RU2837392C1 - Cryptosporidiosis vaccine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to Cryptosporidium gp40 protein for immunization against cryptosporidiosis. Said gp40 protein contains one or more alkylated amino acids, wherein this Cryptosporidium gp40 protein is obtained by incubation of a composition containing Cryptosporidium gp40 protein with binary ethyleneimine. Invention also relates to use of said protein in a vaccine for protection against cryptosporidiosis, as well as to a vaccine against cryptosporidiosis and a method for preparing such a vaccine.

EFFECT: present invention provides high immunogenicity of gp40, which leads to a higher titre of gp40-specific antibodies in the vaccinated target mammal.

7 cl, 5 dwg, 4 tbl, 7 ex

Description

The present invention relates to the field of parasitological vaccines. More specifically, the invention relates to the Cryptosporidium gp40 protein, methods for producing said protein, medical use of this protein as a vaccine against cryptosporidiosis, a vaccine containing the protein, and the production of this vaccine, as well as methods for protecting against cryptosporidiosis.

Cryptosporidia are parasitic protozoa belonging to the phylum Apicomplexa. In mammals, they reside mainly in the intestine and cause cryptosporidiosis, which is mainly characterized by diarrhea, colic, dehydration, secondary infections, etc. Symptoms may range from mild in immunocompetent humans or non-human animals to severe in more vulnerable hosts, such as the young or immunocompromised. In human medicine, the disease is most noticeable in malnourished children and in AIDS patients. In veterinary medicine, the disease of newborn ruminants: sheep, goats, but especially calves, is a serious problem both from the point of view of animal welfare and economic reasons. The main causative agent is Cryptosporidium parvum, which occurs in both zoonotic and anthroponotic genotypes. Many other Cryptosporidia species, such as the human pathogen C. hominis, also cause cryptosporidiosis.

The parasitic stage excreted by infected hosts is the oocyst, which is highly infectious and resistant to environmental conditions and many disinfectants. Thus, infection can spread rapidly through faecal material, such as through close contact or faecal contamination of food or drinking water.

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 neonatal calves. Ideally, an effective vaccine would also be developed, but this has proven very difficult due to the complex life cycle of the parasite and the inability to culture the parasite in vitro. As a result, there are currently no licensed vaccines against cryptosporidiosis.

This is not due to lack of trying, as a large number of experimental cryptosporidiosis vaccines have been tested over time, including live attenuated, killed, lysate, and subunit vaccines. Many proteins from different stages of the parasite have been tested as subunit vaccines. For a review, see Lemieux et al. (2018, Pathogens, vol. 7, DOI: 10.3390/pathogens7010002).

One of the many candidate antigens for a subunit vaccine is the 40 kDa mucin-type glycoprotein from Cryptosporidia: gp40. This protein is found on the surface of motile stages of the parasite and is heavily glycosylated.

It should be noted that the protein name for Cryptosporidia is misleading, and gp40 (or its encoding gene or gene product) has also been called Cpgp40/15 (Cevallos et al., 2000, Inf. & Imm., vol. 68, pp. 4108–4116); gp15/45/60 (Strong et al., 2000, Inf. & Imm., vol. 68, pp. 4117–4134); or S60 (Winter et al., 2000, Funct. Integr. Genomics, vol. 1, pp. 207–217). Also related is the Cp17 protein (Priest et al., 2000, Mol. Biochem. Parasit., vol. 106, pp. 261–271). The differences in reported molecular weight reflect variability in sequence and glycosylation level. 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 acc.nr. U22892). The same applies to the antigens designated "CP15" (GenBank accession no. L34568) or "cp41" (WO 01/040439).

Expression of gp40 in a recombinant expression system and use for (passive) vaccination have been proposed for many years, e.g. in: WO 93/024649, WO 01/040248, WO 01/077293 and US 2002/0081312. However, there is still no functional vaccine.

For newborn ruminants, which are usually infected at a very early age, active vaccination will only provide protection after 3-4 weeks, which is too late in practice. However, a convenient method of passive vaccination against cryptosporidiosis by colostrum transmission is possible, for example by feeding colostrum to a calf from vaccinated cattle. Such vaccination of newborn calves against neonatal diarrhea, also known as calf scour, by colostrum transmission is described, for example, in WO 01/045735 and WO 2011/056175. In addition, this method of protection would be in line with the measures currently applied for other causes of neonatal calf scour, such as Escherichia coli, bovine coronavirus, bovine rotavirus and clostridia. Passive vaccination of humans by feeding (antibodies) to colostrum was also effective, see Lemieux et al., 2018 (above).

Thus, many publications describe the isolation or expression of Cryptosporidium antigens and their study in diagnostics or antigenicity studies. It is also proposed to use some antigens for active or passive vaccination of humans or animals. However, to date, no commercial vaccine has been approved. Therefore, there is a need for a vaccine against cryptosporidiosis that is safe and effective.

Aziridines are organic chemical compounds containing an aziridine ring. These compounds are used in medicine as anticancer agents because of their tendency to damage nucleic acids through an alkylation reaction, causing cross-linking and strand breaks. In biotechnology, this property of aziridines is used to inactivate microorganisms such as bacteria and viruses.

In addition to nucleic acids, aziridines can interact with proteins by alkylating nucleophilic sites on amino acids. For example, by reacting with a sulfur-containing side chain, such as a sulfhydryl or thioether group; with a nitrogen-containing side chain, such as an amino group; or with an oxygen-containing side chain, such as a hydroxyl group.

These reactions can occur with one or more amino acids of the protein; but which amino acids react with aziridine and become alkylated is not entirely clear.

A widely used aziridine for virus inactivation is ethyleneimine (EI), mainly used in the form of binary ethyleneimine (BEI). A review is given in H. Bahnemann (1990, Vaccine, vol. 8, p. 299-303).

Although it is a hazardous chemical, the use of EI is preferable to traditional formalin-based virus inactivation because of its more predictable, i.e., linear, reaction kinetics. Furthermore, because EI preferentially interacts with nucleic acids, it is less detrimental 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).

There are a few reports that describe incubation of protein antigen with a chemical to increase antigenicity, however this only applies to protein cross-linking reactions such as formalin and/or heat treatment (Grovit-Ferbas et al., 2000, J. of Virol., vol. 74, p. 5802-5809). Other incubations of subunit proteins with an inactivating agent have been described only for detoxification of bacterial toxins by formalin. For aziridines, incubation with a subunit protein has not been described except in the context of use for inactivation of microorganisms. Incubation with aziridine has not been described for the Cryptosporidium gp40 protein.

Several papers describe the negative impact on immunogenicity from the action of aziridines on proteins during inactivation reactions and recommend precautions; Chen et al. (1999, J. of Exp. Med., vol. 189, pp. 1757-1764) describe the loss of antigenicity by S-alkylation of cysteines in proteins. To reduce such reactivity, WO 98/51660 describes the development of EI polymers that are even more specific for nucleic acids. Furthermore, WO 98/45415 describes inactivation with EI at acidic pH to reduce the adverse reaction with viral proteins.

The object of the present invention is to overcome the shortcoming of the prior art and to satisfy this need in the art by providing a vaccine against cryptosporidiosis that is safe and effective.

When inventors attempted to develop a cryptosporidiosis vaccine based on recombinantly expressed gp40 protein, initial results were promising: gp40 could be expressed in E. coli as described in the literature and was used with standard adjuvants to vaccinate pregnant heifers. After calving, colostrum containing high levels of gp40-specific antibodies was obtained. When calves were fed the colostrum, they were protected from disease caused by severe infectious immunization with C. parvum. Unfortunately, the level of recombinant expression was low and the expressed gp40 protein had low immunogenicity, so relatively high doses of antigen had to be used. Under these circumstances, production of a cryptosporidiosis vaccine based on recombinantly expressed gp40 was not considered feasible for commercialization.

Another problem was that the gp40 protein appeared to be quite reactive after vaccination: when immunologically effective doses of gp40 were administered to cattle as a standard emulsified vaccine, the overall safety was good and there was no effect on pregnancy, but unacceptable levels of local reactions to vaccination occurred, especially when administered subcutaneously. Also, these local reactions increased after revaccination.

While the use of lower doses of antigen reduced local adverse reactions to vaccination to an acceptable level, unfortunately these doses did not induce sufficient levels of antibodies in the colostrum to provide effective passive vaccination of calves.

Inventors had no idea how to overcome these problems to develop an affordable, safe, and effective cryptosporidiosis vaccine.

It has been unexpectedly found that the object of the invention can be achieved and, therefore, one or more disadvantages of the prior art can be overcome by incubating the gp40 protein with aziridine before its use as a vaccine. It has been found that this incubation significantly increases the immunogenicity of gp40, which leads to a higher titer of gp40-specific antibodies in the vaccinated target mammal, and subsequently also in the colostrum obtained from this vaccinated target; this is compared to immunization with the same amount of gp40 that was not incubated with aziridine.

This discovery makes it possible to reduce the antigenic mass of gp40 used for vaccination by 10-20 times compared to gp40 not incubated with aziridine, while still inducing sufficient levels of antibodies in colostrum to provide effective passive immune protection of the offspring against severe Cryptosporidium infection. In addition to creating an effective cryptosporidiosis vaccine, it is possible to significantly reduce the antigenic mass of gp40 per vaccine dose, which solves two other problems: it reduces the level of local reactions, so that vaccination with an effective dose is now safe. In addition, it makes a cryptosporidiosis vaccine based on recombinantly expressed gp40 economically feasible.

It is known that gp40-specific antibodies can inhibit the invasion of Cryptosporidia parasites into host cells. Therefore, when passively vaccinating against cryptosporidiosis, for example by feeding colostrum to a vaccinated mammal, the increased antibody levels in this colostrum will provide increased local antibody levels in the gastrointestinal tract and provide protection against parasite invasion.

However, it is not known exactly how and why incubation with aziridine increases the immunogenicity of gp40.

Although the inventors do not wish to be bound by any theory or model that might explain these results, they expect that the chemical change in the gp40 protein caused by incubation with aziridine, in particular the alkylation of one or more amino acids, results in a different immune response in the vaccinated human target or non-human target animal compared to the immune response to gp40 that has not been alkylated with aziridine. In addition to the observed increase in the level of anti-gp40 antibodies produced, there is likely to be one or more other changes in the antibody profile produced, such as the avidity and/or specificity of the antibodies produced. In addition, there may be a change in the type or level of activated cellular immune response.

This was not at all obvious from any description of the prior art: in many publications reporting damage to immunogenic epitopes during chemical inactivation, the positive effect of incubation with aziridine on immunogenicity was completely unexpected. Let alone an increase in immunogenicity of this magnitude. Furthermore, no such effect was observed when gp40 was incubated with formalin, Triton X-100, or after gamma irradiation.

Therefore, in one aspect, the invention relates to a Cryptosporidium gp40 protein or an immunogenic portion thereof, characterized in that the gp40 protein and portion thereof comprise one or more alkylated amino acids.

"Protein" is a molecular chain of amino acids as defined herein. Among other things, polypeptide, peptide and oligopeptide are included in the definition of protein. A protein may be of natural or synthetic origin, may be a native or mature protein, a pre- or proprotein or a portion of a protein.

The term Cryptosporidium refers to a genus of parasites, phylum Apicomplexa, and subclass Coccidia. These microorganisms have the characteristic features of their taxonomic class, such as morphological, genomic, and biochemical characteristics, as well as biological characteristics, such as physiological, immunological, or pathological behavior. A large number of species of Cryptosporidium parasites are known. They can infect a wide range of non-human animals, including humans.

A well-known species of Cryptosporidium parasite is C. parvum, which occurs in two genotypes: genotype I, which is thought to be infectious only to humans, and genotype II, which is a proven zoonotic agent. Both genotypes of C. parvum cause cryptosporidiosis, especially in susceptible subjects. Reference for characteristics and action of C. parvum in veterinary medicine: "The Merck veterinary manual" (11th ed., 2016, ISBN-10: 9780911910612).

As is known in the art, the classification of a microorganism into a specific taxonomic group is based on a combination of its features. Thus, the invention also includes other species of Cryptosporidium that belong to this genus. Likewise, this applies to parasites that are subclassified in some way, such as into subspecies, strains, isolates, genotypes, variants, subtypes or subgroups, and the like.

Furthermore, it will be obvious to a person skilled in the art to which the invention pertains that, although a particular parasite according to the invention may currently be classified in such a species or genus, this is a taxonomic classification that may change over time, as new research results may lead to a reclassification into a new or different taxonomic group. However, since this does not change the parasite itself or its antigen repertoire, but only its scientific name or classification, such reclassified parasites remain within the scope of the invention.

