CONJUGATED AFLATOXIN B TO PROTECT AGAINST MYCOTOXICOSIS

Abstract

The present invention pertains to the use of conjugated aflatoxin (AFB) in a method to protect an animal against AFB induced mycotoxicosis, in particular to protect against a decrease in average daily weight gain, immune suppression, icterus, hemorrhagic enteritis as a result of the ingestion of AFB.

Claims

1. A method of actively protecting an animal against aflatoxin B (AFB) induced mycotoxicosis comprising administering to the animal a conjugated AFB.

2. The method according to claim 1, wherein the conjugated AFB is systemically administered to the animal.

3. The method according to claim 2, wherein the conjugated AFB is administered intramuscularly, orally and/or intradermally.

4. The method according to claim 1, wherein the conjugated AFB is administered to the animal at an age of 6 weeks or younger.

5. The method according to claim 4, wherein the conjugated AFB is administered to the animal at an age of 4 weeks or younger.

6. The method according to claim 5, wherein the conjugated AFB is administered to the animal at an age of 1-3 weeks.

7. The method according to claim 1 wherein the conjugated AFB is administered to the animal at least twice.

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. The method according to claim 1, characterised in that the animal is a swine or chicken.

14. A vaccine comprising conjugated AFB, an adjuvant and a pharmaceutically acceptable carrier.

15. The vaccine of claim 14, wherein the adjuvant is an emulsion of water and oil.

16. The vaccine of claim 15, wherein the adjuvant is a water-in-oil emulsion or an oil-in-water emulsion.

17. The vaccine of claim 14, wherein the conjugated AFB comprises AFB conjugated to a protein having a molecular mass above 10.000 Da.

18. The vaccine of claim 14, wherein the conjugated AFB comprises AFB conjugated to keyhole limpet hemocyanin (KLH) or ovalbumin (OVA).

Description

EXAMPLES OF THE INVENTION

[0025] In a first series of experiments (see Examples 1-4) it was assessed whether an active immune response against a mycotoxin can be elicited using a conjugated mycotoxin, and if so, is able to protect the vaccinated animal against a disorder induced by this mycotoxin after ingestion thereof. For the latter a pig model for challenge with DON was used. Thereafter (Examples 5 and 6) it was assessed whether or not the use of conjugated AFB in a vaccine can induce antibodies against aflatoxin in the vaccinated animal.

Example 1: Immunisation Challenge Experiment Using Conjugated DON

[0026] Objective

[0027] The objective of this study was to evaluate the efficacy of conjugated deoxynivalenol to protect an animal against mycotoxicosis due to DON ingestion. To examine this, pigs were immunised twice with DON-KLH before being challenged with toxic DON. Different routes of immunisation were used to study the influence of the route of administration.

[0028] Study Design

[0029] Fourty 1 week old pigs derived from 8 sows were used in the study, divided over 5 groups. Twenty-four piglets of group 1-3 were immunised twice at 1 and 3 weeks of age. Group 1 was immunised intramuscularly (IM) at both ages. Group 2 received an IM injection at one week of age and an oral boost at three weeks of age. Group 3 was immunised intradermally (ID) two times. From 5% weeks of age groups 1-3 were challenged during 4 weeks with DON administered orally in a liquid. Group 4 was not immunised but was only challenged with DON as described for groups 1-3. Group 5 served as a control and only received a control fluid, from the age of 5.5 weeks for 4 weeks.

[0030] The DON concentration in the liquid formulation corresponded to an amount of 5.4 mg/kg feed. This corresponds to an average amount of 2.5 mg DON per day. After four weeks of challenge all animals were post-mortem investigated, with special attentions for the liver, kidneys and the stomach. In addition, blood sampling was done at day 0, 34, 41, 49, 55, 64 (after euthanasia) of the study, except for group 5 of which blood samples were taken only at day 0, 34, 49, and directly after euthanasia.

