IMPROVED HVT-VECTORED ND-IBD VACCINE

Abstract

The present invention regards a new and Improved HVT-vectored ND-IBD vaccine, comprising a recombinant HVT comprising the VP2 gene from IBDV and the F gene from NDV to a target animal. The recombinant HVT can be used in a vaccine for poultry, which displayed good viral vector replication, effective expression of the NDV F—and IBDV VP2 genes, improved immunoprotection against ND and IBD, and improved genetic stability over prior art constructs.

Claims

1. A recombinant DNA expression cassette comprising in the 5′ to 3′ direction and in the following order: a. a murine cytomegalovirus immediate early 1 gene (mCMV-IE1) promoter, b. an infectious bursal disease virus (IBDV) viral protein 2 (VP2) gene, c. a transcription terminator, d. a human cytomegalovirus immediate early 1 gene (hCMV-IE1) promoter, and e. a Newcastle disease virus (NDV) fusion (F) protein gene.

2. The recombinant DNA expression cassette of claim 1, wherein one or more or all of the conditions apply selected from the group consisting of: the mCMV-IE1 gene promoter is a complete promoter; the IBDV VP2 gene encodes a VP2 protein from a classic type IBDV; the transcription terminator comprises both a terminator region and a polyA region; the transcription terminator is derived from simian virus 40 (SV40); the hCMV-IE1 gene promoter is a core promoter; the NDV F gene is from a lentogenic NDV strain; the expression cassette comprises an additional transcription terminator which is located downstream of the NDV F gene; and the additional transcription terminator is derived from the hCMV-IE1 gene.

3. A recombinant DNA molecule comprising the recombinant DNA expression cassette of claim 1.

4. A recombinant herpes virus of turkeys virus (HVT), comprising the recombinant DNA expression cassette of claim 1, wherein the expression cassette is inserted in the Us region of the genome of the recombinant HVT.

5. A host cell comprising the recombinant HVT of claim 4.

6. A method of constructing the recombinant HVT of claim 4, said method comprising the insertion of the recombinant DNA expression into the Us region of the genome of the recombinant HVT.

7. A vaccine for poultry comprising the recombinant HVT of claim 4, and a pharmaceutically acceptable carrier.

8. The vaccine of claim 7, comprising at least one additional immunoactive component.

9. A method of preparing the vaccine of claim 7, said method comprising the steps of: infecting host cells with the recombinant HVT, harvesting the infected host cells, and admixing the harvested infected host cells with a pharmaceutically acceptable carrier.

10-12. (canceled)

13. A method for preventing or reducing infection by IBDV, NDV, or IBDV and NDV, or associated signs of disease, the method comprising the administration of the vaccine according to claim 7 to poultry.

14. A method of vaccinating poultry, comprising the step of inoculating poultry with the vaccine according to any of claim 7.

15. A method of vaccinating poultry, comprising the step of inoculating poultry with the vaccine of claim 8.

16. A method for preventing or reducing infection by IBDV, NDV, or IBDV and NDV, or associated signs of disease in poultry, the method comprising the administration of the vaccine of claim 8 to poultry.

17. A vaccine for poultry comprising the host cell of claim 5 and a pharmaceutically acceptable carrier.

18. The vaccine of claim 17, comprising at least one additional immunoactive component.

19. A recombinant herpes virus of turkeys virus (HVT), comprising the recombinant DNA expression cassette of claim 2, wherein the expression cassette is inserted in the Us region of the genome of the recombinant HVT.

20. A host cell comprising the recombinant HVT of claim 19.

21. A vaccine for poultry comprising the host cell of claim 20 and a pharmaceutically acceptable carrier.

Description

EXAMPLES

1. The Different Expression Cassette Insertions Tested

[0251] In the search for stable and effective recombinant HVT that expressed both NDV F and IBDV VP2, the inventors constructed a series of recombinant HVT constructs with different expression cassettes inserted in Us2, using different elements and orientations. In FIG. 2 a graphic representation is given (not drawn to scale), of the relevant elements of a representative number of similar constructs tested. For comparison the prior art construct HVP309 is also represented. [0252] Variations tested were in the order of the protein genes, in the different promoters used, and in the type of insert gene used.

