Eimeria vaccine with improved efficacy

Abstract

The present invention relates to the fields of veterinary parasitology and -vaccinology; more specifically the invention relates to a composition comprising live Eimeria oocysts and a pharmaceutically acceptable carrier. The composition is characterised in that it comprises a TLR3 agonist. The composition can be used to prepare a vaccine for poultry against Coccidiosis. The vaccine can be applied for example as a coarse spray on day-old chicks. The TLR3 agonist in the vaccine allows for a reduction of the dose of Eimeria oocysts up to 4 fold, while obtaining the same level of protection against challenge as from a vaccine without the TLR3 agonist. Alternatively, when employing a conventional dose of Eimeria oocysts in the vaccine, the TLR3 agonist causes an earlier onset of immunity, resulting in a significant reduction of the intestinal lesion score upon challenge, already at 2 instead of at 3 weeks after vaccination.

Claims

1. A composition comprising live Eimeria oocysts and a pharmaceutically acceptable carrier, wherein the composition further comprises between about 0.3 and about 5% w/v Xanthan gum and a TLR3 agonist.

2. The composition of claim 1, wherein the TLR3 agonist is poly A:U.

3. The composition of claim 1, wherein the live Eimeria oocysts are sporulated oocysts.

4. The composition of claim 1, wherein the live Eimeria oocysts are selected from the group consisting of: E. tenella, E. acervulina, E. maxima, E. mitis, E. necatrix, E. brunetti, E. praecox, E. mivati, E. hagani, E. meleagrimitis 1, E. meleagrimitis 2, E. adenoeides, E. gallopavonis, E. dispersa, and any combination thereof.

5. The composition of claim 1, wherein the composition further comprises between about 0.1 and about 1.6% w/v of a metal salt.

6. The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable colorant.

7. A method for the preparation of a composition comprising live Eimeria oocysts, a pharmaceutically acceptable carrier, and a TLR3 agonist comprising admixing live Eimeria oocysts and a pharmaceutically acceptable carrier, with a TLR3 agonist.

8. The composition of claim 1, comprising 0.3-1.5% w/v Xanthan gum, and having a viscosity of between 200-4000 mPa.Math.s.

9. A method of vaccinating poultry against Coccidiosis, comprising administering the composition of claim 1 to said poultry.

10. The method of vaccinating of claim 9, comprising administering the vaccine by coarse spray onto the poultry and/or onto their surroundings.

11. A method for the reduction of an infection with Eimeria in poultry, or of associated signs of disease, comprising administering the composition of claim 1 to said poultry.

12. A method for the delivery of a TLR3 agonist to an avian gut by an oral route, wherein the TLR3 agonist is admixed with a Xanthan gum.

Description

EXAMPLES

(1) 1. Effect of TLR3 Agonist on Eimeria Vaccine Efficacy; Earlier Onset of Immunity

(2) 1.1. Experimental Outline

(3) This experiment tested the effect on the onset of immunity of Paracox 8 vaccine by the addition of a TLR3 agonist. A dose titration was performed for the TLR3 agonist. Results were directly compared with birds vaccinated with Paracox 8 only.

(4) Two hundred and ten one day-old birds were divided into seven groups of 30 birds. All birds in groups 1-5 were vaccinated with Paracox vaccine, diluted in 0.6% Xanthan Gum, containing 0.1% carmine; by oral gavage of 0.5 ml vaccine. Groups 1-4 each received a different amount of TLR3 agonist. The birds in group 5 were vaccinated with Paracox 8 only and the birds in groups 6 and 7 were not vaccinated (see Table 1). All birds were kept in their groups in separate pens for 14 days post vaccination (PV). Faecal samples were collected on days 7, 14 and 19 from the control birds (Groups 6 and 7) to monitor for a Coccidiosis infection. On day 13 PV prior to challenge, all birds were uniquely identified by numbered tag, then on day 14 PV each bird in groups 1-6 was challenged with 15.000 oocysts of E. tenella wildtype. While this date of challenge was too early for normal Paracox vaccination to reach full protection, it allowed the detection of any enhancement in onset of immunity.