Samples of Cryptosporidium parasites for use in the invention may be obtained from various sources, such as a field isolate from a human or non-human animal in the wild or on a farm, or from various laboratories, (depository) institutions or (veterinary) universities. In addition, much of the genetic information on Cryptosporidium and gp40 is available digitally in publicly available sequence databases such as GenBank NCBL and EBI EMBL. A publicly available database specializing in sequences from Cryptosporidia is CryptoDB, which is available online at: cryptodb.org.

For the purposes of the invention, "gp40" is a protein comprising the amino acid sequence of SEQ ID NO: 1 or a homologue thereof. The same sequence is shown in Figure 1 in IUPAC one-letter code. This particular sequence corresponds to the core sequence of the gp40 protein, starting at amino acid 31 and ending at 220, from GenBank, accession no. AAF78345.1 (Strong et al., 2000, supra). This GenBank entry represents the amino acid sequence of the 60 kDa precursor protein from C. parvum, Iowa strain, which is a bovine isolate in the United States and has genotype II.

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 tract (both in part of gp40) and a C-terminal GPI anchor (in gp15). Following cleavage of the precursor, the two proteins interact and attach to the apical surface region of the merozoite and sporozoite stages of Cryptosporidia, where they participate in host cell attachment and invasion. In nature, gp40 is heavily glycosylated, primarily with O-linked GalNac structures on the amino acids threonine and serine.

The gene encoding the gp40 precursor protein from C. parvum is described as gene accession number: cgd6_1080 in the CryptoDB database (above).

A "homologue" 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 to SEQ ID NO: 1 when aligned with the full length of SEQ ID NO: 1. Sequence alignment should be performed using the "blastp" algorithm with standard parameters from the NCBI BLAST™ alignment software suite, available online at blast.ncbi.nlm.nih.gov.

This variation in the level of sequence identity is necessary to cover the natural variation in the amino acid sequence of the Cryptosporidium gp40 proteins of the invention, which is well known to be quite heterogeneous (see also Strong et al., 2000, supra). For example, even among the gp40 proteins from C. parvum genotype II isolates, there is a variation of up to 23% in the level of amino acid sequence identity compared to the sequence of SEQ ID NO: 1. This is the result of, on the one hand, some distributed variations in the amino acid sequence and, on the other, variations in the length of the polyserine tract. This polyserine tract in Cryptosporidium gp40 can have from 6 to 25 consecutive serines, although all of these are functional gp40 proteins. See, for example, the C. parvum genotype II gp40 homologues in GenBank accession nos. AOA32955.1 (6 serines) and ACR78128.1 (25 serines). When aligned to the full length of SEQ ID NO: 1 using the blastp program, they show 77 and 96% amino acid sequence identity, respectively.

Further variation in the amino acid sequence of gp40 occurs in genotype I isolates of C. parvum. As described 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% and still be functional gp40 proteins.

On the diversity of Cryptosporidium gp40 parvum, see also: Leav et al., 2002, Inf. and Imm., vol. 70, pp. 3881–3890; and: Wu et al., 2003, Appl. Environ. Microbiol., vol. 69, pp. 4720–4726.

Another level of gp40 amino acid sequence variability occurs among gp40 homologues of closely related species such as C. hominis, for which amino acid sequence identity to SEQ ID NO: 1 decreases to approximately 60%.

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

In order to be capable of alkylation by incubation with aziridine according to the invention, the Cryptosporidium gp40 homologue comprises at least one amino acid selected from: cysteine, methionine, serine, threonine, tyrosine, lysine, arginine, valine, glutamic acid and aspartic acid.

The term "comprise" (as well as variations such as "comprising," "comprises," and "comprised") as used herein is intended to mean all of the elements, and in any possible combination possible for the invention, that are encompassed or included in the text section, paragraph, claim, etc. in which the term is used, even if such elements or combinations are not explicitly stated; and without excluding any of such elements or combinations.

Thus, any such text section, paragraph, claim, etc. may also refer to one or more embodiments in which the term “comprising” (or variations thereof) is replaced by terms such as “consists of,” “consisting of,” or “consists essentially of.”

An "alkylated amino acid" according to the invention is an amino acid in which a sulfur, nitrogen or oxygen atom has been alkylated by reaction with an aziridine such that the amino acid has acquired an additional alkyl group compared to the native structural formula of the amino acid. Alkylated amino acids include, for example: the amino acid cysteine in which the sulfhydryl group of the side chain has been alkylated to an alkylene thioether, or the amino acid methionine in which the thioether of the side chain has been alkylated to a tertiary thioether; lysine or arginine in which the amino group of the side chain has been alkylated to an alkylamino group; or serine, threonine, tyrosine, glutamic acid or aspartic acid in which the hydroxyl group of the side chain has been alkylated to an alkyl ether.

The alkylation reaction that occurs upon incubation with aziridine will depend on the availability of such amino acids for the reaction. For example, when two cysteines are linked by a sulfur bridge, they will not be available for alkylation under non-reducing conditions. On the other hand, amino acids, one of whose groups is normally unreactive, can be alkylated at the amino or carboxyl group when such a group is exposed, for example, at the N- or C-terminus of a protein fragment. For example, when the protein gp40 or part of it is initiated with valine, then the exposed amino group at the N-terminus of this valine can become alkylated upon reaction with aziridine.

As will be appreciated by those skilled in the art, a portion of the Cryptosporidium gp40 protein comprising one or more alkylated amino acids according to the invention may equally be used to induce increased levels of gp40-specific antibodies in an immunized target. 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 ensured, inter alia, by a portion comprising one or more alkylated amino acids as described herein.

One skilled in the art can readily determine whether a portion of the gp40 protein or its homologue is such an immunogenic portion by immunizing the target and testing for the formation of gp40-specific antibodies.

In order to provide immunogenic effectiveness according to the present invention, the immunogenic portion of the Cryptosporidium gp40 protein comprising one or more alkylated amino acids has at least 50 consecutive amino acids from SEQ ID NO: 1 and comprises at least one alkylated amino acid selected from: glutamic acid corresponding to amino acid number (a.a. No.) 88 or 94 of SEQ ID NO: 1; and aspartic acid corresponding to a.a. No. 129 of SEQ ID NO: 1.

For the invention, "corresponding" in the feature "amino acid corresponding to amino acid number" refers to an amino acid that is in a comparable position and/or in a comparable amino acid context, as in the case of the amino acid sequence of the said sequence identifier.

Each of the Cryptosporidium gp40 protein or immunogenic portion thereof of the invention may be modified in a variety of ways well known in the art. Examples include glycosylation, lipidation or pegylation. An additional portion of the protein may also be added, such as a carrier, hapten, signal sequence, anchor sequence, marker or label. Any of these modifications may be useful for expression, detection and/or purification of the protein. Such modification may also be useful for adaptation or further enhancement of immunogenicity.

For example, the examples below describe the use of the Cryptosporidium gp40 protein containing one or more alkylated amino acids with a C-terminal 6x-histidine tag. Although this tag does not affect immunogenicity, it allows the gp40 protein to be purified by metal affinity chromatography, such as when the protein is assembled from a recombinant expression system.

The N-terminal coding sequence of the gp40 protein used in the vaccination experiments described in the examples is extended by a portion of the native signal sequence and encodes an N-terminal methionine to initiate its transcription. This improved expression levels in a baculovirus-insect cell expression system without inducing secretion from insect cells.

The amino acid sequence of the Cryptosporidium gp40 protein used in the examples is presented in SEQ ID NO: 3. This protein comprises the complete amino acid sequence of SEQ ID NO: 1 with the additional N- and C-terminal sequences indicated: it is expressed from a recombinant gene that is provided for a His-tag, an additional methionine and a partial signal sequence. In addition, this gene encoding gp40 according to the invention has been codon-optimized with respect to the codon preference of the AcMNPV baculovirus polyhedrin gene. The coding nucleotide sequence is presented in SEQ ID NO: 2.

Alkylation of the Cryptosporidium gp40 protein or an immunogenic portion thereof with aziridine provides increased immunogenicity of the protein portion thereof. 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 induced immune response may be of the humoral and/or cellular type, and protection may be achieved in a variety of ways, as described below. Immunogenicity in relation to vaccination is also referred to as "potency".

An increase in the immunogenicity of a protein can be readily determined by one skilled in the art by conducting a comparative immunization experiment. For the invention, this may mean comparing the immune response in a non-human animal immunized with a Cryptosporidium gp40 protein containing one or more alkylated amino acids of the invention with the response of an animal immunized with a gp40 protein not containing one or more alkylated amino acids, all other parameters and conditions being substantially the same. According to the invention, an increased immunogenicity will result in a higher amount of gp40-specific antibodies produced in the target serum by a Cryptosporidium gp40 protein containing one or more alkylated amino acids, compared to a similar dose of gp40 that does not contain one or more alkylated amino acids. The level and specificity of the serum response can be readily measured using any suitable sero-diagnostic method, such as ELISA, IFT or AlphaLisa. For optimal comparison of the effect of the invention, gp40 that does not contain one or more alkylated amino acids should be incubated with a mock, i.e., incubated under the same conditions as the incubation with aziridine, except that aziridine will not be present.

For example, when cattle are immunized with 10 μg of gp40, either alkylated or mock-treated, in a standard oil adjuvant, the Cryptosporidium gp40 protein containing one or more alkylated amino acids of the invention will induce a gp40-specific antibody titer that is 2-4 Log2 units higher when measured in a standard antibody ELISA using coated gp40, compared to the titer obtained with gp40 that does not contain one or more alkylated amino acids; this increase in gp40-specific antibody titer of up to 16-fold is a result of aziridine alkylation.

For the purposes of the invention, gp40-specific antibodies are antibodies that recognize and bind to gp40 in a "specific" manner. In the art, this means that binding and recognition are correlated with the concentration of the antibody and antigen. Therefore, in the case of specific binding, a dilution of either the antigen or the antibody should exhibit a gradual decrease in the level of binding that can be detected in a standard serological assay.

Details of embodiments and additional aspects of the invention will be described below.

In an embodiment of the Cryptosporidium gp40 protein of the invention, the homologue is a protein comprising an amino acid sequence that has an amino acid sequence that is at least 60% identical to SEQ ID NO: 1, when compared to the full-length SEQ ID NO: 1, as defined herein.

More preferably, the Cryptosporidium gp40 protein homologue of the invention has an amino acid sequence identity of at least 62, 65, 67, 70, 72, 75, 77, 80, 82, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or even at least 99% amino acid sequence identity to SEQ ID NO: 1 when compared to the full-length SEQ ID NO: 1, in the stated order of preference.

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

All these amino acids are well-known chemical compounds whose non-integrated chemical substance (L-isomer) are: Cysteine: CAS Registry No.: 52-90-4; Methionine: CAS No.: 59-51-8; Serine: CAS No.: 56-45-1; Threonine: CAS No.: 80-68-2; Tyrosine: CAS No.: 60-18-4; Lysine: CAS No.: 56-87-1; Arginine: CAS No.: 74-79-3; Valine: CAS No.: 72-18-4; Glutamic Acid: CAS No.: 56-86-0; and Aspartic Acid: CAS No.: 56-84-8.

Alkylation of the sulfhydryl group of the side chain of cysteine yields cysteine having an alkylene thioether, with the alkyleneamino group replacing the hydrogen atom from the sulfhydryl group.

Alkylation of the thioether group of the side chain of methionine yields methionine, which has a tertiary thioether, with the alkyleneamino group replacing the hydrogen atom from the thioether group.

Alkylation of the side chain amino group of arginine or lysine yields arginine or lysine having an alkylene amino group attached to the side chain amino group.

Alkylation of the side chain hydroxy group of serine, threonine, tyrosine, aspartic acid, or glutamic acid yields serine, threonine, tyrosine, glutamic acid, or aspartic acid to which an alkyleneamino is attached as an alkyl ester.

Alkylation of the amino group of valine yields valine, which has an alkyleneamine attached to the amino group.

In a preferred embodiment, the alkyleneamine added by reaction with the aziridine is an ethyleneimine group.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof of the invention, the alkylated amino acid is alkylated with an alkyl group of Formula (2)

(2),

wherein R1 is selected from the group consisting of: H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, brosyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl, wherein each of alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl is optionally 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.

Each of R2' and R2" is independently selected from H and alkyl. Suitably, R2' and R2" are each independently selected from H and C _1-6 alkyl. Preferably, each of R2' and R2" is 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.

