COMBINATION VACCINE FOR PROTECTING SWINE AGAINST VARIOUS DISORDERS

Abstract

The present invention pertains to a vaccine comprising in combination non-replicating immunogen of porcine circo virus type 2 (PCV2), non-replicating immunogen of Mycoplasma hyopneumoniae and conjugated deoxynivalenol (DON) for protecting swine against an infection with porcine circo virus type 2, an infection with Mycoplasma hyopneumoniae and DON induced mycotoxicosis.

Claims

1. A vaccine comprising in combination a non-replicating immunogen of porcine circo virus type 2 (PCV2), a non-replicating immunogen of Mycoplasma hyopneumoniae and a conjugated deoxynivalenol (DON) for protecting swine against an infection with porcine circo virus type 2, an infection with Mycoplasma hyopneumoniae and DON induced mycotoxicosis.

2. A vaccine according to claim 1, characterised in that the non-replicating immunogen of PCV2 is an ORF2 protein of PCV2.

3. A vaccine according to claim 1, characterised in that the non-replicating immunogen of PCV2 is recombinantly expressed ORF2 protein of PCV2.

4. A vaccine according to claim 1, characterised in that the non-replicating immunogen of Mycoplasma hyopneumoniae is a Mycoplasma hyopneumoniae bacterin.

5. A vaccine according to claim 1, characterised in that the non-replicating immunogen of Mycoplasma hyopneumoniae comprises killed whole Mycoplasma hyopneumoniae.

6. A vaccine according to claim 1, characterised in that the conjugated DON is DON conjugated to a protein having a molecular mass above 10.000 Da.

7. A vaccine according to claim 1, characterised in that the conjugated DON is DON conjugated to keyhole limpet hemocyanin (KLH) or ovalbumin (OVA).

8. A vaccine according to claim 1, characterised in that the vaccine comprises a non-replicating immunogen of Lawsonia intracellularis.

9. A vaccine according to claim 1, characterised in that the vaccine comprises killed whole cells of Lawsonia intracellularis.

10. A method of protecting a swine against an infection with porcine circo virus type 2, an infection with Mycoplasma hyopneumoniae and DON induced mycotoxicosis comprising administering to the swine a vaccine comprising a non-replicating immunogen of porcine circo virus type 2 (PCV2), a non-replicating immunogen of Mycoplasma hyopneumoniae and conjugated deoxynivalenol (DON).

11. The method according to claim 10, characterised in that the vaccine is systemically administered to the swine.

12. The method according to claim 10, characterised in that the vaccine is administered intramuscularly or intradermally to the swine.

13. The method according to claim 10, characterised in that the vaccine is administered to the swine at an age of 6 weeks or younger.

14. The method according to claim 10, characterised in that the vaccine is administered to the swine at an age of 4 weeks or younger, preferably at an age of 1-3 weeks.

15. A kit-of-parts comprising in combination a first composition comprising in combination a non-replicating immunogen of porcine circo virus type 2 (PCV2), a non-replicating immunogen of Mycoplasma hyopneumoniae, and a second composition comprising a conjugated deoxynivalenol (DON).

Description

EXAMPLES

[0034] In a first series of experiments (described in Examples 1 through 3 below), the efficacy of a monovalent DON vaccine was tested for safety and efficacy. Thereafter, the efficacy of the trivalent vaccine according to the invention was tested accordingly (Example 4).

Example 1

Immunisation Challenge Experiment Using Conjugated DON

Objective

[0035] The objective of this study was to evaluate the safety and efficacy of conjugated deoxynivalenol to protect swine 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

[0036] Forty 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.

[0037] 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

[0038] Three different immunogenic compositions were formulated, namely Test Article 1 comprising DON-KLH at 50 g/ml in an oil-in-water emulsion for injection (X-solve 50, MSD AH, Boxmeer) which was used for IM immunization; Test Article 2 comprising DON-KLH at 50 g/ml in a water-in-oil emulsion (GNE, MSD AH, Boxmeer) which was used for oral immunization and Test Article 3 comprising DON-KLH at 500 g/ml in an oil-in-water emulsion for injection (X-solve 50) for ID immunisation.

[0039] The challenge deoxynivalenol (obtained from Fermentek, Israel) was diluted in 100% methanol at a final concentration of 100 mg/ml and stored at <15 C. Prior to usage, DON was further diluted and supplied in a treat for administration.

Inclusion Criteria

[0040] Only healthy animals were used. In order to exclude unhealthy animals, all animals were examined before the start of the study for their general physical appearance and absence of clinical abnormalities or disease. Per group piglets from different sows were used. In everyday practice all animals will be immunised even when pre-exposed to DON via intake of DON contaminated feed. Since DON as such does not raise an immune response, it is believed that there is no principle difference between animals pre-exposed to DON and nave with respect to DON.

Results

[0041] None of the animals had negative effects associated with the immunisation with DON-KLH. The composition thus appeared to be safe.