(5) On day 5 post challenge all the challenged birds, and those in group 7 were euthanized and examined post mortem for lesions associated with an E. tenella infection. The birds were observed daily throughout the study for any clinical signs which may have been related to an Eimeria spp. infection.

(6) TABLE-US-00001 TABLE 1 Schedule of vaccinations and challenges of Example 1 Treatment TLR3 agonist Group Vaccine (g/animal dose Challenge 1 Yes 5 Yes 2 10 3 20 4 40 5 0 6 No 0 7 0 No
1.2. Materials & Methods

(7) A commercial batch of Paracox 8 vaccine was used, which was resuspended to give one full dose per 0.5 ml. The vaccine was diluted 1:1 with a 2 concentrated stock of a diluent containing 1.2% Xanthan gum solution, in 75 mM NaCl (i.e. 0.44% w/v), and containing 0.2% w/v Carmine (E 120). The diluent stock had been sterilised by autoclaving at 115 C. for 30 minutes.

(8) The Paracox 8 vaccine dilution was prepared in a safety cabinet. 100 ml of vaccine (containing 200 doses) was prepared for each group; two 1000 dose sachets of the vaccine were shaken and massaged vigorously for 1 minute to ensure resuspension of the oocysts. The contents of the sachets were pooled in a suitable container. For each vaccine 21 ml of the neat pooled Paracox 8 vaccine was added to a sterile glass Duran bottle (appropriately labelled with the group number). A TLR3 agonist stock solution at 10 mg/ml was thawed quickly, and then the appropriate volumes of the agonist and water were added to the vaccine and mixed thoroughly. Next, 50 ml of the 2 diluent stock was added to each Duran bottle (depending on group). All formulations were then thoroughly mixed by shaking for at least 10 minutes prior to use.

(9) In the vaccine samples Groups 1-4 received an amount of the TLR3 agonist stock at 10 mg/ml, to reach the intended amount of agonist; group 1: 100 l, group 2: 200 l, etc.; all these volumes were completed with sterile water up to 29 ml.

(10) TLR3 Agonist:

(11) The TLR3 agonist used was poly A:U, double stranded homopolymer (Sigma Aldrich). It was dissolved from a lyophilised sodium salt to a 10 mg/ml stock solution, and stored at 70 C. until use.

(12) Challenge Materials:

(13) The challenge material was a batch of E. tenella wildtype, sterile sporulated oocysts, stored at 2-8 C. until use. The concentration of the oocysts in the challenge material was determined using a modified Fuchs-Rosenthal counting chamber. From this a dilution was made to give 15.000 oocysts per 0.5 ml challenge dose.

(14) Test Animals:

(15) Experimental animals were Lohmann SPF chicks of mixed sex, day old at vaccination. Birds were placed randomly into groups of 30, in individual pens.

(16) Birds were marked individually with swift tags on day 13 post vaccination.

(17) The birds were vaccinated by administering 0.5 ml of the appropriate vaccine preparation per bird, by oral gavage.

(18) Chicks were challenged with 0.5 ml each of the E. tenella wild type preparation, by oral gavage, all within 1 hour from each other.

(19) Throughout the experiment all birds were observed daily for clinical signs of coccidial infection such as diarrhoea or blood in the faeces, depression or inappetance. No special findings were observed. 3 chicks died from causes unrelated to the experiment: 1 from group 1, and 2 from group 2.

(20) On day 5 post challenge all birds were euthanized, and sent for necropsy. Post mortem, all birds from each group were examined for gut lesions relating to E. tenella.

(21) For the purpose of checking vaccine- or challenge take, faecal collections were made from the floor of the pens of the control birds (groups 6 and 7) on days 7, 14 and 19 post vaccination. Each sample was taken from the litter in the pen, the samples were approximately 10-20 grams and were representative of the whole pen e.g. collected from 4 corners and from the middle of the pen; the samples were fresh and contained the minimum amount of litter as was possible. Each sample was double bagged, labelled, and stored at 2-8 C. until processing. The oocysts were detected as per standard practice, and the numbers were calculated using the Modified McMaster counting technique.

