Conjugated T-2 toxin for protection against mycotoxin infections

Abstract

The present invention relates to the use of conjugated T-2 toxin (T2) in a method for protecting animals from T2-induced mycotoxinosis, in particular for protecting against one or more of the symptoms of reduced average daily weight gain, intestinal damage, skin damage and nasal damage, and thus mycotoxinosis induced by T2 as a result of ingestion of T2.

Description

Technical Field

[0001] The present invention generally relates to protection against mycotoxicosis induced by mycotoxins. In particular, the present invention relates to protection against mycotoxicosis induced by T-2 toxin (type A trichothecene-2 toxin or T2).

Background Art

[0002] Mycotoxins are generally highly diverse secondary metabolites produced in nature by a wide variety of fungi, causing food contamination and resulting in mycotoxicosis in animals and humans. In particular, trichothecene mycotoxins produced by the genus Fusarium are of greater agricultural importance worldwide due to the potential health hazards they cause. These are mainly metabolized and excreted after ingestion, giving rise to over 20 metabolites, with hydroxytrichothecene-2 toxin being the major metabolite. Trichothecenes are deleteriously toxic due to their further potential for local absorption, and their metabolites affect the gastrointestinal tract, skin, kidneys, liver, as well as the immune and hematopoietic progenitor cell lines. Sensitivity to this type of toxin varies from dairy cows to pigs, and the most sensitive endpoints are neurological, reproductive, immunological and hematological effects. The mechanism of action mainly consists of inhibition of protein synthesis and oxidative damage to cells, followed by disruption of nucleic acid synthesis and subsequent apoptosis. Along with regulatory guidelines and recommendations regarding trichothecene mycotoxins, the possible risks, historical significance, toxicokinetics, and genotoxic and cytotoxic effects are generally known.

[0003] T-2 toxin is mainly found in grains such as wheat, corn, barley, rice, soybeans, etc., especially oats and their products. The fungal proliferation and production of T-2 are enhanced in developing countries around the world due to tropical conditions such as high temperature and moisture levels, monsoons, irregular rainfall at harvest time and flash floods. The production of T-2 is enhanced by factors such as the humidity of the substrate, relative humidity, temperature and oxygen availability.

[0004] T-2 is readily absorbed through various routes, including topical, oral, and inhalation. It is claimed to be 400 times more toxic than sulfur mustard as a skin irritant and blister-forming agent. Respiratory ingestion of the toxin demonstrates activity comparable to that of mustard or lewisite. T-2 mycotoxins are unique in that systemic toxicity can arise from any route of exposure, namely skin, oral, or respiratory.

[0005] The toxicity and adverse effects of T-2 vary based on numerous factors, including the animal's age, sex, and overall health, along with the route of administration; exposure time and amount; the dose administered; and the presence of any other mycotoxins. Poisoning often occurs after feeding with feed made from grains, hay, and straw, when animals overwinter outdoors and are contaminated with F. sporotrichiella and F. poae. Exemplary symptoms of T-2-induced mycotoxosis include vomiting, vomiting, skin blistering, loss of appetite, and weight loss.

[0006] Ruminants are known to be relatively resistant to T-2 toxin compared to monogastric animals. In poultry, T-2 toxin has been the causative agent of oral and intestinal lesions, in addition to impaired immune response, disruption of the hematopoietic system, decreased egg production, thinning of eggshells, rejection of feed, weight loss and alteration of feather patterns, abnormal wing positioning, and hissteroid seizures or impaired righting reflexes [49,50]. Poultry have been reported to be relatively less susceptible to trichothecenes than pigs. In pigs, some necrosis is established in the nasal, lip and tongue, gastric mucosa edema and mucosal coating, along with serous hemorrhagic necrotizing ulcerative inflammation of the gastrointestinal tract, and swelling of the head region, especially around the eyelids and larynx, sometimes with paresis or even paralysis. The toxic effects of T-2 toxin usually manifest as foodborne toxic aleukemia (ATA). Symptoms include vomiting, diarrhea, leukopenia, hemorrhage, shock and death. Acute toxic effects are also characterized by massive bleeding along the liver's serosal membrane, as well as the intestines, stomach, and esophagus.

