Patent Publication Number: US-2022233496-A1

Title: Conjugated deoxynivalenol to protect against mycotoxicosis

Description:
GENERAL FIELD OF THE INVENTION 
     The invention in general pertains to protection against mycotoxicosis induced by the mycotoxin deoxynivalenol (DON), also known as vomitoxin. DON is a type B trichothecene which is present predominantly in grains such as wheat, barley, oats, rye, and corn, but also in rice, sorghum, and triticale. The occurrence of deoxynivalenol is associated primarily with  Fusarium graminearum  ( Gibberella zeae ) and  Fusarium culmorum , both of which are important plant pathogens which cause  fusarium  head blight in wheat and  gibberella  or  fusarium  ear blight in corn. A direct relationship between the incidence of  fusarium  head blight and contamination of wheat with deoxynivalenol has been established. The incidence of  fusarium  head blight is strongly associated with moisture at the time of flowering, and the timing of rainfall, rather than the amount, is the most critical factor. Furthermore, DON contents are significantly affected by the susceptibility of cultivars towards  Fusarium  species, previous crop, tillage practices, and fungicide use.  Fusarium graminearum  grows optimally at a temperature of 25° C., whereas  Fusarium culmorum  grows optimally at 21° C.  Fusarium graminearum  therefore being the more common species occurring in warmer climates. 
     DON has been implicated in incidents of mycotoxicoses in both humans and farm animals. The toxin belongs to the class of trichothecenes which are strong inhibitors of protein synthesis. Exposure to DON causes the brain to decrease its uptake of the amino acid tryptophan and, in turn, its synthesis of serotonin. Reduced levels of serotonin are believed to be responsible for the anorexic effects of DON. Irritation of the gastrointestinal tract may also play a role in reducing feed intake, and may also partially explain the high incidence of paraesophageal stomach ulcers observed in sows during feed refusal. 
     Prophylactic treatment of DON induced mycotoxicosis is currently restricted to good agricultural practice to reduce mycotoxins production on crop and control programs of food and feed commodities to ensure that mycotoxin levels remain below certain limits. 
     BACKGROUND OF THE INVENTION 
     Fungi cause a broad range of diseases in animals, involving parasitism of organs and tissues as well as allergenic manifestations. However, other than poisoning through ingestion of non-edible mushrooms, fungi can produce mycotoxins and organic chemicals that are responsible for various toxic effects referred to as mycotoxicosis. This disease is caused by exposure to mycotoxins, pharmacologically active compounds produced by filamentous fungi contaminating foodstuffs or animal feeds. Mycotoxins are secondary metabolites not critical to fungal physiology, that are extremely toxic in minimum concentrations to vertebrates upon ingestion, inhalation or skin contact. About 400 mycotoxins are currently recognized, subdivided in families of chemically related molecules with similar biological and structural properties. Of these, approximately a dozen groups regularly receive attention as threats to animal health. Examples of mycotoxins of greatest public interest and agroeconomic significance include aflatoxins (AF), ochratoxins (OT), trichothecenes (T; including DON), zearalenone (ZEN), fumonisins (F), tremorgenic toxins, and ergot alkaloids. Mycotoxins have been related to acute and chronic diseases, with biological effects that vary mainly according to the diversity in their chemical structure, but also with regard to biological, nutritional and environmental factors. The pathophysiology of mycotoxicoses is the consequence of interactions of mycotoxins with functional molecules and organelles in the animal cell, which may result in carcinogenicity, genotoxicity, inhibition of protein synthesis, immunosuppression, dermal irritation, and other metabolic perturbations. In sensitive animal species, mycotoxins may elicit complicated and overlapping toxic effects. Mycotoxicoses are not contagious, nor is there significant stimulation of 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 vaccine is available for combating mycotoxicoses. 
     A growing body of work is thus focusing in developing vaccines and/or immunotherapy with efficacy against broad fungal classes as a powerful tool in combating mycoses, i.e. the infection with the fungi as such, instead of the toxins, in the prevention of specific fungal diseases. In contrast to mycoses, mycotoxicoses do not need the involvement of the toxin producing fungus and are considered as abiotic hazards, although with biotic origin. In this sense, mycotoxicoses have been considered examples of poisoning by natural means, and protective strategies have essentially focused on exposure prevention. Human and animal exposure occurs mainly from ingestion of the mycotoxins in plant-based food. Metabolism of ingested mycotoxins could result in accumulation in different organs or tissues; mycotoxins can thus enter into the food chain through animal meat, milk, or eggs (carry over). Because toxigenic fungi contaminate several kinds of crops for human and animal consumption, mycotoxins may be present in all kinds of raw agricultural materials, commodities and beverages. The Food and Agriculture Organization (FAO) estimated that 25% of the world&#39;s food crops are significantly contaminated with mycotoxins. At the moment, the best strategies for mycotoxicoses prevention include good agricultural practice to reduce mycotoxins production on crop and control programs of food and feed commodities to ensure that mycotoxin levels stand below predetermined threshold limits. These strategies may limit the problem of contamination of commodities with some groups of mycotoxins with high costs and variable effectiveness. Except for supportive therapy (e.g., diet, hydration), there are almost no treatments for mycotoxin exposure and antidotes for mycotoxins are generally not available, although in individual exposed to AFs some encouraging results have been obtained with some protective agents such as chlorophyllin, green tea polyphenols and dithiolethiones (oltipraz). 