(22) 1.3. Results

(23) Gut lesion scores relating to E. tenella were determined at 5 days post challenge by necropsy. The lesion assessments were carried out blinded with respect to treatment group, with the exception of the control groups 6 and 7. Lesion score results for groups 1-7 are summarised in FIG. 1, panels A through G respectively, as percentage of animal displaying a certain score. Apart from an occasional death, for these groups n=30.

(24) The lesion score results were as expected for the control groups; the unchallenged birds in group 7 all had a lesion score of 0, whereas the unvaccinated, challenged birds of group 6 had high scores of 3 or 4, resembling the lesion score profile of naive birds.

(25) The birds in group 5 (Paracox 8 vaccine only) had scores which were weighted to the higher end of the scoring range. This was not unexpected as the birds were challenged at 14 days post vaccination when this vaccine normally only provides partial protection. However this facilitated observing whether there had been an earlier onset of immunity as result of the addition of the TLR3 agonist.

(26) The birds in groups 1 and 2, which received the low doses of the TLR3 agonist (5 or 10 g/bird respectively), showed a full range of lesion scores with a clearly increased proportion of lower lesion scores. In groups 3 and 4, where the birds received the higher two dose levels of the TLR3 agonist (20 or 40 g/bird respectively), the lesion score profile was not very different compared to the birds vaccinated with Paracox 8 only (group 5).

(27) Faecal collections showed that no oocysts were detected in any of the faecal samples taken. This demonstrated that the birds in group 7 remained uninfected throughout the study. For group 6, also no oocysts were detected in the final collection, however this was probably related to the severe illness of these birds as was demonstrated by their gut lesion scores, preventing normal faeces to form.

(28) 1.4. Conclusions

(29) Of all the TLR3 agonist amounts tested, the birds receiving the lower doses of 5 or 10 g per animal, had the best lesion score results, with a bigger range of scores, and a clear tendency towards the lower lesion scores. This is convincing proof of the induction of an earlier onset of immunity.

(30) In this experiment the higher amounts of TLR3 agonist/animal did not present clear immunological enhancement. This may be an indication that there is an optimum profile in the dose-effect of the TLR3 agonist. However, in another vaccination-challenge experiment (Example 2) a higher dose of TLR3 agonist/animal did show a clear benefit compared to a Paracox 8 vaccine only group.

(31) 2. Effect of TLR3 Agonist on Eimeria Vaccine Efficacy; Earlier Onset of Immunity 2

(32) In an experiment with largely the same setup and procedures as that described in Example 1, amounts of TLR3 agonist of 10 or 20 micrograms/bird were tested for effect on enhancement in onset of immunity.

(33) Groups of 20 chickens were used, which received treatment as displayed in Table 2

(34) TABLE-US-00002 TABLE 2 Schedule of vaccinations and challenges of Example 2 Treatment TLR3 agonist Group Vaccine (g/animal dose Challenge 1 Yes 10 Yes 2 20 3 0 4 No 0 5 0 No
2.1. Results

(35) The lesion score results of this experiment are represented in FIG. 2, in panels A through E, for the groups 1-5 respectively.

(36) The results were as expected for the control groups: the unvaccinatedunchallenged birds of group 5 all had a lesion score of 0, whereas the unvaccinatedchallenged birds had high scores of 3 or 4, indicating these had no protection against the challenge. Challenge severity appeared to be slightly below that applied in Example 1.

(37) The birds receiving Paracox 8 vaccine only, group 3, had a range of scores, but weighted to the higher end of the scoring range. Like in Example 1 this was to be expected because of the early date of challenge (2 weeks) post vaccination.

(38) The treatment groups 1 and 2, had a broad lesion score profile, with a higher proportion of lower lesion scores, than the birds in vaccine group 3, indicating that the TLR3 agonist did enhance the vaccine's immunity in respect of the onset of immunity it induced.

(39) In this experiment, the higher amount of TLR3 agonist (20 g/bird) seemed more effective than the lower amount (10 g/bird) in inducing an earlier onset of immunity.