Each of R3' and R3" is independently selected from the group consisting of H and alkyl. Suitably, R3' and R3" are each independently selected from H and C _1-6 alkyl. Preferably, each of R3' and R3" is 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.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof, alkyl has 1-20 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-6 carbon atoms, and most preferably 1-3 carbon atoms. Preferably, alkenyl, alkynyl and cycloalkyl have 2-20 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms, and most preferably 2-3 carbon atoms. Accordingly, aryl has 5-12 carbon atoms, preferably 5-10 carbon atoms, more preferably 6-10 carbon atoms.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof of the invention, the alkylated amino acid is alkylated with an alkyl group of Formula (2), wherein

- R1 represents H;

- R2' represents H;

- R2' represents ethyl;

- R1 is acetyl;

- R2' is methyl;

- R1 is ethanol;

- R2' isobutyl;

- R2” represents H;

- R3' is H; or

- R3” represents H.

In a preferred embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof of the invention, the alkyl group of Formula (2) has one of the combinations of substituents from the group:

- R1 represents H, R2' represents H, R2” represents H, R3' represents H and R3” represents 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, 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(CH ₃ ) ₃ , R2" is H, R3' is H and R3" is H.

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

As described in the examples, further analyses showed that these gp40 amino acids are preferentially alkylated upon incubation with aziridine.

As described above, the Cryptosporidium gp40 protein of the invention comprises the amino acid sequence of SEQ ID NO: 1 or a homologue thereof. Therefore, the alkylated amino acids preferably comprise the amino acids corresponding to E88, E94 and D129 of SEQ ID NO: 1.

NB: E and D represent the IUPAC single-letter amino acid codes for glutamic and aspartic acids, respectively. Thus: "E88" refers to glutamic acid at amino acid position #88, etc.

In a preferred embodiment, the gp40 protein of the invention comprises a region of SEQ ID NO: 3, which comprises SEQ ID NO: 1. More preferably, gp40 comprises aa Nos. 19-207 of SEQ ID NO: 3; aa Nos. 15-207; 10-207; 5-207; 4-207; 3-207; 2-207; or even 1-207 of SEQ ID NO: 3, in that order of preference.

As described in the examples, one form of the gp40 protein of the invention, found after recombinant expression and incubation with aziridine, contains aa #2-207 of SEQ ID NO: 3. Apparently, the partial signal sequence was not cleaved, but the N-terminal methionine was cleaved, leaving the valine at position 2 (V2) exposed at the N-terminus of gp40. Interestingly, valine was also alkylated upon incubation with aziridine.

Thus, in an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof of the invention and an alkylated amino acid selected from valine, glutamic acid and aspartic acid, valine is valine corresponding to amino acid number (aa#) 2 of SEQ ID NO: 3; glutamic acid is glutamic acid corresponding to aa# 106 or aa# 112 of SEQ ID NO: 3; and/or aspartic acid is aspartic acid corresponding to aa# 147 of SEQ ID NO: 3.

In another aspect, the invention relates to a composition comprising the Cryptosporidium gp40 protein or an immunogenic portion thereof, according to the invention.

As described, the Cryptosporidium gp40 protein and its immunogenic portion of the invention containing alkylated amino acids can be obtained by incubation with aziridine as described herein.

Therefore, in a further aspect of the invention, the Cryptosporidium gp40 protein or immunogenic portion thereof of the invention can be obtained by incubating a composition comprising the Cryptosporidium gp40 protein or immunogenic portion thereof with aziridine.

Similarly, in a further aspect, the invention relates to a method for producing the Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention, wherein the method comprises the step of incubating a composition comprising the Cryptosporidium gp40 protein, or an immunogenic portion thereof, with aziridine.

NB: Aziridines are toxic and mutagenic chemicals that must be stored, handled and disposed of safely and appropriately. Aziridines can be neutralized by incubation with a thiosulfate, such as sodium thiosulfate (Na ₂ S ₂ O ₃ ).

The aziridine ring is a three-membered heterocycle consisting of one amino and two methylene groups. "Aziridine" is an organic chemical compound containing an aziridine ring and has the structural formula (1):

(1).

R1 is selected from the group consisting of: H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, brosyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl, wherein each of alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl is optionally 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.

Each of R2' and R2" is independently selected from H and alkyl. Suitably, R2' and R2" are each independently selected from H and C _1-6 alkyl. Preferably, each of R2' and R2" is 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.

Each of R3' and R3" is independently selected from the group consisting of H and alkyl. Suitably, R3' and R3" are each independently selected from H and C _1-6 alkyl. Preferably, each of R3' and R3" is 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.

Accordingly, alkyl has 1-20 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-6 carbon atoms, and most preferably 1-3 carbon atoms. Preferably, alkenyl, alkynyl and cycloalkyl have 2-20 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms, and most preferably 2-3 carbon atoms. Accordingly, aryl has 5-12 carbon atoms, preferably 5-10 carbon atoms, more preferably 6-10 carbon atoms.

Widely used aziridines for the inactivation of microorganisms are ethyleneimine (R1=H) and 1-acetylethyleneimine (R1=acetyl).

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the aziridine is an aziridine of Formula (1), wherein R1 is H or is acetyl.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the aziridine is an aziridine of Formula (1), wherein

- R1 represents H;

- R2' represents H;

- R2' represents ethyl;

- R1 is acetyl;

- R2' is methyl;

- R1 is ethanol;

- R2' isobutyl;

- R2” represents H;

- R3' is H; or

- R3” represents H.

The aziridine ring is very reactive and can be opened by reaction of one of the methylenes, for example with a protein, which leads to alkylation of the sulfhydryl group. Cysteine amino acids are easily alkylated by aziridine. At alkaline pH, also the thioester of methionine can be alkylated by aziridine.

In the alkylation of a protein with an aziridine according to the invention, the open ring of the aziridine becomes attached to one or more of its amino acids as an alkyl group. For example, one or more amino acids are alkylated with an alkyl group of formula (2), where in formula (2) the dotted line represents a bond to an amino acid.

(2)

R1 is selected from the group consisting of: H, alkyl, alkylsulfonyl, mesyl, tosyl, nosyl, brosyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl, wherein each of alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl and cycloalkyl is optionally 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.

Each of R2' and R2" is independently selected from H and alkyl. Suitably, R2' and R2" are each independently selected from H and C _1-6 alkyl. Preferably, each of R2' and R2" is 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.

Each of R3' and R3" is independently selected from the group consisting of H and alkyl. Suitably, R3' and R3" are each independently selected from H and C _1-6 alkyl. Preferably, each of R3' and R3" is 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.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the aziridine is an aziridine of Formula (1) having one of the combinations of substituents from the group:

- R1 represents H, R2' represents H, R2” represents H, R3' represents H and R3” represents 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, 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(CH ₃ ) ₃ , R2" is H, R3' is H and R3" is H.

In a preferred embodiment of the aziridine of the invention, one of the conditions selected from:

- ethyleneimine is preferably CAS No. 151-56-4,

- 2-ethylethyleneimine, preferably CAS No. 2549-67-9,

- 1-acetylethyleneimine is preferably CAS No. 460-07-1,

- 2-methylethyleneimine is preferably CAS No. 75-55-8,

- 1-ethylenimine ethanol is preferably CAS No. 1072-52-2 and

- 2-isobutylethyleneimine is preferably CAS No. 3647-37-8.

As described, such incubation with aziridine results in alkylation of the Cryptosporidium gp40 protein or an immunogenic portion thereof, particularly amino acids selected from: cysteine, methionine, serine, threonine, tyrosine, lysine, arginine, valine, glutamic acid, and aspartic acid. This alkylation, in turn, imparts increased immunogenicity to such protein or portion thereof.

For the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as for the method for obtaining the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, "incubation" includes conventional procedures such as combining aqueous compositions containing said components and reacting them 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 under a wide variety of conditions and parameters, all of which are well known to a person skilled in the art of the invention and are readily available for optimization and adaptation by conventional methods, when necessary.

For example, said incubation can be carried out in a wide range of temperatures. Preferably, the incubation with aziridine is carried out at a temperature above zero degrees Celsius, more preferably from 1 to 55°C, from 5 to 50°C, from 10 to 40°C, or even from 15 to 40°C, in that order of preference.

Similarly, incubation with aziridine can be performed over a wide range of pH values. However, incubation at a pH that is not too acidic is most effective, since alkylation of amino acids by aziridine may be less effective at very acidic pH values.

Therefore, in an embodiment of the said method, the incubation with 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 even at a pH above 7.5, in that order of preference.

The upper limit of the pH value for the incubation with aziridine in the method is easily determined in relation to other incubation parameters. Preferably, the incubation with aziridine is carried out at a pH below 12, below 10, or even below 9, in that order of preference.

As is clear to one skilled in the art, since aziridine is consumed during the incubation reaction, its concentration can only be reliably indicated at the beginning of the incubation.

Therefore, in an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the concentration of aziridine at the beginning of the incubation is at least 0.1 mM, 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 even at least 50 millimolar, in the stated order of preference.

The upper limit of the aziridine concentration can be easily determined. In one embodiment, the concentration of aziridine at the beginning of the incubation is less than 1 M, less than 0.5 M, or even less than 0.1 M, in that order of preference.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the duration of incubation of the composition comprising the protein or fragment thereof and aziridine is 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 even at least 36 hours, in order of preference. In an embodiment, the duration is overnight (i.e. 12-18 hours).

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for obtaining the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, an additional step is carried out after incubation with aziridine, wherein this step ensures neutralization of any remaining aziridine.

In a preferred embodiment, the neutralization step is carried out by adding to the incubation mixture, after the incubation reaction, an appropriate amount of thiosulfate and incubating for an appropriate period of time to complete the neutralization. For example: neutralization by adding 10 to 100 mM sodium thiosulfate and incubating for 15 to 90 minutes at a temperature of 15 to 30°C.

In practice, the amount of thiosulfate used for neutralization will be a slight overdose. This serves as a guarantee that when some thiosulfate remains after the neutralization reaction, one can be sure that all the aziridine has been consumed.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the aziridine is selected from: ethyleneimine (R1=H), 2-ethylethyleneimine (R2'=ethyl), 1-acetylethyleneimine (R1=acetyl), 2-methylethyleneimine (R2'=methyl), 1-ethyleneimineethanol (R1=ethanol) and 2-isobutylethyleneimine (R2'=isobutyl).

In a preferred embodiment of the aziridine of the invention, one of the conditions selected from:

- ethyleneimine is preferably CAS No. 151-56-4,

- 2-ethylethyleneimine is preferably CAS No. 2549-67-9,

- 1-acetylethyleneimine is preferably CAS No. 460-07-1,

- 2-methylethyleneimine is preferably CAS No. 75-55-8,

- 1-ethylenimine ethanol is preferably CAS No. 1072-52-2, and

- 2-isobutylethyleneimine is preferably CAS No. 3647-37-8.

Since aziridine is a fairly hazardous chemical, it is preferably used for incubation according to the invention in a diluted form.

For ethyleneimine, this can be conveniently achieved by using the so-called "binary ethyleneimine" (BEI). BEI is the product of the cyclization reaction of bromoethylaminobromide (BEA) under alkaline conditions and moderate heating, e.g. to about 37°C. Alkaline conditions can conveniently be provided by adding, for example, sodium hydroxide. All this is well known in the art, see, for example, Bahnemann 1990 (above).

Thus, in a preferred embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the aziridine is ethyleneimine or is a binary ethyleneimine.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained in accordance with the invention, as well as the method for obtaining the Cryptosporidium gp40 protein or immunogenic portion thereof in accordance with the invention, the additional step is carried out before the incubation with aziridine, and BEI is obtained therefrom.

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

Preferably, BEI is obtained by reacting BEA with a hydroxide.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the gp40 protein or immunogenic portion thereof is derived from C. parvum; more preferably, from C. parvum genotype II.

For the invention, "from C. parvum" means that the amino acid sequence of the gp40 protein or a portion thereof is based on the gp40 protein from the species C. parvum, either directly by isolating the protein or the encoding nucleic acid; or indirectly, such as based on sequence information of such a C. parvum gp40 protein.

The Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention is thus preferably an isolated protein, meaning that it is not in its natural context in or on a living Cryptosporidium parasite.

A composition comprising the Cryptosporidium gp40 protein or an immunogenic portion thereof for use in incubation with aziridine according to the invention can be obtained by various methods, including the isolation of Cryptosporidium parasites obtained from a living infected host organism. The protein or portion thereof can also be obtained in a cell-free transcription system.

However, the most convenient and scalable to industrial production levels is the production of the Cryptosporidium gp40 protein or an immunogenic portion thereof, for use in the invention, by in vitro expression using a recombinant expression system. Such an expression system may use a cell culture system of prokaryotic or eukaryotic cells that have been genetically modified to express the desired protein. Alternatively, the cells may 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 modified Chinese hamster ovary (CHO) cells, bacteria such as E. coli, Bacillus species or Staphylococcus carnosus; or yeast species, such as Saccharomyces cerevisiae or Pichia pastores. Examples of the use of a recombinant virus for expression from a host cell include: baculovirus-insect cell or adenovirus-mammalian cell systems.

