TRIPLE VACCINE AGAINST AVIBACTERIUM PARAGALLINARUM AND AVIAN ENCEPHALOMYELITIS VIRUS AND FOWL POX VIRUS

Abstract

The present invention relates i.a. to an immunogenic composition comprising: a) one or more antigens of avibacterium paragallinarum and one or more antigens of avian encephalomyelitis virus and one or more antigens of fowl pox virus; and b) a pharmaceutically acceptable carrier. Furthermore, the present invention relates to methods for immunizing a subject comprising administering to such subject the immunogenic composition of the present invention. Moreover, the present invention relates to methods of treating or preventing clinical signs caused by avibacterium paragallinarum, avian encephalomyelitis virus and fowl pox virus in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to the present invention.

Claims

1. An immunogenic composition comprising: a) one or more antigens of a bacterin of avibacterium paragallinarum and one or more antigens of a modified live avian encephalomyelitis virus and one or more antigens of a modified live fowl pox virus; and b) a pharmaceutically acceptable carrier

2. An immunogenic composition comprising: a) a bacterin of avibacterium paragallinarum and a modified live avian encephalomyelitis virus and a modified live fowl pox virus; and b) a pharmaceutically acceptable carrier.

3. The immunogenic composition of claim 1 or 2, wherein the bacterin is a whole inactivated bacterin.

4. The immunogenic composition of any one of claims 1 to 3, wherein the immunogenic composition is effective in the treatment and/or prophylaxis of clinical signs caused by avibacterium paragallinarum infection and/or avian encephalomyelitis virus infection and/or fowl pox virus infection in a subject of need.

5. The immunogenic composition of any one of claims 1 to 4, wherein said immunogenic composition is a vaccine.

6. The immunogenic composition of any one of claims 1 to 5, wherein said pharmaceutically acceptable carrier is an adjuvant.

7. The immunogenic composition of any one of claims 1 to 6, wherein said pharmaceutically acceptable carrier is an adjuvant selected from the group consisting of mineral oil, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, and combinations thereof.

8. The immunogenic composition of any one of claims 1 to 7, wherein the antigens of avibacterium paragallinarum and avian encephalomyelitis virus and fowl pox virus act synergistically together.

9. A kit comprising the immunogenic composition of any one of claims 1 to 8.

10. The kit according to claim 9, wherein the bacterin of avibacterium paragallinarum, the modified live avian encephalomyelitis virus and the modified live fowl pox virus are in one or two container(s).

11. A method for immunizing a subject comprising administering to such subject an immunogenic composition of any one of claims 1 to 8.

12. A method of treating or preventing clinical signs caused by avibacterium paragallinarum and/or avian encephalomyelitis virus and/or fowl pox virus in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 8.

13. A method of reducing the mortality and/or the tremors and/or the drop in egg production in a subject of need, in comparison to a subject of a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 8.

14. The method of any one of claims 11 to 13, wherein said subject is poultry.

15. The method of any one of claims 11 to 14, wherein the immunogenic composition is administered once.

16. The method of any one of claims 11 to 15, wherein said immunogenic composition is administered subcutaneously, intramuscularly, intracutaneously, oral or by eye drop.

17. The method of any one of claims 11 to 16, wherein said method results in an improvement in an efficacy parameter selected from the group consisting shorter duration of bacteremia, shorter duration of viremia, a lower bacterial load, a lower viral load, reduced mortality, reduced tremors, reduced ataxia, reduced weakness, reduced weight loss, reduced drop in egg production, reduced lesions, reduced anorexia, reduced inflammation of infra orbital sinuses or combinations thereof, in comparison to a subject of a non-immunized control group of the same species.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0250] FIG. 1. Percentage of birds with pox lesions (on the wing and/or on the thigh) per day post challenge. PC=positive control; NC=negative control

[0251] FIG. 2. Mean AE ELISA titer per Treatment Group. PC=positive control; NC=negative control

EXAMPLES

[0252] The following examples are set forth below to illustrate specific embodiments of the present invention. These examples are merely illustrative and are understood not to limit the scope or the underlying principles of the present invention.