(40) 3. Effect of TLR3 Agonist on Eimeria Vaccine Efficacy; Reduction of Oocysts Per Dose

(41) A further experiment was performed to test the effect of a TLR3 agonist on the efficacy of live Eimeria oocyst vaccination. In this experiment a number of large groups were treated, to assess the effect of the TLR3 agonist on the dose of the vaccine. The set-up and performance in essence was the same as that for the experiments in the Examples 1 and 2, except that: chickens were of commercial layer type; the vaccine used was Paracox 5, the dose of TLR3 agonist/bird was fixed at 7.5 g, the vaccination was administered by coarse spray at 0.2 ml/bird, and the challenge was at day 21 post vaccination and with 4 separate strains.

(42) 3.1. Experimental Outline

(43) 1125 chicks of day-old were divided over 9 groups of 125 animals; one group remained unvaccinated, the other 8 groups received Paracox 5 vaccine in different dosages, and either with or without TLR3 agonist, see Table 3. Vaccine contained Xanthan gum, and was applied as a coarse spray using a standard hatchery spray applicator.

(44) On day 21 post vaccination, 80 birds from each treatment group were allocated to one of four challenge types, leaving 20 birds per species-challenge subgroup. A total of 720 birds were given a 0.5 ml oral gavage of one of four Eimeria species challenges. Five birds from control group 1 were left unchallenged and kept in their original pen for assessment of baseline necropsy assessments on day 28 p.v.

(45) All birds were weighed individually on day 20 p.v. prior to allocation to challenge type, and again on day 28 p.v. prior to necropsies, to determine live bodyweight gain. Bulked faecal samples were collected from day 21 to 25 and from day 25 to 28 p.v. from all pens for oocyst output enumeration. Ten birds per challenge treatment group for E. acervulina, E. maxima and E. tenella challenge types were necropsied for assessment of gut lesions. As E. mitis does not produce pathognomonic gut lesions, ten birds from group 1 (controls) that were challenged with E. mitis were necropsied for parasite status to confirm uptake of challenge.

(46) 3.2. Materials & Methods

(47) Test Animals:

(48) Hyline brown, commercial layer chickens. Of male gender, number tagged individually. All chicks were day-old at day of vaccination. Birds were kept in appropriate pens under climatised and controlled conditions, on bedding of wood shavings. Feed was unmedicated.

(49) Chicks were closely monitored throughout the study; 7 birds died in the first week after vaccination of causes unrelated to the treatment, these were from different groups.

(50) TABLE-US-00003 TABLE 3 Schedule of vaccinations and challenges of Example 3 Treatment TLR3 agonist Group Vaccine dose (X g/bird) Challenge 1 none none Yes, 2 1:20 no one of: E. acervulina, 3 1:20 yes E tenella, E. mitis, or 4 1:10 no E. maxima 5 1:10 yes (n = 20) 6 1:4 no 7 1:4 yes 8 full no 9 full yes
Vaccines:

(51) Sachets of Paracox 5, at a total of 11.000 doses were shaken thoroughly, divided into the various groups in respective dilutions in water, mixed with TLR3 agonist where intended, and all vaccines were mixed 1:1 with a sterile 2 stock of diluent containing Xanthan gum (1.2%), NaCl (75 mM), and carmine (0.2%), as described.

(52) The amounts of the oocysts in the full dose vaccine for groups 8 and 9 were per dose: E. acervulina: 500; E. maxima, strain CP: 200; E. maxima, strain MFP: 100; E. mitis: 1000; and E. tenella: 500 oocysts.

(53) The TLR3 agonist used was poly A:U at a dose of 7.5 g per bird.

(54) Vaccines were administered as a coarse spray, using a Spraycox II machine, according to the manufacturer's instructions: volume and pressure settings were calibrated prior to use; vaccines were shaken well before use; vaccines were administered lowest to highest dose, and groups without agonist prior to groups with agonist, with intermediate rinsing. Vaccine volume was set at 20 ml per 100 birds. Once vaccinated, birds were kept in the tray in which they were vaccinated for at least 30 minutes in a well-lit and warm area before transfer to holding pens.