[0031] Test Articles

[0032] Three different immunogenic compositions were formulated, namely Test Article 1 comprising DON-KLH at 50 g/ml in an oil-in-water emulsion for injection (X-solve 50, MSD AH, Boxmeer) which was used for IM immunization; Test Article 2 comprising DON-KLH at 50 g/ml in a water-in-oil emulsion (GNE, MSD AH, Boxmeer) which was used for oral immunization and Test Article 3 comprising DON-KLH at 500 g/ml in an oil-in-water emulsion for injection (X-solve 50) for ID immunisation.

[0033] The challenge deoxynivalenol (obtained from Fermentek, Israel) was diluted in 100% methanol at a final concentration of 100 mg/ml and stored at <15 C. Prior to usage, DON was further diluted and supplied in a treat for administration.

[0034] Inclusion Criteria

[0035] Only healthy animals were used. In order to exclude unhealthy animals, all animals were examined before the start of the study for their general physical appearance and absence of clinical abnormalities or disease. Per group piglets from different sows were used. In everyday practice all animals will be immunised even when pre-exposed to DON via intake of DON contaminated feed. Since DON as such does not raise an immune response, it is believed that there is no principle difference between animals pre-exposed to DON and nave with respect to DON.

[0036] Results

[0037] None of the animals had negative effects associated with the immunisation with DON-KLH. The composition thus appeared to be safe.

[0038] All pigs were serologically negative for titres against DON at the start of the experiment, During the challenge the groups immunised intramuscular (Group 1) and intradermally (Group 3) developed antibody responses against DON as measured by ELISA with native DON-BSA as the coating antigen. Table 1 depicts the average IgG values on 4 time points during the study with their SD values. Both Intramuscular immunisation and Intradermal immunisation induced significant titres against DON.

TABLE-US-00001 TABLE 1 IgG titres group 1 group 2 group 3 group 4 Group 5 T = 0 <4.3 <4.3 <4.3 <4.3 <4.3 T = 35 11.2 4.86 9.99 4.3 4.19 T = 49 9.56 4.64 8.81 4.71 3.97 T = 64 8.48 4.3 7.56 4.3 3.31

[0039] As depicted in Table 2 all immunised animals, including the animals in Group 2 that showed no significant anti-DON IgG titre increase, showed a significant higher weight gain during the first 15 days compared to the challenge animals. With respect to the challenged animals, all animals gained more weight over the course of the study.

TABLE-US-00002 TABLE 2 weight analysis Average additional weight gain compared weight weight to challenge animals ADG1.sup.1 ADG.sup.2 begin end (grams) group 1 0.67 0.80 11.63 32.29 +1060 group 2 0.64 0.79 12.31 32.13 +760 group 3 0.58 0.82 12.88 32.25 +310 group 4 0.54 0.81 12.69 31.75 0 group 5 0.57 0.80 11.63 31.08 +390 .sup.1average daily weight gain over the first 15 days of the challenge .sup.2average daily weight gain over the last 13 days of the challenge

[0040] The condition of the small intestines (as determined by the villus/crypt ratio in the jejunum) was also monitored. In table 3 the villus/crypt ratio is depicted. As can be seen, the animals in group 3 had an average villus crypt/crypt ratio comparable to the healthy controls (group 5), while the non-immunised, challenged group (group 4) had a much lower (statistically significant) villus crypt ratio. In addition, group 1 and group 2, had a villus/crypt ratio which was significantly better (i.e. higher) compared to the non-immunised challenge control group. This indicates that the immunisation protects against the damage of the intestine, initiated by DON.

TABLE-US-00003 TABLE 3 villus/crypt ratio group 1 group 2 group 3 group 4 group 5 average 1.57 1.41 1.78 1.09 1.71 STD 0.24 0.22 0.12 0.10 0.23

[0041] The general condition of other organs was also monitored, more specifically the liver, the kidneys and the stomach. It was observed that all three test groups (groups 1-3) were in better health than the non-immunised challenge control group (group 4). In table 4 a summary of the general health data is depicted. The degree of stomach ulcer is reported from (no prove of ulcer formation) to ++(multiple ulcers). The degree of stomach inflammation is reported from (no prove of inflammation) to ++/(initiation of stomach inflammation).