[0007] In fact, the prospects for trichothecenes as potential risk factors, decontamination strategies, and future outlooks are comprehensively described in this field. Regarding treatment for T-2-induced mycotoxin disease, this is primarily limited to detection strategies concerning maximum permissible limits in feed and food stocks. Nevertheless, its presence may prove toxic. Currently, treatment of T-2 toxins for inducible injury mainly emphasizes the use of natural substances, probiotics, and amino acids, and the search for a precise antidote to the toxin continues to this day. Therefore, strict regulations and quarantine activities are in place to prevent large-scale unplanned exposures. While it has been mentioned that target animals can be vaccinated against T-2 (see, for example, Manohar V. et al., “Final Report: Development Of Vaccines To The Mycotoxin T-2”, Borriston Laboratories, Maryland, USA, 15 March 1985, AD-A158 544 / 7 / XAB 16p, NTIS database), this has been consistently done using an anti-idiotype vaccination strategy, which induces an antibody response against T-2-specific antibodies. However, this strategy has not been found to be successful in the prophylactic treatment of T2-induced mycotoxins. Therefore, prophylactic treatment of T2-induced mycotoxins is currently limited mainly to good agricultural practices to reduce mycotoxin production in crops and control programs for food and feed products to ensure that mycotoxin levels remain below certain limits.

[0008] Fungi generally cause a wide range of diseases in animals, including organ and tissue parasitism and allergenic expression. However, aside from poisoning from ingesting non-edible mushrooms, fungi can produce mycotoxins and organic chemicals that carry various toxic effects, known as mycotoxin diseases. These diseases are 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 important to the physiology of fungi and are extremely toxic to vertebrates at low concentrations upon ingestion, inhalation, or skin contact. Approximately 400 mycotoxins are currently recognized and subdivided into families of chemically related molecules with similar biological and structural properties. Of these, more than a dozen groups are regularly highlighted as threats to animal health. Mycotoxins of the highest public interest and agroeconomic significance include aflatoxins (AF), ochratoxins (OT), trichothecenes (T; including deoxynivalenol (DON)), zearalenones (ZEA), fumonisin (F), oscillating toxins, and ergot alkaloids. Mycotoxins are associated with acute and chronic diseases, and their biological effects vary mainly depending on the diversity of their chemical structures, but are also related to biological, nutritional, and environmental factors. The pathophysiology of mycotoxin diseases is the result of interactions between mycotoxins and functional molecules and organelles in animal cells, which can lead to carcinogenicity, genotoxicity, inhibition of protein synthesis, immunosuppression, skin irritation, and other metabolic disruption. In susceptible animal species, mycotoxins can induce complex and overlapping toxic effects. Mycotoxin diseases are not contagious and do not significantly stimulate the immune system. Treatment with drugs or antibiotics has little or no effect on the course of the disease. To date, no human or animal vaccines are available to combat mycotoxin infections.

[0009] Therefore, growing research focuses on developing vaccines and / or immunotherapies with efficacy against a broad class of fungi as powerful tools in combating mycoses, i.e., infections caused by the fungi themselves rather than toxins, in the prevention of specific fungal diseases. In contrast to mycoses, mycotoxins do not require the involvement of toxin-producing fungi and are considered abiotic hazards, although they are of biogenic origin. In this sense, mycotoxins are considered an example of poisoning by natural means, and defense strategies have essentially focused on exposure prevention. Exposure to humans and animals occurs primarily from the ingestion of mycotoxins in plant-based foods. The metabolism of ingested mycotoxins can lead to accumulation in different organs or tissues, and thus mycotoxins can enter the human food chain through animal meat, milk, or eggs (carryover). Mycotoxins can be present in all kinds of raw materials, commodities, and beverages, as toxic fungi contaminate several types of crops for human and animal consumption. The Food and Agriculture Organization of the United Nations (FAO) estimates that 25% of the world's food crops are significantly contaminated with mycotoxins. Currently, the best strategies for preventing mycotoxin disease include good agricultural practices to reduce mycotoxin production in crops, as well as control programs for food and feed products to ensure mycotoxin levels remain below predetermined thresholds. These strategies can limit the problem of product contamination by several groups of mycotoxins, which have high costs and variable effectiveness. Aside from supportive therapies (e.g., diet, hydration), there are few treatments for mycotoxin exposure, and antidotes for mycotoxins are generally unavailable. However, in individuals exposed to AF, some promising results have been observed with certain protective agents, such as chlorophyllin, green tea polyphenols, and dithiolthione (oltipraz).

[0010] In this field, several specific vaccination strategies against mycotoxins have been proposed to prevent mycotoxin diseases caused by contamination of important animal-derived foods. These strategies primarily target the prevention of mycotoxin diseases in humans and utilize strategies based on the production of antibodies that can specifically block the initial absorption or bioactivation of mycotoxins, or their toxicity and / or secretion in animal products (such as milk) through immune blockade.