(55) Challenge Materials:

(56) All challenge materials were liquid suspensions of sporulated oocysts from 4 different species: E. acervulina (Ea), E. tenella (Et), E. mitis (Emit), or E. maxima (Emax). The intended number of oocysts were determined using a modified Fuchs-Rosenthal counting chamber, as respectively: Ea: 110{circumflex over ()}6/bird in 1 ml; Et: 310{circumflex over ()}4/bird in 0.5 ml; Emit: 310{circumflex over ()}5/bird in 1 ml; Emax: 110{circumflex over ()}4/bird in 0.5 ml.

(57) Challenge material was administered individually, by oral gavage.

(58) Necropsy and Lesion Scoring:

(59) Staff performing oocyst counting or lesion scoring were blinded from information on the treatments. Lesion scoring used standard criteria, specific for each of the challenge species (Ea, Et, and Emax).

(60) 3.3. Results

(61) Take of Vaccine and Challenge:

(62) Faecal samples of group 1 birds remained negative for oocyst counts prior to challenge indicating that group 1 was coccidia free prior to challenge. Oocyst counts of faecal samples taken from vaccinated groups 2 to 9 were all positive from day 11 p.v., indicating that vaccinal oocyst replication was successful for all vaccinated groups.

(63) Challenge take was also successful with clinical symptoms of Coccidiosis observed from three days post challenge until necropsy. At necropsy on day 28 p.v., birds challenged with E. acervulina, E. maxima and E. tenella had gut lesion scores above 0, and all gut scrapings of control birds challenged with E. mitis were positive for oocysts confirming challenge was effective.

(64) Live Bodyweight Gain:

(65) Results of average live bodyweight gains in grams, from day 20 trough day 28 post vaccination, for the different challenge subgroups, are presented in Table 4. Size of the different subgroups was 20 animals.

(66) For E. acervulina, all dilutions of vaccine had a significantly better (p<0.01) average live bodyweight gain compared with the unvaccinated-challenged control group, with the exception of the 1/20 dilution (group 2), which had a significantly lower average bodyweight gain. However this dramatically recovered in the presence of TLR3 agonist.

(67) Of the birds in the vaccinated subgroups receiving one of the other challenge species (E. tenella, E. mitis, or E, maxima), most had significantly better average live weight gains compared to their unvaccinated-challenged group. However no effect of vaccine dilution or the presence of TLR3 agonist could be detected.

(68) It is assumed that the vaccine doses applied, even in the diluted samples still contained too high doses of oocysts to be able to observe an effect on bodyweight gain. In a repeat experiment higher vaccine dilutions will be used.

(69) TABLE-US-00004 TABLE 4 Average live bodyweight gains in grams, from day 20 trough day 28 post vaccination, for the different challenge subgroups (n = 20) E. E. Group acervulina E. tenella E. mitis maxima 1 - unvacc. - no TLR3 agonist 63.7 73.3 50.4 53.7 2 - 1/20 dose 36.7 84.0 72.3 92.3 3 - 1/20 dose + TLR3 agonist 88.1 84.2 73.1 90.7 4 - 1/10 dose 85.6 77.6 60.2 76.5 5 - 1/10 dose + TLR3 agonist 83.5 87.4 72.6 no data 6 - 1/4 dose 88.8 91.2 70.6 83.4 7 - 1/4 dose + TLR3 agonist 93.2 84.2 74.5 81.6 8 - full dose 85.3 84.5 71.5 86.4 9 - full dose + TLR3 agonist 93.0 83.0 72.0 82.6
Gut Lesion Scores:

(70) Results of average gut lesion scores by challenge type and treatment group are represented in Table 5. for these groups n=20.

(71) Only 50% of the unvaccinated control birds challenged with E. acervulina had lesion scores over 2.

