ATTENUATED IBV WITH EXTENDED CELL CULTURE AND TISSUE TROPISM

Abstract

The present invention relates i.a. to an IBV (infectious bronchitis virus) deposited with the BVR of IZSLER under accession number DPS RE RSCIC 16, any descendant IBV thereof and any IBV having all of the identifying characteristics of the deposited IBV. Further, the present invention relates to an immunogenic composition comprising said deposited IBV.

Claims

1. An IBV (infectious bronchitis virus) deposited with the BVR of IZSLER under accession number DPS RE RSCIC 16, any descendant IBV thereof or any IBV having all of the identifying characteristics of the IBV deposited under DPS RE RSCIC 16.

2. An IBV (infectious bronchitis virus) deposited with the BVR of IZSLER under accession number DPS RE RSCIC 16, any attenuated descendant IBV thereof having an extended cell culture or tissue tropism and being protective against virulent M41 challenge or any attenuated IBV having an extended cell or tissue tropism and being protective against virulent M41 challenge.

3. The IBV of claim 1, wherein the IBV is attenuated.

4. The IBV of any one of claim 1, wherein the IBV is non-recombinant.

5. The IBV of any one of claim 1, wherein the IBV is of a Massachusetts genotype or serotype.

6. The IBV of any one of claim 1, wherein the IBV has an extended cell or tissue tropism.

7. The IBV of claim 1, wherein the IBV is infecting and/or replicating in a cell line or cell selected from the list consisting of: primary chicken embryo cells from lung or liver or primary chicken fibroblasts, a chicken embryo fibroblast cell line, a duck embryonic stem cell line, a human embryonic kidney cell line, a baby hamster kidney cell line, an African green monkey kidney cell line, a rabbit kidney cell line, a canine kidney cell line, a chicken liver cell line, a bovine kidney cell line, a porcine kidney cell line and an insect cell line.

8. The IBV of claim 1, wherein the IBV is infecting and/or replicating in at least one cell line selected from the list consisting of: DF-1 (Douglas Foster), EB66 (duck embryonic stem cell line), PBS-12, PBS-12SF (PBS-12 serum free), BHK21 (baby hamster kidney), HEK 293T (human embryonic kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male hepatoma), MDCK (Madin-Darby canine kidney), MDBK (Madin-Darby bovine kidney), PK15 (porcine kidney), PK2A, SF9, SF21 and SF+ (Spodoptera frugiperda).

9. The IBV of claim 1, wherein the attenuation of the IBV is increased compared to an IBV without extended cell or tissue tropism.

10. A plasmid comprising a nucleotide sequence encoding the IBV of claim 1.

11. A cell comprising the IBV or plasmid of claim 1.

12. An immunogenic composition comprising the IBV of claim 1.

13. The immunogenic composition of claim 12 wherein the immunogenic composition is a vaccine.

14. The immunogenic composition of claim 12 wherein the immunogenic composition is a modified live vaccine.

15. A method for immunizing a subject comprising administering to such subject the immunogenic composition of claim 12.

16. A method of treating or preventing clinical signs caused by IBV in a subject of need, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of claim 12.

17. A method of reducing ciliostasis 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 the immunogenic composition of claim 12.

18. The method of claim 15, wherein said subject is a chicken.

19. The method of claim 15, wherein the immunogenic composition is administered once.

20. The method of claim 15, wherein said immunogenic composition is administered subcutaneously, intramuscularly, oral, in ovo, via spray, via drinking water or by eye drop.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0275] FIG. 1.: Immunofluorescence staining for IB66HP-infected EB66® cells.

[0276] FIG. 2.: Immunofluorescence staining for IB66HP 72 hours after infection of different cell lines. Negative controls were included but are not shown.

EXAMPLES

[0277] 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

Adaption of IBV to Cells and In Vitro and In Vivo Characterization

[0278] An attenuated IBV M41 is used for 10 serial passages in EB66® cells. The material harvested after each passage is characterized by EID.sub.50 and TCID.sub.50. Indeed, infective titers are detected already in the first passage after inoculation of EB66® cells with an attenuated IBV Mass seed stock. Results for EID.sub.50 (infectivity for embryonated eggs) and TCID.sub.50 (infectivity for cell culture) titer determination for the first 10 passages and are summarized in table 1 and detection of immunofluorescence signal for IB66HP infected EB66® cells in FIG. 1. The results confirm that the IBV is adapted to cell culture in EB66® cells. The IBV is efficiently adapted after one passage in EB66® cells, whereas the infectivity for embryonated eggs is not affected. A passage 4 of the IBV (IB66HP) is deposited at the BVR of IZSLER (Biobanking of Veterinary Resources of the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna “Bruno Ubertini”) under accession number DPS RE RSCIC 16.

TABLE-US-00001 TABLE 1 Summary of EID.sub.50 and TCID.sub.50 titers throughout IB66HP passaging in EB66 ® cells. Passage 1 2 3 4 5 6 7 8 9 10 EID.sub.50/ml 5.6 9 7.1 5.3 7 7 6.8 7.5 6.6 6.6 TCID.sub.50/ml 1 6.5 4.25 5.5 6.3 6.32 4.6 7 6 5.4
From table 1 and the other experiments further below, it is appearent that IB66 HP is phenotypically stable since the extended cell culture/tissue tropism and attenuation remains over time (over passage).