Therefore, in an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, an additional step is carried out to obtain a composition comprising the gp40 protein or portion thereof by means of a recombinant expression system; preferably, by means of a baculovirus-insect cell expression system.

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

More preferably, the baculovirus-insect cell expression system produces the Cryptosporidium gp40 protein by expressing the recombinant DNA sequence SEQ ID NO: 2 as a heterologous insert for the recombinant baculovirus.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for obtaining the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, wherein the composition comprises the gp40 protein or portion thereof (before incubation with aziridine) obtained using a baculovirus-insect cell expression system, said composition is collected from an insect cell culture, for example, as a whole culture or as a portion of such a culture, for example, a supernatant or pellet after centrifugation of an insect cell culture, or a filtrate or retentate after filtration.

More preferably, the composition comprising the Cryptosporidium gp40 protein or its immunogenic portion is a supernatant or filtrate of a culture of a baculovirus-insect cell expression system. The supernatant may be obtained after allowing the culture to settle under gravity, such as overnight, or by centrifugation; the filtrate is what passes through the filter during filtration.

In an embodiment of the Cryptosporidium gp40 protein or an immunogenic portion thereof obtained according to the invention, as well as the method for obtaining the Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention, wherein the composition comprises the gp40 protein or a portion thereof obtained by means of a recombinant expression system, after obtaining and collecting said composition from the expression system and before incubation with aziridine, an additional step is carried out, wherein the step comprises purifying said composition. Preferably, the purification is carried out by means of column chromatography. More preferably, the gp40 protein or a portion thereof comprises a tag facilitating the purification of said composition by means of column chromatography, such as a histidine tag for metal affinity chromatography.

Since incubation with aziridine is also an effective means for chemically inactivating a microorganism, the production of an alkylated protein or an alkylated immunogenic portion thereof according to the invention can be conveniently combined with the inactivation of a microorganism, such as a virus or bacteria.

Therefore, in an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for producing the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, the composition comprising the Cryptosporidium gp40 protein or immunogenic portion thereof also comprises a microorganism. The microorganism may, for example, be a C. parvum parasite inactivated by aziridine; although this inactivates the parasite, it also causes alkylation of the gp40 protein. Preferably, the microorganism is a virus or a bacterium, such as those used in a recombinant expression system.

The conditions for inactivating many microorganisms using aziridine are well known in the art. Any desired adaptation or optimization can be easily accomplished by conventional methods.

In an embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, as well as the method for obtaining the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention, one or more conditions selected from the group consisting of:

- incubation with aziridine is carried out at a temperature above zero degrees Celsius, more preferably from 1 to 55°C, from 5 to 50°C, from 10 to 40°C or even from 15 to 40°C, in that order of preference;

- incubation with 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 even at a pH above 7.5 in that order of preference;

- incubation with aziridine is carried out at a pH below 12, below 10 or even below 9, in that order of preference;

- the concentration of aziridine at the beginning of the incubation is at least 0.1 mM, 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 even at least 50 millimolar, in that order of preference;

- the concentration of aziridine at the beginning of incubation is below 1 mol, below 0.5 mol or even below 0.1 mol, in the order of preference indicated;

- the duration of incubation of the composition comprising the protein or fragment thereof and the aziridine is 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 even at least 36 hours, in the stated order of preference; in an embodiment, the duration is overnight (i.e. 12-18 hours);

- after incubation with aziridine, an additional step is carried out which ensures the neutralization of any remaining aziridine; in a preferred embodiment, the neutralization is carried out by adding an appropriate amount of thiosulfate to the incubation mixture;

- aziridine is selected from: ethyleneimine, 2-ethylethyleneimine, 1-acetylethyleneimine, 2-methylethyleneimine, 1-ethyleneimineethanol and 2-isobutylethyleneimine;

- for aziridine, one of the conditions selected from:

○ ethyleneimine is preferably CAS No. 151-56-4,

○ 2-ethylethyleneimine is preferably CAS No. 2549-67-9,

○ 1-Acetylethyleneimine is preferably CAS No. 460-07-1,

○ 2-methylethyleneimine is preferably CAS No. 75-55-8,

○ 1-Ethylene imine ethanol is preferably CAS No. 1072-52-2, and

○ 2-isobutylethyleneimine is preferably CAS No. 3647-37-8;

- aziridine is ethyleneimine or binary ethyleneimine;

- prior to incubation with aziridine, an additional step is carried out in which BEI is obtained; then an appropriate amount of the obtained BEI is used as aziridine for incubation with a composition containing the Cryptosporidium gp40 protein or an immunogenic portion thereof; preferably, BEI is obtained as a result of the reaction of BEA with hydroxide;

- the gp40 protein or an immunogenic portion thereof is obtained from C. parvum; more preferably, from C. parvum genotype II;

- an additional step is carried out to obtain a composition containing the protein or part thereof, which is to be incubated with aziridine at a later stage of the method, by means of a recombinant expression system; preferably, by means of a baculovirus-insect cell expression system; more preferably, the step is obtaining, by means of a baculovirus-insect cell expression system, the Cryptosporidium gp40 protein by expressing the recombinant DNA sequence SEQ ID NO: 2 as a heterologous insert;

- a composition containing the Cryptosporidium gp40 protein or its immunogenic portion, which is obtained by means of a baculovirus-insect cell expression system, is collected from an insect cell culture, for example, as a whole culture, or as a supernatant or pellet after centrifugation of an insect cell culture, as a filtrate or retentate;

- a composition containing the Cryptosporidium gp40 protein or its immunogenic portion is a supernatant or filtrate of a culture of a baculovirus-insect cell expression system;

- an additional step is carried out after obtaining and collecting the composition comprising the protein or a portion thereof using a recombinant expression system, wherein the step comprises purifying said composition; preferably, purifying by column chromatography; more preferably, the Cryptosporidium gp40 protein or an immunogenic portion thereof comprises a tag facilitating purification of said composition by column chromatography, such as a histidine tag for metal affinity chromatography; and

- a composition comprising the Cryptosporidium gp40 protein or an immunogenic portion thereof also comprises a microorganism; preferably, the microorganism is a virus or a bacterium.

Thus, in a preferred embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof obtained according to the invention, one or more or all of the features selected from:

- aziridine is ethyleneimine or is a binary ethyleneimine,

- Cryptosporidium gp40 is derived from Cryptosporidium parvum,

- a composition containing the Cryptosporidium gp40 protein or its immunogenic portion is a supernatant or filtrate from a culture of a baculovirus-insect cell expression system, and

- the said supernatant or filtrate is purified by column chromatography.

Similarly, in a preferred embodiment of the method for producing the Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention, one or more or all of the features selected from:

- aziridine is ethyleneimine or is a binary ethyleneimine,

- Cryptosporidium gp40 is derived from Cryptosporidium parvum,

- a composition containing the Cryptosporidium gp40 protein or its immunogenic portion is a supernatant or filtrate from a culture of a baculovirus-insect cell expression system, and

- the said supernatant or filtrate is purified by column chromatography.

As described, the invention provides an immunogen for a safe and effective vaccine against cryptosporidiosis. Cryptosporidium gp40 or an immunogenic portion thereof according to the invention, containing one or more alkylated amino acids, are particularly useful in this regard, since they have increased immunogenicity.

Therefore, in another aspect, the invention relates to a Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention, or that which can be produced according to the invention, or that which can be produced by the method of the invention, for use in a vaccine for protecting a target human or a target animal other than a human from cryptosporidiosis.

Similarly, in another aspect, the invention relates to the use of the Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention, or obtainable according to the invention, or obtainable by the method according to the invention, for the production of a vaccine for protecting a target human or a target animal other than human from cryptosporidiosis.

It is well known that a "vaccine" is a composition comprising an immunogen and a pharmaceutically acceptable carrier. The immunogen elicits an immunological response in the vaccinated target, wherein the response is effective in providing protection against disease or infection or its sequelae. The protection involves a reduction in the burden or duration of replication of the pathogen against which vaccination is directed. This in turn results in a reduction in the number, intensity or severity of lesions, associated symptoms and clinical signs of disease caused by the pathogen in the vaccinated target.

Determining the efficacy of the vaccine of the invention is within the skill of a medical practitioner and can be accomplished, for example, by monitoring the immunological response after vaccination or by checking the occurrence of clinical symptoms or mortality after infectious immunization, for example by monitoring signs of disease in targets, clinical assessments, serological parameters, or by re-isolating a control pathogen and comparing these results with the response to a control vaccination observed in sham-vaccinated animals. Many methods for measuring and characterizing relevant pathogens, their symptoms and diseases are known in the art.

The Cryptosporidium gp40 protein or immunogenic portion thereof of the invention for use in a vaccine of the invention, as well as their use for the manufacture of a vaccine of the invention, are effective in protecting against "cryptosporidiosis". This term refers to a set of symptoms caused by infection of a susceptible target with the Cryptosporidium parasite. Such symptoms mainly concern diarrhea, but some species of the parasite can also cause respiratory disease. The symptoms of veterinary cryptosporidiosis are described in "The Merck veterinary manual" (above).

For the invention, "protection... against cryptosporidiosis" concerns a reduction in the severity and/or duration of diarrhea caused by the Cryptosporidium parasite. The vaccine of the invention is effective in this regard, as shown in the Examples section.

Protection is generated in the vaccinated target itself. In addition, when a target mammal is immunized, protection can be provided indirectly by colostrum transfer. This involves collecting milk from a vaccinated pregnant mammal around the time of parturition and feeding that milk (or antibodies derived from it) to a target human or non-human target animal.

The result of this protection is the restoration of the general health of the (passively) vaccinated targets. In veterinary medicine, protection against cryptosporidiosis results in improved economic performance in (passively) vaccinated animals, reflected in one or more of the following parameters: reduced mortality, better average daily gain, improved feed conversion, improved milk yield, improved reproduction and/or reduced health care costs.

In addition to reducing the symptoms of the disease, especially diarrhea, the protection concerns the reduction of the probability of infection with Cryptosporidium parasites by reducing the excretion of oocysts by vaccinated targets into the environment of the herd or population, as well as within the geographic area. Consequently, the protection of the invention also leads to a reduction in the prevalence of Cryptosporidium parasites.

In a preferred embodiment of the Cryptosporidium gp40 protein or immunogenic portion thereof of the invention, for use in a vaccine of the invention, as well as for use in the manufacture of a vaccine of the invention, the vaccine is intended to protect ruminants; more preferably, to protect newborn cattle by colostrum transfer.

For the purposes of the invention, "ruminant" refers to any ruminant animal related to veterinary medicine or commercial livestock farming. Preferably, it refers to cows, goats, sheep or deer. More preferred are cattle, goats and sheep. The most preferred ruminant animal is cattle.

"Cattle" according to the invention is cattle European cow (Bos taurus), zebu cattle (Bos indicus), buffalo, bison, yak or aurochs. Cattle may be of any type: dairy or beef, or parent stock for a dairy or beef type.

To achieve protection against cryptosporidiosis as described, the Cryptosporidium gp40 protein or an immunogenic portion thereof of the invention comprising one or more alkylated amino acids is formulated and administered as a vaccine.

Therefore, in a further aspect, the invention relates to a vaccine against cryptosporidiosis for a target human or a target animal other than a human, wherein said vaccine comprises the Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention, or can be produced according to the invention, or can be produced by the method according to the invention, and a pharmaceutically acceptable carrier.

A "pharmaceutically acceptable carrier" assists in the production, administration and/or preservation of the vaccine without causing (severe) side effects. Such a carrier may be an aqueous solution such as water, a buffer or a culture medium.

A preferred pharmaceutically acceptable carrier for the vaccine of the invention is an insect cell culture medium or buffer such as saline, PBS or 50 mM HEPES.

In addition, the pharmaceutically acceptable carrier may contain further additives and excipients such as a filler, stabilizer, preservative or adjuvant. Details and examples are well known, for example, they are described in such reference books 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).

In addition, the vaccine according to the invention may contain an adjuvant. In particular for subunit vaccines, this further enhances the immune response of the target against the subunit antigen.

Therefore, in an embodiment, the vaccine according to the invention is characterized in that it contains an adjuvant.

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

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

Handbook of adjuvants, their use and action: “Vaccine adjuvants” (Methods in molecular medicine, vol. 42, D. O’Hagan ed., 2000, Humana press, NJ, ISBN: 0896037355).

In an embodiment of the vaccine of the invention, the adjuvant is one or more adjuvants 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.

