RU2848644C1 - Conjugated fumonisin for protection against mycotoxicosis - Google Patents

Abstract

FIELD: veterinary medicine and pharmaceuticals.

SUBSTANCE: method for protecting animals from FUM-induced mycotoxicosis, involving the use of conjugated fumonisin (FUM).

EFFECT: technical result is the protection of animals from mycotoxicosis caused by fumonisin.

14 cl, 11 dwg, 7 tbl

Description

LEVEL OF TECHNOLOGY

The invention generally relates to protection against mycotoxicosis caused by mycotoxins. Specifically, the invention relates to protection against mycotoxicosis caused by fumonisin (FUM). Fumonisin is a mycotoxin produced by the fungus Fusarium verticillioides , a common contaminant of corn and corn products, and the closely related fungus Fusarium proliferatum. Aspergillus niger has recently been found to produce fumonisins in grapes, wine, and dried grapes, but only in low concentrations. The term "fumonisin" actually represents a group of at least 15 very closely related mycotoxins classified into four groups (A, B, C, and P), of which fumonisin B1 (FB1) is the most common in animal feed, accounting for 70%-80% of the total fumonisin content and (along with fumonisins B2 and B3) appears to be the major fumonisin due to its toxic properties. It is most important in veterinary medicine as a cause of porcine pulmonary edema, equine leukoencephalomalacia, and liver damage in both horses and pigs. Fumonisin is structurally similar to sphingosine, the major long-chain base of cellular sphingolipids, and has been demonstrated to be a competitive inhibitor of sphinganine (sphingosine) N-acyltransferase (ceramide synthase (CerS). Inhibition of this enzyme by fumonisin results in disruption of sphingolipid metabolism, resulting in a strong increase in sphinganine, a less strong increase in sphingosine, resulting in a change in the sphinganine-to-sphingosine ratio, along with a decrease in the content of complex sphingolipids in animal serum and tissues, which is generally accepted as the mechanism of action for fumonisin toxicity in most species. Clinical signs associated with fumonisin toxicity will vary significantly between species depending on the primary target organ, and safe levels of fumonisin in feed vary widely between species. Diagnosis of fumonisin toxicity depends on the detection Characteristic lesions in affected animals, along with the detection of fumonisin in feed. No specific treatment for fumonisin toxicity in animals has been described, other than removing the source of contaminated grain. In mild cases, clinical signs resolve after discontinuing fumonisin. However, if animals already exhibit neurological symptoms or respiratory distress (particularly pigs), the prognosis is poor.

Preventive treatment of mycotoxicosis caused by FUM is currently limited to good agricultural practices aimed at reducing mycotoxin formation in crops and food and feed control programs to keep mycotoxin levels below certain limits.

Fungi in general cause a wide range of diseases in animals, including parasitic infestations of organs and tissues, as well as allergic reactions. However, in addition to poisoning from inedible mushrooms, fungi can produce mycotoxins and organic chemicals responsible for various toxic effects, known as mycotoxicosis. This disease is caused by exposure to mycotoxins, pharmacologically active compounds produced by filamentous fungi that contaminate food or animal feed. Mycotoxins are secondary metabolites that are not critical to fungal physiology and are extremely toxic to vertebrates at minimal concentrations when ingested, inhaled, or through skin contact. Currently, approximately 400 mycotoxins are known, divided into families of chemically related molecules with similar biological and structural properties. Of these, approximately a dozen groups regularly attract attention as a threat to animal health. Examples of mycotoxins of greatest public concern and agroeconomic importance include aflatoxins (AF), ochratoxins (OT), trichothecenes (T; including deoxynivalenol, abbreviated DON), zearalenone (ZEA), fumonisin (F), tremorgenic toxins, and ergot alkaloids. Mycotoxins are associated with acute and chronic diseases, the biological effects of which vary largely due to the diversity of their chemical structure and to biological, nutritional, and environmental factors. The pathophysiology of mycotoxicosis results from the interaction of mycotoxins with functional molecules and organelles in the animal cell, which can lead to carcinogenicity, genotoxicity, inhibition of protein synthesis, immunosuppression, skin irritation, and other metabolic disturbances. In susceptible animal species, mycotoxins can cause complex and overlapping toxic effects. Mycotoxicoses are not contagious and do not significantly stimulate the immune system. Treatment with medications or antibiotics has little or no effect on the disease. Currently, there is no vaccine for humans or animals to combat mycotoxicosis.