[0253] In more detail: the IBDV VP2 gene was connected to the following promoters: [0254] the mCMV 1E1 gene promoter (including enhancer—and core regions), [0255] the Long Terminal Repeat (LTR) promoter from the 3′ terminal end of Rous Sarcoma Virus (Schmidt-Ruppin D strain), and [0256] the gB gene promoter from pseudorabies virus, from vaccine strain Bartha (GenBank acc. nr: BK001744)

[0257] Also, the VP2 gene was tested in a native and in a codon optimised version, using the HVT codon table. [0258] Further in a number of constructs, the heterologous genes were in reversed order to each other.

[0259] All these different recombinant HVT were constructed, transfected, and amplified. Next they were selected by testing for expression in vitro on CEF cells, and for expression and vireamia in vivo by inoculation into experimental animals.

2. Vireamia and Serology of Different Recombinant HVT Constructs

[0260] In an animal trial the vireamia and serological responses induced by the various recombinants HVT constructs were tested. Animal experiments were performed essentially as described in WO 2013/057.235. In short: one day-old SPF layer chickens were vaccinated intramuscularly and kept in isolators under negative pressure. At 10, 24 and 38 days post vaccination, HVT virus was re-isolated from spleen (10 and 38 days) or from blood samples (24 days; peripheral blood lymphocytes: PBL), to test vireamia. Serological responses were determined in blood samples taken at 37 days post vaccination. Results are presented in Table 2 and in FIGS. 3 and 4.

[0261] All HVT recombinants replicated in the vaccinated chickens. Vireamia levels of HVP364 and its mirrored construct HVP367 were low compared to the other recombinants. A portion of the plaques of both viruses from vireamia at 10 and 24 days, showed no expression of VP2 and/or F in IF-assays. Vireamia levels at 38 days were therefore not performed, and HVP364 and HVP367 were excluded from further studies. All other recombinants showed expression of VP2 and F in all plaques at all-time points tested.

[0262] The serological responses induced upon inoculation in experimental animals were determined: for NDV F Elisa values were not discriminatory, therefore heamagglutination (HI) against NDV (Clone 30) was used as a selection tool; for IBDV neutralisation (VN) against D78 was used; FIG. 4. [0263] Even though HVP364 and HVP367 showed non-expressing plaques, antibody induction was measured. The recombinant HVT HVP362 gave excellent vireamia, but no serum response against IBDV VP2. Similarly, HVP361 and HVP366 did give seroconversion against NDV, but very little or none at all against IBDV. Consequently, these three recombinants were also excluded from further studies. Surprisingly, only the HVP360 construct gave good vireamia and serology levels, and this recombinant HVT was therefore selected for further studies.

3. Characterisation of Recombinant HVT: HVP360

3.1. Introduction

[0264] HVP360 is a recombinant HVT according to the invention, and expresses both the IBDV VP2 gene and the NDV F gene. An HVT FC-126 based cosmid set was used to insert its expression cassette in the Us2 gene locus on the HVT genome. [0265] In HVP360, the VP2 gene of IBDV was isolated from the classic type F52/70 strain and is driven by the IE1 gene promoter from mCMV strain ATCC VR-194. The F gene originated from NDV vaccine strain Clone 30 and is driven by the IE1 gene promoter from hCMV strain AD169. Also HVP360 contains the SV40 termination signal and the hCMV IE1 gene terminator, as defined herein. FIG. 1 shows a schematic view of the expression cassette in HVP360, with all elements drawn to scale, and flanking sequences of the HVT Us2 gene. In this example, the construction and characterization of HVP360 is described

3.2. Materials and Methods

3.2.1.Construction of HVP Recombinants

[0266] For the construction of HVP360, insertion of the recombinant DNA expression cassette into the HVT Us2 gene locus was performed with a set of overlapping cosmid-derived DNA fragments from HVT vaccine strain FC-126 that, after transfection into CEF, regenerated infectious virus, as described in WO 2013/057.235. Also a pBR322 based plasmid was used as transfer vector. Where possible unique restriction enzyme digestion sites were used; when not available these were introduced by PCR directed insertion of a synthetic linker sequence that comprised such a unique site.