TABLE-US-00004 TABLE 4 General health data Stomach Liver colour Stomach ulcer inflammation Kidneys Group 1 Normal-yellow Pail Group 2 Normal +/ Normal Group 3 Normal +/ +/ Normal Group 4 Pail ++ ++/ Pail Group 5 Normal + ++/ Normal

Example 2: Effect of Immunisation on DON Levels

[0042] Objective

[0043] The objective of this study was to evaluate the effects of immunization with a DON conjugate on the toxicokinetics of DON ingestion. To examine this, pigs were immunised twice with DON-KLH before being fed toxic DON.

[0044] Study Design

[0045] Ten 3 week old pigs were used in the study, divided over 2 groups of 5 pigs each. The pigs in Group 1 were immunised IM twice at 3 and 6 weeks of age with DON-KLH (Test Article 1; example1). Group 2 served as a control and only received a control fluid. At the age of 11 weeks the animals were each administered DON (Fermentek, Israel) via a bolus at a dose of 0.05 mg/kg which (based on the daily feed intake) resembled a contamination level of 1 mg/kg feed. Blood samples of the pigs were taken juts before DON administration and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 h post DON administration.

[0046] Inclusion Criteria

[0047] Only healthy animals were used.

[0048] Analysis of DON in Plasma

[0049] Plasma analysis of unbound DON was done using a validated LC-MS/MS method on an Acquity UPLC system coupled to a Xevo TQ-S MS instrument (Waters, Zellik, Belgium). The lower limit of quantification of DON in pig plasma using this method is 0.1 ng/ml.

[0050] Toxicokinetic Analysis

[0051] Toxicokinetic modeling of the plasma concentration-time profiles of DON was done by noncompartmental analysis (Phoenix, Pharsight Corporation, USA). Following parameters were calculated: area under the curve from time zero to infinite (AUC.sub.0.fwdarw.), maximal plasma concentration (C.sub.max), and time at maximal plasma concentration (t.sub.max).

[0052] Results

[0053] The toxicokinetic results are indicated in table 5 here beneath. As can be seen immunisation with DON-KLH decreases all toxicokinetic parameters. As it is unbound DON that is responsible for the exertion of toxic effects, it may be concluded that immunisation with DON-KLH will reduce the toxic effects caused by DON by reducing the amount of unbound DON in the blood of animals.

TABLE-US-00005 TABLE 5 Toxicokinetic parameters of unbound DON Toxicokinetic parameter DON-KLH Control AUC.sub.0.fwdarw. 77.3 23.6 187 33 C.sub.max 12.5 2.7 30.8 2.5 t.sub.max 1.69 1.03 2.19 1.07

Example 3: Serological Response Against Various DON Conjugates

[0054] Objective

[0055] The objective of this study was to evaluate the efficacy of different conjugated deoxynivalenol products.

[0056] Study Design

[0057] Eighteen 3 week old pigs were used in the study, divided over 3 groups of six pigs each. The pigs of group 1 were immunised twice intramuscularly at 3 and 5 weeks of age with DON-KLH (using Test Article 1 of Example 1). Group 2 was immunised correspondingly with DON-OVA. Group 3 served as a negative control. All animals were checked for an anti-DON IgG response at 3 weeks of age, 5 weeks of age and 8 weeks of age.

[0058] Results

[0059] The serological results are indicated here below in the table in log 2 antibody titre.

TABLE-US-00006 TABLE 6 anti-DON IgG response Test Article 3 weeks 5 weeks 8 weeks DON-KLH 3.5 6.6 8.3 DON-OVA 3.3 3.9 11.8 Control 4.8 3.3 3.3

[0060] It appears that both conjugates are suitable to raise an anti-DON IgG response. Also, a response appears be induced by one shot only.

