising a Cryptosporidium gp40 protein with binary ethyleneimine.

9. The method of claim 1, wherein the Cryptosporidium gp40 is from Cryptosporidium parvum. - the composition comprising the Cryptosporidium gp40 protein is a supernatant or filtrate from a baculovirus-insect cell expression system culture; and - the supernatant or filtrate is purified by column chromatography; The method according to claim 8 , wherein one or more or all of the features selected from the following are applied:

10. A Cryptosporidium gp40 protein according to any one of claims 1 to 7, or obtainable by the method according to claims 8 or 9, for use in a vaccine for the protection of human or non-human animal targets against cryptosporidiosis.

11. 10. Use of a Cryptosporidium gp40 protein according to any one of claims 1 to 7 or obtainable by the method according to claims 8 or 9 for the manufacture of a vaccine for the protection of human or non-human animal targets against cryptosporidiosis.

12. 10. A vaccine for human or non-human animal targets against cryptosporidiosis, said vaccine comprising a Cryptosporidium gp40 protein according to any one of claims 1 to 7 or obtainable by the method according to claim 8 or 9, and a pharma- ceutically acceptable carrier.

13. The vaccine of claim 12, characterized in that the vaccine comprises an adjuvant.

14. 14. The vaccine according to claim 12 or 13, characterized in that the vaccine comprises at least one additional immunoactive component.

15. A method for the manufacture of a vaccine according to any one of claims 12 to 14, comprising the step of formulating a Cryptosporidium gp40 protein according to any one of claims 1 to 7 or obtainable by a method according to claims 8 or 9 into a vaccine.

16. A method for producing colostrum containing antibodies against Cryptosporidium gp40 protein, obtainable by the method according to any one of claims 1 to 7 or according to claims 8 or 9, comprising the steps of: a. vaccinating a pregnant non-human mammal at least once with the vaccine according to any one of claims 12 to 14; b. collecting colostrum from the mammary gland of said non-human mammal; A method comprising:

17. The method of claim 16 , wherein the target is a ruminant.

18. Colostrum obtainable by the method of claim 16 or 17 for use in the protection of human or non-human animal targets against cryptosporidiosis.

19. A method for the protection of a non-human animal target against cryptosporidiosis, comprising at least one administration to said non-human animal target of a vaccine according to any one of claims 12 to 14.

20. 20. A method for the protection of a non-human animal target against cryptosporidiosis, comprising feeding said non-human animal target with colostrum obtainable by the method of claim 16 or 17 or as described in claim 18.

21. 21. The method of claim 19 or 20, wherein the target is a ruminant.

Images