VACCINATION FOR PROTECTING POULTRY AGAINST A POULTRY PATHOGEN

Abstract

The invention pertains to a vaccine comprising non-live antigen of a poultry pathogen and a mucoadhesive adjuvant, for use in boosting an immune response in a poultry animal directed against the poultry pathogen by administering the vaccine mucosally to the poultry animal. The invention also pertains to a vaccine comprising a liquid pharmaceutically acceptable carrier, a non-live antigen of a poultry pathogen and a mucoadhesive adjuvant, as well as a method of boosting an immune response in a poultry animal, which immune response is directed against a poultry pathogen, by administering a vaccine mucosally to the poultry animal, the vaccine comprising a non-live antigen of the said poultry pathogen and a mucoadhesive adjuvant.

Claims

1. A method of boosting an immune response in a poultry animal, which immune response is directed against a poultry pathogen, by administering a vaccine mucosally to the poultry animal, the vaccine comprising a non-live antigen of the said poultry pathogen and a mucoadhesive adjuvant.

2. The method according to claim 1, characterised in that the immune response in the poultry animal is a result of a natural infection with the poultry pathogen or the administration of a previous vaccine directed against the pathogen.

3. The method according to claim 1, characterised in that the immune response in the poultry animal is a result of a previous vaccination directed against the pathogen, by administering a previous vaccine parenterally or mucosally.

4. The method according to claim 1, characterised in that the immune response in the poultry animal is a result of a previous vaccination directed against the pathogen, by administering a previous vaccine comprising a non-live antigen of the poultry pathogen parenterally or by administering a previous vaccine comprising a live or non-live antigen of the poultry pathogen mucosally.

5. The method according to claim 2, characterised in that the vaccine comprising a non-live antigen of a poultry pathogen and a mucoadhesive adjuvant is administered within 2-6 weeks after administration of the previous vaccine.

6. The method according to claim 1, characterised in that the vaccine is administered ocular (OC) and/or intranasally (IN).

7. The method according to claim 1, characterised in that the vaccine is administered by spraying the vaccine on the poultry animal.

8. The method according to claim 1, characterised in that the mucoadhesive adjuvant has a molecular weight above 10,000 Da.

9. The method according to claim 1, characterised in that the mucoadhesive adjuvant has a molecular weight above 10,000 Da and below 1,000,000 Da.

10. The method according to claim 1, characterised in that the mucoadhesive adjuvant has a molecular weight above 10,000 Da and below 300,000 Da.

11. The method according to claim 1, characterised in that the mucoadhesive adjuvant has a pKa value below 6.

12. The method according to claim 1, characterised in that the mucoadhesive adjuvant has a pKa value between 3 and 5.

13. The method according to claim 1, characterised in that the mucoadhesive adjuvant is chosen from an alginate and a polyalkylene oxide blockcopolymer.

14. The method according to claim 1, characterised in that the mucoadhesive adjuvant is present in the vaccine in an amount between 1 and 10% w/w.

15. The method according claim 1, characterised in that the poultry pathogen is a viral pathogen.

16. The method according to claim 1, characterised in that the poultry pathogen is chosen from Newcastle Disease Virus (NDV), Avian Reo Virus (ARV), Infectious bursal disease virus (IBDV) and Salmonella typhimurium.

17. A vaccine comprising a liquid pharmaceutically acceptable carrier, a non-live antigen of a poultry pathogen and a mucoadhesive adjuvant.

18. (canceled)

Description

Example 1

[0029] Object of the Study

[0030] Newcastle disease (ND) is a highly contagious viral disease of poultry that causes economical losses worldwide. In the field, the majority of birds are vaccinated against this virus by live priming followed by a booster vaccination. Mass spray application for the live primo vaccination is commonly applied. The booster vaccination is commonly applied via the intramuscular route. An inactivated antigen in a mucosal, preferably a spray, method for the booster vaccination would be most convenient for the farmer to apply to the flock.

[0031] This study aimed at establishing an effective vaccination regime with a live prime vaccine followed by a booster vaccine comprising of an inactivated antigen that can be applied via the mucosal route, for example via a spray application. In particular the efficacy of spray vaccination needs to be assessed, since the useful antigen fraction applied by spray with an actual impact is highly influenced through losses by settlement, drift and evaporation. Therefore, different vaccine formulations comprising of inactivated antigen combined with a mucoadhesive adjuvant were tested. The model antigen that was used in this study is NDV Clone 30, as NDV is a respiratory viral disease for which mucosal (in particular spray) vaccination would be of interest.