     Vaccination against particular mycotoxins has been proposed to prevent mycotoxicosis in livestock and contamination by mycotoxins in important foods of animal origin with a strategy based on the production of antibodies that could specifically block initial absorption or bioactivation of mycotoxins, their toxicity and/or secretion in animal products (such as milk) by immuno-interception. 
     The production of vaccines for protection against mycotoxicoses however are very challenging, principally related to the wide range of structures, chemical properties, and toxicity associated with mycotoxins. Mycotoxins are low molecular weight, usually nonproteinaceous molecules, which are not ordinarily immunogenic (haptens), but can potentially elicit an immune response when attached to a large carrier molecule such as a protein. Methods for conjugation of mycotoxins to protein or polypeptide carrier and optimization of conditions for animal immunization have been extensively studied, with the purpose of producing monoclonal or polyclonal antibodies with different specificities to be used in immunoassay for screening of mycotoxins in products destined for animal and human consumption. Coupling proteins used in these studies included bovine serum albumin (BSA), keyhole limpet haemocyanin (KLH), thyroglobulin (TG) and polylysine, among others. In the past decades, many efforts have been made for developing mycotoxin derivatives that can be bound to proteins while retaining enough of the original structure so that antibodies produced will recognize the native toxin. Through these methods, antibodies against many mycotoxins have been made available, demonstrating that conjugation to proteins may be an effective tool for immunization. The application of this strategy for human and animal vaccination, thus to arrive at protection while being safe for the recipient, has not been successful so far due to the toxic properties of the molecules that might 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 potential release of the 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). In analogy with toxoid vaccines, which may confer a state of protection against the pathological effects of bacterial toxins, a reasonable approach to the development of vaccines against mycotoxin may be based on conjugated “mycotoxoids”, defined as modified form of mycotoxins, devoid of toxicity although maintaining antigenicity (Giovati L et al,  Anaflatoxin B 1  as the paradigm of a new class of vaccines based on “Mycotoxoids ”, in Ann Vaccines Immunization 2(1): 1010, 2015). Given the nonproteinaceous nature of mycotoxins, the approach for conversion to mycotoxoids should rely on chemical derivatization. The introduction of specific groups in strategic positions of the related parent mycotoxin may lead to formation of molecules with different physicochemical characteristics, but still able to induce antibodies with sufficient cross-reacting to the native toxin. The common rationale for mycotoxin vaccination would thus be based on generating antibodies against the mycotoxoid with an enhanced ability to bind native mycotoxin compared with cellular targets, neutralizing the toxin and preventing disease development in the event of exposure. A potential application of this strategy has been demonstrated in the case of mycotoxins belonging to the AF group (Giovati et al, 2015), but not for any of the other mycotoxins. Moreover, the protective effect has not been demonstrated against mycotoxicosis of the vaccinated animal as such, but only against carry over in dairy cows to their milk, so as to protect people that consume the milk or products made thereof from mycotoxicosis. 
     OBJECT OF THE INVENTION 
     It is an object of the invention to provide a method to protect an animal against mycotoxicosis induced by deoxynivalenol, one of the most widely spread mycotoxins in animal feed. 
     SUMMARY OF THE INVENTION 
     In order to meet the object of the invention it has been found that conjugated deoxynivalenol (DON) is suitable for use in a method to protect an animal against DON induced mycotoxicosis. It was found that there was no need to convert the DON into a toxoid, the conjugated toxin appeared to be safe for the treated host animal. Also, it was surprising to see that the immune response induced was strong enough to protect the animal itself against mycotoxicosis after ingestion of DON post treatment. Such actual protection by inducing an immune response against the mycotoxin itself has not been shown in the art for any mycotoxin, let alone for the highly abundant and extremely toxic compound deoxynivalenol. 
     Definitions 