Light (or white) liquid paraffin oil such as Drakeol® 6VR (Penreco), Marcol® 52 (Exxon Mobile) and Klearol® (Sonneborn) are widely used as mineral oil adjuvants in veterinary vaccines. An alternative is a pre-blended mineral oil/emulsifier mixture such as the Montanide® range from Seppic, France.

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

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.

A widely used aluminum salt is aluminum hydroxide, such as Alhydrogel™ (Brenntag Biosector), Rehydragel™ (Reheis), and Rehsorptar™ (Armour Pharmaceutical).

The oil adjuvant vaccine 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 emulsion (o/w/o).

More preferred is a vaccine according to the invention in which the adjuvant is oil and which is formulated as a water-in-oil emulsion.

Thus, in an embodiment of the vaccine of the invention, the vaccine is formulated as a water-in-oil emulsion.

In a preferred embodiment of the vaccine of the invention comprising an adjuvant, the adjuvant comprises an oil; preferably, the oil is light paraffin oil.

More preferably, the adjuvant also comprises an aluminum salt; the aluminum salt is preferably aluminum hydroxide.

In a preferred embodiment of the vaccine according to the invention, said vaccine is intended for vaccination of pregnant mammals. Preferably, the pregnant mammal is a ruminant animal, more preferably, cattle.

This would allow colostrum to be collected from the mammal in question around the time of parturition, and this colostrum could then be used to provide passive protection against cryptosporidiosis to a human or non-human animal by feeding that colostrum or the antibodies it contains.

The vaccine of the invention can be administered to a target human or a target non-human by various routes.

In one embodiment, the vaccine of the invention is administered parenterally, i.e. through the skin, for example: intramuscularly, intraperitoneally, intradermally, submucosally or subcutaneously. The preferred route of administration is intradermal, intramuscular or subcutaneous.

The volume per dose of the vaccine according to the invention can be selected according to the characteristics of the specific vaccine used, the characteristics of the target and the intended route of administration. Parenteral injections are usually carried out at a dose of 0.01-10 ml/target. For an adult representative of cattle, the preferred volume per dose is 0.5 ml for subcutaneous administration and 1-2 ml for intramuscular administration.

The advantage of the aziridine alkylation according to the invention is that a smaller amount of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the 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 providing a good level of protection. The selection of the amount of gp40 protein or portion thereof per dose can be made by a person skilled in the art based on the characteristics of the vaccine and the target.

Therefore, in an embodiment, the vaccine of the invention comprises 0.01 to 50 μg of the Cryptosporidium gp40 protein or immunogenic portion thereof of the invention per dose of the vaccine. Preferably, the vaccine comprises 0.05 to 20 μg, 0.1 to 10 or even 0.1 to 5 μg of the Cryptosporidium gp40 protein or immunogenic portion thereof of the invention per dose, in this order of preference.

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

In a variant of the vaccine according to the invention, where the vaccine is intended for vaccination of cattle, the vaccine contains less than 10 μg of the Cryptosporidium gp40 protein or its immunogenic part according to the invention per dose. Preferably, the vaccine according to the invention intended for cattle contains less than 9, 8, 7, 6, 5, 4, 3 or even less than 2 μg of the Cryptosporidium gp40 protein or its immunogenic part according to the invention per dose.

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

The amount of Cryptosporidium gp40 protein or immunogenic portion thereof of the invention per dose can be assayed in the final vaccine emulsion. This can be done using standard biochemical laboratory procedures, such as breaking the emulsion and testing the aqueous phase by SDS-PAGE. The amount of protein is then determined by comparison with a known amount of a reference protein, such as albumin. See, for example, The Protein Protocols Handbook, 2nd edition, September 2002, ed. J.M. Walker, Humana Press Inc., Totowa, NJ; Chapter 29, p. 237-242.

Preferably, the amount of the Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention is determined by an antigen mass ELISA as described below. Alternatively, the protein can be quantified by liquid chromatography, such as HPLC, or by mass spectrometry.

Preferably, the amount of Cryptosporidium gp40 protein or immunogenic portion thereof according to the invention per dose is determined in the aqueous phase of the vaccine according to the invention prior to mixing with the aluminium salt and/or prior to emulsification with the oil phase.

The administration schedule of the vaccine according to the invention is selected based on the expected type of protection: vaccination to provide active protection to the vaccinated target itself or vaccination to form colostrum for passive vaccination.

Active vaccination of the target can mobilize humoral and cellular immune response, and can be timed at a convenient time. It can be carried out regardless of the age, weight, sex, immunological status and other parameters of the vaccinated target. Active vaccination can be carried out as a single dose, which can be followed, for example, by revaccination in about two weeks with subsequent annual revaccinations.

However, passive vaccination by colostrum transfer is almost exclusively antibody-mediated. Therefore, vaccination for the formation of such colostrum must be timed to coincide with the expected date of parturition in order to obtain high levels of gp40-specific antibodies in the colostrum. The duration of pregnancy of different mammal species is known, so the timing of this vaccination can be selected and optimized. For the production of colostrum in cows, a pregnant cow is preferably vaccinated twice with the vaccine according to the invention: revaccination in the period of 1-4 weeks before the expected calving, and primary vaccination 1-12 weeks before revaccination. Preferably: revaccination is carried out 2-4 weeks before the expected calving, and primary vaccination, 2-6 weeks before revaccination. If the cow has already received primary and revaccination during a previous pregnancy, it may be sufficient to make only one vaccination for subsequent pregnancies in the period of 1-12 weeks before the expected calving; preferably 2-8 weeks before the expected calving.

Preferably, the method, time and volume of administration of the vaccine according to the invention are integrated into existing vaccination schedules with other vaccines that may be required by the target human or target animal, in order to reduce stress for the target and to reduce labor costs. These other vaccines can be administered simultaneously, in parallel or sequentially, and in a manner compatible with their licensed use.

In a preferred embodiment, the vaccine of the invention is administered to pregnant female cattle, and is administered in combination with other vaccines intended to protect newborn calves from neonatal diarrhea by colostrum transfer. Examples of such other vaccines are: Guardian® (Merck Animal Health), an I/O emulsion containing a mineral oil adjuvant, which is administered subcutaneously; and: Rotavec® Corona (MSD Animal Health), an I/O emulsion that contains both mineral oil and an aluminum salt as adjuvant, and is administered intramuscularly. A preferred method of combination is the so-called "associated non-mixed" use.

Depending on the target animal, the intended disease, the route of administration, etc., it may be necessary to adapt the elements of the vaccine according to the invention or its administration. This is within the capabilities of a person skilled in the art and typically involves fine-tuning the effectiveness of the vaccine. This can be achieved by adapting, for example, the vaccine dose, amount, frequency of administration, route of administration, excipients, etc.

It goes without saying that the mixing of other compounds such as stabilizers, carriers, adjuvants, diluents, emulsions and the like with the vaccines of the invention is also within the scope of the invention. Such additives are described in known reference books such as: "Remington" and "Veterinary Vaccinology" (both above).

The vaccine of the present invention may benefit from the creation of additional combinations with additional immunoactive components. This may serve to enhance the existing immune protection or to extend it to other pathogens.

Therefore, in one embodiment, the vaccine of the invention comprises at least one additional immunoactive component.

Such an "additional immunoactive component" may be an antigen, an immunostimulatory substance, an adjuvant or immunomodulator, a cytokine, another vaccine, or any combination thereof. This provides advantages in terms of cost, efficacy, and target quality of life. Alternatively, the vaccine of the invention itself may be added to another vaccine.

General methods and considerations applicable to the manufacture of vaccines according to generally recognized pharmaceutical manufacturing standards are described, for example, in government guidelines and regulations (Pharmacopoeia, 9CFR) and in well-known reference books (Veterinary Vaccinology and Remington, both above). Typically, such vaccines are prepared sterile and are prepared using pharmaceutical grade excipients.

Such preparations will include microbiological tests for sterility and absence of extraneous agents; and may include in vivo or in vitro studies to confirm efficacy and safety. Once testing for quality, quantity, sterility, safety and efficacy is complete, the vaccine may be released for sale. All of this is well known to those skilled in the art.

Therefore, in a further aspect, the invention relates to a method for producing a vaccine according to the invention, wherein the method comprises the step of formulating the Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention, or in the form in which it can be obtained according to the invention, or in which it can be obtained by the method according to the invention, into a vaccine.

Such formulation may include simple mixing of the gp40 protein or its immunogenic portion with a pharmaceutically acceptable carrier. It may also include mixing with an adjuvant as described, including emulsification, and/or mixing with at least one additional immunoactive component.

An embodiment of the cryptosporidiosis vaccine of the invention is colostrum produced by a pregnant mammal immunized with the vaccine of the invention. This colostrum is very effective for passively vaccinating a human target or a non-human target animal against cryptosporidiosis. The colostrum can be fed or given to the target to drink and provides intestinal, mucosal and/or systemic immune protection against cryptosporidiosis. Alternatively, antibodies obtained from such colostrum can be administered.

Colostrum can be obtained by vaccinating pregnant mammals before parturition with the vaccine of the invention.

Therefore, in a further aspect, the invention relates to a method for producing colostrum comprising antibodies against the Cryptosporidium gp40 protein or an immunogenic portion thereof according to the invention, or obtainable according to the invention, or obtainable by the method according to the invention, wherein the method comprises the steps of:

a. vaccinating a pregnant mammal at least once with a vaccine according to the invention, and

b. collecting colostrum from the mammary glands of the said mammal.

As is known, “colostrum” is the milk secreted by the mammary glands of a mammal in the period preceding childbirth.

Preferably, the colostrum of the invention is milk secreted in the period from 1 day to 4 days after birth; more preferably, from 1 hour to 72 hours after birth.

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

In an embodiment of the method for producing colostrum according to the invention, the target is a ruminant animal; preferably, the target is cattle as defined herein.

The colostrum of the invention comprises antibodies against the Cryptosporidium gp40 protein or against an immunogenic portion thereof of the invention, wherein the antibodies have a higher concentration and/or differ in avidity or specificity compared to antibodies in the colostrum of a mammal vaccinated with the gp40 protein or a portion thereof that does not contain one or more alkylated amino acids.

Vaccination of pregnant mammals is preferably carried out in accordance with the vaccination schedule described herein.

The collection of colostrum is preferably carried out in the period from 1 day before parturition to 4 days after parturition, more preferably from the day of parturition and for at least 3 days, as described herein.

In a further aspect, the invention relates to colostrum obtained by the method for producing colostrum according to the invention, for use in protecting a target human or a target animal other than a human from cryptosporidiosis.

In a preferred embodiment of colostrum for use according to the invention, protection is achieved by feeding the target colostrum or antibodies obtained from colostrum.

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

In a further aspect, the invention relates to a method for protecting a target human or a target animal other than a human from cryptosporidiosis, wherein said method comprises administering to said target at least a single vaccine of the invention.

In another aspect, the invention relates to a method for protecting a target human or a target animal other than a human from cryptosporidiosis, wherein said method comprises feeding colostrum obtained by the method of the invention or colostrum of the invention to said target.

The method of protection using colostrum according to the invention also relates to passive vaccination of a target using a preparation of gp40-specific antibodies that have been purified from colostrum obtained by the method according to the invention or from colostrum according to the invention.

When the passive protection method relates to cattle, a further distinction can be made between the various methods of animal husbandry applied to cattle for various purposes: calves of dairy cattle are usually taken from their mothers on the day of birth, so that vaccination of these calves by administering colostrum is carried out by actively collecting colostrum and feeding it to the calves during the first week of their life. Preferably, the dairy calves will receive colostrum according to the invention for at least 3 days, more preferably at least 4 or even at least 5 days, in that order of preference. The first dose of colostrum should be given to the calves within 8 hours of birth, preferably within 6 or even within 4 hours of birth.

Colostrum feeding can be done once or twice a day; preferably, feeding is done once a day.

In beef cattle, due to the smaller volume of milk produced, the concentration of antibodies in the colostrum is higher than in dairy cattle. Also, beef cattle calves are usually left with their mothers after birth. In this case, feeding with colostrum for the method of protection according to the invention is carried out by allowing the calves to swallow the colostrum by sucking. Preferably, beef calves are allowed to suck colostrum from a cow vaccinated as described herein for at least 3 days, more preferably at least 4 or even at least 5 days, in the stated order of preference. Access of calves to colostrum should be ensured within 8 hours after birth, preferably within 6 or even within 4 hours after birth.

In an embodiment of both methods of protection according to the invention, the target is a ruminant animal; preferably, the target is cattle as defined herein.

The invention will be further described by means of the following non-limiting examples.