[0011] However, the production of vaccines for protection against mycotoxin diseases is extremely difficult, mainly due to the fact that mycotoxins themselves are small, non-immunogenic molecules, and the toxicity associated with mycotoxins that does not make their use as antigens in healthy subjects risk-free. Mycotoxins are low molecular weight, usually non-proteinogenic molecules, and not usually immunogenic (haptens), but can potentially induce an immune response when bound to larger carrier molecules such as proteins. Methods for conjugating mycotoxins to protein or polypeptide carriers and optimizing conditions for animal immunization have been widely studied with the aim of producing monoclonal or polyclonal antibodies with different specificities for use in immunoassays to screen for mycotoxins in products destined for animal and human consumption. Coupling proteins used in these studies have included, among others, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), thyroglobulin (TG), and polylysine. Over the past few decades, much effort has been made to develop mycotoxin derivatives that can bind to proteins while retaining a good amount of the original structure so that the produced antibodies can recognize the natural toxins. These methods have made antibodies against many mycotoxins available, demonstrating that protein conjugation can be an effective tool for antibody production. Therefore, the application of this strategy for human and animal vaccination to achieve protection while remaining safe for recipients has not been successful so far due to the toxic properties of molecules that may be released in vivo. For example, conjugation of toxins such as T-2 to protein carriers has been shown to result in unstable complexes with the potential release of the active form of the free toxin (Chanh et al, Monoclonal anti-idiotype induces protection against the cytotoxicity of the trichothecene mycotoxin T-2, in J Immunol. 1990, 144:4721-4728).Similar to toxoid vaccines that can provide protection against the pathological effects of bacterial toxins, a rational approach to developing vaccines against mycotoxins may be based on conjugated “mycotoxins,” defined as modified forms of mycotoxins that retain antigenicity while lacking toxicity (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-proteinogenic nature of mycotoxins, approaches for conversion to mycotoxins should rely on chemical derivatization. By introducing specific groups at strategic locations in the relevant parent mycotoxin, molecules with different physicochemical characteristics can be formed, while still inducing antibodies that adequately cross-react to native toxins. Therefore, the general rationale for mycotoxin vaccination would be based on producing antibodies against mycotoxins with enhanced ability to bind to natural mycotoxins compared to cellular targets, thereby neutralizing the toxins and preventing disease development in case of exposure. The applicability of this strategy has been demonstrated for mycotoxins belonging to the AF group (Giovati et al, 2015), but not for any other mycotoxins. Furthermore, the preventive effect has not been demonstrated against mycotoxinosis in the vaccinated animals themselves, but only against carryover to the milk of dairy cows in order to protect people who consume milk or products made from it from mycotoxinosis. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Manohar V.et al., “Final Report:Development Of Vaccines To The Mycotoxin T-2”, Borriston Laboratories,Maryland,USA,15 March 1985,AD-A158 544 / 7 / XAB 16p,NTIS database [Non-Patent Document 2] Chanh et al, Monoclonal anti-idiotype induces protection against the cytotoxicity of the trichothecene mycotoxin T-2, in J Immunol.1990,144:4721-4728 [Non-Patent Document 3] 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 [Overview of the Initiative] [Problems that the invention aims to solve]

[0013] The object of the present invention is to provide a method for protecting animals from mycotoxin disease induced by T-2 toxin, an important mycotoxin in animal feed. [Means for solving the problem]

[0014] To satisfy the objectives of the present invention, conjugated T-2 toxin (T2) was found to be suitable for use in a method of protecting animals from T2-induced mycotoxinosis. It was found that it was not necessary to convert T2 to a toxoid, and the conjugated toxin appeared to be safe for the treated host animal. Furthermore, it was surprising to find that the immune response induced against small molecules such as mycotoxins was strong enough to protect the animals themselves from mycotoxinosis after ingestion of the mycotoxin following treatment. Such actual protection of animals by inducing an immune response against the mycotoxin itself has not been demonstrated in the art for any mycotoxin.

[0015] definition Mycotoxinosis is a disease caused by exposure to mycotoxins. Clinical signs, target organs, and consequences depend on the inherent toxic characteristics of the mycotoxin, as well as the amount and duration of exposure, and the health status of the exposed animal.

[0016] Protecting against mycotoxin disease means preventing or reducing one or more negative physiological effects of mycotoxins in animals, such as reduced mean daily weight gain, intestinal damage, skin damage, and nasal damage.

[0017] T-2 toxins (also known as T-2 mycotoxins, T-2 fusariotoxins, insariotoxins, or trichothecenes) are mycotoxins that commonly possess a tetracyclic sesquiterpenoid 12,13-epoxytricoteca-9-ene ring, which is responsible for their toxicological activity. Their chemical structures are characterized by a hydroxyl group at the C-3 position, acetyloxy groups at the C-4 and C-15 positions, a hydrogen atom at the C-7 position, and an ester-bonded isovaleryl group at the C-8 position (in place of the carbonyl group in other types of trichothecenes such as deoxynivalenol), as shown in Formula 1 below. [ka]

[0018] Conjugated molecules are molecules to which an immunogenic compound is bound via a covalent bond. Typically, the immunogenic compound is a large protein such as KLH, BSA, or OVA.