Thus, increasing research is focused on developing vaccines and/or immunotherapies effective against broad classes of fungi as a potent tool in the fight against mycoses—that is, fungal infections per se, rather than toxins—and in the prevention of specific fungal diseases. Unlike mycoses, mycotoxicoses do not require the participation of a toxin-producing fungus and are considered abiotic hazards, despite being biotic in origin. In this sense, mycotoxicosis is considered an example of natural poisoning, and protective strategies primarily focus on preventing exposure. Exposure to humans and animals occurs primarily through the ingestion of mycotoxins in plant-based foods. Metabolism of ingested mycotoxins can lead to accumulation in various organs or tissues; thus, mycotoxins can enter the human food chain through animal meat, milk, or eggs (transferred). Because toxigenic fungi contaminate several types of crops for human and animal consumption, mycotoxins can be present in all types of agricultural raw materials, commodities, and beverages. The Food and Agriculture Organization (FAO) estimates that 25% of the world's food crops are significantly contaminated with mycotoxins. Currently, the best strategies for preventing mycotoxicosis include good agricultural practices aimed at reducing mycotoxin formation in crops and food and feed monitoring programs to ensure mycotoxin levels remain below predetermined thresholds. These strategies can limit the problem of commodity contamination by certain groups of mycotoxins, but with high costs and variable effectiveness. Apart from supportive care (e.g., diet, hydration), there is little treatment for mycotoxin damage, and antidotes against mycotoxins are generally unavailable, although some encouraging results have been obtained in individuals affected by AF with some protective agents such as chlorophyllin, green tea polyphenols, and dithiolethiones (oltipraz).

In the art, specific vaccination strategies against certain mycotoxins have been proposed, mainly for the prevention of mycotoxicosis by contamination of important food products of animal origin, with a strategy based on the production of antibodies that can specifically block the initial absorption or bioactivation of mycotoxins, their toxicity and/or secretion into products of animal origin (such as milk) by immune interception, aimed mainly at the prevention of mycotoxicosis in humans.

However, producing vaccines to protect against mycotoxicosis is a very complex task, primarily due to the fact that mycotoxins themselves are small, non-immunogenic molecules, and the toxicity associated with mycotoxins makes their use as antigens in healthy subjects unsafe. Mycotoxins are small, typically non-protein molecules (haptens) that are not typically immunogenic but can potentially elicit an immune response when attached to a large carrier molecule, such as a protein. Methods for conjugating mycotoxins to a protein or polypeptide carrier and optimizing conditions for animal immunization have been extensively studied to produce monoclonal or polyclonal antibodies with varying specificities for use in immunoassays to screen for mycotoxins in products intended for animal and human consumption. The binding proteins used in these studies included bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), thyroglobulin (TG), and polylysine, among others. In recent decades, considerable effort has been devoted to developing mycotoxin derivatives that can bind to proteins while retaining enough of the original structure to allow the resulting antibodies to recognize the native toxin. These methods have resulted in the availability of antibodies against many mycotoxins, demonstrating that protein conjugation can be an effective tool for antibody production. Application of this strategy to human and animal vaccination, aimed at providing protection while being safe for the recipient, has so far been unsuccessful due to the toxic properties of the molecules that can be released in vivo. For example, conjugation of toxins such as T-2 to a carrier protein has been shown to result in the formation of unstable complexes with the potential release of free toxin in its active form (Chanh et al, Monoclonal anti-idiotype induces protection against the cytotoxicity of the trichothecene mycotoxin T-2 , in J Immunol. 1990, 144: 4721–4728). By analogy with toxoid vaccines, which can confer protection against the pathological effects of bacterial toxins, a reasonable approach to developing mycotoxin vaccines may be based on conjugated "mycotoxoids," defined as modified forms of mycotoxins that lack toxicity while retaining antigenicity (Giovati L et al, "Anaflatoxin B1 as the paradigm of a new class of vaccines based on 'Mycotoxoids' ," in Ann Vaccines Immunization 2(1): 1010, 2015). Given the non-protein nature of mycotoxins, the approach to conversion into mycotoxoids should be based on chemical derivatization. The introduction of specific groups into strategic positions of a related parent mycotoxin can result in molecules with different physicochemical characteristics that are still capable of inducing antibodies with sufficient cross-reactivity to the native toxin. Thus, the general rationale for mycotoxin vaccination should be based on the development of antibodies against mycotoxoids with enhanced binding capacity to the native mycotoxin compared to cellular targets, neutralizing the toxin and preventing disease development upon exposure. The potential application of this strategy has been demonstrated for mycotoxins belonging to the AF group (Giovati et al, 2015), but not for any other mycotoxins. Furthermore, a protective effect has not been demonstrated against mycotoxicosis in the vaccinated animal per se, but only against transfer from dairy cows into their milk, thereby protecting people consuming milk or milk products from mycotoxicosis.