Serial Passaging in EB66® Cells

[0279] EB66® cells are seeded at a density of 10.sup.6 cells/ml into a 250 ml shaker flask with a total volume of 50 ml in GRO I medium (SIGMA, cat.: 141530C) supplemented with glutamine and 1 ml CHO feed (SIGMA, cat: 1615). The cells are infected with a 1/10 dilution of attenuated IBV Mass seed stock for the first passage which is incubated for 96 hours. For the following passages 2 to 10 inoculation with the harvested culture is performed with a 1/10 dilution of the previous passage into freshly seeded EB66® cells which are subsequently incubated for 48 to 96 hours under the same cultivation conditions. A passage 4 (supernatant) was generated for deposit by inoculation of 4*10.sup.5 cells/ml with a 1/250 dilution of passage 3 and was harvested 72 hours post infection and he titer of the harvested material is determined by the 50% embryo infectious dose (EID.sub.50) and immunofluorescence tissue culture infectious dose 50 (TCID.sub.50) assay which are 10.sup.5.75/ml and 10.sup.7.33/ml, respectively.

Determination of the 50% Embryo Infectious Dose (EID.SUB.50.)

[0280] For the determination of the EID.sub.50 titer 10-day old embryonated SPF chicken eggs are inoculated with 100 μl 10-fold serial dilution per egg into the allantoic cavity in 6 replicates per dilution. Infected eggs are incubated for 7 days and candling is conducted every 24 h in order to determine mortality. Eggs with dead embryos prior to 24 hours of incubation are not included in the evaluation. The EID.sub.50/mL is calculated as described by Reed & Muench.

Determination of the 50% Tissue Culture Infectious Dose (TCID.SUB.50.)

[0281] For the determination of the TCID.sub.50, 2.5 to 3*10.sup.5 cells with a viability of at least 90% are seeded per well into a 96 well plate one day prior to infection with a 10-fold dilution series of passaged IBV Mass. Medium is removed from the cells and they are infected with 100 μl per well in 5 replicates per dilution. After 72 hours of static incubation at 37° C. and 7.5% CO.sub.2, medium is removed and cells are fixed for 15 min at 4° C. with 50 μl 80% acetone per well. A washing step with 1×PBS is conducted and followed by incubation with 50 μl per well of a 1:1000 dilution of anti-IBV Mass antiserum (Charles River Laboratories, Cat.: 10100454) at 37° C. for 1 hour. The plates are washed twice with 1×PBS before 50 μl of a 1:2000 dilution of Alexa 488-conjugated anti-chicken antibody (Invitrogen, Cat.: A11039) are added and incubated at 37° C. for 1 hour. Subsequently, a final wash with 1×PBS is conducted and 100 μl of 1×PBS are added into each well followed by the evaluation in an inverted fluorescence microscope. The infectious titer of IBV in EB66® cells is determined according to the formula of Reed & Muench.

Infectivity of IB66HP for Different Cell Lines

[0282] Different cell lines are seeded into 96 well plates to reach 70-80% confluence the next day. Cells are infected with a 10-fold dilution series of an IB66HP P8 set to a titer of 10.sup.5 to 10.sup.5.75 TCID.sub.50/ml derived from titration on EB66® cells. Medium is removed from the cells and they are infected with 100 μl per well in 4 replicates per dilution. After 72 hours the TCID.sub.50 titer is determined. Indeed, infectivity of IB66HP for different primary cells and cell lines is detected and results are listed in table 2 and 3 and shown in FIG. 2.

TABLE-US-00002 TABLE 2 IB66HP has an extended tissue tropism. TCID.sub.50 determination in various cell lines for IB66HP set to a titer of 10.sup.5 TCID.sub.50 prior to titration. Cell Species Tissue Log TCID.sub.50/ml EB66 duck Embryonic stem 5 cell BHK hamster Kidney 4 MA104 African green Kidney 2 monkey Vero EU African green Kidney 2.67 monkey RK13 rabbit Kidney 3 CEK Chicken embryo Primary kidney 5.33 CEH Chicken embryo Primary liver 3.67 CEL Chicken embryo Primary lung 6

TABLE-US-00003 TABLE 3 IB66HP has an extended tissue tropism. TCID.sub.50 determination in various cell lines for IB66HP set to a titer of 10.sup.5.75 TCID.sub.50 prior to titration. Cell Species Tissue Log TCID.sub.50/ml SF9 Fall armyworm Pupal ovarian tissue 5.6 CEF Chicken embryo Primary fibroblasts 4.5 MDCK Dog kidney 5.38

Conclusion Example 1

[0283] The attenuated IBV M41 was adapted to cell culture. The deposited strain as well as descendants show an extended cell or tissue tropism as they were able to infect in primary cells and a broad range of different cell lines.