[0019] Adjuvants are nonspecific immunostimulants. In principle, any substance that can support or amplify a specific process in a cascade of immunological events, ultimately leading to a better immunological response (i.e., an integrated physical response to an antigen, particularly one mediated by lymphocytes, typically involving recognition of the antigen by specific antibodies or previously sensitized lymphocytes), can be defined as an adjuvant. Adjuvants generally are not required for the specific process to occur, but simply support or amplify it. Adjuvants can generally be classified according to the immunological event they induce. In particular, the first class, including ISCOM (immunostimulatory complexes), saponins (or their fractions and derivatives, e.g., QuilA), aluminum hydroxide, liposomes, cocreates, and polylactic acid / glycolic acid, promotes antigen uptake, transport, and presentation by APCs (antigen-presenting cells). In particular, the second class, which includes oil emulsions (w / o, o / w, w / o / w, or o / w / o), gels, polymer microspheres (Carbopol), nonionic block copolymers, and possibly aluminum hydroxide, provides a depot effect. In particular, the third class, which includes CpG-rich motifs, monophosphoryl lipid A, mycobacteria (muramyl dipeptide), yeast extracts, and cholera toxin, is based on the recognition of conserved microbial structures, so-called pathogen-associated microbial patterns (PAMPs), defined as signal 0. In particular, the fourth class, which includes oil emulsion surfactants, aluminum hydroxide, and hypoxia, is based on stimulating the immune system's ability to distinguish between dangerous and harmless (not necessarily the same as self and non-self) substances. In particular, the fifth class, which includes cytokines, is based on co-stimulatory molecules on APCs, upregulation of signal 2.

[0020] A vaccine, in the context of the present invention, is configured for application to animals and contains one or more antigens in an immunologically effective amount (i.e., typically in combination with a pharmaceutically acceptable carrier (i.e., a biocompatible medium, i.e., a medium that does not induce a significant adverse reaction in the target animal after administration and can present 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 commonly used to obtain a lyophilized vaccine (based on sugars and / or proteins)), and can stimulate the immune system of the target animal sufficiently to at least reduce the negative effects of challenge by disease-inducing agents. It may contain an immunostimulant (adjuvant) and induces an immune response for treating a disease or disorder upon administration to an animal, i.e., helps prevent, ameliorate, or cure a disease or disorder.

Embodiments for Carrying Out the Invention

[0021] In a further embodiment of the present invention, conjugated T2 is administered systemically to animals. For example, local administration via the gastrointestinal tract (oral or anal cavity) or mucosal tissues of the eye (e.g., when immunizing chickens) is known to be an effective route for inducing an immune response in various animals, but systemic administration has been found to result in an immune response sufficient to protect animals from T2-induced mycotoxicosis. In particular, effective immunization has been found to be achievable upon intramuscular, oral, and / or intradermal administration.

[0022] The age of administration is not critical, but it is preferred to administer before the animal can ingest feed contaminated with a significant amount of T2. Thus, the preferred age at administration is 6 weeks or less. Even more preferred is 4 weeks or less, for example, 1 - 3 weeks of age.

[0023] In yet another embodiment of the present invention, conjugated T2 is administered to animals at least twice. While many animals (particularly pigs, chickens, and ruminants) are generally susceptible to immunization with only one injection of an immunogenic composition, two injections are considered preferable for economically viable protection against T2. This is because, in practice, naturally occurring T2 is not immunogenic, and therefore the animal's immune system is not induced to produce anti-T2 antibodies by natural exposure to T2. Thus, the animal's immune system becomes entirely dependent on the administration of conjugated T2. The time between the two shots of conjugated T2 can be any time between one week and one to two years. For young animals, a plan of prime immunization at, for example, one to three weeks of age followed by a booster dose 1 to four weeks later, typically one to three weeks later, for example, two weeks later, is considered sufficient. Older animals may require booster doses every few months (e.g., 4, 5, or 6 months after the last dose), or annually or semi-annually, as is known from other commercially applied immunization regimens for animals.

[0024] In yet another embodiment, conjugated T2 is used in a composition comprising conjugated T2 and an adjuvant. An adjuvant can be used if the conjugate itself cannot induce an immune response to obtain a predetermined level of protection. While conjugate molecules are known that can adequately stimulate the immune system without additional adjuvants such as KLH or BSA, the use of additional adjuvants may be advantageous. This may eliminate the need for booster administration or allow for longer administration intervals. It all depends on the level of protection required in the specific situation. Types of adjuvants that have been shown to induce a good immune response to T2 when conjugated T2 is used as an immunogen include water- and oil-based emulsions, such as water-in-oil emulsions or oil-in-water emulsions. The former is commonly used in poultry, while the latter is commonly used in animals more susceptible to adjuvant-induced site reactions, such as pigs and ruminants.