OBJECT OF THE INVENTION

The object of the invention is to create a method for protecting an animal from mycotoxicosis caused by fumonisin, an important mycotoxin in animal feed.

ESSENCE OF THE INVENTION

To achieve the objective of the invention, it was discovered that conjugated fumonisin (FUM) is suitable for use in a method for protecting animals from FUM-induced mycotoxicosis. It was found that there was no need to convert FUM into a toxoid; the conjugated toxin was safe for the treated host animal. Furthermore, it was surprising to find that the immune response induced against a small molecule, such as a mycotoxin, was strong enough to protect the animal itself from mycotoxicosis after ingesting the mycotoxin after treatment. Such effective animal protection by inducing an immune response against the mycotoxin itself has not been demonstrated in the art for any mycotoxin.

Definitions

Mycotoxicosis is a disease resulting from exposure to a mycotoxin. Clinical signs, target organs, and outcome depend on the intrinsic toxic properties of the mycotoxin, the amount and duration of exposure, and the health of the exposed animal.

Protection against mycotoxicosis means preventing or reducing one or more of the negative physiological effects of mycotoxin in an animal, such as decreased average daily weight gain, intestinal damage, liver damage, and kidney damage.

The term fumonisin actually refers to a group of at least 15 closely related mycotoxins classified into four groups designated A, B, C, and P, of which fumonisin B1 (FB1) is the most commonly found in animal feed. Fumonisins are polyhydroxyalkylamines esterified with two carboxylic acids and differ in the presence and position of the free hydroxyl groups. The A-series fumonisins are acetylated at the amino group, while the B-series are free amines. The chemical structure of fumonisin B1 (CAS No. 116355-83-0) is shown below:

Other fumonisins can be obtained using CAS Nos. 116355-84-1, 1422359-85-0, 136379-60-7, etc. The main fumonisin-producing species are Fusarium verticillioides, Fusarium proliferatum, Fusarium fujikuroi, Fusarium globosum, Fusarium nygamai , and Fusarium subglutinans , all of which are included in the Gibberella fujikuroi species complex. Recent studies have shown that some strains of A. niger and A. welwitschiae , as well as Fusarium oxysporum and Alternaria alternata , are also capable of producing fumonisins.

A conjugate molecule is a molecule to which an immunogenic compound is covalently linked. Typically, the immunogenic compound is a large protein, such as KLH, BSA, or OVA.

Adjuvantis a non-specific immunostimulating agent. In principle, any substance that can promote or enhance a specific process in the cascade of immunological events ultimately leading to a superior immunological response (i.e., the body's complex response to an antigen, particularly mediated by lymphocytes and typically including antigen recognition by specific antibodies or presensitized lymphocytes) can be defined as an adjuvant. An adjuvant is generally not required for the specific process to occur, but simply promotes or enhances it. In general, adjuvants can be classified according to the immunological phenomena they elicit. The first class, which includes, among others, ISCOMs (immunostimulating complexes), saponins (or their fractions and derivatives, such as Quil A), aluminum hydroxide, liposomes, cochleates, and polylactic/glycolic acid, facilitates the uptake, transport, and presentation of antigens by APCs (antigen-presenting cells). The second class, including, among others, oil emulsions (W/O, O/W, W/O/W, or O/W/O), gels, polymer microspheres (Carbopol), nonionic block copolymers, and most likely also aluminum hydroxide, provide a depot effect. The third class, including CpG-rich motifs, monophosphoryl lipid A, mycobacteria (muramyl dipeptide), yeast extracts, and cholera toxin, is based on the recognition of conserved microbial structures, the so-called pathogen-associated microbial patterns (PAMPs), defined as signal 0. The fourth class, including, in particular, oil emulsion surfactants, aluminum hydroxide, and hypoxia, is based on stimulating the immune system's ability to distinguish between dangerous and harmless (which does not necessarily have to be the same as self and non-self). The fifth class, which includes cytokines in particular, is based on the positive regulation of costimulatory molecules, signal 2, on the APC.

In the sense of the present invention, a vaccine is a composition suitable for administration to an animal, comprising one or more antigens in an immunologically effective amount (i.e., capable of stimulating the immune system of the target animal sufficiently to at least reduce the negative effects of a challenge with a disease-causing agent), typically in combination with a pharmaceutically acceptable carrier ( i.e. , a biocompatible medium, i.e. , a medium that, upon administration, does not cause significant adverse reactions in the animal subject, capable of presenting the antigen to the immune system of the host animal after administration of the vaccine), such as a liquid containing water and/or any other biocompatible solvent, or a solid carrier such as is commonly used to produce lyophilized vaccines (based on sugars and/or proteins), optionally containing immunostimulatory agents (adjuvants), which, when administered to an animal, induce an immune response to treat a disease or disorder, i.e., help prevent, alleviate, or cure a disease or disorder.

