CRYPTOSPORIDIOSIS VACCINE

Abstract

The invention is based on the finding that incubating a Cryptosporidium gp40 protein with an aziridine, significantly increases its immunogenicity. When used as a vaccine, this allows a reduction of the dose, which improves economic feasibility and safety. Consequently the aziridine-treated gp40 can now be used as a safe and effective subunit-vaccine for humans or non-human-animals against Cryptosporidiosis. Specifically for new-born ruminants a vaccination by way of colostral transfer was found to be very effective in reducing clinical signs of Cryptosporidiosis, especially diarrhoea.

Claims

1. A Cryptosporidium gp40 protein or an immunogenic part thereof, wherein that the gp40 protein and the part thereof comprise one or more alkylated amino acids.

2. The gp40 protein or the immunogenic part thereof of claim 1, wherein the alkylated amino acid is alkylated with an alkyl group of Formula (2): ##STR00004## 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 the alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl, and cycloalkyl is optionally substituted with a substituent selected from the group consisting of: carbonyl, hydroxyl, alkyl, and haloalkyl; wherein R2′ and R2″ are each independently selected from H and alkyl; and wherein R3′ and R3″ are each independently selected from the group consisting of H and alkyl.

3. The gp40 protein or the immunogenic part thereof of claim 1, wherein the alkyl group of Formula (2), has one of the combinations of substituents selected from the group consisting of: 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(CH3)3, R2″ is H, R3′ is H, and R3″ is H.

4. The gp40 protein or the immunogenic part thereof of claim 1, wherein the alkylated amino acid is one or more selected from the group consisting of: cysteine, methionine, serine, threonine, tyrosine, lysine, arginine, valine, glutamic acid, and aspartic acid.

5. The gp40 protein or the immunogenic part thereof of claim 4, wherein the alkylated amino acid is one or more selected from the group consisting of: valine, glutamic acid, and aspartic acid.

6. The gp40 protein or the immunogenic part thereof of claim 5, wherein the valine is a valine corresponding to that of amino acid number (aa. no.) 2 of SEQ ID NO: 3; the glutamic acid is a glutamic acid corresponding to that of aa. no. 106 or to that of aa. no. 112 of SEQ ID NO: 3; and/or the aspartic acid is an aspartic acid corresponding to that of aa. no. 147 of SEQ ID NO: 3.

7. The Cryptosporidium gp40 protein or the immunogenic part thereof, of claim 1, obtainable by incubating a composition comprising a Cryptosporidium gp40 protein or an immunogenic part thereof, with an aziridine.

8. The Cryptosporidium gp40 protein or the immunogenic part thereof, of claim 7, wherein one or more or all of the features are applied, selected from the group consisting of: the aziridine is ethylenimine or is binary ethylenimine, the Cryptosporidium gp40 is from Cryptosporidium parvum, the composition comprising the Cryptosporidium gp40 protein or the immunogenic part thereof is the supernatant or filtrate from a baculovirus-insect cell expression system culture, and said supernatant or filtrate is purified by column chromatography.

9. A method for the preparation of the Cryptosporidium gp40 protein or of the immunogenic part thereof, of claim 1, the method comprising the step of incubating a composition comprising a Cryptosporidium gp40 protein, or an immunogenic part thereof, with an aziridine.

10. The method of claim 9 wherein one or more or all of the features is applied, selected from the group consisting of: the aziridine is ethylenimine or is binary ethylenimine, the Cryptosporidium gp40 is from Cryptosporidium parvum, the composition comprising the Cryptosporidium gp40 protein or the immunogenic part thereof is the supernatant or filtrate from a baculovirus-insect cell expression system culture, and said supernatant or filtrate is purified by column chromatography.

11. (canceled)

12. (canceled)

13. A vaccine for a human- or non-human animal target against Cryptosporidiosis, said vaccine comprising the Cryptosporidium gp40 protein or the immunogenic part thereof of claim 1, and a pharmaceutically acceptable carrier.

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

15. The vaccine of claim 13, wherein the vaccine comprises at least one additional immunoactive component.

16. (canceled)

17. A method for the production of colostrum comprising antibodies against the Cryptosporidium gp40 protein or the immunogenic part of claim 1, the method comprising the steps of: a. vaccinating a mammal at least once with the vaccine of claim 13, wherein said mammal is pregnant; b. collecting colostrum from the mammary glands of said mammal after parturition.

18. The method of claim 17, wherein the mammal is a ruminant.

19. A method of protecting a human- or non-human animal target against Cryptosporidiosis comprising administering colostrum obtainable by the method of claim 17, to said human- or non-human animal target.

20. A method of protecting a human- or non-human animal target against Cryptosporidiosis, said method comprising administering to said target at least once the vaccine of claim 13.

21. The method of protecting a human- or non-human animal target against Cryptosporidiosis of claim 19, wherein said administering is performed by feeding the colostrum to said human- or non-human animal target.

22. The method of claim 20, wherein the target is a ruminant.

23. The method of claim 22, wherein the ruminant is a bovine.

Description

EXAMPLES

Example 1: Production of Cryptosporidiosis Vaccine

1.1. The Recombinant Protein Construct

[0302] The recombinant gp40 protein for use in vaccination studies was constructed starting from the core amino acid sequence of SEQ ID NO: 1, which derives from an C. parvum, strain Iowa, genotype II. This sequence was extended with a C-terminal 6× Histidine tag, to allow for purification by Nickle column chromatography.

[0303] For Cryptosporidium gp40 protein, the cleavage of the N-terminal signal sequence is known to be different in an expression system where cleavage is at amino acid no. 20; as compared to the native gp40 protein which starts at amino acid no. 31, see O'Connor et al. (2007, Mol. Bioch. Parasit., vol. 152, p. 149-158). For the invention, the N-terminal gp40 signal sequence was partially restored, by using amino acids 14-30 from GenBank entry AAF78345.1.