Example 4: Serological Response Against DON Conjugate in Chickens

[0061] Objective

[0062] The objective of this study was to evaluate the serological response of DON-KLH in chickens.

[0063] Study Design

[0064] For this study 30 four week-old chickens were used, divided over three groups of 10 chickens each. The chickens were immunized intramuscularly with DON-KLH. Group 1 was used as a control and received PBS only. Group 2 received DON-KLH without any adjuvant and group 3 received DON-KLH formulated in GNE adjuvant (available from MSD Animal Health, Boxmeer). A prime immunization was given on day 0 with 0.5 ml vaccine into right leg. On day 14, chickens received a comparable booster immunization into the left leg.

[0065] Blood sampling took place at day 0 and 14, as well as on day 35, 56, 70 and 84. Serum was isolated for the determination of IgY against DON. At day 0 and 14 blood samples were isolated just before immunisation.

[0066] Results

[0067] The serological results are depicted in table 7 in log 2 antibody titre. The PBS background has been subtracted from the data.

TABLE-US-00007 TABLE 7 anti-DON IgY response Vaccine Day 0 Day 14 Day 35 Day 56 Day 70 Day 84 DON-KLH 0 0 0.6 1.2 1.1 1.2 DON-KLH in GNE 0 1.9 6.5 6.0 6.7 7.7

[0068] As can be seen, the conjugated DON also induces an anti-DON titre in chickens. GNE adjuvant increases the response substantially but appears to be not essential for obtaining a net response as such.

Example 5: Serological Response Against AFB Conjugate in Swine

[0069] Objective

[0070] The aim of this experiment was to assess whether or not the use of conjugated AFB in a vaccine can induce antibodies against aflatoxin in vaccinated swine.

[0071] Study Design

[0072] The vaccine contained Aflatoxin 1 (AFB1) conjugated to Bovine Serum Albumin (BSA). The conjugate was mixed with a mineral oil-containing adjuvant (XSolve 50) at a final concentration of 50 g/ml, and applied by intramuscular (IM) administration. In the experiment, 2 groups of 6 animals were used at three weeks of age. Group one received a PCV vaccine, Porcilis PCV (as a negative control) and Group 2 the AFB1-BSA vaccine. All primes were at three weeks of age and the boosters were at seven weeks of age. The animals were monitored for 12 weeks after start of the study.

[0073] Results

[0074] All pigs were serologically negative for titres against AFB1 at the start of the experiment (i.e. a titre of 3.5 or below). Titres developed as indicated here below In Table 8.

TABLE-US-00008 TABLE 8 IgG titres against AFB1 Group T = 7 weeks T = 10 weeks T = 12 weeks 1 3.5 3.7 4.6 2 3.7 8.1 6.8

[0075] There appeared to be a slight titre increase in the negative control group, possibly induced by AFB1 in the feed. The titres in the conjugated AFB group however rose significantly stronger, showing a good induction of an immune response against AFB in these swine.

Example 6: Serological Response to AFB1 in Chickens

[0076] Objective

[0077] The objective of this study was to evaluate the serological response to AFB1-KLH in chickens.

[0078] Study Design

[0079] For this study 30 four week-old chickens were used, divided over three groups of 10 chickens each. The chickens were immunized intramuscularly with AFB1-KLH. Group 1 was used as a control and received PBS only. Group 2 received AFB1-KLH without any adjuvant and group 3 received AFB1-KLH formulated in GNE adjuvant (available from MSD Animal Health, Boxmeer). A prime immunization was given on day 0 with 0.5 ml vaccine into right leg. On day 14, chickens received a comparable booster immunization into the left leg.

[0080] Blood sampling took place at day 0 and 14, as well as on day 35, 56, 70 and 84. Serum was isolated for the determination of IgY against AFB1. At day 0 and 14 blood samples were isolated just before immunisation.

[0081] Results

[0082] The serological results are depicted in table 9 in log 2 antibody titre. The PBS background has been subtracted from the data.