Examples

Example 1: Production of a vaccine against cryptosporidiosis

1.2. Recombinant protein construct

Recombinant gp40 protein for use in vaccination studies was constructed starting from the major amino acid sequence of SEQ ID NO: 1, which was obtained from C. parvum, Iowa strain, genotype II. This sequence was supplemented with a C-terminal 6x histidine tag to allow purification by Nickel column chromatography.

It is known that for the Cryptosporidium gp40 protein, the cleavage of the N-terminal signal sequence differs in the expression system, where the cleavage occurs at amino acid number 20; compared to the native gp40 protein, which starts at amino acid number 31, see O'Connor et al. (2007, Mol. Bioch. Parasit., vol. 152, p. 149-158). For the invention, the N-terminal signal sequence of gp40 was partially reconstructed using amino acids 14-30 of GenBank entry AAF78345.1.

In addition, an N-terminal methionine is added to initiate transcription. This assembly can be expressed efficiently in a recombinant baculovirus-insect cell expression system, although it does not allow secretion or glycosylation. Expression is therefore cytoplasmic, and gp40 can be collected from the culture supernatant at the end of the culture period, when most of the insect cells have been lysed.

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

Expression of Rgp40His was confirmed by SDS-Page of insect cell culture supernatant, which showed the expressed protein as a band of approximately 32 kDa. Further tests were performed by Western blotting and immunofluorescence analysis using a bovine polyclonal anti-gp40 antiserum and an anti-His monoclonal antibody. The recombinant baculovirus was established as a vaccine stock virus after several tests for genetic correctness and stability, and sterility.

1.2. Expression and collection

Rgp40His is produced from Sf9 insect cells infected with a recombinant baculovirus primer. Cells are cultured in the commercial animal compound-free insect cell culture medium SF900II™. Small scale cultures of 0.5-2 liters are carried out in laboratories; large scale production runs of up to 500 liters of culture are carried out in pre-production facilities. In general, the following scheme is used: pure Sf9 cells are produced in increasing cultures. Once sufficient pure cells are obtained, the cells are concentrated and replated in fresh medium at 1.6×10 ⁶ cells/mL. They are infected with an moi of 0.002 and incubated for 5 days at 28°C. At the end of the culture, the supernatant is collected; at small scale by centrifugation, at large scale by deep filtration distillation. For small scale expression, the recombinant baculovirus is then inactivated by incubation with 0.1% Triton® X-100 for 1 hour at room temperature followed by gamma irradiation at a commercial facility. For large scale production, all subsequent processing is performed in a limited use facility using closed connections and the majority of the recombinant baculovirus is recovered by various filtration and purification steps. Any remaining virus is then destroyed by aziridine treatment.

For purification, the collected rgp40His is loaded onto a Ni-Sepharose column. The column is then washed and rgp40His is eluted using imidazole. The imidazole is then removed by diafiltration against 50 mM HEPES buffer at pH 7.5. rgp40His is then sterile filtered through 0.2 μm membrane filters and incubated with aziridine. BEI is prepared by combining equal volumes of 1.09 M BEA and 1.91 M NaOH. This gives a stock solution of 545 mM BEI. BEI is then added to the rgp40His-containing composition to 33 mM. The mixture with BEI is incubated at room temperature for 24 hours. Sodium thiosulfate is then added at a concentration of 33 mM. It is incubated for an hour at room temperature and the pH value is measured at 7.1.

For comparative experiments, Cryptosporidium gp40, which does not contain one or more alkylated amino acids, was prepared in the same manner except that mock incubation buffer was added in place of aziridine.

1.3. Determination of antigen mass

The amount of rgp40His protein is initially measured by SDS-PAGE in a range of dilutions of known amounts of the reference protein. The bands are then quantified by densitometry of the stained gels. This test is later changed to a more sensitive one: the competitive antigen array ELISA, which has been fully validated for specificity, robustness, linearity and accuracy. Briefly, the antigen array ELISA is performed as follows: the wells of a microtiter plate are coated with purified rgp40His (non-alkylated) at 50 ng/well, overnight in coating buffer and at 4°C. The following day, the plate is washed, then coated with casein-containing buffer for 1 hour at 37°C, and then washed again. On separate microtiter plates, the rgp40His test sample of unknown concentration is serially diluted in ELISA buffer (containing 0.05% polysorbate 80) and a fixed amount of anti-gp40 mouse monoclonal antibody is added. This is preincubated for 1 hour at 37°C, then this mixture is transferred to coated plates and incubated for 1 hour at 37°C in ELISA buffer. The plates are then washed and the amount of gp40 monoclonal antibody that has bound to the plate is determined by incubation with peroxidase-conjugated goat anti-mouse IgG in ELISA buffer for 1 hour at 37°C. Peroxidase is visualized by enzymatic conversion of tetramethylbenzidine for 15 minutes at room temperature, which is quenched with sulfuric acid. The optical density of the yellow color in each well is measured at 450 nm. The amount of peroxidase conjugate is inversely proportional to the amount of antigen in the test sample. The data are processed by the software using the Logit-Log algorithm for at least 3 sampling points from the dilution series. Also, samples are measured in at least two replicates, and appropriate positive and negative controls are taken during testing. The gp40 antigen mass value in the test sample in micrograms/mL is then calculated based on the values of the standardized reference sample.

1.4. Formulation into a vaccine

The column purified rgp40His, incubated with aziridine or mock incubated, is then formulated into an emulsion vaccine for further use by combining with suitable adjuvants and emulsifying using well known methods and materials. Briefly, rgp40His is placed in sterile saline (0.85% w/v sodium chloride) to the desired concentration. In parallel, the oil phase is obtained from ISA™ 70 VG (Seppic, France) by sterile filtration. Both phases are combined and emulsified using a Silverson™ or Dispax™ homogenizer to a water-in-oil emulsion. The gradual increase in temperature during homogenization is controlled and maintained below 50°C. The resulting emulsion is stored at 4°C until filling into suitable containers. The filled product undergoes multiple stability and sterility tests before release.

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

Long-term stability testing of the 1- and 2-year shelf life at 4°C is ongoing. However, the results of the intermediate and accelerated stability tests are indicative: for 15 months at 4°C and for 3 weeks at 30 or 37°C. The quality of the emulsion remains good throughout, and the average change in the detected antigen mass is within 10% of the original composition. This is a good indicator of long-term stability under refrigeration conditions.

Example 2: Vaccination-challenge trial settings

Several animal models have been used over the years to develop and optimize an effective vaccination-challenge model to evaluate the efficacy of different formulations being tested as cryptosporidiosis vaccines. The following setup has proven to be most indicative of in vivo efficacy:

Vaccines were prepared as described above: the cryptosporidiosis gp40 protein was expressed in a baculovirus-insect cell expression system and harvested at the end of the culture, purified (or not) by metal affinity chromatography, and incubated (or not) with aziridine. The protein was then formulated with an adjuvant, either oil or oil and aluminum salt, and emulsified as a W/O emulsion.

2.

2.1 Animal model

For passive vaccination studies, target animals are healthy Holstein-Friesian dairy cows of approximately one year of age, selected from both heifers and multiparous cows. Cows are in the last trimester of pregnancy and their serum is tested for background levels of gp40-specific antibodies only. Cows are treated on the farm where they live, so acclimatization is not required. Feed and water are provided according to standard cattle husbandry practices. Animals are identified with a unique identification number using an ear tag.

2.2. Vaccinations

Vaccines containing 10 mg rgp40His/dose, either aziridine-treated or sham-treated, emulsified IM with mineral oil in a volume of 2 ml and administered intramuscularly in the neck are administered. Timing is as follows: booster 3 weeks before the expected calving date and primary 6 weeks before the booster. Cows are also vaccinated with Rotavec® Corona 4 weeks before the expected calving date to prevent neonatal diarrhea in calves from pathogens other than C. parvum. Serum samples are collected at specified time points and tested by ELISA for antibodies.

After calving, colostrum is collected at the first, second and third milkings; on average, cows receive 4-5 litres of colostrum per milking. Colostrum is pooled for each vaccination group tested, for each milking time point and aliquoted into 250 ml jars before pasteurisation using a water bath for 30-45 minutes at 56°C. Aliquots are then divided and stored frozen until use. Thawing is monitored using a 43°C water bath or hot tap water, checking the water temperature regularly. Colostrum is warmed by hand (i.e. approximately 40°C) when feeding to the calves.

Newborn calves are collected at calving, housed individually, and fed a 3-liter bolus of anti-gp40 colostrum from one of the vaccinated groups within 4 hours of birth. Calves are then challenged with live C. parvum oocysts 2 hours after the first feed.

Group size is 5-10 calves and all experiments include an unprotected challenge group that receives mock colostrum. The use of unprotected, non-challenged indicators is found to be uninformative and is not considered in later experiments. Calves included in the studies weigh at least 30 kg at birth.

Calves are kept in an isolated room, in individual boxes. Contact between calves is not allowed. Keepers change gloves and shoes before handling each animal.

Each individual animal pen consists of two compartments. One contains wood shavings and the other consists of a wire mesh over a horizontal smooth surface plate over a waste collection channel. Calves are kept on wood shavings for the first three days of life. At the end of the third day, calves are transferred to the second compartment. The smooth surface plate allows the consistency of feces to be assessed for diarrhea. This plate is cleaned after each counting time point to allow further assessment of the fecal material that is collected between each counting time point.

Calves are fed twice daily; a feed consisting of colostrum (first feeding) or colostrum and milk replacer, beginning with the second feeding, is given daily according to the manufacturer's specifications after the first feeding of colostrum to the calf.

2.3. Control infections

The challenge inoculum is prepared fresh before administration using 1×10 ⁶ live oocysts of C. parvum, Iowa/calf strain. It is given to calves orally in 50 ml milk replacer. Subsequently, calves receive colostrum (gp40- or challenge vaccine) in milk replacer twice daily: 0.25-0.5 liters of colostrum per 2-2.5 liters of milk per day for several days.

This challenge dose was chosen to induce recurrent and persistent diarrhea that was severe (but temporary) in all unprotected challenge groups.

Live oocysts were purchased fresh from Waterborne Inc., New Orleans, USA. Oocysts were freshly shed, no older than 2 months before the start of the animal experiment. The viability of the parasite by the excystation method (test criterion 50-100%), plus the infectivity of the HCT-8 cell layer were sufficient both before and at the end of the experiment.

Following challenge, calves are observed for 14 days, with diarrhea typically developing between days 4 and 11, with peak severity occurring between days 5 and 10. Antibody levels are measured in colostrum and serum of calves on day 1 pre-feeding and day 3 post-feeding.

2.4. Evaluation of effects

Several methods have been tested over time to best quantify and evaluate the impact of vaccination on challenge. Initially only bulls were used and feces from all calves were bagged for analysis of quantity and consistency. Data proved to be inaccurate as sick calves lay down and material was lost. Most clinically useful was the twice daily recording of a number of observable assessment parameters, namely general health, dehydration, rear appearance and diarrhea score. The diarrhea scoring parameters used were based on the Wisconsin-Madison scale as described by S. McGuirk, 2008 (Vet. Clin. North Am. Food Anim. Pract., vol. 24, p. 139-153). The parameters used for the observations are presented in Table 1. With some experience, caregivers were able to assign scores consistently.

When diarrhea becomes noticeable, a test for Cryptosporidia in feces is performed using a commercial testing kit: BIO K 306™ - Rainbow Calf Scours (Bio-X Diagnostics, Belgium), which uses liquid chromatography on special test strips.

Oocyst enumeration of faecal samples is performed to determine the reduction in shedding. Oocyst enumeration is performed as follows: a 50 ml sample is taken from the collected faecal material and stored at 2-8°C until use. The sample is serially diluted and the sample is placed on a microscope slide and air dried for 1 hour. Slides are stained according to the Ziehl-Neelsen (acid-fast) method and red-stained oocysts are counted using light microscopy. Results are reported as the number of oocysts per gram of faecal material.