[0304] In addition an N-terminal methionine was added to initiate transcription. This assembly could be expressed efficiently in a recombinant baculovirus-insect cell expression system, although it did not allow for secretion or glycosylation. Consequently, expression was cytoplasmatic, and gp40 could be harvested from the culture supernatant at the end of the culturing period when most insect cells were lysed.

[0305] The exact amino acid sequence of the Cryptosporidium gp40 protein used in vaccination experiments is that of SEQ ID NO: 3. This was expressed from a recombinant DNA that had been codon-optimised based on the codon-preference of the AcMNPV baculoviral polyhedrin gene; the rec. DNA sequence used was that of SEQ ID NO: 2. This recombinant gene was inserted as a BamH1-EcoR1 fragment into a pVL1393 plasmid-based baculovirus transfervector, which drives expression from the polyhedrin promoter. Stably transfected recombinant baculovirus was 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.

[0306] Rgp40His expression was confirmed by SDS-Page of insect cell culture supernatant, which displayed the expressed protein as a band of about 32 kDa. Further tests were done by Western blot and immunofluorescence assay using a bovine polyclonal anti-gp40 antiserum, and an anti-His monoclonal antibody. The recombinant baculovirus was laid down as master seed virus after several tests for genetic correctness and -stability, and sterility.

1.2. Expression and Harvesting

[0307] Rgp40His was produced from Sf9 insect cells infected with the recombinant baculovirus seed. The cells were cultured in commercial animal-compound free insect cell culture medium SF900II™. Small scale cultures of 0.5-2 litres were done in laboratories; large scale production runs up to 500 L cultures were done in pre-production facilities. In general the following schedule was applied: clean Sf9 cells were produced in cultures of increasing volume. When enough clean cells were produced, the cells were concentrated and reseeded in fresh medium at 1.6×10{circumflex over ( )}6 cells/ml. These were infected with an m.o.i. of 0.002, and incubated for 5 days at 28° C. At the end of the culture, the supernatant was harvested; at small scale by centrifugation, at large scale this was by clarification by deep-filtration. When expressed at small scale, the recombinant baculovirus was then inactivated using an incubation with 0.1% Triton® X-100 for 1 hour at room temperature, followed by gamma-irradiation by a commercial company. When produced at large scale, all downstream processing was done in contained facilities and using closed connections, and the bulk of the recombinant baculovirus was removed by the different filtration and purification steps. Any remaining virus was then killed-off during the aziridine treatment.

[0308] For purification, the harvested rgp40His was loaded onto a Ni-Sepharose column. Next the column was washed, and rgp40His was eluted using imidazol. Next the imidazol was removed by diafiltration against 50 mM HEPES buffer at pH 7.5. Next the rgp40His was sterile filtered over 0.2 μm membrane filters, and incubated with aziridine. BEI was prepared from combining equal volumes of 1.09 M BEA and 1.91 M NaOH. This generated a stock-solution of 545 mM BEI. Then the BEI was added to the composition comprising the rgp40His to 33 mM. The mixture with BEI was incubated at room temperature for 24 hours. Next sodium-thiosulphate was added at 33 mM. This was incubated for an hour at room-temperature, and pH was measured to be 7.1.

[0309] For comparative experiments, Cryptosporidium gp40 that did not comprise one or more alkylated amino acids, was produced in the same way, except that a buffer was added instead of an aziridine, for a mock incubation.

1.3. Antigenic Mass Determination

[0310] The amount of rgp40His protein was initially measured by SDS-PAGE along a dilution range of known amounts of a reference protein. Using densitometry of stained gels, bands were then quantified. This test was replaced later by a more precise one: a competitive antigenic mass ELISA, that was fully validated for specificity, robustness, linearity, and precision. In short the antigenic mass ELISA was performed as follows: wells of a micro-titration plate were coated with purified rgp40His (not alkylated), at 50 ng/well, overnight in coating buffer and at 4° C. Next day the plate was washed, post-coated with a casein-containing buffer for 1 hour at 37° C., and then washed again. On separate microtitration plates a test sample of rgp40His of unknown concentration was serially diluted in ELISA buffer (containing 0.05% polysorbate 80), and a fixed amount of an anti-gp40 mouse monoclonal antibody was added. This was pre-incubated for 1 hour at 37° C., then this mixture was transferred to the coated plates, and incubated for 1 hour at 37° C., in ELISA buffer. Plates were then washed, and the amount of gp40 monoclonal antibody that was bound to the plate was detected by incubation with a peroxidase-conjugated goat-anti-mouse IgG, in Elisa buffer for 1 hour at 37° C. The peroxidase was visualised by enzymatic conversion of tetramethylbenzidine for 15 minutes at room temperature, which reaction as stopped using sulphuric acid. The optical density of the yellow colour in each well was measured at 450 nm. The amount of peroxidase-conjugate is inversely correlated to the amount of antigen in the test sample. This was processed by a software program using a Logit-Log algorithm, for at least 3 sample points from a dilution series. Also samples were measured at least in duplicate, and appropriate positive and negative controls were taken along in the test. A value for the antigenic mass of gp40 in the test sample was then calculated in micrograms/ml, based on standardised reference sample values.

1.4. Formulation into a Vaccine

[0311] The rgp40His, column-purified, and aziridine-incubated or mock-incubated, was then formulated as an emulsion vaccine for further use, by combination with suitable adjuvants and emulsification, all using well-known methods and materials. In short: the rgp40His was taken up into sterile saline (0.85% w/v sodium chloride) to the required concentration. In parallel the oil-phase was prepared of ISA™ 70 VG (Seppic, France) by sterile filtration. Both phases are combined and emulsified using a Silverson™ or Dispax™ homogeniser, into a water-in-oil emulsion. The gradual increase of temperature during the homogenisation is monitored and kept to below 50° C. The resulting emulsion was stored at 4° C. until filling into appropriate containers. The filled product is subjected to a variety of tests for stability and sterility before release.