[0267] The viral DNA fragments from the cosmid vectors and from the transfer plasmid were excised by digestion with appropriate restriction enzymes. The linear DNA fragments were then transfected into CEF cells by means of calcium phosphate precipitation. After DNA had entered the cell, infectious HVT virus was regenerated by homologous recombination between the overlapping sequences of the DNA fragments, thereby generating an intact HVT FC-126 genome, comprising the expression cassette in Us2. This virus construct was called HVP360.

[0268] Progeny of the transfected cultures was amplified once on fresh CEF and checked for the presence of HVT expressing VP2 and F by immunofluorescence assay (IFA) using monoclonal antisera against these antigens. Recombinant virus was isolated by single-plaque purification: monolayers of infected CEF were covered with agarose in culture medium, when HVT CPE was clearly visible. Several plaques were picked randomly and passaged two times on CEF before harvesting and storage as cell associated virus preparation.

[0269] Two HVP360 parallel plaque isolates (A1 and B1) were each passaged fifteen times in consecutive CEF cell cultures, and screened for expression of VP2 and F by IFA at different passage levels.

3.2.2.DNA Analyses

[0270] For detailed characterisation of the HVP360 construct, several DNA analyses were performed on plasmid DNA of the transfer vector, and on total DNA of CEF cultures infected with FC-126 or with HVP360 passage 5 from both parallel isolates. Sequence analysis and Southern blot analysis of the coding nucleotide sequence of the inserted cassette and HVT Us2 flanking regions were performed to confirm correct integration into Us2, and genetic stability upon passaging. Southern blot analysis was also performed on the full genome of HVP360 to confirm correct recombination at the overlapping regions of the HVT DNA fragments from the cosmid set that were used to reconstruct the virus.

[0271] HVT DNA was isolated from CEF cell cultures infected with HVP360 or control parental HVT FC-126 that had also been assembled from a set of cosmids as used for HVP360 but without Us2 expression cassette; this was used as control virus in subsequent experiments, and called HVT FC-126/435. Virus stocks were passaged once on CEF and total DNA was isolated using the Easy-DNA™ kit (Invitrogen). Plasmid DNA of the transfervector was isolated from E. coli cultures transformed with the transfer vector, and DNA was isolated using the Quantum Prep™ Plasmid Midiprep kit (Bio-Rad).

[0272] 3.2.3. Characterisation by Southern Blot

[0273] Southern blots were performed for an in depth analysis of the genome structure of HVP360 to verify that virus assemblage was exactly as intended and no unintended insertions or deletions had occurred during the virus regeneration.

[0274] HVT viral DNA was digested with restriction enzymes PvuI and AatII; or with BamHI, KpnI, Bg/II and EcoRI. Transfer plasmid DNA was digested with restriction enzymes PvuI and AatII. [0275] After digestion DNA fragments were loaded in multiple parallel lanes on 0.7% agarose/TAE gels, electrophoresed, and transferred onto a nitrocellulose membrane. Blots were cut in identical pieces and hybridized individually with one of the .sup.32P labelled HVT probes and a probe that detects the DNA size marker (Smartladder™, Eurogentec). After 16 h incubation, excess probe was removed in two wash steps and the blot exposed to an X-ray film. After developing the autoradiogram, DNA restriction fragments specifically hybridizing with the probe were visible.

[0276] To detect if any parts of the cloning plasmids had been incorporated in the recombinant HVT, a probe was made by digestion of the plasmid pBR322 into smaller fragments with HaeIII. These fragments were labelled with .sup.32P. All cloning vectors used in HVT reconstruction are derivatives of pBR322, and will be detected by this probe if vector sequences are present. Also, the HVP360 transfer plasmid was used in one lane of the Southern blots as positive control for the detection of plasmid sequences.