Table 1: Monitoring parameters for assessing signs of cryptosporidiosis in calves

Grade General health Dehydration Rear view Diarrhea assessment 0 Ordinary:
The calf is alert, hungry, and watches its keepers. It may stretch when standing. The calf eats greedily, often twitching its tail as it eats. Not dehydrated: (eyes are normal, skin is elastic (fur is smooth). Clean butt, tail and legs. Normal:
The feces retain their shape. The feces may be pasty, but do not flow down an inclined surface. 1 Slightly depressed:
The calf gets up and drinks without coaxing, but not greedily. The calf shows some attention to the keepers. Maybe:
questionable eyes, dull/glazed coat. The tail and rear are dirty with a small amount of sticky feces or dry fecal matter. Minor:
in the form of a puddle rather than a pancake. Sufficient water content to flow slowly over or down an inclined surface. 2 Moderate depression:
The calf must be coaxed to drink, otherwise it will not consume all the milk. It pays little attention to the caretaker until it is touched. Moderately:
eyes too deep, or dull/cloudy coat. The tail and rear are very dirty but not wet, drying. Moderate:
feces with sufficient water content to flow easily over or down an inclined surface, leaving behind adhering material. 3 Severe depression:
The calf needs to be coaxed to stand up, may have difficulty standing or standing, does not pay attention to caretakers when touched, may refuse food, and has no sucking reflex. Strong:
The eyes are very deep-set and the coat is dull/faded. The tail, back and legs are dirty and wet from watery diarrhea. Heavy:
some or all of the faeces are very watery. The faeces may run off, leaving little or no trace on a smooth, sloping surface (a calf may have very watery faeces followed by some solid matter, and may have severe diarrhea). 4 Dead or Dying:
The calf does not react or barely reacts to stimuli and lies on its side. -- -- --

Example 3: Results of vaccination-challenge experiments

3.

3.1 Recombinant expression of gp40:

It was found that the baculovirus-insect cell expression system with the applied optimizations: partial signal sequence and adaptation of codon usage, yields more protein than the previously used E. coli expression system. However, when vaccinated with unpurified gp40 obtained from insect cell cultures, i.e. only centrifuged and inactivated with Triton X-100 and gamma irradiation, the immunogenicity of the standard dose of 20 μg was not as good as for the same dose of similarly inactivated and column-purified gp40. Therefore, His-tag fusion constructs and Nickle column purification are used as a standard.

3.2. Composition of the vaccine

The oil and oil+aluminum adjuvants used induce effective antibody titres in pregnant cows and subsequently in their colostrum. However, in cattle not previously vaccinated with gp40, a single vaccination was insufficient and a prime/boost vaccination schedule was required to induce antibody levels in colostrum that protect against severe infection, as used in these trials. Typically, naive cattle have gp40-specific antiserum titres of 9-12 Log2 in the antibody ELISA. Therefore, a titre of up to 10 Log2 is considered background levels. Following vaccination with 10 micrograms of purified gp40, which does not contain one or more alkylated amino acids, in an emulsion IM vaccine, this titre increased to 12-13 Log2 after primary vaccination and to 15-17 Log2 after booster vaccination.

Colostrum containing gp40-specific antibodies with titers of about 20 Log2 is collected from these cows. Therefore, doses of 10 μg rgp40His vaccine that were not incubated and purified with aziridine are used in priming/boosting experiments. When used in combination with the Rotavec Corona vaccine, no effect on the vaccination efficacy against rotavirus, coronavirus or E. coli with the gp40 vaccine is observed, see also figure 2. It is concluded that these vaccines can be effectively combined.

There is no difference in feeding colostrum once or twice a day, so once a day is used thereafter. It has also been found that feeding colostrum for 5 days is sufficient.

3.3. Testing Rainbow scour

All animals that received the control inoculum and had diarrhea scores of 2 or 3 tested positive for the presence of Cryptosporidium.

3.4. Adverse reactions

While vaccination of cattle with emulsified rgp40His vaccines is generally safe, i.e. no systemic effects or effects on pregnancy are observed, they cause local reactions at the vaccination site: they are palpable and signs of inflammation were found at autopsy. This appeared to be due to the presence of the gp40 protein, since a similar emulsion vaccine, Rotavec Corona, does not cause such local reactions.

The only way to reduce these reactions was to reduce the dose of gp40, but this was only possible while maintaining efficacy when using the Cryptosporidium gp40 protein containing one or more alkylated amino acids of the invention.

Example 4: Effect of incubation with aziridine in vaccination-challenge experiments

4.

4.1. Effect on antibody titers

In a vaccination experiment with groups of 5 pregnant cattle each, as described in Example 3 above, cows receive two doses of 10 µg rgp40His in a W/O emulsion with oil+aluminum adjuvate. gp40 is column purified and either incubated with or without aziridine. A group receiving only Rotavec Corona vaccine of the same composition serves as a negative control. Another group receives both gp40 vaccine lacking one or more alkylated amino acids and Rotavec Corona vaccine. Serum antibody titers are measured weekly and determined by antibody ELISA. Titers of 10 Log2 or less are set equal to 10 and considered background. The seroconversion profiles over time are similar for all gp40 vaccines. Only Rotavec Corona vaccine does not induce antibodies against gp40. The titer results for the combination vaccine show that there is no interference between the two types of vaccines.

When containing the same amount of gp40 antigen, a vaccine containing Cryptosporidium gp40 protein containing one or more alkylated amino acids induces antibody titers in cattle that are significantly higher than for the non-alkylated gp40 vaccine: 3-4 Log2 higher after primary vaccination, and 2 Log2 higher after booster vaccination; this is shown in Figure 2. The titers obtained in colostrum after vaccination with alkylated gp40 were found to be very high, 24 Log2.

4.2. Effect on hair loss

The effect on oocyst shedding by calves after challenge with passive vaccination was modest but significant. A 10-fold reduction in the total number of oocysts per 100 grams of faeces between days 4 and 14 was typically found: from 10 ⁹ oocysts in unvaccinated challenged calves to 10 ⁸ in vaccinated calves. However, as the fecal count was also significantly reduced by the shorter duration of diarrhea found in calves: 2 days in gp40 vaccinated calves treated with BEI compared to 6 days in unvaccinated calves. Thus, the overall reduction in oocyst shedding by vaccination is certainly significant.

4.3. Impact on safety

gp40 protein in combination with mineral oil adjuvant was highly reactive; this was most evident with subcutaneous vaccination and with the prime/boost vaccination schedule. This effect was not enhanced by incubation of gp40 protein with aziridine.

A vaccine containing a dose of 10 µg gp40 (alkylated or not), adjuvanted with mineral oil and administered as a primary and booster vaccine to adult Holstein cows by the subcutaneous route, causes a swelling at the site of vaccination up to 10 cm in diameter and up to 2 cm thick. However, apart from appearing heavy, vaccinated cows show no signs of discomfort from this local reaction: there is no fever, and there is no effect on appetite or pregnancy.

However, it was decided that such swelling, even asymptomatic and temporary, was undesirable.

Fortunately, the invention described herein allows the gp40 protein dose to be reduced to a safe level while maintaining good immunological efficacy: when the protein amount per dose is adjusted to 1 μg of alkylated gp40 protein, the swelling after two subcutaneous doses is typically less than 5 cm in diameter and 1 cm in height, generally only 2 cm in diameter. This is considered acceptable. It is important to note that such a 10-fold reduction in protein amount affects the vaccination efficacy, since it was still possible to obtain fairly high titers of antibodies to gp40 in colostrum.

4.4 Effect on diarrhea

In the challenge experiment, diarrhea severity is assessed based on the parameters presented in Table 1, last column. The results for the two most relevant groups of calves are presented in Figure 3. These calves receive colostrum from mothers either vaccinated with the BEI-treated gp40 vaccine or with the untreated gp40 vaccine. Challenge is performed on day 1, after the first bolus of colostrum, as described. The average daily diarrhea score per group is determined and plotted for the 14-day experiment.

As can be clearly seen from Figure 3, the severity of diarrhea is much lower and much shorter in the group fed with colostrum from cows vaccinated with BEI-gp40. The difference is striking and of great importance: in the rgp40His+BEI vaccine group, significant diarrhea (score greater than 1.0) is observed only for 2 days (on days 7 and 8). However, in the rgp40His (gp40 mock-treated with aziridine) vaccine group, significant diarrhea is observed for 6 days (on days 6-11). However, diarrhea in the rgp40His+BEI vaccine group is also much less severe: the maximum score was 1.2 compared to the maximum score of 2.8 in the sham BEI vaccine group.

In conclusion:

A truly effective vaccine against cryptosporidiosis has been found that can significantly reduce both the severity and duration of diarrhea caused by severe infection with Cryptosporidium parasites.

A model of passive vaccination of newborn calves by feeding them colostrum from vaccinated mothers also proved to be highly effective.

Example 5: Ongoing Research

5.

5.1 Dose determination

Ongoing dose-finding studies are testing doses of 0.1-0.2-0.5-1-2.5 and 5 μg of the alkylated gp40 protein of the invention.

Initial results indicate that 1 μg alkylated gp40 protein per dose in a prime/boost vaccination regimen induces adequate levels of gp40-specific antibodies in pregnant cows. Currently, a 1 μg dose with mineral oil adjuvant administered twice subcutaneously induces a titre of 19 Log2 units by ELISA 2 weeks post-prime, which declines to 18 at 4 weeks p.v. when a booster is given. Booster vaccination then increases the titre again to 22 at 6 weeks p.v., which declines slightly to a titre of 21 Log2 at 8 weeks post-prime. At calving, colostrum Ab levels are 1-2 Log2 higher than serum Ab levels.

Given that a colostrum titer of 17-18 Log2 would be sufficient to protect calves, the optimal vaccination protocol aims to achieve a titer of 15-16 Log2 in the vaccinated mother cow. Therefore, even a vaccine dose of 0.5 μg of the alkylated gp40 protein of the invention would be sufficient to achieve such titer levels in the mother with a prime/boost vaccination regimen.

Alternatively, the possibility of achieving this level of protective titre in colostrum by single vaccination of pregnant ruminants using 0.5 or 1 μg of alkylated gp40 protein per dose is being investigated.

Example 6: Mass spectrometric analysis

To determine which amino acids were alkylated by incubation with aziridine, LC-MS/MS analyses of BEI-incubated and non-incubated rgp40His were performed. The protein was digested in parallel with different enzymes, and the detected peptides were analyzed for the presence of the 42 Da adduct, which corresponds to alkylation by the ethyleneimine molecule.

6.1 Materials and methods

6.1.1 Protein samples tested

Two lots of rgp40His having different initial protein concentrations were used: lot 01K with 4.1 mg/ml and lot 12G with 1.0 mg/ml of BCA. Both of these lots were incubated with BEI as described. A lot of rgp40His protein not incubated with aziridine was used as a control; lot 2DJ, 0.62 mg/ml, and this lot was nanofiltered to remove any residual baculovirus.

6.1.2 Analysis of intact gp40 mass

Protein quantification was performed by BCA (Pierce) using the manufacturer's protocol. Approximately 6-8 µg of rgp40His sample was analyzed by liquid chromatography-mass spectrometry (LC-MS) using an Agilent 1290 LC and an Agilent 6545 QTOF MS. A MassPrep™ Online Desalting Cartridge (Waters) was used at a flow rate of 0.4 mL/min at 60°C. 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 from 5% to 80% Buffer B over 5 min and maintained at 80% Buffer B for 1 min. Equilibration occurred at 5% Buffer B with a total run time of 8 min. The column eluent is electrosprayed onto the MS using an ESI Dual AJS source (typically at 4 kV). MS scans are recorded between 100 and 3200 m/z at a rate of 1 spectrum/sec.

6.1.3 gp40 peptide mapping

Mapping of the rgp40His peptide is performed by digestion with trypsin, chymotrypsin or GluC (Staphylococcus aureus Protease V8) enzymes at an enzyme:protein ratio of 1:50, incubating for 1 hour. Then the enzyme is added again at the same ratio and digestion is continued overnight. Samples are cleaned up using Sep-Pak™ tC18 sorbent cartridges, 1 ^cm3 , 50 mg (Waters). Peptide mixtures are eluted with 0.1% v/v formic acid in 90% acetonitrile. The solvent is evaporated in a Speedvac™ at room temperature. Peptide sample mixtures are reconstituted in 0.1% formic acid.

Approximately 30 μg of the enzymatic digest was analyzed by LC-MS as described. Peptide mixtures were separated on an Agilent AdvanceBio™ Peptide Map, C18, 1.2 x 150 mm, 2.7 μm, at 0.4 mL/min at 60°C. LC buffers A and B were as described. The gradient used was 2%-45% buffer B for 110 min, then 45%-95% buffer B for 5 min and held at 95% buffer B for 10 min. Equilibration was performed using 2% buffer B, total run time was 145 min. Column eluent was electrosprayed onto the MS using an ESI Dual AJS source (typically at 4 kV). MS scans are recorded in the frequency range from 100 to 1700 m/z at 4 spectra/sec. MS/MS scans are recorded in the range from 150 to 3200 m/z at 2 spectra/sec. A maximum of 5 precursor ions per cycle are selected for MS/MS at a threshold of 10,000 counts. Precursor ions are excluded after 5 repetitions of 1 min. The selection width is set to medium (4 amu).