ADDITIONAL VARIATIONS OF THE INVENTION

In another embodiment of the invention, conjugated FUM is administered to an animal systemically. Although topical administration, such as through the gastrointestinal mucosa (oral or anal cavity) or into the eyes (e.g., during immunization of chickens), is known to be an effective way to induce an immune response in various animals, systemic administration has been found to elicit an adequate immune response to protect animals from FUM-induced mycotoxicosis. In particular, it has been found that effective immunization can be achieved by intramuscular, oral, and/or intradermal administration.

The age of administration is not critical, although it is preferable that administration occur before the animal can ingest feed contaminated with significant amounts of FUM. Therefore, the preferred age at administration is 6 weeks or younger. An age of 4 weeks or younger is also preferred, such as 1-3 weeks of age.

In another embodiment of the invention, conjugated FUM is administered to the animal at least twice. Although many animals (particularly pigs, chickens, and ruminants) are generally susceptible to immunization with only one injection of the immunogenic composition, two injections are considered preferable for economically viable protection against FUM. This is because, in practice, the animal's immune system will not be triggered to produce anti-FUM antibodies upon natural exposure to FUM simply because naturally occurring FUM is not immunogenic. Thus, the animal's immune system is entirely dependent on the administration of conjugated FUM. The time between two injections of conjugated FUM can range from 1 week to 1-2 years. It is believed that for young animals, a primary immunization schedule, for example, at 1-3 weeks of age, followed by a booster 1-4 weeks later, typically 1-3 weeks later, for example, 2 weeks later, will be sufficient. Older animals may require booster vaccinations every few months (e.g. 4, 5, 6 months after the last injection) or annually or every two years, as is known from other commercially used animal immunization schedules.

In another embodiment, conjugated FUM is used in a composition containing an adjuvant in addition to the conjugated FUM. An adjuvant can be used if the conjugate alone is unable to induce an immune response to achieve the desired level of protection. Although conjugated molecules are known to sufficiently stimulate the immune system without an additional adjuvant, such as KLH or BSA, the use of an additional adjuvant may be advantageous. This could eliminate the need for a booster dose or extend the interval for its administration. This depends on the level of protection required in a particular situation. A type of adjuvant that has been shown to induce a good immune response against FUM when using conjugated FUM as an immunogen is a water-oil emulsion, such as a water-in-oil emulsion or an oil-in-water emulsion. The first type is usually used in poultry, and the second is usually used in animals that are more prone to local reactions caused by the adjuvant, such as pigs and ruminants.

In another embodiment, the conjugated FUM comprises FUM conjugated to a protein with a molecular weight greater than 10,000 Da. Such proteins, particularly keyhole limpet hemocyanin (KLH) and ovalbumin (OVA), have been found to induce an adequate immune response in animals, particularly pigs and chickens. A practical upper limit for the protein may be 100 MDa.

With regard to protection against mycotoxicosis, it was found, in particular, that when using the invention, the animal is considered to be protected from a decrease in average daily weight gain, pulmonary edema, liver, heart and kidney damage, thus one or more of these signs of mycotoxicosis induced by FUM.

The invention will now be explained with the help of the following examples.

EXAMPLES OF INVENTION

The first series of experiments (see examples 1-4) evaluate whether a conjugated mycotoxin can elicit an active immune response against a mycotoxin, and if so, whether vaccinated animals can be protected from disease caused by this mycotoxin after ingestion. For the latter, a porcine DON challenge model is used. Subsequently (example 5), the use of conjugated FUM in a vaccine is evaluated to induce antibodies against fumonisin in vaccinated animals.

Example 1: Immunization experiment using conjugated DON

Target

The aim of this study is to evaluate the efficacy of conjugated deoxynivalenol in protecting animals from mycotoxicosis caused by DON ingestion. To test this, pigs are immunized twice with DON-KLH before being challenged with toxic DON. Different immunization routes are used to study the effect of administration.

Study design

The study involved 40 1-week-old pigs from 8 sows divided into 5 groups. Twenty-four piglets from groups 1–3 were immunized twice at 1 and 3 weeks of age. Group 1 was immunized intramuscularly (IM) at both ages. Group 2 received an IM injection at 1 week of age and an oral booster dose at 3 weeks of age. Group 3 was immunized intradermally (ID) twice. Beginning at 5.5 weeks of age, groups 1–3 were challenged with DON administered orally in liquids for 4 weeks. Group 4 was not immunized but only challenged with DON as described for groups 1–3. Group 5 served as a control and received only the control liquid, beginning at 5.5 weeks of age for 4 weeks.