Example 1

Determination of the Interference of a Vaccine Against Avian Infectious Coryza with the Efficacy of a Modified Live Vaccine Against Fowl Pox and Avian Encephalomyelitis in Poultry

Study Objective

[0253] Determination of the interference of an inactivated infectious avian coryza vaccine (avibacterium paragallinarum) with the efficacy of a live attenuated vaccine against fowl pox (FP) and a live attenuated vaccine against avian encephalomyelitis (AE) in poultry.

Experimental Design

[0254] For SPF-laying-type chickens (21 days of age at DO) are divided randomly into four Treatment Groups (TGs): [0255] TG01 (20 animals): vaccination with live AE-FP vaccine added to an inactivated coryza vaccine (triple vaccine) followed by a FP challenge. This group serves to detect interference of the coryza vaccine with the efficacy of the AE-FP vaccine. [0256] TG02 (20 animals): vaccination with live AE-FP vaccine (bivalent vaccine), followed by a FP challenge. This group serves to demonstrate the efficacy of the live AE-FP vaccine. [0257] TG03 (10 animals): not vaccinated but exposed to FP virus (FVP). This group serves to determine the challenge with FPV and thereby validate the animal trial (challenge control). [0258] TG04 (5 animals): not vaccinated but inoculated with sterile water. This group serves to control the absence of antibodies to AE and FPV and the absence of development of pox and the validity of the study.

[0259] The experimental unit was the Treatment Groups (TG).

[0260] The vaccinated animals receive one dose of the vaccines tested in the different combinations. The vaccination routes are the registered routes for the vaccines (table 1). Challenge is done three weeks after vaccination; TG01, TG02 and TG03 with a pathogenic pox strain through the wing web and the feather follicle method and TG04 (control group) is inoculated with sterile water. Thereafter and until the end of the study, the birds are observed daily and between days 29-35 are checked for the appearance of clinical signs due to fowl pox. At the end of the study, the birds are euthanized by inhalation of a O.sub.2—CO.sub.2 gas mixture. Blood samples are taken in all birds on days 0, 21 and 42. The blood is examined for the presence and absence of antibodies to AE.

TABLE-US-00001 TABLE 1 Experimental design of the study Check Treatment No. of Blood vaccinal Pox Blood Observation Blood Group SPF Vaccination sampling* pox challenge sampling** pox sampling** (TG) layers D 0 D 8 D 21 D 29-D 35 D 42*** TG01 20 Inactivated Yes Yes Yes Yes Yes Yes Coryza vaccine + Live AE + FP vaccine (subcutaneously) TG02 20 Live AE + Yes Yes Yes Yes Yes Yes FP vaccine (wing web) TG03 10 No Yes Yes Yes Yes Yes Yes TG04 5 No Yes Yes No Yes Yes Yes *For ELISA AE **For ELISA AE ***Or earlier when bird is euthanized for animal welfare reasons

[0261] The primary parameters in this study are: [0262] 1. the antibody response to the AE virus determined by using a standard commercially available ELISA test; [0263] 2. the determination of antibody titers to Coryza A using Hemagglutination Inhibition test; [0264] 3. the presence or absence of pox development at the inoculation sites (wing web and side of the thigh).

Materials and Methods

[0265]

TABLE-US-00002 TABLE 2 Bivalent Vaccine against avian encephalomyelitis and fowl pox Generic name of Modified live virus vaccine against active ingredient avian encephalomyelitis and fowl pox consisting of the Calnek 1733 strain of the AE virus and the Beaudette strain of FPV, originated in Specific Pathogen Free (SPF) chicken embryos. The product contains a minimum titer for AE: 10.sup.2.5 EID.sub.50 and FP: 10.sup.2.5 EID.sub.50 per dose. Manufacturer Boehringer Ingelheim Vetmedica Storage conditions 2 to 7° C. Protect from direct sunlight and do not freeze.

TABLE-US-00003 TABLE 3 Trivalent Vaccine against avian infectious coryza and avian encephalomyelitis and fowl pox Generic name of Trivalent Inactivated water in oil active ingredient vaccine against FP and AE (see table 2) and avian infectious coryza containing strains of Avibacterium paragallinarum of serogroups A, B and C; the minimum titer/dose/ serotype: 10.sup.8.0CFU Manufacturer Boehringer Ingelheim Vetmedica Storage conditions 2 to 7° C. Protect from direct sunlight and do not freeze.