6.1.4 MS data analysis

Intact analyses

LC peaks containing protein fragments were deconvoluted using MassHunter™ Qualitative Analysis B.07.00 software. The deconvolution algorithm was “maximum entropy”; mass range was 16,000–23,000 Da, m/z range was 800–2000. Input subtraction was enabled and input factor was set to 7.00. Adduct was set to “proton” and isotope width to “automatic”.

Peptide mapping

Agilent QTOF MS/MS data files are converted to MFG files using MassHunter software. For each data file, a compound list is created using the “Compound Search by Automated MS/MS” function. “MS/MS Extraction” is enabled with extraction of average MS/MS spectra for all collision energies. Otherwise, default settings are used. The compound list is exported to MGF with “Deisotope Calculation” enabled, “Isotope Model” set to peptide, and “Constrained Assigned Charge State” set to maximum 7. MFG files are probed against the recombinant Cryptosporidium parvum gp40 protein sequence SEQ ID NO: 3 in the Mascot™ database. No enzyme restriction is set.

Ethylene imine (43.04 Da, C ₂ H ₅ N) is selected as the variable modification for all amino acids. The MS tolerance is set at 25 ppm, and the MS/MS tolerance is set at 0.1 Da. The peptide charge is set at 2+, 3+, and 4+.

MGF files were also searched against the host cell protein database (Sf9, Spodoptera frugiperda, Uniprot 201909127, with 26506 entries) using parameters identical to those described above.

6.2 Results

In gp40 incubated with BEI, about a quarter of the protein contains no EI adduct, one quarter contains one adduct, and the remainder contains two or more adducts, the frequency of which rapidly decreases.

Intact mass analysis revealed no evidence of glycosylation or other post-translational modifications. In addition, no significant database matches were found for Sf9 host cell proteins.

For protein fragment analysis, a multiple digestion approach is used using trypsin, chymotrypsin or GluC digest. Together, they generate a complete set of overlapping peptides with good sequence coverage and very reliable assignment of EI adducts. The top 4 modification sites are subjected to additional manual analysis.

The PepMap results show complete coverage of the gp40 sequence, but limited coverage of the His-tag tail sequence. However, it is very unlikely that the EI adducts are located on histidines. The coverage of the various peptide fragments is presented in Table 2.

In Table 2, result 1 (*) for the total number of EI alkylated peptides in the native protein sample is a false positive.

Table 2: Peptide mapping results

Tested batch of gp40 protein Peptide IDs Unique EI Modification Sites Dominant site EI modification trypsin chymotrypsin GluC Total EI alkylated 01K-BEI 201 64 134 50 31 4 12G-BEI 317 223 135 117 62 4 2DJ (native) 207 156 83 1* 0 0

PepMap analysis allows identification of EI adducts for specific sites. Identification of EI modification sites can be done by automated database searching, since the adducts to be detected are known in advance: each EI adduct increases in size by 42 Da, but the resolution of the methods used is accurate to ±1 Da, so the weight increase of 41-43 Da is analyzed. Selection against several EI adducts for a single amino acid is performed using MS analysis software.

From the pooled data of both batches incubated with aziridine, the gp40 amino acids 2V, 106E, 112E and 147D (numbered as in SEQ ID NO: 3) were found to be the dominant alkylation sites. The results are shown in Figure 4. Several other amino acids were also found to be alkylated, although with (significantly) lower frequencies: cysteine, methionine, serine, threonine, tyrosine, lysine and arginine. Thus, interestingly, aspartic acid (D) and glutamic acid (E) were found to be the dominant modification sites with lower frequencies for the other amino acids.

The Cryptosporidium gp40 protein used in these assays is recombinantly expressed gp40-His with the amino acid sequence SEQ ID NO: 3. However, the dominant protein found in all samples is rgp40His without the initiator methionine, which has a major molecular weight of 20.9 kDa. The missing N-terminal methionine exposes the valine at position 2 to alkylation.

Example 7: Vaccination-challenge trials using low-dose vaccines

As described above in Example 5, dose determination experiments were conducted using the vaccines of the invention with very low doses of BEI-gp40. Pregnant heifers were actively vaccinated and calves were passively vaccinated and challenged. The settings and performance were essentially the same as described above. Specifically, the experiments were conducted as follows: First, hyperimmune colostrum was produced by vaccinating pregnant heifers and collecting their colostrum from the first and second milking. The amount of alkylated gp40 protein used per dose per animal was: 0.4 or 1.5 μg. The vaccines were formulated as a water-in-oil emulsion as described above, using oil (ISA 70) + aluminum (Alhydrogel) as adjuvants. The vaccine was administered subcutaneously as a prime and booster vaccination 7 weeks and 3 weeks before the expected calving date, respectively. Serum and colostrum samples are tested for specific IgG antibody response against gp40 using ELISA as described above.

Newborn calves are then fed this colostrum for 5 days and then challenged with C. parvum. Following challenge, diarrhea and other clinical assessments are performed and recorded as a health score according to the Wisconsin-Madison scale as described by McGuirk (above).

7.1 Production of hyperimmune sera using low-dose vaccines

Pregnant heifers (Holstein-Friesian cows) are used in two groups: Group 1: 11 animals are vaccinated twice subcutaneously with the adjuvanted BEI-rgp40his-subunit vaccine with 1.5 μg gp40 per dose per animal in a volume of 2 ml/dose. Vaccinations are carried out approximately seven and three weeks before the expected calving date. In Group 2, twelve animals remain unvaccinated to obtain control colostrum. All animals are also vaccinated with Rotavac® Corona vaccine (MSD AH) according to the manufacturer's instructions.

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

The serology results of vaccinated cows are presented in Table 3 as the mean anti-gp40 ELISA titer per group with their standard deviations.

Table 3: Anti-gp40 IgG titers (Log2) in bovine serum

Group gp40 dose day 0 (1 vaccination) 4 n p/v 1 1 n p/v 2 Avg. CO Avg. CO Avg. CO 1 1.5 10.2 1,2 16.8 2.2 17.5 1.3 2 -- 9.9 0.9 -- -- -- --

At the beginning of the study, the average titers of IgG antibodies to gp40 were comparable for both groups and were at the background level.

As can be seen from Table 3, the results show a marked increase in serum IgG titers against gp40 in Group 1 after the first and second vaccination. However, both titers indicate that protective levels of antibodies will be generated in colostrum.

After calving, the first two milkings from each cow were collected and saved for subsequent use in the passive vaccination/challenge experiment. The first milking was performed within 6 hours after calving, and the second milking was performed within 20 hours after calving. On average, approximately 5 liters of colostrum were collected per milking per cow. The colostrum IgG ELISA results are presented in Table 4 as group means with their standard deviations.

Table 4: Anti-gp40 IgG titers (2Log) in colostrum

Group Milking Avg. CO 1 1 21.4 1.6 2 20.1 1,2 2 1 13.8 1.5 2 11.6 1.8

Colostrum titers show that protective levels of colostrum anti-gp40 antibodies are produced in the colostrum of the vaccinated group and are significantly higher than the titer in the colostrum of unvaccinated control animals (p-value < 0.001).

7.2 Passive vaccination-challenge trial using hyperimmune colostrum

In a subsequent experiment, hyperimmune colostrum, produced as described in section 7.1 above, is used to passively vaccinate newborn calves. This allows the protection provided by this colostrum to be tested against a challenge with C. parvum parasites following colostrum feeding for 5 days. Groups of eight newborn calves are used; as the calves were born on different days, they are given a testing schedule with different start dates. The calves are Holstein-Friesian, weigh at least 34 kg, are no more than 4 hours old at the start of the experiment, and are not fed colostrum prior to the start of the experiment.

All calves receive 3 litres of colostrum within 4 hours of birth and a combination of colostrum and milk replacer once daily for five consecutive days (Groups 1 and 2). All calves are challenged orally with 10 ⁴ C. parvum oocysts 2-4 hours after the first colostrum feed. Faecal consistency and health parameters of each calf are assessed twice daily for fourteen days.

The division into groups is as follows:

1. n=8, colostrum from cows vaccinated with 1.5 µg BEI-gp40 for 5 days

2. n=8, colostrum from unvaccinated cows, for 5 days

Blood samples were taken from the calves at the beginning of the experiment and on the third day to confirm their uptake of anti-gp40 antibodies.

Following challenge, daily health checks are performed on each calf for 14 days to determine health parameters according to Table 1 above, including twice daily (morning and afternoon) faecal consistency assessment.

Fecal material from each animal that has a diarrhea score of 2 or 3 at any time is tested once daily for the presence of C. parvum or other enteric pathogens using the commercial Rainbow Calf Scours 4™ test (BIO-K 288) until at least one test is confirmed positive for C. parvum.

The results of serological studies show the following titers of IgG in serum against gp40: on the first day, all titers are at the initial level of 8.8 Log2. On the third day of colostrum feeding, the average titers in the serum of calves are: group 1: 20.6 ± 0.4, group 2: 10.9 ± 0.7.

Rainbow test scores indicate that all diarrhea is caused by C. parvum infection. Most calves in Group 1 (colostrum from cows vaccinated with 1.5 µg BEI-gp40 for 5 days) had C. parvum in their faeces on day 8 s.c., and most calves in Group 2 (colostrum from unvaccinated cows for 5 days) had C. parvum in their faeces on day 6 s.c.

The results of the health assessments are presented in Figure 5. These assessments include fecal consistency assessments and are defined as in Table 1 above, according to the Wisconsin-Madison scale (see above). As can be seen, calves fed colostrum from gp40-vaccinated cows were significantly better able to cope with a severe C. parvum parasite challenge, as they did not become as sick as calves fed colostrum without anti-gp40 antibodies.

7.3 Conclusion

It can be concluded that even doses of 1.5 μg of alkylated gp40 protein per animal vaccine dose can induce levels of anti-gp40 antibodies in colostrum that may passively effectively protect against severe C. parvum infection.

Designations on drawings

Figure 1

The amino acid sequence of the Cryptosporidium gp40 protein of the present invention in IUPAC single letter code corresponds to SEQ ID NO: 1.

Figure 2

Graphical representation of measured titers of gp40-specific antibodies in the serum of vaccinated cattle in Log2 ELISA units. Details are given in Examples 3 and 4.

Test groups:

rgp40His=Cryptosporidium gp40 protein vaccine that has not been alkylated;

rgp40His+BEI=Cryptosporidium gp40 protein vaccine comprising one or more aziridine-alkylated amino acids of the invention;

RC vaccine = Rotavec Corona vaccine

rgp40His+RC=double-vaccinated group that received both the Rotavec Corona vaccine and the Cryptosporidium gp40 protein vaccine that was not alkylated.

Figure 3

Results of an experiment on vaccination of calves in relation to the severity of diarrhea.

The horizontal axis represents days after challenge.

The vertical axis represents the average daily diarrhea rate for the rgp40His vaccine group and the rgp40His+BEI vaccine group.

The details of the experiment are described in Examples 3 and 4.

Figure 4

Results of mass spectrometric analysis of Cryptosporidium gp40 protein incubated with aziridine. The graph shows the amino acid sequence of gp40 (shown as SEQ ID NO: 3 herein) on the horizontal axis and the number of times a particular amino acid from gp40 was found to be alkylated upon incubation with aziridine on the vertical axis. The experimental details are described in Example 6.

Figure 5

Results of health assessment in a vaccination experiment using a very low dose of gp40 protein to produce hyperimmune colostrum. The horizontal axis shows days post-challenge. The vertical axis shows the Wisconsin-Madison health score. Details are described in Example 7.

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SEQUENCE LIST

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<---

Claims (7)

1. A Cryptosporidium gp40 protein for immunization against cryptosporidiosis, characterized in that the gp40 protein contains one or more alkylated amino acids, and that this Cryptosporidium gp40 protein is obtained by incubating a composition containing the Cryptosporidium gp40 protein with binary ethyleneimine. 2. The Cryptosporidium gp40 protein according to claim 1, wherein Cryptosporidium gp40 is derived from Cryptosporidium parvum. 3. Use of the Cryptosporidium gp40 protein according to claim 1 or 2 in a vaccine for protecting a target human or a target animal other than a human against cryptosporidiosis. 4. A vaccine against cryptosporidiosis for a target human or a target animal other than a human, wherein said vaccine comprises the Cryptosporidium gp40 protein according to claim 1 or 2 and a pharmaceutically acceptable carrier. 5. The vaccine according to item 4, characterized in that the vaccine contains an adjuvant. 6. A vaccine according to item 4 or 5, characterized in that the vaccine contains at least one additional immunoactive component. 7. A method for producing a vaccine according to any one of claims 4-6, comprising the step of formulating the Cryptosporidium gp40 protein according to claim 1 or 2 into a vaccine.