[0025] In another embodiment, conjugated T2 includes T2 conjugated to a protein having a molecular weight greater than 10,000 Da. Such proteins, particularly keyhole limpet hemocyanin (KLH) and ovalbumin (OVA), have been shown to induce appropriate immune responses in animals, especially pigs and chickens. The practical upper limit for the protein may be 100 MDa.

[0026] With regard to protection against mycotoxin disease, it has been found that, in particular, the use of the present invention is thought to protect animals from one or more of these signs of mycotoxin disease induced by T2, including reduced average daily weight gain, liver damage, and damage to the intestinal tract, especially the stomach.

[0027] The present invention will now be further explained using the following examples.

[0028] [Examples] In the first series of experiments (see Examples 1-4), we evaluated whether an active immune response to mycotoxins could be induced using conjugated mycotoxins, and, if so, whether vaccinated animals could be protected from mycotoxin-induced injuries after ingestion. For the latter, a pig model for the DON challenge was used. Subsequently (Example 5), we evaluated whether the use of conjugated T2 in vaccines could induce antibodies against T-2 toxin in vaccinated animals.

[0029] [Example 1] Immunotherapy challenge experiment using conjugated DON the purpose The objective of this study was to evaluate the effectiveness of conjugated deoxynivalenol in protecting animals from mycotoxin infections caused by DON ingestion. To investigate this, pigs were immunized twice with DON-KLH before being challenged with toxic DON. The effects of different immunization routes were studied.

[0030] Research design Forty one-week-old piglets, derived from eight sows, were divided into five groups for the study. Twenty-four piglets from groups 1-3 were immunized twice, at one and three weeks of age. Group 1 was immunized 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 immunized twice intradermally (ID). Groups 1-3, at 5 1 / 2 weeks of age, were challenged for four weeks with oral administration of DON in liquid. Group 4 was not immunized but was challenged only with DON as described for groups 1-3. Group 5 served as a control and was administered only the control solution for four weeks starting at 5.5 weeks of age.

[0031] The DON concentration in the liquid formulation was equivalent to 5.4 mg / kg of feed. This corresponds to an average daily intake of 2.5 mg of DON. After a 4-week challenge, all animals were post-mortem, with special attention paid to the liver, kidneys, and stomach. Furthermore, blood samples were collected on days 0, 34, 41, 49, 55, and 64 (after euthanasia) of the study, except for group 5. Blood samples from group 5 were collected only on days 0, 34, 49, and immediately after euthanasia.

[0032] Test items Three different immunogenic compositions were formulated: Test Item 1, an oil-in-water emulsion for injection (X-solve50, MSD AH, Boxmeer) used for IM immunization containing 50 μg / ml of DON-KLH; Test Item 2, an oil-in-oil emulsion (GNE, MSDAH, Boxmeer) used for oral immunization containing 50 μg / ml of DON-KLH; and Test Item 3, an oil-in-water emulsion for injection (X-solve50) used for ID immunization containing 500 μg / ml of DON-KLH.

[0033] Challenged deoxynivalenol (obtained from Fermentek, Israel) was diluted in 100% methanol to a final concentration of 100 mg / ml and stored at <-15°C. Before use, DON was further diluted and supplied in preparation for administration.

[0034] Inclusion Criteria Only healthy animals were used. To exclude unhealthy animals, all animals were examined for their general physical appearance and the absence of clinical abnormalities or diseases before the start of the study. Different sow piglets were used for each group. In the daily implementation, all animals were immunized by ingesting DON-contaminated feed, even if they had been previously exposed to DON. Since DON itself does not trigger an immune response, there is considered to be no fundamental difference between animals previously exposed to DON and animals naive to DON.

[0035] result None of the animals exhibited adverse effects associated with immunization by DON-KLH. Therefore, the composition appeared to be safe.

[0036] All pigs were serologically negative for DON titer at the start of the experiment. However, during the challenge, the intramuscular immunization group (Group 1) and the intradermal immunization group (Group 3) developed an antibody response to DON, as measured by ELISA using natural DON-BSA as the coated antigen. Table 1 shows the mean IgG values ​​at four time points during the experiment, along with their standard deviations. Both intramuscular and intradermal immunization induced significant titers against DON.

[0037] Table 1 IgG Titer [Table 1]

[0038] As shown in Table 2, all immunized animals, including those in Group 2 which did not show a significant increase in anti-DON IgG titer, showed significantly higher weight gain during the first 15 days compared to challenged animals. Regarding the challenged animals, all of them gained more weight throughout the course of the study.

[0039] Table 2 Weight analysis [Table 2]

[0040] The condition of the small intestine (determined by the villi / crypt ratio in the jejunum) was also monitored. Table 3 shows the villi / crypt ratios. As can be seen from the figure, animals in group 3 had an average villi / crypt ratio comparable to that of healthy controls (group 5), while the unimmunized challenged group (group 4) had a much lower (statistically significant) villi / crypt ratio. Furthermore, groups 1 and 2 had significantly better (i.e., higher) villi / crypt ratios compared to the unimmunized challenged control group. This indicates that immunization protects against intestinal damage initiated by DON.