[0312] Depending on the intended geographical market or the combination with other antigens, batches of the emulsion were also prepared that included a further adjuvant, specifically aluminium-hydroxide. In that case: the aqueous rgp40His preparation was first combined with a heat-sterilised suspension of 3% w/v Alhydrogel® (Brenntag) in saline, and left to absorb to the aluminium for 30-60 minutes at room temperature while stirring. Subsequently this mixture of antigen and aluminium was then emulsified with the oil phase as described above.

[0313] Long term stability tests of 1- and 2-year shelf-life at 4° C. are ongoing. Indicative however are the results of intermediate and expedited stability tests: for 15 months at 4° C., and for 3 weeks at 30 or 37° C. Emulsion quality remained to be good throughout, and the average change in antigenic mass found was within 10% from the start composition. This is a good indication of long-term stability under cooled conditions.

Example 2: Set-Up of the Vaccination-Challenge Trials

[0314] Over a number of years, several trials in experimental animals were performed to develop and optimise a model for effective vaccination-challenge trails to be able to evaluate the potency of the various compositions that were tested as Cryptosporidiosis vaccines. The following set-up proved most indicative of in vivo potency:

[0315] Vaccines were prepared as described above: Cryptosporidiosis gp40 protein was expressed in the baculovirus-insect cell expression system and was harvested at the end of the culture, purified (or not) by metal-affinity chromatography, and incubated (or not) with an aziridine. The protein was then formulated with an adjuvant, either an oil, or an oil and an aluminium salt, and emulsified as a W/O emulsion.

2.1. Animal Model

[0316] For passive vaccination studies, target animals were healthy dairy cattle, of Holstein-Friesian type, from about one year old and selected from both heifers and multiparous cows. The cows were in their last trimester of pregnancy and their serum was tested for having only background levels of gp40-specific antibodies. The cows were treated at the farm where they lived, therefore no acclimatisation was necessary. Feed and water were provided according to standard cattle management practice. Animals were marked by unique life number, via ear tag.

2.2. Vaccinations

[0317] Vaccines were administered, containing 10 mg rgp40His/dose, either aziridine treated or mock treated, emulsified as W/O with mineral oil, in a 2 ml volume, and administered intra-muscularly in the neck. Timing was: booster at 3 weeks before expected calving date, and priming at 6 weeks before booster. The cows also received a vaccination with Rotavec® Corona vaccine at 4 weeks before expected calving date, to prevent new-born diarrhoea in the calves from pathogens other than C. parvum. Serum samples were taken at specific time points and tested by antibody ELISA.

[0318] After calving, colostrum was collected as the first, second and third milking; on average 4 to 5 litres of colostrum could be obtained per milking per cow. Colostrum was pooled per vaccination test group, per milking timepoint and aliquoted in 250 ml jars, before pasteurization using a water bath for 30-45 minutes at 56° C. The aliquots were then divided and kept frozen until use. Thawing was controlled by using a water bath set at 43° C. or using hand warm tap water whilst regularly checking the water temperature. The colostrum was hand warm (i.e. approximately 40° C.) when given to the calves.

[0319] Neonatal calves were collected at calving, housed individually, and fed a 3 litre bolus of anti-gp40 colostrum, from one of the vaccine groups, within 4 hours of birth. Next the calves received a challenge dose of live C. parvum oocysts at 2 hours after the first feed.

[0320] Group sizes were 5-10 calves, and a non-protected challenged group was included in all experiments, which group received mock colostrum. The use of non-protected non-challenged sentinels was found not to be informative, and was left out in later experiments. Calves included in the studies were at least 30 kg bodyweight at birth.

[0321] The calves were kept in an isolated facility, in single-animal boxes. Contact between calves was not allowed. Caretakers changed gloves and footwear before treating each animal.

[0322] Each single animal box consisted of two compartments. One contained wood shavings and one consisted of a metal grid above a horizontal smooth surface plate above the waste collection channel. Calves were kept on wood shavings during the first three days of life. At the end of day three the calves were moved to the second compartment. The smooth surface plate provided the ability to assess faecal consistency, to determine the diarrhoea score. This plate was cleaned after each scoring time point in order for the next scoring to be on the faecal material that was produced between each scoring time point.

[0323] The calves received feeding twice daily; consisting of colostrum (first feed) or colostrum and milk replacer from second feed onward was given daily according to the manufacturers specifications after the colostrum was first given to the calf.

2.3. Challenges

[0324] The challenge inoculum was prepared fresh before administration, with 1×10{circumflex over ( )}6 live C. parvum, strain Iowa oocysts/calve. This was given to calves orally in 50 ml milk replacer. Subsequently the calves received twice daily feeding of (gp40- or control-vaccine) colostrum in milk replacer: 0.25 to 0.5 litres of colostrum in a total of 2-2.5 litres of milk per day, for several days.

[0325] This challenge dose was selected to cause a repeatable and consistent diarrhoea, that was severe (but transient) in all of the unprotected challenge controls.

[0326] Live oocysts were purchased fresh from Waterborne Inc., New Orleans, USA. The oocysts were freshly shed, no older than 2 months before the start of the animal experiment. The parasite viability by means of excystation (test criterion is 50-100%), plus infectivity of an HCT-8 cell layer, were found to be sufficient both before the start, and at the end of the experiment.

[0327] After challenge, calves were monitored for 14 days, at which diarrhoea typically developed between days 4-11, with a peak in severity from days 5-10. Antibody levels were measured in the colostrum and in the calves' serum at day 1 before feeding and at day 3 of feeding.