The DON concentration in the liquid composition corresponds to 5.4 mg/kg of feed. This corresponds to an average daily intake of 2.5 mg of DON. After four weeks of exposure, all animals undergo postmortem examination, with particular attention to the liver, kidneys, and stomach. Additionally, blood samples are collected on days 0, 34, 41, 49, 55, and 64 (post-euthanasia) of the study, with the exception of Group 5, for which blood samples are collected only on days 0, 34, 49, and immediately after euthanasia.

Tested products

Three different immunogenic compositions are prepared, namely test article 1, containing DON-KLH in an amount of 50 μg/ml in an oil-in-water emulsion for injection (X-solve 50, MSD AH, Boxmeer), which is used for i/m immunization; test article 2, containing DON-KLH in an amount of 50 μg/ml in a water-in-oil emulsion (GNE, MSD AH, Boxmeer), which is used for oral immunization, and test article 3, containing DON-KLH in an amount of 500 μg/ml in an oil-in-water emulsion for injection (X-solve 50) for i/d immunization.

The deoxynivalenol challenge (obtained from Fermentek, Israel) is diluted with 100% methanol to a final concentration of 100 mg/ml and stored at <-15°C. Before use, DON is further diluted and delivered in a treat for administration.

Inclusion criteria

Only healthy animals are used. To exclude unhealthy animals, all animals are examined for their general appearance and the absence of clinical abnormalities or diseases before the study begins. Piglets from different sows are used in each group. In routine practice, all animals should be immunized, even if they were previously exposed to DON through consumption of DON-contaminated feed. Since DON itself does not elicit an immune response, it is believed that there is no fundamental difference between animals previously exposed to DON and those naive to DON.

Results

No adverse effects associated with DON-KLH immunization were observed in any of the animals. Therefore, the composition is safe.

All pigs were serologically negative for anti-DON titers at the beginning of the experiment. During the challenge, the groups immunized intramuscularly (Group 1) and intradermally (Group 3) developed an anti-DON antibody response, measured by ELISA with native DON-BSA as the coating antigen. Table 1 presents the mean IgG values at four time points during the study, along with their SD values. Both intramuscular and intradermal immunizations induced significant anti-DON titers.

Table 1 IgG titers

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

As shown in Table 2, all immunized animals, including those in Group 2 that did not show a significant increase in anti-DON IgG titers, demonstrated significantly greater weight gain during the first 15 days compared to control animals. As for the challenged animals, all animals gained more weight over the course of the study.

Table 2 weight analysis

ADG1 ¹ ADG ² Starting weight Final weight Average additional weight gain compared to control animals (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

¹ average daily weight gain during the first 15 days of challenge

² average daily weight gain over the last 13 days of challenge

Small intestinal health (as measured by the villus/crypt ratio in the jejunum) is also monitored. Table 3 shows the villus/crypt ratio. As can be seen, animals in Group 3 have an average villus/crypt ratio comparable to the healthy control (Group 5), while the unimmunized, challenged group (Group 4) has a significantly lower (statistically significant) villus/crypt ratio. Furthermore, in Group 1 and Group 2, the villus/crypt ratio is significantly better (i.e., higher) compared to the unimmunized, challenged control group. This indicates that immunization protects against DON-induced intestinal damage.

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 CO 0.24 0.22 0.12 0.10 0.23

The general health of other organs, particularly the liver, kidneys, and stomach, is also monitored. All three test groups (Groups 1–3) were found to be in better health than the unimmunized challenge control group (Group 4). Table 4 provides a summary of the general health data. The severity of gastric ulceration is indicated from - (no signs of ulcer formation) to ++ (multiple ulcers). The severity of gastric inflammation is indicated from - (no signs of inflammation) to ++/- (onset of gastric inflammation).

Table 4 General health data

Liver color Stomach ulcer Inflammation of the stomach Kidneys Group 1 Normal-yellow - - Pale Group 2 Normal +/-- - Normal Group 3 Normal +/- +/-- Normal Group 4 Pale ++ ++/- Pale Group 5 Normal + ++/- Normal

Example 2: Effect of immunization on DON levels

Target

The aim of this study is to evaluate the effect of DON conjugate immunization on the toxicokinetics of oral DON. To test this, pigs were immunized twice with DON-KLH before being fed toxic DON.