[0041] Table 3 Villus / crypt ratio [Table 3]

[0042] The overall health of other organs, more specifically the liver, kidneys, and stomach, was also monitored. All three study groups (groups 1-3) were observed to be in better health than the non-immune challenge control group (group 4). Table 4 provides a summary of the general health data. The severity of gastric ulcers was reported from - (no evidence of ulcer formation) to ++ (multiple ulcers). The severity of gastric inflammation was reported from - (no evidence of inflammation) to ++ / - (onset of gastric inflammation).

[0043] Table 4 General Health Data [Table 4]

[0044] [Example 2] The effect of immunization on DON levels the purpose The objective of this study was to evaluate the effect of immunization with a DON conjugate on the toxicological effects of DON ingestion. To investigate this, pigs were immunized twice with DON-KLH before administering toxic DON.

[0045] Research design Ten 3-week-old pigs were used in the study and divided into two groups of five pigs each. Group 1 pigs were immunized twice with DON-KLH (Test Item 1; Example 1) at 3 weeks and 6 weeks of age. Group 2 served as a control and received only the control fluid. At 11 weeks of age, each animal was administered a bolus of DON (Fermentek, Israel) at a dose of 0.05 mg / kg, which was similar to the contamination level of 1 mg / kg feed (based on daily feed intake). Blood samples were taken from the pigs using a jack before DON administration and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 hours after DON administration.

[0046] Inclusion Criteria Only healthy animals were used.

[0047] Analysis of DON in plasma Plasma analysis of unbound DON was performed using a validated LC-MS / MS method with an Acquity® UPLC system connected to a Xevo® TQ-SMS instrument (Waters, Zellik, Belgium). The lower limit of quantification for DON in porcine plasma using this method is 0.1 ng / ml.

[0048] Toxicokinetic analysis Toxicological modeling of the plasma concentration-time profile of DON was performed using non-compartmental analysis (Phoenix, Pharsight Corporation, USA). The following parameter was calculated: Area under the curve (AUC) from time 0 to infinity. 0→∞ ), maximum plasma concentration (C max ), and the time to maximum plasma concentration (t max ).

[0049] result The toxicological results are shown in Table 5 below. As can be seen, immunization with DON-KLH reduces all toxicological parameters. Since unbound DON is responsible for the toxic activity, it can be concluded that immunization with DON-KLH reduces the toxic effects caused by DON by reducing the amount of unbound DON in the animals' blood.

[0050] Table 5. Toxicogenic parameters of unbound DON [Table 5]

[0051] [Example 3] Serological responses to various DON conjugates the purpose The objective of this study was to evaluate the efficacy of different conjugated deoxynivalenol products.

[0052] Research design Eighteen 3-week-old pigs were used in the study and divided into three groups of six pigs each. Group 1 pigs were intramuscularly immunized twice with DON-KLH (using test item 1 of Example 1) at 3 weeks and 5 weeks of age. Group 2 pigs were immunized in accordance with DON-OVA. Group 3 served as the negative control. All animals were checked for anti-DON IgG responses at 3 weeks, 5 weeks, and 8 weeks of age.

[0053] result The serological results are shown below in the log2 antibody titer table.

[0054] Table 6 Anti-DON IgG response [Table 6]

[0055] Both conjugates appear suitable for increasing the anti-DON IgG response. Furthermore, a response seems to be induced with only a single shot.

[0056] [Example 4] Serological response in chickens the purpose The objective of this study was to evaluate the serological response to DON-KLH in chickens.

[0057] Research design For this study, 30 four-week-old chickens were used and divided into three groups of 10 chickens each. The chickens were intramuscularly immunized with DON-KLH. Group 1 was used as a control and received PBS only. Group 2 received DON-KLH without adjuvant, and Group 3 received DON-KLH formulated with GNE adjuvant (available from MSD Animal Health, Boxmeer). On day 0, the right leg was primed with 0.5 ml of vaccine. On day 14, the left leg was booster immunized with an equivalent amount.

[0058] Blood samples were collected on days 0 and 14, and on days 35, 56, 70, and 84. Serum was isolated for the measurement of IgY against DON. Blood samples were isolated on days 0 and 14 immediately before immunization.

[0059] result Serological results are shown in Table 7 as log2 antibody titers. PBS background has been subtracted from the data.

[0060] Table 7 Anti-DON IgY response [Table 7]

[0061] As can be seen, conjugated DON also induces anti-DON titers in chickens. GNE adjuvants substantially increase the response, but do not appear to be essential for obtaining the net response itself.