2.4. Scoring of Effects

[0328] Several methods were tested over time to best quantify and assess the effect of the vaccination on the challenge infection. Initially only bull calves were used, and faeces were collected in bags from all calves to be analysed for amount and consistency. This proved inaccurate as calves would lie down when sick, and material got lost. Most clinically relevant proved to be the twice daily recording of a number of observational scoring-parameters, specifically for: general health, dehydration, backside appearance, and diarrhoea score. The diarrhoea scoring parameters used, were based on the Wisconsin-Madison scale as described in S. McGuirk, 2008 (Vet. Clin. North Am. Food Anim. Pract., vol. 24, p. 139-153). The parameters used for the observations are represented in Table 1. With some experience, animal caretakers were able to determine the scores consistently.

[0329] When diarrhoea became prominent, a test for Cryptosporidia in faeces was applied, which was done using a commercial test kit: BIO K306™—Rainbow Calf Scours (Bio-X Diagnostics, Belgium), which employs fluid chromatography on specific test strips.

[0330] Counting of oocysts in faecal samples was done to determine reduction of shedding. Oocysts counting was as follows: from collected faeces a 50 ml sample was taken and stored at 2-8° C. until use. The sample was serially diluted, and a sample was put on a diagnostic microscopic slide and allowed to dry by air for 1 hour. The slides were stained according to Ziehl-Neelsen (acid fast) method and red-stained oocysts were counted by light-microscopy. Results were expressed as oocysts per gram faeces.

TABLE-US-00001 TABLE 1 Observational parameters for the scoring of signs of Cryptosporidiosis in calves General Backside Score health Dehydration appearance Diarrhoea score 0 Normal: not clean Normal: The calf dehydrated: backside, faeces retain form. is alert, (eyes normal, tale and The faeces may hungry, and elastic skin legs. be pasty but do watches the (smooth fur). not flow across caretakers. a tilted surface. It may stretch when it gets up. The calf will eat greedily, often twitching its tail as it eats. 1 Mildly possibly: tale and Mild: depressed: doubtful backside form is a puddle, The calf eyes, dirty with not a patty. rises and dull/glazed some Sufficient water drinks fur. sticky content to slowly without faeces or flow across or coaxing, dry faecal down a tilted but not material. surface. greedily. The calf pays some attention to the caretakers. 2 Moderately moderately: tale and Moderate: depressed: eyes too backside faeces with The calf deep, or very dirty sufficient water must be dull/ but not content to easily coaxed to tarnished wet, flow across or drink or does fur. drying. down a tilted not consume surface, while all the leaving some milk. It adherent material. pays little attention to the caretaker until touched. 3 Severely severe: tale, Severe: depressed: eyes lie backside part or all of The calf very deep, and faeces are very must be and dull/ legs dirty watery. Faeces coaxed to get tarnished and wet can drain away up, may have fur. from leaving little or difficulty watery no residual on a rising diarrhoea. smooth tilted or standing, surface (a calf does not may have very pay attention watery faeces to caretakers followed by some when solid material touched, and still have may refuse severe diarrhoea). to eat, no suckling reflex. 4 Dead or — — — moribund: The calf does not or hardly respond to stimuli, lateral recumbence.

Example 3: Results of the Vaccination-Challenge Experiments

[0331] 3.1. Recombinant Expression of pp40:

[0332] The baculovirus-insect cell expression system with the optimisations applied: the partial signal sequence, and the codon-usage adaptation, was found to yield more protein that the previously used E. coli expression system did. However, when vaccinating with crude gp40 harvest from insect cell-cultures, i.e. only centrifuged and inactivated with Triton X-100 and gamma-irradiation, the immunogenicity for a standard 20 μg dose was not as good as for a similar dose of similarly inactivated and column purified gp40. Therefore His-tag fusion constructs and Nickle column purification were used as standard.

3.2. Set-Up of Vaccine Formulation

[0333] The oil− and the oil+ aluminium adjuvants used, both induced effective antibody titres in the pregnant cows, and subsequently in their colostrum. However, for cattle that had not previously received a vaccination with gp40, a single vaccination did not suffice, and prime-boost vaccination regimen was required for inducing the levels of antibodies in colostrum that were protective against the severe challenge as applied in these trials. Commonly naïve cattle had antiserum titres of gp40-specific antibodies of between 9 and 12 Log 2 in the antibody ELISA. Therefore, a titre of up to 10 Log 2 was considered to be the background level. After vaccination with a dose of 10 micrograms of purified gp40 that did not comprise one or more alkylated amino acids in W/O emulsion vaccine this titre increased to 12-13 Log 2 after the prime, and to 15-17 Log 2 after the booster vaccination.

[0334] From these cows colostrum could be collected that contained gp40-specific antibody titres of about 20 Log 2. Consequently, vaccine doses of 10 μg of rgp40His that was not aziridine-incubated and purified were used in prime and boost experiments. When applied in combination with Rotavec Corona vaccine, no effect was measured on the efficacy of the vaccination against rotavirus, coronavirus, or E. coli by the gp40 vaccination, see also FIG. 2. It was concluded that these vaccines could effectively be combined.

[0335] There was no difference in the feeding of colostrum once or twice a day, so once daily was applied going forward. Also feeding of colostrum for a total of 5 days was found to suffice.

3.3. Rainbow Scour Test

[0336] All animals that received challenge inoculum and had a diarrhoea score of 2 or 3, tested positive in the test for the presence of Cryptosporidium.