[0277] To check for correct assembly at the overlapping regions of the cosmid inserts and the repeat regions of the HVT genome, primer pairs were designed to hybridise in these relevant regions and probes were obtained by PCR on HVT FC-126 viral DNA prepared from infected CEF cell cultures. Amplicons were digested into smaller fragments with Sau3AI, labelled with .sup.32P and used as probes in the Southern blot hybridization. [0278] Next the various probes were hybridised to HVP360 DNA that had been digested with BamHI, KpnI, BgIII or EcoRI.

[0279] The restriction fragment lengths detected in the Southern blots, were compared to those expected as based on the published sequence for HVT strain FC-126 (GenBank acc. nr. AF291866).

3.2.4.Characterisation by Sequence Analysis

[0280] To confirm correct insertion and stability of the coding sequences, a complete DNA sequence analysis was done on the expression cassette and on the HVT Us2 flanking regions. [0281] To allow the DNA sequencing, specific DNA fragments of the HVT's were amplified by PCR using specific primers. Amplicons were purified using the Qiaquick™ kit (Qiagen). Next, PCR sequencing was performed on these amplicons, using the Big Dye Terminator™ v.3.1 Cycle Sequencing kit (Applied Biosystems), according to the manufacturer's instructions. Sequencing was done using a 3500 series Genetic Analyzer™ (Applied Biosystems). Sequence readings were analysed using Sequencher™ v. 5.0 software (Gene Codes Corporation). [0282] A contiguous sequence was assembled from overlapping sequence readings. Any ambiguities were resolved by repeating sequencing reactions and compiling multiple sequence readings.

3.2.5.Characterisation of Expression by IFA

[0283] After transfection, plaque purification and serial passaging, isolates for both parallel isolates of HVP360, from passage levels 5, 10 and 15 were monitored for the maintained expression of the inserted genes by IFA. CEF monolayers were infected with the recombinant isolates, incubated for 2-3 days until CPE was clearly visible, and then fixated with 80% ethanol. Expression of IBDV VP2 or NDV F was detected with monoclonal antibodies as first reagent, and a fluorescein isothiocyanate (FITC) labelled conjugate as secondary antibody, and read by UV microscopy.

3.3. Results

3.3.1.Results of Southern Blot Hybridizations

[0284] Genetic homogeneity and -stability was confirmed by Southern blot analysis using specific probes. Blots hybridized with a plasmid pBR322 probe on lanes containing restricted DNA from strain HVP360 and FC-126, gave no signal with the plasmid probe. However the plasmid probe did react positively with lanes containing restricted DNA from the transfer plasmid, showing fragments specific for the plasmid backbone as predicted. Also, plasmid probe was positive for most bands of the DNA size marker.

[0285] The same blot was then hybridized with a probe specific for the Us2 insertion locus, again revealing the restriction fragments as predicted. [0286] As expected, a different—expected—banding pattern was observed for the genome region where the expression cassette has been inserted.

[0287] Hybridizations showed that the viral genome of HVP360 was reassembled correctly and matched the pattern observed for the parent HVT cosmid-reconstructed strain FC-126/435.

[0288] In the hybridizations with probes that detected overlapping sequences and repeat regions, the patterns for HVP360 and FC-126 were found to be largely identical, although in some regions the pattern found differed slightly from the predicted Southern blot hybridization pattern for the junction between unique long and terminal repeat region, based on published DNA sequence for HVT FC-126. However the pattern in these regions is identical for both recombinant—and paternal virus strains. [0289] Consequently, these differences were caused by differences in the sequence of the viral genome of the parental strain HVT FC-126 and the published sequence, and not by rearrangements during assemblage of the virus genome by the cosmid reconstruction technology.

3.3.2. Results of Characterisation by Sequence Analysis

[0290] The entire DNA sequence of inserted cassette and flanking regions of the transfer plasmid used was determined by PCR-sequencing. The consensus sequence of the insert in the viral genome of HVP360 was aligned for both isolates A1 and B1, and compared with the sequence in the transfer plasmid, as well as with the sequence of the insertion region of parent strain FC-126. The result of the alignment confirms that the sequence inserted in HVP360 is identical for isolates A1 and B1. [0291] In addition, this sequence was shown to be identical to the original expression cassette in the transfer plasmid. Also, flanking regions of the Us2 insertion locus of the transfer plasmid, of HVP360 A1 and B1, and of FC-126 were all shown to be identical in DNA sequence.