[0062] [Example 5] Serological response to T2 conjugate the purpose The purpose of this experiment was to evaluate whether the use of conjugated T2 in vaccines can induce antibodies against T-2 toxin in vaccinated animals.

[0063] Research design For this purpose, a vaccine containing T-2 toxin conjugated to keyhole limpet hemocyanin (T2-KLH) was used. The conjugate was mixed with an oil-in-water emulsion adjuvant (XSolve50, MSD Animal Health, The Netherlands) at a final concentration of 115 μg / ml for intramuscular (IM) administration or 1150 μg / ml for intradermal (ID) administration.

[0064] In this experiment, the DON vaccine described above was also used as a positive control. Subsequently, vaccines containing other conjugated mycotoxins were formulated and used. In particular, zearalenone (ZEA) conjugated with keyhole limpet hemocyanin (ZEA-KLH) and fumonisin (FUM) conjugated with KLH (T2-KLH) were formulated into vaccines. The conjugates were mixed with oil-in-water emulsion adjuvants (XSolve) as described herein at final concentrations of 50 μg / ml for intramuscular (IM) administration or 500 μg / ml for intradermal (ID) administration, respectively.

[0065] In the experiment, six groups of five animals each were used for vaccination at three weeks of age. Group 1 received two intradermal doses of 0.2 ml of FUM-KLH, Group 2 received two doses of 0.2 ml of ZEA-KLH, Group 3 received two doses of 2.0 ml of X-Solve50 DON-KLH IM, Group 4 received two doses of 2.0 ml of FUM-KLH IM, Group 5 received two doses of 2.0 ml of ZEA-KLH IM, and finally Group 6 received two doses of 2.0 ml of T2-KLH IM. A control group of three piglets was also included and did not receive vaccination. All primes were three weeks old, and boosters were five weeks old. The animals were monitored for 14 weeks after the start of the study.

[0066] result All pigs were serologically negative for titers against FUM, ZEA, T2, and DON at the start of the experiment, and all vaccinated groups expressed antibody titers. The obtained log2 titers are shown in Table 8 below. As can be seen, high levels of antibody elevation were achieved against each conjugated mycotoxin. This supports the idea that the vaccine can be effectively used against the corresponding mycotoxin, as shown above for DON-induced mycotoxin.

[0067] Table 8 IgG Titer [Table 8]

[0068] [Example 6] Response to T2 conjugates in chickens the purpose The purpose of this experiment was to evaluate whether the use of conjugated T2 in vaccines could induce protective antibodies against T2 in chickens.

[0069] Research design For this purpose, a vaccine containing T2 conjugated to keyhole limpet hemocyanin (T2-KLH) was used in accordance with Example 5. The conjugate was mixed with the oil emulsion adjuvant at a final concentration of 50 μg / ml in the same mineral oil used in Example 5, and alternatively in an equivalent non-mineral oil emulsion.

[0070] A flock of 15 chickens was used in the study. Three groups of 5 animals each were used. Group 1 was used as a negative control and administered PBS solution, Group 2 was vaccinated with T2-KLH mixed with a mineral oil-containing adjuvant, and Group 3 was vaccinated with a non-mineral oil-containing adjuvant. Chickens were intramuscularly vaccinated with 0.5 ml of vaccine at T=8 and T=22 (chickens were included in the study at T=0 for acclimatization).

[0071] result All chickens were serologically negative for titer against T2 at the start of the experiment (T=0, data not shown), and all vaccinated groups expressed antibody titers. The obtained log2 titers are shown in Table 9 below. As can be seen, antibody induction using non-mineral oil appeared to be better, but both groups were able to elevate antibody against conjugated T2 to high levels. This supports the common understanding that the type of adjuvant is not essential on its own for a sufficient increase in the immune response, but the actual level of immune response elevation may be adjuvant-dependent.

[0072] Table 9 Antibody titers against T2 in chickens [Table 9]

[0073] Serum samples from this study were further tested in an in vitro potency assay, with cells (Caucasian human colon adenocarcinoma cells) incubated with the toxin alone, the toxin combined with serum from a pool of ELISA-positive animals, and the toxin combined with serum from PBS injection (negative animals). Cell viability was measured by adding CCK8 and reading the optical density at 450 nm, and Table 10 shows the results.

[0074] When comparing positive serum from groups 1 and 2, an increase in OD450 values ​​(cell viability) can be observed compared to negative serum at the same dilution (2x or 4x). Furthermore, OD increased compared to when serum was not added in combination with T2. This indicates that positive (vaccinated animals) serum can at least partially neutralize the effects of the toxin. Negative serum cannot, thus demonstrating a protective effect against the vaccine-induced immune response.

[0075] Table 10 Neutralization data for chicken IgY on cells [Table 10]

[0076] [Example 7] Defense against T2 challenges in pigs the purpose The purpose of this experiment was to evaluate whether the use of conjugated T2 in vaccines could induce protection against T2 challenge in pigs.