3.4. Side-Reactions

[0337] While the vaccinations of cattle with emulsified vaccines of rgp40His where generally safe, i.e. no systemic effects were observed, or effects on pregnancy, they did induce local reactions at the site of the vaccination: these were palpable, and upon necropsy signs of inflammation were found. This proved to be related to the presence of the gp40 protein, as a vaccine in a similar emulsion, the Rotavec Corona vaccine, did not induce such local reactions.

[0338] The only way to reduce those reactions was a reduction of the dose of gp40, however that was only possible while maintaining efficacy, when employing the Cryptosporidium gp40 protein comprising one or more alkylated amino acids of the invention.

Example 4: Effect of the Aziridine Incubation in Vaccination-Challenge Experiments

4.1. Effect on Antibody Titres

[0339] In the vaccination experiment of groups of 5 pregnant cattle each, as described in Example 3 above, the cows received two doses of 10 μg rgp40His in an oil+aluminium adjuvated W/O emulsion. The gp40 was column purified, and either aziridine incubated or not. As negative control served a group receiving only the Rotavec Corona vaccine, having the same formulation. A further group received both a vaccination with gp40 that did not comprise one or more alkylated amino acids and one with Rotavec Corona vaccine. Serum antibody titres were measured weekly, and determined with the antibody ELISA. Titres at or below 10 Log 2 were set at 10, and considered background. Seroconversion profiles over time for all the gp40 vaccinates were similar. The Rotavec Corona vaccine only, induced no antibodies against gp40. The titre results for the combination vaccine show there is no interference between the two vaccine types.

[0340] While containing the same amount of gp40 antigen, the vaccine comprising Cryptosporidium gp40 protein comprising one or more alkylated amino acids induced antibody titres in cattle that were significantly higher than for the non-alkylated gp40 vaccine: 3 to 4 Log 2 higher after prime vaccination, and 2 Log 2 higher after the booster; this is represented in FIG. 2. The resulting colostrum titres of alkylated gp40 vaccination were found to be very high, at 24 Log 2.

4.2. Effect on Shedding

[0341] The effect on the shedding of oocysts by calves after challenge infection, as a result of passive vaccination, was modest, but significant. Typically a 10 fold reduction of the total amount of oocysts per 100 gram of faeces, between days 4 to 14 was found: from 10{circumflex over ( )}9 oocysts in non-vaccinated—challenged calves, to 10{circumflex over ( )}8 in vaccinated calves. However because also the faecal amounts were much reduced resulting from the shorter duration of diarrhoea found in the calves: 2 days in calves vaccinated with BEI-treated gp40, against 6 days in non-vaccinated calves. Therefore the overall reduction of oocyst shedding by vaccination was certainly relevant.

4.3. Effect on Safety

[0342] The gp40 protein in combination with a mineral oil adjuvant, turned out to be quite reactive; this was most apparent upon vaccination by subcutaneous route, and when applying a prime-boost regime. This effect was not aggravated by aziridine incubation of the gp40 protein.

[0343] A vaccine having a dose of 10 μg gp40 (alkylated or not), with mineral oil adjuvant, and given as prime and boost vaccination of adult Holstein cows by subcutaneous route, induced a swelling at the inoculation site up to 10 cm in diameter and up to 2 cm thick. Apart from looking severe however, the vaccinated cows did not show any signs of suffering from this local reaction: no fever was observed, and there was no effect on appetite or on the pregnancy.

[0344] Nevertheless it was decided that such swelling, even when asymptomatic and transient, was not desirable.

[0345] Fortunately, the invention as described herein allowed a reduction of the dose of gp40 protein to safe levels, while maintaining good immunological efficacy: upon adaptation of the amount of protein per dose to 1 μg of the alkylated gp40 protein, swelling after two doses given subcutaneous was typically below 5 cm in diameter and 1 cm high, mostly only 2 cm in diameter. This was considered to be acceptable. Importantly, this reduction in amount of protein by 10 fold did not affect the efficacy of vaccination, in that still sufficiently high titres of gp40 antibodies could be obtained in the colostrum.

4.4. Effect on Diarrhoea

[0346] From the vaccination-challenge experiment, the severity of diarrhoea was scored based on the parameters as presented in Table 1, last column. The results for the two most relevant groups of calves are presented in FIG. 3. These calves received colostrum either from mothers vaccinated with BEI-treated gp40 vaccine, or with non-treated gp40 vaccine. Challenge was given at day one, after the first bolus of colostrum, as described. The average daily score of diarrhoea per group was determined, and plotted over the course of the 14 day experiment.

[0347] As can clearly be seen from FIG. 3, the severity of diarrhoea was much lower, and much shorter for the group receiving colostrum from BEI-gp40 vaccinated cows. The difference is impressive and highly relevant: the rgp40His+BEI vaccine group only had significant diarrhoea (score above 1.0) for 2 days (on days 7 and 8). However the rgp40His vaccine group (gp40 mock treated with aziridine) had significant diarrhoea for 6 days (on days 6-11). Whereby the diarrhoea in the rgp40His+BEI vaccine group was also of much lesser severity: maximal score was 1.2, as compared to a maximum score of 2.8 in the mock-BEI-treated vaccine group.

In Conclusion:

[0348] A truly effective Cryptosporidiosis vaccine has been found, which is capable of significantly reducing both the severity and the duration of diarrhoea caused by a heavy challenge infection with Cryptosporidium parasites.

[0349] Also, the model of passive vaccination of newborn calves, by the feeding of colostrum from vaccinated mothers, was found to be highly effective.

Example 5: Ongoing Studies

5.1. Dose Finding

[0350] In ongoing dose finding studies, vaccine doses of 0.1-0.2-0.5-1-2.5 and 5 μg of the alkylated gp40 protein according to the invention are being tested.