TABLE-US-00004 TABLE 4 Challenge material Generic name of active ingredient FPV Manufacturer GD (Gezondheidsdienst voor Dieren, Deventer, the Netherlands) Potency 10.sup.7.1 EID.sub.50 per vial (1 mL) Storage conditions <−60° C.

TABLE-US-00005 TABLE 5 Media Generic name of active ingredient Sterile water Manufacturer GD Storage conditions 2-8° C.

TABLE-US-00006 TABLE 6 Details of the vaccination and challenge procedure Dose Treatment No. of Vaccine Volume Challenge [log.sub.10 Volume Group birds (D 0) Dose Route [mL] (D 21) n EID.sub.50] Route [mL] TG01 20 Inactivated 1 s.c. 0.5 FPV 20 4.1 Wing web 0.01 infectious coryza with live AE + FP feather 0.1 vaccine follicles TG02 20 Live 1 Wing 0.01 FPV 20 4.1 Wing web 0.01 AE + FP web vaccine feather 0.1 follicles TG03 10 — — — — — FPV 10 4.1 Wing web 0.01 feather 0.1 follicles TG04 5 — — — — — Mock 5 — Wing web 0.01 feather 0.1 follicles

TABLE-US-00007 TABLE 7 Adminstration of the Vaccine Dosing: The inactivated infectious coryza vaccine and the live attenuated AE - FP vaccine is administered s.c. in a volume of 1 dose 0.5 mL once on D0. The live attenuated AE - FP vaccine alone is administered by the wing web method in a volume of 1 dose 0.01 mL once on D0. Equipment for s.c.: A 1 ml sterile syringe will be used administering the mounted with a 23Gx1″ or equivalent needle Vaccine: wing web method: using a double needle for wing web application Administration: s.c.: dorsal side of the neck wing web method: double needle penetrates the skin fold of the wing s.c. = subcutaneously

TABLE-US-00008 TABLE 8 Adminstration of the Challenge material Dosing: 10.sup.4.1 EID.sub.50 per chicken (see Administration) Equipment: Double needle for wing web Cotton swab for rubbing feather follicles Administration: 10.sup.4.1 EID.sub.50 is administered on D21 by: dilute the stock of the challenge strain (10.sup.7.1 EID.sub.50) 1:100 in cold sterile, demineralized water to a titer of 10.sup.5.1 EID.sub.50 per mL (=10.sup.4.1 per 0.11 mL) intracutaneous administration according to the wing web method (double needle penetrates the skin fold of the wing): 1 dose of 0.01 mL per animal in this way is inoculated in one wing. feather follicle method: five feathers are pulled out on the side of the upper leg. Then, 0.1 mL poxvirus is rubbed gently at the feather follicles

Observations

[0266] After challenge, the chickens are observed twice during the day for clinical signs. All signs and mortality are recorded and stored in the study file.

Sampling

[0267] Blood samples for serology are taken by puncturing the wing vein at days 0, 21 and 42. The blood is collected in serum-gel-tubes and let to clot for at least 2 hours at room temperature. Thereafter, serum is collected by centrifuging the tubes (4700 g; 10-15 minutes; room temperature) and aliquoted in double portions and stored at <−16° C. for further processing

Hemagglutination Inhibition Test

[0268] The presence of antibodies to Coryza serotype A was assessed in the sera of the birds taken at D42 using Hemagglutination Inhibition test using Kume strain A1 antigen. Twofold serial dilutions of sera were made in PBS, using round bottom microtiter plates. To each cup, equal amounts of hemagglutinating units (50 μl, 4 units) and 0.75% formalinized chicken erythrocytes (50 μl) were added. After incubation for 45 minutes at room temperature, the results were read as the maximum serum dilution that completely inhibited hemagglutination. Formalinized chicken erythrocytes were prepared by incubating chicken erythrocytes (washed twice in PBS) with PBS with 3% formaldehyde for 18 h at 4° C. Subsequently, the erythrocytes were washed once in PBS and suspended in PBS to make a 50% suspension which is diluted to 0.75% just before the test is done (Jacobs et al., 1992).