[0032] Materials and Methods In this study 7 groups (n=13/group) with 1-day old SPF chickens were used. In addition, at the start of the experiment at T=0, an additional 5 hatch mates were used for obtaining reference samples. At T=0 weeks post vaccination (wkpv) all groups received a primo vaccination with live NDV clone 30 (0.1 ml via ocular route). At T=6 wkpv the animals from groups 2-7 received a booster vaccination. Group 2 served as control in which the animals received the booster via an 0.5 ml intramuscular (i.m.) injection at a full dose (100%,) of killed (inac) NDV with GNE adjuvant (MSD Animal Health). Group 3 received a booster formulation applied via the oculo-nasal route (40% inac NDV with 5% w/v Synperonic F127 (poloxamer 407; Croda health Care) mucoadhesiveve adjuvant; 0.2 ml/animal). To reduce potential impact of loss by spray application (evaporation, settlement) groups 4-7 received the same antigen input per booster formulation but blended within different dosage volumes.

[0033] Animals from groups 4 and 5 received different booster formulations applied via fine spray with a total volume of 1 mL per animal. Animals from group 6 and 7 received 2 mL per animal. Groups and treatment are indicated in Table 1.

TABLE-US-00001 TABLE 1 Vaccination scheme Group Prime vaccination Booster vaccination 1 Live NDV 2 Live NDV Killed NDV, GNE adjuvant, IM, 0.5 ml 3 Live NDV Killed NDV, 5% Synperonic F127, OC/IN, 0.2 ml 4 Live NDV Killed NDV, 10% Synperonic F127, spray, 1 ml 5 Live NDV Killed NDV, 5% Synperonic F127, spray, 1 ml 6 Live NDV Killled NDV, 10% Synperonic F127, spray, 2 ml 7 Live NDV Killed NDV, 5% Synperonic F127, spray, 2 ml

[0034] T=2 wkpb, T=4 wkpb blood was withdrawn from all animals. To determine serology, sera samples were analyzed for the presence of anti-NDV hemagglutination inhibiting antibodies. For the characterization of peripheral blood mononuclear cells (PBMC), the cells were isolated using Sepmate (STEMCELL Technologies) 15 mL tubes and counted using Neubauer counting chamber. CD4 and CD8a cells were assessed using APC and Pacific Blue fluorochromes of Southern Biotech.

[0035] Results

[0036] The serology results are indicated here below in table 2. As can be seen, the 2 log antibody titers for group 1 remained at a low level of about 5, whereas the antibody level for the boosted groups rose to a very high level of about 12 for the IM group, as expected. However, also the groups boosted with the killed vaccine applied mucosally each showed a significant boosting effect to levels of about 7-8, corresponding to levels for adequate protection against NDV.

TABLE-US-00002 TABLE 2 2log Antibody titers 0, 2 and 4 weeks post booster vaccination Group At booster 2 wpb 4wpb 1 6 5 5 2 5 12 12 3 6 8 7 4 5 7 7 5 6 8 7 6 6 7 7 7 5 8 8

[0037] In table 3 the percentages of 004 and CD8a cells of the total cell population are provided. This is another indication that by applying the vaccine of the invention, a significant boosting effect of the immunity can be induced.

TABLE-US-00003 TABLE 3 percentages of CD4 and CD8 cells of the total cell population Group CD4 CD8a 1 12 8 2 31 17 3 35 21 4 23 37 5 25 36 6 25 20 7 25 29

Example 2

[0038] Object of the Study

[0039] The object was to further assess the effects of a mucoadhesive adjuvant in poultry vaccination, by using next to the mucoadhesive adjuvant of Study 1, two other mucoadhesive adjuvants. The antigen was changed to inactivated IBDV to assess efficacy for another poultry antigen.

[0040] Materials and Methods

[0041] In this second study, 5 groups (N=10/group) of 3 week old SPF chickens were used. Group 1 served as a control groups which received no vaccination. At T=0, all animals of groups 2 to 5 received a primo vaccination according to the information as provided in table 4, using inactivated strain D78 of IBDV as antigen. These vaccines were applied either intramuscularly in the leg (group 2) at 0.5 ml/dose (100 units) or applied ocular/nasal (groups 3-5) at 0.2 ml/dose (5000 units). All chickens of groups 3-5 received a booster vaccination according the formulations and routes as indicated in table 4, and at the same time blood was taken for serological analysis. Group 2 received only a primo vaccination, because GNE adjuvant is known for a prolonged antigen release, not necessitating a booster vaccination. Two weeks after the booster vaccination, all chickens were exposed to a challenge infection with IBDV CS89 strain virus, which was applied via eye drop. At the same time, blood was taken for serological analysis. After challenge the chickens were monitored for clinical signs of disease or death for a period of 11 days including the challenge day.

TABLE-US-00004 TABLE 4 Vaccination scheme Group Prime vaccination Booster vaccination 1 2 Inac D78, 0.5 ml IM, GNE 3 Inac D78, 0.2 ml OC/IN, Inac D78, 0.2 ml OC/IN, chitosan chitosan 4 Inac D78, 0.2 ml OC/IN, Inac D78, 0.2 ml OC/IN, alginate alginate 5 Inac D78, 0.2 ml OC/IN, Inac D78, 0.2 ml OC/IN, SynperonicF127 SynperonicF127

[0042] Results

[0043] The serology results are indicated here below in table 5. As can be seen, the 2 log antibody titers for group 1 remained below detection level of 4.0, whereas the antibody level for the boosted groups rose to a very high level of about 10 for the IM group, as expected. However, also the groups boosted with the killed vaccine applied mucosally each showed a significant boosting effect to levels of about 7-9, corresponding to levels for adequate protection against IBDV.