[0351] Initial results indicate that 1 μg of the alkylated gp40 protein per dose, in a prime-boost regime, will induce sufficient level of gp40 specific antibody in a pregnant bovine. So far, a 1 μg dose, with mineral oil adjuvant, and given twice by subcutaneous route induced a titre of 19 Log 2 Elisa units, at 2 weeks after the priming vaccination, which dropped to 18 at 4 weeks p.v., when the booster vaccination was given. The booster then increased the titre again to 22 at 6 weeks p.v., which reduced slightly to a titre of 21 Log 2 at 8 weeks after the priming vaccination. At calving, colostrum antibody values were found to be 1-2 Log 2 higher than serum Ab levels.

[0352] Considering that a colostrum titre of 17-18 Log 2 will suffice to protect calves, the optimal vaccination protocol aims to reach a titre of 15-16 Log 2 in the vaccinated bovine mother. Consequently, even a vaccine dose of 0.5 μg of the alkylated gp40 protein according to the invention will suffice to reach such titre levels in the mother with a prime-boost regime.

[0353] Alternatively, it is being investigated if such protective titre level in colostrum can be achieved by giving only a single vaccination to pregnant ruminants, using 0.5 or 1 μg of alkylated gp40 protein per dose.

Example 6: Mass-Spectrometry Analysis

[0354] To determine which amino acids were alkylated by aziridine incubation, LC-MS/MS analyses were performed of BEI-incubated- and non-incubated rgp40His. The protein was digested in parallel by different enzymes, whereby the peptides detected were analysed for having an adduct of 42 Da, corresponding to an alkylation by an ethylenimine molecule.

6.1 Materials and Methods

[0355] 6.1.1 Protein Samples Tested

[0356] Two batches of rgp40His were used, having a different initial concentration of the protein: batch 01K at 4.1 mg/ml, and batch 12 G at 1.0 mg/ml, by BCA. Both these batches were BEI-incubated as described. As control, a batch of rgp40His protein that was not aziridine-incubated was used; batch 2DJ, at 0.62 mg/ml, and this batch was nano-filtered to remove any residual baculovirus.

[0357] 6.1.2 Intact-Mass Analyses of gp40

[0358] Protein amount was determined by BCA (Pierce) using the manufacturer's protocol. Approximately 6-8 μg of rgp40His sample was analysed by liquid-chromatography and mass-spectrometry (LC-MS) using an Agilent 1290 LC and Agilent 6545 QTOF MS. A MassPrep™ Online Desalting cartridge (Waters) was used at 0.4 ml/min flowrate at 60° C. LC buffer A consisted of 0.1% v/v formic acid in water, and buffer B of 0.1% formic acid in acetonitrile. The gradient was 5%-80% buffer B in 5 min, and was kept at 80% buffer B for 1 min. Equilibration occurred at 5% buffer B, with total run time of 8 min. The column eluent was electrosprayed into the MS using the Dual AJS ESI source (typically at 4 kV). MS scans were recorded between 100 and 3200 m/z at 1 spectra/sec.

[0359] 6.1.3 Peptide Mapping of gp40

[0360] Peptide mapping of rgp40His was performed by digestion with the enzymes: trypsin, chymotrypsin or GluC (Staphylococcus aureus Protease V8) at an enzyme:protein ratio of 1:50, by incubation for an hour. Subsequently enzyme was added again, in the same ratio, and digestion was continued overnight. Sample clean-up was performed by Sep-Pak™ tC18, 1 cc, 50 mg sorbent cartridges (Waters). The peptide mixtures were eluted with 0.1% v/v formic acid in 90% acetonitrile. The solvent was evaporated in a Speedvac™ at room temperature. Peptide sample mixtures were reconstituted in 0.1% formic acid.

[0361] Approximately 30 μg of enzyme digest was analysed by LC-MS as described. Peptide mixtures were separated using an Agilent AdvanceBio™ Peptide Map, C18, 1.2×150 mm, 2.7-micron at 0.4 mL/min at 60° C. LC buffers A and B were as described. The gradient used was 2%-45% buffer B in 110 min followed by 45%-95% buffer B in 5 min, and kept at 95% buffer B for 10 min. Equilibration was done with 2% buffer B, with a total run time of 145 min. The column eluent was electrosprayed into the MS using the Dual AJS ESI source (typically at 4 kV). MS scans were recorded between 100 and 1700 m/z at 4 spectra/sec. MS/MS scans were recorded between 150 and 3200 m/z at 2 spectra/sec. A maximum of 5 precursor ions per cycle were selected for MS/MS at an abundance threshold of 10000 counts. Precursor ions were excluded after 5 repeats for 1 min. Isolation width was set to Medium (4 amu).

[0362] 6.1.4 MS Data Analyses

[0363] Intact Analyses

[0364] LC peaks containing protein fragments were deconvoluted using MassHunter™ Qualitative Analysis B.07.00 software. The deconvolution algorithm was ‘maximum entropy’; the mass range was 16,000-23,000 Daltons, m/z range 800-2000. Baseline subtraction was turned on, and the baseline factor was set to 7.00. Adduct was set to ‘proton’, and the isotope width to ‘automatic’.

[0365] Peptide Mapping

[0366] Agilent QTOF MS/MS data files were converted to MFG files using the MassHunter software. For each data file a compounds list was created using the ‘Find Compounds by Auto MS/MS’ function. ‘Extract MS/MS’ with Extract average MS/MS spectra for all collision energies was enabled. Otherwise default settings were applied. The compounds list was exported to MGF with ‘Compute deisotope’ enabled, ‘Isotope model’ set to peptide and ‘Limited assigned charge state’ set to a maximum of 7. MFG files were searched against the Cryptosporidium parvum gp40 recombinant protein of SEQ ID NO: 3 database in Mascot™. Enzyme restriction was set to none.