Study design

The study involved ten three-week-old pigs divided into two groups of five pigs each. Pigs in Group 1 were immunized twice intramuscularly at 3 and 6 weeks of age with DON-KLH (test article 1; example 1). Group 2 served as a control and received only the control liquid. At 11 weeks of age, each animal was administered DON (Fermentek, Israel) via a bolus at a dose of 0.05 mg/kg, which (based on daily feed intake) corresponds to a contaminant level of 1 mg/kg of feed. Blood samples were collected from pigs immediately before DON administration and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after DON administration.

Inclusion criteria

Only healthy animals are used.

Plasma DON Analysis

Unbound DON in plasma is analyzed 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 porcine plasma by this method is 0.1 ng/mL.

Toxicokinetic analysis

Toxicokinetic modeling of the plasma DON concentration-time dependence is performed using non-compartmental analysis (Phoenix, Pharsight Corporation, USA). The following parameters are calculated: area under the curve from zero time to infinity (AUC_0→∞), maximum plasma concentration (C_max) and the time of maximum plasma concentration (t_max).

Results

The toxicokinetic results are presented in Table 5 below. As can be seen, immunization with DON-KLH reduces all toxicokinetic parameters. Since unbound DON is responsible for the toxic effects, it can be concluded that immunization with DON-KLH reduces DON-induced toxic effects by reducing the amount of unbound DON in the animals' blood.

Table 5 Toxicokinetic parameters of unbound DON

Toxicokinetic parameter DON-KLH Control AUC _0→∞ 77.3 ± 23.6 187 ± 33 C _max 12.5 ± 2.7 30.8 ± 2.5 t _max 1.69 ± 1.03 2.19 ± 1.07

Example 3: Serological response to different DON conjugates

Target

The aim of this study is to evaluate the efficacy of different deoxynivalenol conjugated products.

Study design

The study involved eighteen three-week-old pigs, divided into three groups of six pigs each. Group 1 pigs were immunized twice intramuscularly at 3 and 5 weeks of age with DON-KLH (using test article 1 from Example 1). Group 2 was immunized with DON-OVA. Group 3 served as a negative control. All animals were tested for anti-DON IgG responses at 3, 5, and 8 weeks of age.

Results

Serological results are shown in the table below as log2 antibody titer.

Table 6. anti-DON IgG response

Tested product 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

Both conjugates appear to be effective in enhancing the anti-DON IgG response. Furthermore, the response appears to be elicited by a single administration.

Example 4 Serological response in chickens

Target

The aim of this study is to evaluate the serological response of DON-KLH in chickens.

Study design

This study utilized 30 four-week-old chickens, divided into three groups of 10 chickens each. The chickens were immunized intramuscularly with DON-KLH. Group 1 served as a control and received PBS alone. Group 2 received DON-KLH without any adjuvant, and Group 3 received DON-KLH formulated with GNE adjuvant (available from MSD Animal Health, Boxmeer). Primary immunization was administered on day 0 with 0.5 ml of vaccine in the right leg. On day 14, the chickens received a comparable booster immunization in the left leg.

Blood samples are collected on days 0 and 14, as well as on days 35, 56, 70, and 84. Serum is isolated for the determination of IgY against DON. On days 0 and 14, blood samples are collected immediately before immunization.

Results

Serological results are presented in Table 7 as log2 antibody titers. PBS background has been subtracted from the data.

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

As can be seen, conjugated DON also induces anti-DON titers in chickens. The GNE adjuvant significantly increases the response, but does not appear to be essential for achieving a net response.

Example 5: Serological response to FUM conjugate in pigs

Target

The aim of this experiment is to evaluate whether the use of conjugated FUM in a vaccine can induce anti-fumonisin antibodies in the vaccinated animal.

Study design

For this purpose, a vaccine containing fumonisin B1 conjugated with keyhole limpet hemocyanin (FUM-KLH) is used. The conjugate is mixed with an adjuvant in the form of an oil-in-water emulsion (XSolve 50, MSD Animal Health, The Netherlands). at a final concentration of 50 mcg/ml for intramuscular (i.m.) administration or 500 mcg/ml for intradermal (i.d.) administration.

The DON vaccine, as described above, was also used in the experiment as a positive control. Vaccines containing other conjugated mycotoxins were also formulated and used. Specifically, zearalenone (ZEA) conjugated to keyhole limpet hemocyanin (ZEA-KLH) and T-2 mycotoxin (T2 toxin) conjugated to KLH (T2-KLH) were included in the vaccines. The conjugates are mixed with an oil-in-water emulsion adjuvant (XSolve) as above at a final concentration of 50 μg/mL for intramuscular (i.m.) administration or 500 μg/mL for intradermal (i.d.) administration for ZEA-KLH and DON-KLH and 115 (i.m.) or 1150 μg/mL (i.d.) for T2-KLH, respectively.