     Mycotoxicosis is the disease resulting from exposure to a mycotoxin. The clinical signs, target organs, and outcome depend on the intrinsic toxic features of the mycotoxin and the quantity and length of exposure, as well as the health status of the exposed animal. 
     To protect against mycotoxicosis means to prevent or decrease one or more of the negative physiological effects of the mycotoxin in the animal, such as a decrease in average daily weight gain. 
     Deoxynivalenol (also known as vomitoxin or VOM) is a mycotoxin produced by the fungus  Fusarium graminearum , which causes  Fusarium  head blight (FHB), or scab, of small grains. DON can cause feed refusal and vomiting. The molecular formula of the basic compound is C 15 H 20 O 6 . 
     A conjugated molecule is a molecule to which an immunogenic compound is coupled through a covalent bond. Typically the immunogenic compound is a large protein such as KLH, BSA or OVA. 
     An adjuvant is non-specific immunostimulating agent. In principal, each substance that is able to favor or amplify a particular process in the cascade of immunological events, ultimately leading to a better immunological response (i.e. the integrated bodily response to an antigen, in particular one mediated by lymphocytes and typically involving recognition of antigens by specific antibodies or previously sensitized lymphocytes), can be defined as an adjuvant. An adjuvant is in general not required for the said particular process to occur, but merely favors or amplifies the said process. 
     FURTHER EMBODIMENTS OF THE INVENTION 
     In a further embodiment of the invention, the conjugated DON is systemically administered to the animal. Although local administration, for example via mucosal tissue in the gastro-intestinal tract (oral or anal cavity) or in the eyes (for example when immunising chickens) is known to be an effective route to induce an immune response in various animals, it was found that systemic administration leads to an adequate immune response for protecting animals against a DON induced mycotoxicosis. It was found in particular that effective immunisation can be obtained upon intramuscular, oral and/or intradermal administration. 
     The age of administration is not critical, although it is preferred that the administration takes place before the animal is able to ingest feed contaminated with substantial amounts of DON. Hence a preferred age at the time of administration of 6 weeks or younger. Further preferred is an age of 4 weeks or younger, such as for example an age of 1-3 weeks. 
     In yet another embodiment of the invention the conjugated DON is administered to the animal at least twice. Although many animals (in particular swine chickens, ruminants) in general are susceptible for immunisation by only one shot of an immunogenic composition, it is believed that for economic viable protection against DON two shots are preferred. This is because in practice the immune system of the animals will not be triggered to produce anti-DON antibodies by natural exposure to DON, simply because naturally occurring DON is not immunogenic. So, the immune system of the animals is completely dependent on the administration of the conjugated DON. The time between the two shots of the conjugated DON can be anything between 1 week and 1-2 years. For young animals it is believed that a regime of a prime immunisation, for example at 1-3 weeks of age, followed by a booster administration 1-4 weeks later, typically 1-3 weeks later, such as 2 weeks later, will suffice. Older animals may need a booster administration every few months (such as 4, 5, 6 months after the last administration), or on a yearly or biannual basis as is known form other commercially applied immunisation regimes for animals. 
     In still another embodiment the conjugated DON is used in a composition comprising an adjuvant in addition to the conjugated DON. An adjuvant may be used if the conjugate on itself is not able to induce an immune response to obtain a predetermined level of protection. Although conjugate molecules are known that are able to sufficiently stimulate the immune system without an additional adjuvant, such as KLH or BSA, it may be advantageous to use an additional adjuvant. This could take away the need for a booster administration or prolong the interval for the administration thereof. All depends on the level of protection needed in a specific situation. A type of adjuvant that was shown to be able and induce a good immune response against DON when using conjugated-DON as immunogen is an emulsion of water and oil, such as for example a water-in-oil emulsion or an oil-in-water emulsion. The former is typically used in poultry while the latter is typically used in animals who are more prone to adjuvant induced site reactions such as swine and ruminants. 
     In again another embodiment the conjugated DON comprises DON conjugated to a protein having a molecular mass above 10,000 Da. Such proteins, in particular keyhole limpet hemocyanin (KLH) and ovalbumin (OVA), have been found to be able and induce an adequate immune response in animals, in particular in swine and chickens. A practical upper limit for the protein might be 100 MDa. 
     Regarding the protection against mycotoxicosis, it was found in particular that using the invention, the animal is protected against a decrease in average daily weight gain, liver damage, stomach ulcers and/or kidney damage, thus one or more of these signs of mycotoxicosis. 
     The invention will now be further explained using the following examples. 
    