TABLE-US-00005 TABLE 5 2log Antibody titers at booster vaccination and challenge Group At booster vaccination At challenge 1 4.0 4.0 2 6.9 10.0 3 4.1 6.6 4 5.3 8.7 5 4.0 9.1

[0044] In table 6 the survival data after challenge are provided. As can be seen, the serology outcome corresponds to the survival data, confirming that serology corresponds to protection against the viral infection.

TABLE-US-00006 TABLE 6 Survival data Group Survival percentage 1 20% 2 100% 3 60% 4 90% 5 90%

Example 3

[0045] Object of the Study

[0046] The object was to further assess the effects of a mucoadhesive adjuvant in poultry vaccination, by using the two most promising mucoadhesive adjuvants of Study 2 (i.e. alginate and the polyalkylene oxide Synperonic), and changing the antigen to inactivated ARV, while at the same time using spray vaccination instead of direct OC/IN vaccination.

[0047] Materials and Methods

[0048] In total 3 groups are included in this study, using 1-week old SPF layer chickens (n=10/group). At DO all animals of groups 1-3 receive a live Reo 2177 vaccination using the commercial vaccine Nobilis Reo 2177 (MSD Animal Health) according to the information as provided in table 7. This primo vaccine is applied intramuscularly in the leg. At D28, all groups receive a booster vaccination as indicated in the table. The dose of antigen in the spray vaccines is ten times as high to account for any antigen that does not arrive at the mucosal surfaces of the chickens (but instead on the feathers, ground, walls etc.). In order to measure the efficacy by establishing the serological response as consequence of the booster vaccinations, blood is withdrawn from all animals at D28 (at booster vaccination), D42 (2 weeks after the booster vaccination) and D56 (4 weeks after the booster vaccination).

TABLE-US-00007 TABLE 7 Vaccination scheme Group Prime vaccination Booster vaccination 1 Nobilis Reo, 0.2 ml, IM Inac Reo, GNE, 0.3 ml, IM 2 Nobilis Reo, 0.2 ml, IM Inac Reo, Synperonic, 2 ml, spray 3 Nobilis Reo, 0.2 ml, IM Inac Reo, alginate, 2 ml, spray

[0049] Results

[0050] The serology results of the antibodies against ARV are indicated here below in table 8. As can be seen, the 2 log antibody titers for Group 1 are very high, but those of Groups 2 and 3, receiving the booster vaccination via a spray are at a corresponding level, indicating that an adequate immune response against ARV is induced. In particular the data using the mucoadhesive polyalkylene oxide provide indicate that a level of protection can be arrived at, that equals protection arrived at via IM vaccination.

TABLE-US-00008 TABLE 8 2log Antibody titers 2 weeks and 4 weeks after prime and boost vaccination Group At booster 2 weeks after booster 4 weeks after booster 1 6 8.6 9.5 2 6.5 9.5 9.0 3 5 7.6 7.0

Example 4

[0051] Object of the Study

[0052] The object was to further assess the effects of a mucoadhesive adjuvant in poultry vaccination, by using the alginate mucoadhesive adjuvants of Studies 2 and 3, but now in a set up to assess protection against a bacterial infection, namely Salmonella Typhimurium, in a prime-boost regimen with an inactivated multivalent Salmonella vaccine by the ocular/nasal route.

[0053] Materials and Methods

[0054] In total 2 groups are included in this study, using 40 one-day-old SPF chickens (n=20/group). At DO the animals in group 2 receive a prime vaccination with a multivalent Salmonella vaccine containing inactivated Salmonella bacteria of serovar Enteritidis, Typhimurium and Infantis and 1% alginate via the OC/IN route. At D14, the animals of group 2 receive a booster vaccination (same vaccine, same route). At D21 the animals of group 1 (control) and group 2 are challenged with virulent Salmonella typhimurium by oral gavage. At days 24, 26, 28, 31 and 35 cloacal swabs are collected from all birds. Half of the birds are euthanized of 7 days post challenge to collect spleen and liver samples as well as caecal swabs. The other half is euthanised at 14 days post challenge to collect the same.

[0055] Results

[0056] The results as a mean for all days of sample collection, are indicated here below in table 9 as a percentage of positive samples out of the total number of samples. Protection against Salmonella infection is shown.

TABLE-US-00009 TABLE 9 Results of Salmonella vaccination Samples controls vaccinates Cloaca 74 54 Caecum alone 85 55 Spleen/liver/caecum 73 52