3.3.3.Results of Characterisation of Expression by IFA

[0292] Plaque purified virus of HVP360 for both parallel isolates, and from all three passage levels 5, 10 and 15, was screened by IFA, for expression of VP2 and F. All plaques tested showed full expression of the F and VP2 genes. This confirmed functional and stable expression of VP2 and F, up to (at least) cell passage level 15.

[0293] 3.4. Conclusions

[0294] HVP360 is a recombinant HVT expressing both IBDV VP2 and NDV F. Detailed characterization by IFA, Southern blot analysis on viral DNA, and DNA sequencing of the insert and flanking regions, confirmed that two independent HVP360 isolates A1 and B1 both had correctly integrated the expression cassette in the Us2 region of HVT FC-126 and functionally express IBDV VP2 and NDV F genes in infected cultures of CEF during 15 subsequent passages in CEF cells after plaque purification.

[0295] The expression cassette and total genome of HVP360 isolates A1 and B1 are correct in that no deletions, rearrangements, or additional foreign sequences were detected in restriction digestion patterns obtained after Southern blot hybridization with a series of HVT specific genomic probes. Only one copy of the insert is integrated in the Us2 region.

[0296] After detailed sequence analysis of the expression cassettes and the flanking regions of the Us2 insertion locus, it was concluded that HVP360 A1 and B1 are identical, to each other, to the sequence in the transfer vector, and to the parent virus FC-126.

4. Vaccination-Challenge Trial with HVP360

[0297] A vaccination-challenge experiment was performed with recombinant HVT HVP360. In short: one day-old SPF layers were vaccinated intramuscularly with either of the parallel isolates HVP360 A1 or B1. At 3 or at 4 weeks post vaccination, the induced protection was measured against a severe challenge infection with either IBDV or NDV: for IBDV challenge strain CS89 was inoculated at 3000 CID50 in 0.1 ml, by ocular route to each eye; for NDV strain Herts 33/56 was given at 6 Log 10 ELD50 in 0.2 ml per animal, by im route. The level of vireamia of the vaccine and of the control virus was determined by virus reisolation from spleens from inoculated animals. Results are presented in Table 3.

[0298] The level of challenge protection was determined as follows: [0299] For NDV challenge the ratio of live-vs-dead animals was determined at 2 weeks after the challenge at 3 or 4 weeks p.v. For example: 18 survivors out of 20 animals in the group gives an NDV protection score of 90%. [0300] For IBDV challenge, the clinical symptoms of the bursa at necropsy 10 days after challenge are given a score between 0 and 5, representing none up to severe lymphocyte depletion. Animals with a score of 0-2 are considered protected, while a score of 3 -5 is not protected. The IBD protection is the percentage of animals that was protected (bursa clinical score 0-2) from the number of animals in the group.

[0301] The results showed that in this experiment, im vaccination with HVP360 protected chickens of one day-old against ND at 3 weeks after vaccination, for 59 to 73%. At 4 weeks ND protection was 79 to 93%. Protection against IBD at 3 to 4 weeks after vaccination was 93 to 100%.

TABLE-US-00002 TABLE 3 Vireamia, serology, and challenge protection against IBDV and NDV, in SPF layers vaccinated im at 1 day-old with HVP360. Avg. NDV IBDV vireamia HI-NDV VN-IBDV protection protection dose (1) (2 Log) (2 Log) (%) (%) Vaccine (pfu) 10 d 25 d 25 d 25 d 21 d 28 d 21 d 28 d 360 A1 1880 176 17 3.1 6 73 79 94 100 360 B1 1820 158 13 3.4 6.6 59 93 94 93 FC-126/435 2440 404 36 0.9 0 0 0 0 0 (1) Vireamia is expressed in pfu/5 × 10{circumflex over ( )}6 spleen cells

5. Onset—and Duration of Immunity Against ND and Against IBD

[0302] In a subsequent vaccination-challenge experiment, using HVP360 as vaccine, and challenging with NDV or with IBDV, the onset of immunity and the duration of immunity were determined. HVP360 vaccine virus was at passage level 13; animals were SPF layers, 1 day old; vaccination route was: subcutaneous; vaccination dose was between 1500 and 2500 pfu/animal dose of 0.2 ml. Challenge virus was either IBDV CS89 or NDV Herts 33/56. Control animals were inoculated sc with HVT FC-126/435.