(72) Vaccinated-challenged birds had no significant difference in the distribution of their lesion scores. Eighty percent of the unvaccinated control birds challenged with E. maxima had lesion scores over 2. There was a significant (p<0.05) difference in the distribution of lesions for all vaccinated birds (groups 2-9) compared to the unvaccinated birds (all p values <0.03), irrespective of vaccine dilution or the presence of TLR3 agonist. Ninety percent of the unvaccinated control birds challenged with E. tenella had lesion scores over 2. Although for the standard vaccine in the absence of TLR3 agonist (group 8) the percentage of birds with lesion scores over 2 was reduced to 60%, the change in the overall distribution was not statistically significant (p=0.07). There was however a significant reduction in the distribution of the lesion scores with vaccine diluted 1/4 (group 6; p=0.001). At higher dilutions of vaccine ( 1/10, group 2, and 1/20, group 4) 70% of birds had lesion scores over 2, and the distributions were not significantly different from the unvaccinated challenged control group. The presence of TLR3 agonist reduced the percentage of birds with lesion scores over 2 to 20% (group 5, 1/10 dose) and 50% (group 3, 1/20 dose) respectively, resulting in a lesion score distribution that was statistically significantly different from the control group for group 5 (p=0.003), and almost statistically significant for group 3 (p=0.0509).

(73) 3.4. Discussion and Conclusions

(74) There were no significant differences in bird bodyweights between control and vaccinated treatment groups for each challenge type on day 20 p.v. Faecal samples from the unvaccinated controls (group 1) confirmed that group 1 remained coccidia free prior to challenge. Oocyst counts from faecal samples taken from vaccinated treatment groups 2-9 were all positive from day 11 p.v. indicating vaccinal oocyst uptake and re-cycling was successful for all vaccinated groups. Recounting of challenge materials used, showed that challenge dose was within 2% of target.

(75) At necropsy on day 28 p.v., unvaccinated control birds challenged with E. acervulina, E. maxima or E. tenella had gut lesion scores over 2 in 50%, 80% and 90% of the birds respectively. Also all gut scrapings of control birds challenged with E. mitis were positive for oocysts.

(76) Although the lesion scores obtained following challenge with E. acervulina were relatively mild, high levels of oocyst output and a significant effect on weight gain was seen in the unvaccinated control group, thus confirming that all challenges were effective. Vaccine dilution showed a variable effect on efficacy depending upon the challenge species and the parameter being measured. Dilution of vaccine up to 1/20 had no effect on any of the efficacy parameters investigated for either E. maxima or E. mitis, indicating that these antigens are significantly in excess within the Paracox 5 formulation. In contrast, for E. tenella reduced levels of protection in terms of both reduction in oocyst output and lesion score was observed at vaccine dilutions of 1/10 or higher. Similarly for E. acervulina, when vaccine was diluted 1/20, no protection was observed against reduction in weight gain. Gut lesions were too mild to demonstrate a definitive outcome. In all cases where reduced protection was observed due to vaccine dilution, the inclusion of TLR3 agonist in the vaccine formulation was beneficial.

(77) Overall these results prove that the inclusion of a TLR3 agonist in the vaccine formulation may increase vaccine potency at lower antigen doses up to 4 fold, while obtaining the same level of protection against challenge.

(78) TABLE-US-00005 TABLE 5 Average gut lesion scores by challenge type and treatment group (n = 20) Group E. acervulina E. tenella E. maxima 1 - unvacc. - no TLR3 agonist 1.5 2.9 2.1 2 - 1/20 dose 1.5 2.2 0.9 3 - 1/20 dose + TLR3 agonist 0.9 1.7 1.2 4 - 1/10 dose 1.0 2.0 1.2 5 - 1/10 dose + TLR3 agonist 1.3 1.0 1.0 6 - 1/4 dose 1.0 0.6 0.8 7 - 1/4 dose + TLR3 agonist 1.4 0.3 1.2 8 - full dose 1.5 2.0 0.9 9 - full dose + TLR3 agonist 1.1 0.2 0.9

LEGEND TO THE FIGURES

(79) FIG. 1 displays the results of the vaccination-challenge experiment described in Example 1. Panels A through G present the lesion scores after vaccination-challenge treatment for the groups 1-7 respectively.

(80) The horizontal axis presents the lesion score number 0-4, and the vertical axis the percentage of the birds displaying that lesion score in that group. For these groups n=30.

(81) FIG. 2

(82) Results of lesions scores that resulted from the vaccination-challenge experiment of Example 2. Panels A-E depict the results of groups 1-5 respectively. Axes used are the same as in FIG. 1. For these groups n=20.