TABLE-US-00009 TABLE 9 Anti-AFB1 IgY response Vaccine Day 0 Day 14 Day 35 Day 56 Day 70 Day 84 AFB1-KLH 0.0 0.9 1.9 0.2 0.4 0.6 AFB1-KLH in GNE 0.0 5.5 5.8 4.3 4.0 4.4

[0083] As can be seen, the conjugated AFB1 induces an anti-AFB1 titre in chickens. GNE adjuvant increases the response substantially but appears to be not essential for obtaining a net response as such.

Example 7: Protective Effect of AFB1 Vaccination

[0084] An in vitro potency test was performed to establish the protective effect of the serum of vaccinated animals. In this test C6 cells (glioma cell line from rats) were used, and the CCK8 (Cell counting kit 8; Dojindo Laboratories) was used to measure the viability of the cells as a response to aflatoxin. For this test it was established, via a dose response curve, that Aflatoxin 1 is toxic on these cells starting from a concentration of 20 g/ml. In the assay as described here beneath, a concentration of 80 g/ml was selected to assess whether reduction of the toxic effect could be accomplished by the use of serum of vaccinated animals.

[0085] For this assay C6 cells were seeded in a 96 plate, and the wells (all with the same density form the same stock) were incubated with 80 g/ml Aflatoxin, alone, in combination with the serum of pigs vaccinated with Aflatoxin B1-KLH conjugate or in combination with serum from sero-negative animals for AFB1. All the sera were heat inactivated at 56 C. for one hour. It was observed that the positive serum incubations resulted in a higher OD450 value (corresponding to the presence of live cells) compared to the negative serum. This shows that the positive serum increased the viability of the cells and thus, that the AFB1 antibodies raised in the pigs by vaccination are able to neutralise the toxicity of AFB1 on the cells.

Example 8: Serological Response to AFB1 in Fish

[0086] Objective

[0087] The objective of this study was to evaluate the serological response to AFB1-KLH in fish (Tilapia; Oreochromis sp).

[0088] Study Design

[0089] For the experiment a total of 100 tilapia fish were used (weighing on average 20 g), divided into two groups of fifty fish. The first group was injected intraperitoneally (IP) with 0.05 ml an AFB1-KLH mycotoxin vaccines in GNE adjuvant. The AFB1-KLH was present at a final concentration of 12.5 g per dose. The second group was injected with standard vaccine dilution buffer (SVDB) to serve as a negative control. The fish were observed for three weeks and were then given a booster immunisation using the same vaccines as used for primary vaccination. All vaccinated fish were observed for another 2 weeks before blood sampling.

[0090] Results

[0091] The serological results are depicted in table 9 in log 2 antibody titre.

TABLE-US-00010 TABLE 9 Anti-AFB1 IgM response Vaccine T = 2 weeks after booster AFB1-KLH 16.6 Neg. control <1.7

[0092] As can be seen, the conjugated AFB1 induces an anti-AFB1 titre in the fish. In view of the results of example 7, it is believed that the fish are protected against an AFB1 challenge.

Example 9: Protective Effect of AFB1 Vaccination

[0093] In line with Example 7, an in vitro potency test/neutralization assay was performed with the sera raised as described in Example 8. For this, C6 cells were seeded at 2.0*10.sup.4 cells/ml, 100 l per well, and grown at 37 C., 5% CO.sub.2 for 3 days. Cells were incubated for 48 h, with a combination of 20 g/ml AFB1 and 32 diluted serum derived from fish vaccinated with AFB1-KLH in GNE or vaccinated with a placebo. Percentages of live cells were determined by microscopical evaluation.

[0094] Serum from fish vaccinated with AFB1-KLH in GNE contains antibodies against AFB1 (see Example 8) and appeared to be protected against AFB1 damage in C6 cells. Serum from fish vaccinated with placebo did not contain antibodies against AFB1 (see Example 8) and did not protect against AFB1 damage.

TABLE-US-00011 TABLE 10 Percentage live cells after incubation with AFB1 and diluted fish serum Positive serum Negative serum AFB1 95 65