Results

A. Coryza A (Avibacterium Paragallinarum)

[0269] All birds of groups TG02, TG03 and TG04 that had not been vaccinated with the inactivated coryza vaccine had no detectable levels of antibodies against Coryza serotype A. This shows the high specificity of the test.

[0270] In group TG01 (live AE+FP added to inactivated coryza vaccine) antibody levels against Coryza serotype A were detected at expected levels (data not shown). These results show that there is no indication of a negative interference for the coryza vaccine component, what was expected. Any negative interference was predominantly expected for any or both of the live vaccine components.

B. Fowl Pox.

[0271] The result of the vaccination is shown in FIG. 1 (Percentage of birds with Fowl Pox lesions post challenge).

[0272] The study is valid because the negative control group (TG04) shows no pox lesions whereas the birds of the positive control group (TG03) all develop pox lesions. The same is concluded for the avian encephalitis part of the study as the non-vaccinated groups (TG03 and TG04) remain ELISA negative and both AE vaccinated groups (TG01 and TG02) become positive for AE antibodies.

[0273] The level of protection against pox for both groups vaccinated with the live attenuated AE−FP vaccine is shown in FIG. 1. All challenged non-vaccinated birds show pox lesions during a week or more; most of the vaccinated birds (approximately 75%) does not show any pox lesions post challenge; approximately 25% of the birds show minor lesions (lesions classified as small (size 2-3 mm)) for 1 to 3 days. The protection level in TG01 (inactivated coryza+AE−FP vaccines) is 96% and in TG02 (live AE−FP vaccine) is 92%, thus, protection level in TG01 is slightly higher. At 21 d.p.c., no lesions are present in both vaccinated groups whereas 89% of the birds of TG03 still show pox lesions.

[0274] These results show that there is, surprisingly, no indication of a negative interference by the inactivated coryza vaccine on the efficacy of the live attenuated FP vaccine. However, there is even a slightly positive synergistic effect for the pox vaccine efficacy in the triple vaccine combination which is surprising.

C. Avian Encephalomyelitis (AE) Virus

[0275] Table 9 and FIG. 2 show the mean AE ELISA titres for all groups on day of vaccination and at 21 and 42 d.p.v.

TABLE-US-00009 TABLE 9 Mean AE ELISA titer per Treatment Group NUMBER CHALLENGE MEAN TREATMENT OF VACCINATION STRAIN AE ELISA TITER GROUP BIRDS D 0 D 21 D 0 D 21 D 42 TG01 20 Inactivated Fowl Pox 3 1146 1778 Coryza + live Virus AE + FP vaccines TG02 20 Live AE + FP Fowl Pox 1 806 1980 vaccine Virus TG03 10 — Fowl Pox 0 1 19 Virus TG04 5 — — 0 0 0

[0276] The mean titer development for the AE component is shown in FIG. 2. At 21 and 42 days post vaccination, the percentage of AE antibody ELISA positive birds of the group that had been vaccinated with the live attenuated AE−FP vaccine added to the inactivated coryza vaccine (TG01) is 81% and 95% respectively. The percentages of positives for the birds vaccinated with the live attenuated AE-Pox vaccine is respectively 57% and 90% on the same days (TG02). Thus, the percentage of antibody positive birds in TG01 is slightly higher. These results show that there is no indication of a negative interference by inactivated coryza vaccine on the efficacy of the live attenuated AE−FP vaccine. However, there is even a slightly positive synergistic effect for the AE vaccine efficacy (percentage of AE antibody positive birds is increased) in the triple vaccine combination which is surprising.

CONCLUSION

[0277] In summary, it can be concluded that there is no detectable interference with the efficacy of the live attenuated AE (avian encephalomyelitis) and FP (fowl pox) vaccine when added to the inactivated coryza vaccine (avibacterium paragallinarum). The data provided herein show that animals vaccinated with the triple vaccine are protected against fowl pox challenge and that animals produce high antibody titer against AE. There is even a slightly positive synergistic effect, for both the pox vaccine and AE vaccine efficacy, in the triple vaccine combination which is surprising. Often there is a negative effect expected on vaccine efficacy when combining vaccines to end up with a more complex vaccine composition in general. In particular, this would be expected when combining modified live vaccines with a bacterin comprising an adjuvant since the adjuvant may interfere with the activity of the modified live vaccine components.