[0042] All pigs were serologically negative for titres against DON at the start of the experiment, During the challenge the groups immunised intramuscular (Group 1) and intradermally (Group 3) developed antibody responses against DON as measured by ELISA with native DON-BSA as the coating antigen. Table 1 depicts the average IgG values on 4 time points during the study with their SD values. Both Intramuscular immunisation and Intradermal immunisation induced significant titres against DON.

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

[0043] As depicted in Table 2 all immunised animals, including the animals in Group 2 that showed no significant anti-DON IgG titre increase, showed a significant higher weight gain during the first 15 days compared to the challenge animals. With respect to the challenged animals, all animals gained more weight over the course of the study.

TABLE-US-00002 TABLE 2 weight analysis Average additional weight gain compared to weight weight challenge ADG1.sup.1 ADG.sup.2 begin end 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 .sup.1average daily weight gain over the first 15 days of the challenge .sup.2average daily weight gain over the last 13 days of the challenge

[0044] The condition of the small intestines (as determined by the villus/crypt ratio in the jejunum) was also monitored. In table 3 the villus/crypt ratio is depicted. As can be seen, the animals in group 3 had an average villus crypt/crypt ratio comparable to the healthy controls (group 5), while the non-immunised, challenged group (group 4) had a much lower (statistically significant) villus crypt ratio. In addition, group 1 and group 2, had a villus/crypt ratio which was significantly better (i.e. higher) compared to the non-immunised challenge control group. This indicates that the immunisation protects against the damage of the intestine, initiated by DON.

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

[0045] The general condition of other organs was also monitored, more specifically the liver, the kidneys and the stomach. It was observed that all three test groups (groups 1-3) were in better health than the non-immunised challenge control group (group 4). In table 4 a summary of the general health data is depicted. The degree of stomach ulcer is reported from (no prove of ulcer formation) to ++ (multiple ulcers). The degree of stomach inflammation is reported from (no prove of inflammation) to ++/ (initiation of stomach inflammation).

TABLE-US-00004 TABLE 4 General health data 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

[0046] 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

[0047] 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

[0048] Only healthy animals were used.

Analysis of DON in Plasma

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

Toxicokinetic Analysis

[0050] Toxicokinetic modeling of the plasma concentration-time profiles of DON was done by noncompartmental analysis (Phoenix, Pharsight Corporation, USA). Following parameters were calculated: area under the curve from time zero to infinite (AUC.sub.0.fwdarw.), maximal plasma concentration (C.sub.max), and time at maximal plasma concentration (t.sub.max).

Results

[0051] The toxicokinetic results are indicated in table 5 here beneath. As can be seen immunisation with DON-KLH decreases all toxicokinetic parameters. As it is unbound DON that is responsible for the exertion of toxic effects, it may be concluded that immunisation with DON-KLH will reduce the toxic effects caused by DON by reducing the amount of unbound DON in the blood of animals.

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

Example 3

Serological Response Against Various DON Conjugates

Objective

[0052] The objective of this study was to evaluate the efficacy of different conjugated deoxynivalenol products.

Study Design

[0053] 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

[0054] The serological results are indicated here below in the table in log2 antibody titre.

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

[0055] 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

Efficacy of Various Combination Vaccines

Objective

[0056] The aim of the study was to determine whether it is possible to combine the vaccination against DON, with the vaccinations against PCV2, Mhyo and optionally Lawsonia intracellularis.

Study Design

[0057] A herd of 64 one-week old piglets, derived from 12 sows, was divided over 8 groups of 8 piglets each. Groups 1 to 3 were used for intradermal (ID) vaccination, using the IDAL (MSD Animal Health) device, in each case administering 0.2 ml per shot. The piglets from Group 1 received the monovalent DON-KLH vaccine as used in Example 1 (Test Article 3) as a positive control in a prime-boost scheme. Group 2 (denoted PM) received as a first vaccination a monovalent DON-KLH vaccine (same level of DON antigen as Group 1) in an oil-in-water emulsion (comprising squalene and vitamin E-acetate) and a second vaccination with a vaccine containing the three antigens of the invention, viz. non-replicating PCV2 immunogen (in this case baculovirus expressed ORF2 protein of PCV2 at the same level as in Porcilis PCV ID), non-replicating Mhyo immunogen (in this case an Mhyo bacterin at the same level as in Porcilis M Hyo ID ONCE), and the DON-KLH in the same adjuvant. Group 3 was the negative control for DON ID, receiving only Porcilis PCV M Hyo at three weeks of age.

[0058] Groups 4 to 8 were used for intramuscular (IM) vaccination, using a standard hypodermic syringe, in each case administering 2 ml per shot. Group 4 was the positive control for the intramuscular vaccination receiving the monovalent DON-KLH vaccine (Example 1, Test Article 1) in X-solve 50 two times. Group 5 received as a first shot a monovalent DON-KLH vaccine adjuvanted with Emmunade (MSD Animal Health), and as a second shot DON-KLH mixed with Porcilis PCV M Hyo and Porcilis Lawsonia at three weeks in the same adjuvant (i.e. the commercial three-way Porcilis PCV2 M Hyo Lawsonia combination vaccine, denoted PML in this application).The DON-KLH level was at the same level as in Test Article 1 of Example 1. Group 6 received the monovalent DON-KLH vaccine in X-solve 50 as a prime vaccination and a non-mixed associated combination vaccination with the same monovalent DON-KLH vaccine and the separate PML vaccine as a booster. Group 7 was the negative control group (PML alone). Group 8 was the negative control receiving a DON challenge.