[0367] Ethyleneimine (43.04 Da, C2H5N) was set as variable modification for all amino acids. MS tolerance was set to 25 ppm and MS/MS tolerance to 0.1 Da. Peptide charge was set to 2+, 3+ and 4+.

[0368] MGF files also were searched against the host cell protein database (Sf9, Spodoptera frugiperda, Uniprot 201909127, with 26,506 entries) using identical settings as described above.

[0369] 6.2 Results

[0370] In the BEI-incubated gp40, about one quarter of the protein did not contain an El-adduct, one quarter had one adduct, and the rest had two or more adducts, in rapidly declining frequency.

[0371] Intact-mass analysis revealed no evidence of glycosylation or other post-translational modifications. Also, no significant database hits were found for Sf9 host cell proteins.

[0372] A multiple digestion approach was applied to analyse protein fragments, using digestion with trypsin, chymotrypsin or GluC. Together these generated a full set of overlapping peptides, with good sequence coverage, and a highly confident assignment of El-adducts. The top 4 modification sites were given an additional manual analysis.

[0373] The PepMap results showed full sequence coverage of gp40, but limited sequence coverage of the His-tag tail. However it is very unlike that the El adducts are located at the histidines. The coverage of the various peptide fragments is presented in Table 2.

[0374] In Table 2 the 1 (*) result for total El-alkylated peptides in the native protein sample is a false positive score.

TABLE-US-00002 TABLE 2 Peptide mapping results Peptide ID's Unique EI- Dominant gp40 protein Total EI- modification EI-modif. batch tested Trypsin Chymotrypsin GluC alkylated sites sites 01K-BEI 201 64 134 50 31 4 12G-BEI 317 223 135 117  62 4 2DJ (native) 207 156 83   1 * 0 0

[0375] The PepMap analysis allowed for the site-specific identification of El-adducts. The identification of El-modification sites could be done using automated database searching, as there was prior knowledge of the adducts to be detected: each El-adduct increases size by 42 Da, but the resolution of the methods applied had an accuracy of ±1 Da, so weight-increases of 41-43 Da were analysed. Selection against multiple El-adducts on one amino acid was done by the MS-analysis software.

[0376] From the combined data of both aziridine-incubated batches, the gp40 amino acids: 2V, 106E, 112E, and 147D (numbered as in SEQ ID NO: 3) appeared to be the dominant sites of alkylation. Results are presented in FIG. 4. Some other amino acids were found to be alkylated as well, though to (much) lower frequency: cysteine, methionine, serine, threonine, tyrosine, lysine, and arginine. Thus, interestingly, aspartic acid (D) and glutamic acid (E) appeared to be the dominant sites of modification, with minor frequencies for other amino acids.

[0377] The Cryptosporidium gp40 protein used in these tests was the recombinant-expressed gp40-His, with the amino acid sequence of SEQ ID NO: 3. However, the dominant protein detected in all samples was rgp40His without initiating methionine, which has a basic molecular weight of 20.9 kDa. The missing N-terminal methionine, opened-up the valine at position 2 for alkylation.

Example 7: Vaccination-Challenge Trials with Low-Dose Vaccines

[0378] As indicated in Example 5 above, dose-finding experiments were done, using vaccines according to the invention with very low doses of BEI-gp40. Pregnant heifers were actively vaccinated, and calves were passively vaccinated and challenged. The setup and performance was essentially as described above. Specifically the experiments were performed as follows: first, hyper-immune colostrum was generated by vaccinating pregnant heifers and collecting their colostrum from the first and the second milking. The amounts of alkylated gp40 protein used per animal dose were: 0.4 or 1.5 μg. Vaccines were formulated as water-in-oil emulsion as described above, using oil (ISA 70)+aluminium (Alhydrogel) as adjuvants. The vaccine was administered by subcutaneous route as prime and boost, at 7 weeks and at 3 weeks before expected calving date, respectively. Serum and colostrum samples were tested for specific IgG antibody response against gp40 using ELISA, as described above.

[0379] Next, newborn calves were fed with this colostrum for 5 days, and were then challenged with C. parvum parasites. After challenge the scores for diarrhoea and other clinical scores were determined, and reported as health scores, according to the Wisconsin-Madison scale as described in McGuirk (supra).

7.1 Generation of Hyper-Immune Sera with Low-Dose Vaccines

[0380] Pregnant heifers (Holstein Friesian cows) were used, in two groups: group 1: 11 animals were vaccinated twice via the subcutaneous route with adjuvated BEI-rgp40his subunit vaccine with 1.5 μg gp40 per animal dose, in a 2 ml volume/dose. Vaccinations were given at approximately seven and three weeks before expected calving date. In group 2, twelve animals remained unvaccinated for the production of control colostrum. All animals also received a vaccination with Rotavac® Corona (MSD AH) vaccine according to the manufacturer's instructions.

[0381] Blood samples were taken just before each vaccination (on the same day) and 1 week after the second vaccination from each animal of group 1, and once from animals of group 2 one week before vaccination with Rotavec® Corona vaccine.

[0382] The results of the serology from the vaccinated cows are presented in Table 3, as the average anti-gp40 Elisa titre per group, with their standard deviations.

TABLE-US-00003 TABLE 3 Anti-gp40 IgG titres (Log2) of cow sera dose of day 0 (1st vacc.) 4 w p.v.1 1 w p.v.2 Group gp40 Avg. SD Avg. SD Avg. SD 1 1.5 10.2 1.2 16.8 2.2 17.5 1.3 2 — 9.9 0.9 — — — —

[0383] At the start of the study the average IgG antibody titres against gp40 were comparable for both groups and were at background level.

[0384] As is evident from Table 3, the results show a marked increase of serum IgG titres against gp40 in group 1 after the first and the second vaccinations. Both titres however indicated that protective levels of colostrum antibodies would be generated.