Example 2

Determination of IB66HP Safety and Efficacy In Vivo

[0284] The attenuated IBV Mass allantoic fluid stock (P0) and passages 5 (P5) and 10 (P10) in EB66® cells are used to assess their safety and efficacy in 1-day-old SPF layer chickens against virulent M41 challenge. Throughout the whole study chickens are housed in isolation units and are provided with water and commercial balanced feed formula ad libitum. Chickens are observed daily for clinical signs. Three groups of chickens are vaccinated with P0, P5 and P10 each, at a dose of 10.sup.4 TCID.sub.50/chicken via eye drop. One group is treated with placebo and serves as challenge control group. Seven days post vaccination seven animals per group vaccinated with P0, P5 or P10 are euthanized to assess the safety via scoring of the tracheal ciliostasis. For this, the trachea is removed and cut into transversal sections of which 3 of the lower, 4 of the middle and 3 of the upper part are used for safety assessment. All rings are evaluated by light microscopy for beating of the cilia. Each ring is scored individually as described in table 4.

TABLE-US-00004 TABLE 4 Ciliostasis scoring scheme for the assessment of safety Score Ciliar activity [%] 0 100 1 75-99 2 50-74 3 25-49 4  0-24

[0285] Twenty-one days post vaccination all remaining chickens are challenged with virulent IBV M41 with a dose of 10.sup.25 EID.sub.50/chicken via eye drop. Efficacy is assessed at seven days post vaccination by ciliostasis scoring as described in table 4. A ring is recorded as normal if more than 50% of the internal ring shows vigorous ciliar movement (Score 2 and lower). A ring is recorded as positive for ciliostasis if less than 50% of the cilia are beating (Score 3 and 4). An animal is considered protected if not fewer than 9 out of 10 rings show normal ciliar activity. Throughout the whole study, all animals are observed daily for clinical signs such as depression, respiratory, digestive or neurological signs, locomotive damage, prostration or ruffled feathers.

[0286] The aim of the study is to determine whether the cell culture adaptation and cell passage of IB66HP has an effect on safety and efficacy compared to the parental IBV M41att allantoic fluid stock when applied to 1-day-old chickens. The assessment of safety by clinical signs and ciliostais scoring of tracheal explants revealed that P5 and P10 of IB66HP in Eb66® cells have an improved safety profile compared to IBV M41att. The ciliostasis score are strongly reduced for IB66HP P5 and P10 in contrast to IBV M41att (table 5). In addition, clinical signs in the animals of groups vaccinated with IB66HP P5 and P10 were reduced compared to the animals vaccinated with M41att. Conclusively, IB66HP is more attenuated than M41att and has an improved safety profile for vaccination of 1-day-old chickens.

TABLE-US-00005 TABLE 5 Results for assessment of safety for IB66HP EB66 ® cell passage 5 (P5) and 10 (P10) in comparison to the allantoic fluid stock of M41att 5 days post vaccination. The mean ciliostasis score per group is calculated by adding up the sum score of the individual chickens per group and dividing the group sum by the number of animals (highest possible score 40, lowest possible score 0). For not affected animals, at least 9 of the 10 tracheal explants show normal ciliar activity (score ≤2). Mean ciliostasis #Animals/not Vaccination score affected M41att P0 30.6 5/1 IB66HP P5 8.2 5/5 IB66HP P10 6.4 5/5 — 0.4 5/5

[0287] All animals of the non-vaccinated control group show clinical signs and a high ciliostasis score after challenge with virulent IBV M41. In contrast, the EB66® cell passaged IB66HP P5 and P10 are able to reduce clinical signs and the damage of the trachea as efficiently as M41att (table 6).

TABLE-US-00006 TABLE 6 Results for assessment of efficacy for IB66HP EB66 ® cell passage 5 (P5) and 10 (P10) in comparison to the allantoic fluid stock of M41att 7 days post challenge. The mean ciliostasis score per group is calculated by adding up the sum score of the individual chickens per group and dividing the group sum by the number of animals (highest possible score 40, lowest possible score 0). For not affected animals, at least 9 of the 10 tracheal explants show normal ciliar activity (score ≤2). % animals Mean ciliostasis #animals/Not with normal Vaccination Challenge score affected ciliostasis M41att P0 M41 2.2 7/7 100 IB66HP P5 M41 0.1 9/9 100 IB66HP P10 M41 3 10/9  90 — M41 35.9 9/0 0

[0288] In summary, IB66HP is an attenuated IBV strain that efficiently infects and replicates in different cell lines and tissue cells. Surprisingly, it has an improved safety for application in 1-day-old chickens compared to the parental egg-restricted IBV M41att strain. Furthermore, IB66HP maintains vaccine efficacy. In contrast, the only other known cell culture adapted and highly attenuated IBV strain Beaudette does not confer sufficient protection after challenge infection. Thus, IB66HP is the first described IBV that is attenuated, has an extended cell culture or tissue tropism and displays efficacy against virulent challenge after application as a vaccine. Further, IB66HP has been shown to be phenotypically stable.