In the experiment, 6 groups of 5 animals (pigs) at the age of three weeks are used for vaccination. Group 1 receives 0.2 ml of FUM-KLH twice intradermally, group 2 receives 0.2 ml of ZEA-KLH twice, group 3 is vaccinated with 2.0 ml of DON-KLH intramuscularly in X-Solve 50 twice, group 4 receives 2.0 ml of FUM-KLH intramuscularly twice, group 5 receives 2.0 ml of ZEA-KLH twice intramuscularly and, finally, group 6 is vaccinated with 2.0 ml of T2-KLH intramuscularly twice. There is a control group of three piglets, where the control group does not receive vaccination. Primary vaccination is carried out at the age of three weeks, and booster vaccination at the age of five weeks. Animals are observed for 14 weeks after the start of the study.

Results

All pigs were serologically negative for titers against FUM, ZEA, T2, and DON at the beginning of the experiment, and antibody titers developed in all vaccinated groups. The resulting log2 titers are presented in Table 8 below.

Table 8 IgG titers

Group T=0 T=28 T=42 T=56 T=70 T=84 T=91 1 <3.3 12.2 11.1 9.9 8.5 7.1 6.7 2 <4.3 10.1 8.8 8.6 6.7 6.0 5.4 3 <4.3 10.5 9.5 8.5 7.6 6.5 6.6 4 <3.3 15.4 14.7 13.1 12.6 10.6 10.1 5 <4.3 12 10.9 11.5 8.8 8.1 8.0 6 <3.3 13.5 12.6 11.4 10.3 9.1 8.9 FUM control <3.3 <3.3 <3.3 <3.3 <3.3 <3.3 <3.3 ZEA control <4.3 <4.3 <4.3 <4.3 <4.3 <4.3 <4.3 T2 control <3.3 <3.3 <3.3 <3.3 <3.3 <3.3 <3.3 DON control <4.3 <4.3 <4.3 <4.3 <4.3 <4.3 <4.3

As can be seen, antibodies can be produced at high levels against each of the conjugated mycotoxins. This confirms that the vaccine can be effectively used against the corresponding mycotoxicosis, as demonstrated above for DON-induced mycotoxicosis.

Example 6: Serological response to FUM conjugate in chickens

Target

The aim of this experiment is to evaluate whether the use of conjugated FUM in a vaccine can induce anti-fumonisin antibodies in chickens.

Study design

For this purpose, a vaccine containing fumonisin B1 conjugated to keyhole limpet hemocyanin (FUM-KLH) is used in accordance with Example 5. The conjugate is mixed with an oil emulsion adjuvant using the same mineral oil used in Example 5, or alternatively, in a comparable non-mineral oil emulsion, both at a final concentration of 50 μg/ml.

The study involved a group of 15 chickens. Three groups of five animals were used. Group 1 served as a negative control and was administered a PBS solution. Group 2 was vaccinated with FUM-KLH mixed with a mineral oil-containing adjuvant. Group 3 was vaccinated with a mineral oil-free adjuvant. Chickens were vaccinated intramuscularly with 0.5 ml of vaccine at T=8 and T=22 (birds were included in the study at T=0 for acclimatization).

Results

All chickens were serologically negative for FUM titers at the start of the experiment, and antibody titers were developed in all vaccinated groups. The resulting log2 titers are presented in Table 9 below. As can be seen, antibodies to conjugated fumonisin were produced at high levels in both groups. This supports the general understanding that the type of adjuvant is not essential for enhancing an adequate immune response per se.

Table 9 Antibody titers against FUM in chickens

Group T=8 T=22 T=36 T=50 T=71 1 PBS <6.1 6.1 6.8 6.6 6.7 2 Mineral oil FUM-KLH <6.1 14.7 16.0 15.8 15.0 3 Non-mineral oil FUM-KLH <6.1 17.8 16.9 15.8 14.3

Example 7: Protection against FUM infection in pigs

Target

The aim of this experiment is to evaluate whether the use of conjugated FUM in a vaccine can induce protection against fumonisin challenge in pigs.