    
     EXAMPLES OF THE INVENTION 
     Example 1: Immunisation Challenge Experiment Using Conjugated DON 
     Objective 
     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. 
     Study Design 
     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. 
     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. 
     Test Articles 
     Three different immunogenic compositions were formulated, namely Test Article 1 comprising DON-KLH at 50 μg/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 μg/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 μg/ml in an oil-in-water emulsion for injection (X-solve 50) for ID immunisation. 
     The challenge deoxynivalenol (obtained from Fermentek, Israel) was diluted in 100% methanol at a final concentration of 100 mg/ml and stored at &lt;−15° C. Prior to usage, DON was further diluted and supplied in a treat for administration. 
     Inclusion Criteria 
     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 naïve with respect to DON. 
     Results 
     None of the animals had negative effects associated with the immunisation with DON-KLH. The composition thus appeared to be safe. 
     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 1 
               
             
            
               
                   
               
               
                 IgG titres 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 group 1 
                 group 2 
                 group 3 
                 group 4 
                 Group 5 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 T = 0 
                 &lt;4.3 
                 &lt;4.3 
                 &lt;4.3 
                 &lt;4.3 
                 &lt;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 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 2 
               
             
            
               
                   
               
               
                 weight analysis 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Average  
               
               
                   
                   
                   
                   
                   
                 additional  
               
               
                   
                   
                   
                   
                   
                 weight gain  
               
               
                   
                   
                   
                   
                   
                 compared  
               
               
                   
                   
                   
                   
                   
                 to challenge 
               
               
                   
                   
                   
                 weight  
                 weight  
                 animals  
               
               
                   
                 ADG1 1   
                 ADG 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 
               
               
                   
               
               
                   1 average daily weight gain over the first 15 days of the challenge 
               
               
                   2 average daily weight gain over the last 13 days of the challenge 
               
            
           
         
       
     
     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 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 
               
               
                   
                   
               
            
           
         
       
     
     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 4 
               
             
            
               
                   
               
               
                 General health data 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Liver  
                 Stomach  
                 Stomach  
                   
               
               
                   
                   
                 colour 
                 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 
     Objective 
     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. 
     Study Design 
     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; example 1). 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. 
     Inclusion Criteria 
     Only healthy animals were used. 
     Analysis of DON in Plasma 
     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. 
     Toxicokinetic Analysis 
     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 0-∞ ), maximal plasma concentration (C max ), and time at maximal plasma concentration (t max ). 
     Results 
     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 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 Against Various DON Conjugates 
     Objective 
     The objective of this study was to evaluate the efficacy of different conjugated deoxynivalenol products. 
     Study Design 
     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. 
     Results 
     The serological results are indicated here below in the table in log 2 antibody titre. 
     
       
         
           
               
             
               
                 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 
               
               
                   
                   
               
            
           
         
       
     
     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 in Chickens 
     Objective 
     The objective of this study was to evaluate the serological response of DON-KLH in chickens. 
     Study Design 
     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. 
     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. 
     Results 
     The serological results are depicted in table 7 in log 2 antibody titre. The 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, 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.