Acceleration of vector virus induced immune response in avians

Abstract

The addition of an oligodeoxynucleotide that is an avian TLR21 agonist, to an avian Herpesvirus vector vaccine, provides an acceleration of the immune response against the antigen that is expressed and delivered by the Herpesvirus vector.

Claims

1. A vaccine comprising a live Herpesvirus of turkeys (HVT) vector, an oligodeoxynucleotide, and a pharmaceutically acceptable carrier, wherein the live HVT vector comprises a heterologous nucleotide sequence encoding an antigen from a micro-organism that is pathogenic to avians; and wherein the oligodeoxynucleotide is an avian Toll-like receptor (TLR) 21 agonist.

2. A method of administering a vaccine against a micro-organism that is pathogenic to an avian comprising administering the vaccine of claim 1 to the avian.

3. The vaccine of claim 1, wherein the avian TLR21 agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

4. A method for preparing the vaccine of claim 3, comprising the admixing the live HVT vector, the TLR21 agonist, and a pharmaceutically acceptable carrier.

5. A composition comprising the live HVT vector and the TLR21 agonist of claim 3.

6. A vaccine for avians comprising the composition of claim 5, and a pharmaceutically acceptable carrier.

7. A method for preparing a vaccine comprising admixing the composition of claim 5 and a pharmaceutically acceptable carrier.

8. A method of administering a vaccine against a micro-organism that is pathogenic to an avian comprising administering the vaccine of claim 3 to the avian.

9. The vaccine of claim 3, wherein the avian TLR21 agonist comprises the nucleotide sequence of SEQ ID NO: 1.

10. A composition comprising the live HVT vector and the TLR21 agonist of claim 9.

11. A vaccine for avians comprising the composition of claim 10 and a pharmaceutically acceptable carrier.

12. A method of administering a vaccine against a micro-organism that is pathogenic to an avian comprising administering the vaccine of claim 9 to the avian.

13. The vaccine of claim 3, wherein the avian TLR21 agonist comprises the nucleotide sequence of SEQ ID NO: 4.

14. A composition comprising the live HVT vector and the TLR21 agonist of claim 13.

15. A vaccine for avians comprising the composition of claim 14 and a pharmaceutically acceptable carrier.

16. A method of administering a vaccine against a micro-organism that is pathogenic to an avian comprising administering the vaccine of claim 13 to the avian.

Description

EXAMPLES

Example 1: Synthesis and Purification of TLR21 Agonists

(1) Oligodeoxynucleotides for use in experiments for the present invention, were ordered from BioSpring (Frankfurt a. M., Germany), and were received purified and lyophilised at an indicated amount. These were diluted in a suitable buffer, depending on the experimental use. The concentration was checked occasionally by size exclusion chromatography combined with UV monitoring, but was found to be correct in all cases.

Example 2: Avian Herpesvirus Vector

(2) The avian Herpesvirus vector used for some of the experiments, HVT-F-VP2, was based on HVT, and comprised the F gene from NDV, as well as the VP2 gene from IBDV, both inserted into the Us2 region. Construction of the vector, production on cells, quantification, etc., were all as described in WO 2013/057.235.

Example 3: In Vitro Tests of Avian TLR21 Agonist Activity

(3) The use of the HEK293 cell-line transformed with a chicken TLR21 receptor, for the testing and qualification of avian TLR21 agonist candidates has been described e.g. in WO 2012/089.800. However in brief: HEK293 cells were transfected with an NFkappaB activation reporter plasmid: pNiFty2-SEAP (InvivoGen, San Diego, Calif., USA). Clonal cell lines were selected, and used for the transfection with a chicken TLR21 gene. Again clonal cell lines were selected. These were used for the detection of NFkappaB activation upon incubation with different concentrations of avian TLR21 agonistic oligodeoxynucleotides. Read out was by colorimetric detection of the secreted SEAP (secreted embryonic alkaline phosphatase) activity, by OD 405 nm spectrophotometry. This was plotted as mOD405 nm/min, as function of a gradient of the agonist's concentration. The relevant results were those with a high rate of colour formation, at a relatively low agonist concentration.

Example 4: In Vivo Test of Avian TLR21 Agonist Activity on Avian Herpesvirus Vector Induced Immunity

4.1. Introduction

(4) This experiment was designed to test the in vivo effect of a TLR21 agonist: X4-1-minG (EC50=1.9 nM) on avian Herpesviral vector induced immunity in chickens. Different time-points for the administration of the TLR21 agonist to vector vaccinated birds were tested, for one amount of the agonist.