[0385] After parturition, the first two milkings were collected from each cow and stored for later use in the passive vaccination/challenge experiment. First milking was collected within 6 hours post partum, and the second milking within 20 hours p.p. On average, about 5 litres of colostrum was collected per cow per milking. Colostrum IgG Elisa results are presented in Table 4 as averages per group with their standard deviations.

TABLE-US-00004 TABLE 4 Anti-gp40 IgG titres (2Log) in colostrum Group Milking Avg. SD 1 1 21.4 1.6 2 20.1 1.2 2 1 13.8 1.5 2 11.6 1.8

[0386] The colostrum titres showed that protective levels of anti-gp40 colostral antibodies were produced in colostrum from the vaccinated group, and this was significantly higher than the titre in colostrum from the non-vaccinated control animals (p-value<0.001).

7.2 Passive Vaccination-Challenge Trial Using Hyper-Immune Colostrum

[0387] In a subsequent experiment, the hyperimmune colostrum generated as described in section 7.1 above, was used for passive vaccination of newborn calves. This allowed to test the protection provided by that colostrum against a challenge infection with C. parvum parasites, after colostrum feeding for 5 days. Groups of eight newborn calves were used; as the calves were born on different days, they progressed though the trial schedule from different starting dates. Calves were Holstein-Friesian, of at least 34 kg, were not older than 4 hours at the start of the experiment, and had not received any colostrum prior to the trial.

[0388] All calves received 3 litres of colostrum within 4 hours after birth and a combination of colostrum and milk replacer once a day for five consecutive days (groups 1 and 2). All calves were challenged orally with 10{circumflex over ( )}4 C. parvum oocysts 2 to 4 hours after the first feed of colostrum. Faecal consistency and health scores of each calf were assessed twice daily for fourteen days.

[0389] Group division was as follows:

[0390] 1. n=8, colostrum from 1.5 μg BEI-gp40 vaccinated cows, for 5 days

[0391] 2. n=8, colostrum from non-vaccinated cows, for 5 days

[0392] Blood samples were taken from the calves at the start of the experiment and on day three to confirm their uptake of anti gp40 antibodies.

[0393] After challenge, daily health checks were performed of each calf for 14 days, to determine health scores according to Table 1 above, including scoring of faecal consistency twice daily (morning and afternoon).

[0394] Faecal material from every animal that had a diarrhoea score of 2 or 3 at any given time point, was tested once a day for presence of C. parvum or other enteric pathogens using the commercial Rainbow Calf Scours 4™ test (BIO-K 288), until at least one test was confirmed positive for C. parvum.

[0395] The serology results showed the following serum IgG titres against gp40: at day one all titres were at base line level of 8.8 Log 2. At day three of colostrum feeding the average titres in the calf sera were: group 1: 20.6±0.4, and group 2: 10.9±0.7.

[0396] Rainbow test scores showed that all diarrhoea was due to C. parvum infection. Most calves from group 1 (colostrum from 1.5 μg BEI-gp40 vaccinated cows, for 5 days) showed C. parvum in their faeces at day 8 p.c., and most calves from group 2 (colostrum from non-vaccinated cows, for 5 days) at 6 days p.c.

[0397] The results of the health scores are presented in FIG. 5. These scores included the faecal consistency scores, and were determined as outlined in Table 1 above, according to the Wisconsin-Madison scale (supra). As is clear, the calves receiving colostrum from gp40 vaccinated cows were much better capable of dealing with a severe challenge infection of C. parvum parasites, as they did not get nearly as sick as calves receiving colostrum without anti-gp40 antibodies.

7.3 Conclusion

[0398] The conclusion can be drawn that even doses of alkylated-gp40 protein of 1.5 μg per animal dose of vaccine, can induce levels of anti-gp40 antibodies in colostrum that can passively protect effectively against a severe C. parvum challenge infection.

Legend to the Figures

[0399] FIG. 1

[0400] Amino acid sequence of the Cryptosporidium gp40 protein for the invention, in one-letter IUPAC code, corresponding to SEQ ID NO: 1.

[0401] FIG. 2

[0402] Graphical representation of the measured titres of gp40-specific antibodies, in sera of vaccinated cattle, indicated in Log 2 Elisa units. Details are given in Examples 3 and 4.

[0403] Test groups were: [0404] rgp40His=vaccine of Cryptosporidium gp40 protein that had not been alkylated; [0405] rgp40His+BEI=vaccine of Cryptosporidium gp40 protein comprising one or more aziridine-alkylated amino acids according to the invention; [0406] RC vaccine=Rotavec Corona vaccine [0407] rgp40His+RC=dual vaccinated group, received both Rotavec Corona vaccine, and vaccine of Cryptosporidium gp40 protein that had not been alkylated.

[0408] FIG. 3

[0409] Results of a vaccination-challenge experiment in calves, regarding severity of diarrhoea.

[0410] Horizontal axis presents days after challenge infection.

[0411] Vertical axis indicates the average daily diarrhoea score, for the group receiving rgp40His vaccine, and the group receiving rgp40His+BEI vaccine.

[0412] Experimental details are described in Examples 3 and 4.

[0413] FIG. 4

[0414] Results of mass-spectrometry analysis on Cryptosporidium gp40 protein that was incubated with aziridine. The graph depicts on the horizontal axis the amino acid sequence of gp40 (as presented in SEQ ID NO: 3 herein), and on the vertical axis the number of times a specific amino acid from gp40 was found to be alkylated by the incubation with aziridine. Experimental details are described in Example 6.

[0415] FIG. 5

[0416] Results of the health scoring of the vaccination-challenge experiment using a very low dose of gp40 protein to generate hyperimmune colostrum. On the horizontal axis are days post challenge. The vertical axis presents the health score according to the Wisconsin-Madison scale. Details are described in Example 7.