TABLE-US-00003 TABLE 4 Protection by HVP360 against challenge infection with NDV or IBDV, in 1 day old chicks by subcutaneous inoculation with a dose of about 1500-2500 pfu/animal. % protection against ND at Vaccine 2 w 3 w 4 w 6 w 8 w HVP360 20 68 90 100 100 HVT FC-126/435 0 0 0 0 0 % protection against IBD at Vaccine 2 w 3 w 4 w 6 w 8 w HVP360 90 95 100 100 100 HVT FC-126/435 0 5 0 0 0

[0303] The results showed that after vaccination with HVP360, more than 90% protection is obtained against challenge with NDV at 4 weeks after vaccination at day old by sc route. This meets the PhEur 0450 monograph requirements for a live ND vaccine.

[0304] Even better was the protection achieved against challenge with IBDV: more than 90% protection is obtained against challenge at 2 weeks after vaccination with HVP360. This meets the PhEur 0587 monograph requirements for an IBD vaccine.

[0305] Also these results demonstrate that the duration of immuno-protection proceeds until (at least) 8 weeks post vaccination at a level of 100% protection against ND, and against IBD.

6. Testing of Dose-Response Against ND, and Different Routes of Administration

[0306] In an animal trial, vaccination with different doses of HVP360 was applied, and different routes were tested: in ovo (io) and subcutaneous (sc), to test the response from these doses and these routes against challenge infection with NDV. In addition the vireamia levels of HVP360 vaccine and of FC-126 control virus upon reisolation from inoculated animals in the various treatment groups were determined. [0307] After vaccination of 18 day-old embryonated eggs of SPF layers (io), or one day-old SPF layers by sc route, animals were challenged at 3 or at 4 weeks old with NDV Herts 33/56. Vaccine/control virus was re-isolated from spleens or blood samples at 4, 11 or 17 days to determine the vireamia levels reached. Results are presented in Table 5. Vireamia is represented in two ways: once as number of birds positive for vaccine/control virus re-isolation out of the total number of birds in that group, and once as average virus pfu per 2×10̂6 spleen cells. [0308] In the column ‘dose’ the actual inoculation dose is presented, determined by back-titration of rests of the inocula after the vaccination.

[0309] Subcutaneous vaccination appeared to be little dependant on the dose used; doses between 500 and 2500 pfu/animal all reached satisfactory immunoprotection levels.

[0310] Ultimately, both routes (io and sc) could raise the same protection at 3 and 4 weeks, of 65-70% and 84-90% respectively.

LEGEND TO THE FIGURES

[0311] FIG. 1

[0312] Schematic view of the insert section of a preferred recombinant DNA molecule according to the invention, comprising the expression cassette and Us2 gene flanking sequences, that was used to generate

[0313] HVP360, a preferred recombinant HVT according to the invention. The elements of the expression cassette are drawn to scale.

[0314] Abbreviations (from left to right): 5′ US2: flanking upstream sequences from the HVT Us2 gene; mIE: murine CMV IE1 gene promoter; VP2: IBDV VP2 gene; term: SV40 transcription terminator; hIE: human CMV IE1 gene core promoter; F: NDV F gene; term: hCMV IE1 gene terminator; 3′ US2: flanking downstream sequences from the HVT Us2 gene.

[0315] FIG. 2:

[0316] Graphic representation (not drawn to scale), of the relevant elements of a representative number of recombinant HVT constructs tested. For comparison the prior art construct HVP309 is also represented.

[0317] FIG. 3:

[0318] Vireamia levels of different HVT recombinants at 10, 24 and 38 days post vaccination of one day-old SPF layers, as average per group.

[0319] FIG. 4:

[0320] Serological responses induced by the different HVT recombinants, at 37 days post vaccination of one day-old SPF layers, as average per group.