4.2. Materials and Methods

4.2.1. Experimental Design

(5) Seven (7) groups of 1 day-old SPF White Leghorn chicks (n=25/group) were vaccinated once s.c. at the base of the neck with HVT-F-VP2 vector (expressing the NDV-F and the IBDV-VP2 antigens): groups 3-9. Group 2 was only s.c. injected with TLR21 agonist at T=0, and group 1 was the unvaccinated challenge control.

(6) At several time points post vaccination a TLR21 agonist was s.c. injected at the base of the neck according to the treatment schedule for grouping and dosing. Blood samples were taken before vaccination (T=0) from 20 hatchmates via bleeding, and at T=2 weeks post-vaccination from 5 randomly picked vaccinated/injected birds from groups 2-9 after which these 40 animals were taken out of the experiment, and formed a new n=20/group. Sera were used to determine the anti-NDV and anti-IBDV antibody titres. After bloodsampling the 25 non-vaccinated control animals (group 1) were divided at random over the 8 vaccinated/injected groups, i.e. 3 control animals were placed into each group (n=23/group; 1 group with n=24). Subsequently all animals were challenged via the intramuscular (i.m.) route with 0.2 ml (6.0 log 10 EID50) of the NDV Herts 33/56 strain.

4.2.2. Biosafety

(7) Chickens were kept in isolators, under negative pressure, and with Hepa filtered air in/out, to contain the genetically modified vectors and the virulent challenge virus.

4.2.3. Test Materials

(8) avian TLR21 agonist: X4-1-minG

(9) Vector vaccine: HVT-F-VP2, passage 6.

(10) Challenge material: Live NDV Herts 33/56, at a titre of: 9.6 log 10 EID50 per ml.

(11) Dosing: 1 dose equals 0.2 ml. The TLR21 agonist was injected at 0.1 μg per dose.

4.2.4. Test Animals

(12) White Leghorn Chicken, specific pathogen free, of mixed sex, 1 day old at the start of the experiment. Chicks were numbered individually by wing-tag. The 25 control chickens of group 1, were numbered with a different coloured tag.

4.2.5. Food and Water

(13) Food and water was available to the animals ad libitum.

4.2.6. Grouping and Dosing

(14) TABLE-US-00002 Vector vaccine s.c. injection of 0.1 ml X4-l-minG (0.2 ml s.c. at (0.1 μg/dose) at different time Group No. T = 0) points post vaccination (p.v.) 1 25 none none 2 25 none T = 0 3 25 HVT-F-VP2 none 4 25 HVT-F-VP2 T = 0 (incorporated into the vaccine) 5 25 HVT-F-VP2 T = 1 day p.v. 6 25 HVT-F-VP2 T = 3 days p.v. 7 25 HVT-F-VP2 T = 6 days p.v. 8 25 HVT-F-VP2 T = 8 days p.v. 9 25 HVT-F-VP2 T = 10 days p.v. 20 hatchmates were bled at T = 0

4.2.7. Vaccination

(15) The animals from the groups 3-9, were vaccinated s.c. with 0.2 ml vector vaccine at the base of neck at the age of 1 day old. Group 2 was injected with only 0.1 ml TLR21 agonist at T=0.

(16) At several time points post vaccination 0.1 ml TLR21 agonist was s.c. injected at the base of the neck in the animals of groups 5-9 according to the “Grouping and dosing” Table. The 25 control animals from group 1, which had a differently coloured number tag, were not vaccinated.

4.2.8. Challenge

(17) At 2 weeks post vaccination all remaining animals in each group were challenge-infected via the injection of 0.2 ml Live NDV Herts 33/56 (6.0 log 10 EID50 per chicken) via the i.m. route in the right leg muscle.

4.2.9. Blood Sampling

(18) Blood samples for serology were taken before vaccination (T=0) from 20 hatchmates via bleeding, and at T=2 weeks post-vaccination from 5 randomly picked vaccinated/injected birds from all vaccinated/injected groups. After bloodsampling, these 40 animals (8 groups of 5 animals) were taken out of the experiment. All blood samples were transported to a laboratory for processing and analysis.

(19) After bloodsampling, but before challenge, the 25 non-vaccinated control animals were divided at random over the 8 vaccinated/injected groups, i.e. 3 control animals were placed in each group (n=23/group, leaving one group with n=24) to serve as sentinels.