[0077] Research design For this purpose, the same vaccine containing T2 conjugated to keyhole limpet hemocyanin (T2-KLH) in two different adjuvants was used, as described in Example 6, one of which was mineral oil-based and the other non-mineral oil-based. This study used a group of 24 pigs. Eight piglets in Group 1 were vaccinated with T2-KLH, but four animals in the first subgroup received a vaccine based on a mineral oil-containing adjuvant, and the second subgroup received an alternative vaccine. Both vaccines were administered intramuscularly in 2 ml doses at a concentration of 50 μg / ml. The animals received a prime vaccine at 7-12 days of age (T=0) and a booster vaccine at 21-26 days of age (T=14). Group 2 was not vaccinated but was challenged with T2 and served as a positive control. Group 3 was neither vaccinated nor challenged and served as a negative control. Sixteen challenged piglets (groups 1 and 2), approximately 5.5 weeks old, were administered 1.15 mg / kg of T2 daily in liquid form for 4 weeks (0.56 mg / day): the pigs received 0.19 mg of T2 / day in 16 ml of liquid in the first week, 0.39 mg / day in 32 ml of liquid in the second week, 0.72 mg / day in 45 ml of liquid in the third week, and 0.93 mg of T2 / day in 60 ml of liquid in the fourth week. Antibody titers were monitored over time. At the end of the study, the piglets' intestines, skin, and nose were evaluated.

[0078] result All piglets were serologically negative for T2 titer at the start of the experiment. During the challenge, T2-KLH vaccination resulted in an antibody response against T2, as shown in Table 11, which shows IgG levels at six time points during the study.

[0079] Table 11 IgG titer against T2 in pigs [Table 11]

[0080] For all animals, the growth rate per piglet compared to the starting weight at the time of the challenge was determined. Vaccination did not negatively affect growth. In contrast, vaccinated animals showed a slight increase in growth compared to challenge animals. Furthermore, vaccinated animals showed better health when examining the piglets' intestines, skin, and nose.

[0081] Table 12 shows the percentage of animals per group that had a percentage increase in weight during the challenge from the starting weight of the challenge, and further, the percentage of animals with damage to specific organs. All of this demonstrates the successful use of conjugated T2 in a method of protecting animals from T2-induced mycotoxin disease.

[0082] Table 12 Piglet body weight and organ scores [Table 12]

[0083] Improved gut health was observed in vaccinated animals with a higher (healthy) villi / crypt ratio compared to challenged animals, as shown in Table 13.

[0084] Table 13 Villus / crypt ratio [Table 13]

Claims

1. A method for treating a non-human animal with conjugated T-2 toxin (T2) to protect the non-human animal from T2-induced mycotoxin, wherein the conjugated T-2 toxin comprises T2 conjugated to an immunogenic compound via covalent bond.

2. A method according to Claim 1, for protecting a non-human animal from one or more of the clinical signs of T2-induced mycotoxin disease, selected from the group consisting of reduced weight gain, intestinal injury, skin injury, and nasal injury.

3. The method according to claim 1 or 2, characterized in that the conjugated T2 is systemically administered to the non-human animal.

4. The method according to claim 3, characterized in that the conjugated T2 is administered intramuscularly, orally, and / or intradermally.

5. The method according to any one of claims 1 to 4, characterized in that the conjugated T2 is administered to the non-human animal at 6 weeks of age or younger.

6. The method according to claim 5, characterized in that the conjugated T2 is administered to the non-human animal at four weeks of age or younger.

7. The method according to claim 6, characterized in that the conjugated T2 is administered to the non-human animal at 1 to 3 weeks of age.

8. The method according to any one of claims 1 to 7, characterized in that the conjugated T2 is administered to the non-human animal at least twice.

9. The method according to any one of claims 1 to 8, characterized in that the conjugated T2 is used in a composition comprising the conjugated T2 in addition to an adjuvant.

10. The method according to claim 9, characterized in that the adjuvant is an emulsion of water and oil.

11. The method according to claim 10, characterized in that the adjuvant is a water-in-oil emulsion or an oil-in-water emulsion.

12. The method according to any one of claims 1 to 11, characterized in that the conjugated T2 includes T2 conjugated to a protein having a molecular mass of more than 10,000 Da.

13. The method according to claim 12, characterized in that the conjugated T2 comprises T2 conjugated to keyhole limpet hemocyanin (KLH) or ovalbumin (OVA).

14. The method according to any one of claims 1 to 13, characterized in that the non-human animal is a pig or a chicken.

15. A vaccine comprising conjugated T2, an adjuvant, and a pharmaceutically acceptable carrier, wherein the conjugated T2 comprises T2 conjugated to an immunogenic compound to which it is covalently bound.

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