Study design

For this purpose, the same vaccines containing fumonisin B1 conjugated to keyhole limpet hemocyanin (FUM-KLH) are used in two different adjuvants, one based on mineral oil and the other based on non-mineral oil, as described in Example 6. A group of 24 pigs is used in the study. The first group of 8 piglets is vaccinated with FUM-KLH, while the first subgroup of 4 animals receives a vaccine based on mineral oil containing an adjuvant, and the second subgroup receives an alternative vaccine. Both vaccines are administered intramuscularly in an amount of 2 ml at a concentration of 50 μg/ml. Animals are primarily vaccinated at the age of 7-12 days (T = 0), revaccinated at the age of 21-26 days (T = 14). Group 2 is not vaccinated, but is challenged with fumonisin B1 and used as a positive control. Group 3 is neither vaccinated nor challenged and serves as a negative control. Sixteen challenged piglets (Groups 1 and 2) approximately 5.5 weeks old receive 13 mg/kg FUM daily for four consecutive weeks, corresponding to 5.99 mg/day. FUM is administered in a liquid formulation: piglets receive 2.41 mg FUM/day in 16 ml of liquid during the first week, 5.0 mg/day in 32 ml of liquid during the second week, 7.2 mg/day in 45 ml of liquid during the third week, and 9.3 mg FUM/day in 60 ml of liquid during the fourth week. Antibody titers are monitored over time. At the end of the study, the liver, lungs, kidneys, and intestines are assessed.

Results

All piglets were serologically negative for FUM titers at the beginning of the experiment. During the challenge, FUM-KLH-vaccinated piglets developed an anti-FUM antibody response, as shown in Table 10, which shows IgG values at six time points during the study.

Table 10 IgG titers against FUM in pigs

Group T=0 T=28 T=33 T=40 T=47 T=55 1a Mineral oil FUM-KLH <3.3 15.8 15.4 14.5 13.7 12.7 1b Non-mineral oil FUM-KLH <3.3 16.9 16.4 15.4 14.5 13.6 2 Positive control <3.3 <3.3 <3.3 <3.3 <3.3 <3.3 3 Negative control <3.3 <3.3 <3.3 <3.3 <3.3 <3.3

All vaccinated animals demonstrated improved growth during the challenge compared to unvaccinated control animals, resulting in growth comparable to or greater than that of healthy control animals, as determined by measuring the percentage of growth per piglet compared to the initial weight during the challenge. Furthermore, vaccinated animals demonstrated better health when examining the liver, kidneys, and intestines.

Table 11 shows the proportion of animals in the group with a % weight gain during the challenge compared to the initial challenge weight. Additionally, the percentage of animals with damage to a specific organ is shown. All this demonstrates that conjugated fumonisin can be successfully used to protect animals from FUM-induced mycotoxicosis.

Table 11 Piglet weight and organ scores

Group Weight gain Damage to the jejunum Liver damage Kidney damage 1a 305% 25 25 100 1b 316% 0 50 75 2 288% 62.5 87.5 100 3 306% 12.5 0 62.5

Claims (14)

1. A method for protecting an animal from mycotoxicosis induced by FUM, including the use of conjugated fumonisin (FUM). 2. The method according to claim 1 for protecting an animal from one or more clinical signs of FUM-induced mycotoxicosis, wherein said clinical signs of FUM-induced mycotoxicosis are selected from the group consisting of decreased weight gain, intestinal damage, liver damage, and kidney damage. 3. The method according to paragraph 1 or 2, characterized in that in this method the conjugated FUM is systemically administered to the animal. 4. The method according to paragraph 3, characterized in that in the method the conjugated FUM is administered intramuscularly, orally and/or intradermally. 5. The method according to any one of claims 1 to 4, characterized in that in this method the conjugated FUM is administered to an animal aged 6 weeks or younger. 6. The method according to claim 5, characterized in that in this method the conjugated FUM is administered to an animal aged 4 weeks or younger. 7. The method according to claim 6, characterized in that in this method the conjugated FUM is administered to an animal aged 1-3 weeks. 8. The method according to any of the preceding claims, characterized in that in this method the conjugated FUM is administered to the animal at least twice. 9. The method according to any of the preceding claims, characterized in that in the method the conjugated FUM is used in a composition containing an adjuvant in addition to the conjugated FUM. 10. The method according to claim 9, characterized in that in this method the adjuvant is an emulsion of water and oil. 11. The method according to claim 9, characterized in that in this method the adjuvant is a water-in-oil emulsion or an oil-in-water emulsion. 12. The method according to any of the preceding claims, characterized in that in this method the conjugated FUM comprises FUM conjugated to a protein having a molecular weight greater than 10,000 Da. 13. The method according to claim 11, characterized in that in this method the conjugated FUM comprises FUM conjugated with keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). 14. The method according to any of the preceding claims, characterized in that the animal is a pig or a chicken.