4.2.10. Observation for Clinical Signs

(20) Pre-Challenge:

(21) Chickens were observed daily for the presence of clinical signs of disease or other abnormalities, by a qualified person. Chickens showing pain or discomfort, which was considered to be non-transient in nature or likely to become more severe, were sacrificed for animal welfare reasons. Chickens that died or were sacrificed pre-challenge were not submitted for post mortem examination, as no high or unexpected mortalities occurred.

(22) Post-Challenge

(23) For 14 days post-challenge all chickens in all groups were scored daily for the occurrence of clinical evidence of NDV infection or mortality. Data were recorded per animal on special forms. The following score system was used: 0. No occurrence of clinical evidence of Newcastle disease. 1. Occurrence of clinical evidence of Newcastle disease, with central nervous signs like: Clonic spasm, muscular tremors, torticollis, opisthotonos, or paralysis of legs or wings. 2. Mortality caused by NDV challenge. In case animals were sacrificed for animal welfare reasons this was indicated on the form.

4.3. Results

(24) TABLE-US-00003 s.c. injection of 0.1 ml Vector vaccine X4-l-minG (0.1 μg/dose) (0.2 ml s.c. at at different time points % survival Gr. No. T = 0) post vaccination (p.v.) at 2 w. p.v. 1 20 — none None 2 20 — T = 0 None 3 20 HVT-F-VP2 none 20 4 20 HVT-F-VP2 T = 0 35 (incorporated into the vaccine) 5 20 HVT-F-VP2 T = 1 day p.v. 20 6 20 HVT-F-VP2 T = 3 days p.v. 45 7 20 HVT-F-VP2 T = 6 days p.v. 30 8 20 HVT-F-VP2 T = 8 days p.v. 25 9 20 HVT-F-VP2 T = 10 days p.v. 10

4.4. Conclusions

(25) The administration of an avian TLR21 agonist, simultaneous with, or shortly after the administration of an avian Herpesvirus vector provided for a significant acceleration of the immune response against the heterologous antigen that was expressed and delivered by the vector. This is demonstrated by the percentage of survivors of a severe NDV challenge infection, that was given quite early—namely at two weeks—after the vector vaccination.

(26) While none of the chickens survived that were unvaccinated (group 1) or received only agonist (group 2), and only 20% of the vector vaccinates (group 3) survived, the survival of vaccinates receiving both vector and agonist was mostly higher, giving 30, 35 and even 45% challenge survivors (groups 4, 6, and 7). The 20% survival in the group receiving the agonist at 2 days p.v. (group 5) is probably the result of a variability in the trial. After 8 days p.v. the agonist could no longer accelerate the immune response to the vector-expressed heterologous antigen (group 9).

(27) Consequently, the agonist alone could not induce an immune protection. Also, at this early time after vaccination, the vector vaccine alone had only established a modest immunity against the NDV-F antigen. However, with the combined administration of vector and agonist, an early onset of immunity could be achieved, resulting in a challenge survival rate that more doubled.

Example 5: Further In Vivo Test of Avian TLR21 Agonist Activity on Avian Herpesvirus Vector Induced Immunity

(28) A further animal trial in chickens was performed, in essentially the same way as described in Example 4, but with some modifications: the main difference being that all vaccinations comprised both the avian Herpesvirus vector and the oligodeoxynucleotide in the same inoculation, and these were thus administered at the same time (day 0).

(29) Other variations from the protocol of Example 4 were that another avian TLR21 agonist was used: X4-pent (SEQ ID NO: 4) (EC50=430 pM), and that this agonist was tested in three different amounts/dose.

5.1. Results

(30) TABLE-US-00004 Amount of % survival at Gr. No. Vector vaccine X4-pent 2 w. p.v. 1 20 none none None 2 20 HVT-F-VP2 none 10 3 20 HVT-F-VP2 0.1 μg 45 4 20 HVT-F-VP2 1.0 μg 20 5 20 HVT-F-VP2  20 μg 20

5.2. Conclusions

(31) Again, the addition of an avian TLR21 agonist demonstrated significant acceleration of the immune response against the heterologous antigen expressed and delivered by the avian Herpesvirus vector: at two weeks post vaccination, up to 45% of vaccinates that received both the vector vaccine and the agonist (group 3) were protected against a severe NDV challenge infection. Whereas none of the unvaccinated chickens were protected (group 1), and only 10% of the vaccinates receiving only the vector vaccine (group 2).

(32) Notably: the higher amounts of the agonist per dose (1 or 20 μg—groups 4 and 5) did not improve the immune acceleration, as both provided less immune acceleration than the lowest amount of 0.1 μg per animal dose (group 3).