[0059] In each of the above cases the first vaccination was administered in the right side of neck, when the piglets were one week of age and the second vaccination in the left side of neck, at three weeks of age. Challenge (Groups 2, 4, 5 and 8) took place as described here above in Example 1 using DON mixed with fluid. In the first two weeks of challenge the DON was administered in the mornings and in the evenings, and in the second two weeks of challenge the DON was administered in the morning, afternoon and evening. The dosing was such that in the first week the piglets receive 1 mg DON per day, in the second week they received 2 mg DON per day, in the third week they received 3 mg DON per day and in the fourth week they received 4 mg DON per day.

Inclusion Criteria

[0060] Only healthy animals were used. In order to exclude unhealthy animals, they were examined before the start of the study (general physical appearance and absence of clinical abnormalities or disease).

Results

[0061] None of the animals had negative effects associated with the various vaccinations. The compositions thus appeared to be safe.

[0062] All groups receiving a vaccine comprising conjugated DON seroconverted after vaccination (see Table 7). The ID titers were slightly lower than the IM titers. In the ID vaccinated groups the group with the combined vaccination with PM had higher titers than the group vaccinated with DON alone. This is despite the fact that the combined DON+PM group (ID) only received 30% of the DON dose. Also it was noted that the combined DON+PM (ID) group had slower decreasing titers compared to the DON alone (ID) group. Intramuscular it was observed that the results for the groups that received DON alone, combined with PML (mixed) and combined with PML (non-mixed) were very similar. This implies that for the serology against DON, neither combining with the other antigens, nor mixing appears to have a significant effect.

TABLE-US-00007 TABLE 7 DON serology (log2 titres) Group T = 0 T = 28 T = 64 1 <4.3 8.7 5.0 2 <4.3 9.6 6.3 3 <4.3 <4.3 <4.3 4 <4.3 11.1 7.4 5 4.5 10.2 6.6 6 <4.3 10.6 7.4 7 <4.3 <4.3 <4.3 8 4.4 4.4 <4.3

[0063] Protection against DON challenged was measured in the intestine by determining the villus/crypt ratio (see Table 8). The ID group (Group 2) had the highest ratio, this ratio was the same as the healthy controls in the study as described in Example 1. Both the monovalent DON IM group (Group 4), and the 4-way DON, PCV, Mhyo, Lawsonia group (Group 5) had significantly healthier intestine then the non-vaccinated challenged animals (Group 8). The values arrived at for the combination vaccine (Group 2 and 5) where even better than for the group that received the monovalent DON vaccine (Group 4) indicating that adequate protection against DON challenge was arrived at with both combination vaccines, independent of the vaccination route and the presence of additional Lawsonia antigen.

TABLE-US-00008 TABLE 8 villus/crypt ratio Group 2 Group 4 Group 5 Group 8 Group 5, Example 1 average 1.74 1.50 1.65 1.27 1.71

[0064] Next to this, a reduction of stomach ulcers was observed in all vaccinated groups when compared to control Group 8. These data, and the data regarding the condition of the liver are depicted here below in Table 9.

TABLE-US-00009 TABLE 9 General health data Stomach ulcers Liver damage Group 2 +/ Mild Group 4 Normal to mild Group 5 Mild Group 8 +/ Middle to heavy

[0065] Thus, also at the level of stomach ulcers and liver damage it could be seen that the combination vaccine protected against DON challenge.

[0066] For showing protection against the other pathogens (PCV2, Mycoplasma hyopneumoniae and Lawsonia intracellularis) serology was measured for the IM groups at the end of the study. As is known for these existing antigens of existing commercial vaccines (Porcilis range), a positive serology after IM vaccination indicates protection against infection with the corresponding pathogen after IM as well as ID vaccination with the same antigen. The results are indicated in Table 10. For Mhyo a negative/positive test was used. For PCV and Lawsonia an Elisa titre was measured.

TABLE-US-00010 TABLE 10 Serology against PCV, Mhyo and Lawsonia Group 4 Group 5 Group 6 Group 7 PCV <2.0 9.5 12.1 11.5 Mhyo negative positive positive positive Lawsonia <3.9 5.2 5.9 5.8

[0067] The results show that each of the antigens elicited a positive serology, indicating that protection against the corresponding pathogens was arrived at.

Conclusion

[0068] This study shows that DON vaccination is not impacted by simultaneous vaccination with non-replicating immunogens of PCV, Mhyo and Lawsonia, and also, that the conjugated DON antigen does not negate the protection that can be arrived using each of these three antigens.