Recombinant expression of PCV2b ORF2 protein in insect cells

Abstract

The present invention relates to the field of veterinary vaccines, in particular to porcine vaccines against PCV2 and associated diseases. Specifically the invention relates to the finding that a mutation is required in PCV2b ORF2 protein, to prevent its nuclear accumulation upon expression in insect cells; the mutation introduces a Proline at amino acid position 131. This allows efficient expression in insect cells, easy harvesting, and generates large amounts of virus-like particles. The VLPs are highly effective in vaccines for porcines for reduction of infection by PCV2 or of associated signs of disease.

Claims

1. An insect cell comprising a heterologous nucleic acid comprising: a. a nucleotide sequence encoding an ORF2 protein from porcine circovirus 2 of genotype 2b (PCV2b), and b. a transcription control sequence that is operatively linked to said nucleotide sequence, wherein the nucleotide sequence encodes a mutant PCV2b ORF2 protein having a Proline at amino acid position number 131.

2. The insect cell of claim 1, wherein the transcription control sequence is selected from the group consisting of a baculovirus p10 gene promoter and a baculovirus polyhedrin gene promoter.

3. The insect cell of claim 1, wherein the heterologous nucleic acid is comprised in a recombinant baculovirus genome.

4. A method of producing a mutant PCV2b ORF2 protein having a Proline at amino acid position number 131, comprising culturing the insect cell of claim 1; wherein the mutant PCV2b ORF2 protein is produced; and whereby an insect cell culture is formed.

5. The method of claim 4, further comprising harvesting the mutant PCV2b ORF2 protein from the insect cell culture.

6. The method of claim 5, wherein said insect cell is a baculovirus-insect cell.

7. The method of claim 4, wherein said insect cell is a baculovirus-insect cell.

8. A virus-like particle (VLP) of a mutant PCV2b ORF2 protein, wherein the mutant PCV2b ORF2 protein has a Proline at amino acid position number 131.

9. A vaccine for porcine animals for reducing infection by PCV2 or reducing associated signs of disease, comprising a mutant PCV2b ORF2 protein in a pharmaceutically acceptable carrier and an adjuvant, wherein the mutant PCV2b ORF2 protein has a Proline at amino acid position number 131.

10. The vaccine of claim 9, wherein the mutant PCV2b ORF2 protein is in the form of a virus-like particle.

11. The vaccine of claim 9, further comprising an antigen from a micro-organism that is pathogenic to porcine animals.

12. A method of reducing an infection by PCV2 or reducing associated signs of disease in a porcine animal, comprising administering to said porcine animal the vaccine of claim 9.

13. A process of preparing a vaccine of claim 9, comprising admixing a mutant PCV2b ORF2 protein having a Proline at amino acid position number 131, with a pharmaceutically acceptable carrier and an adjuvant, thereby forming an admixture.

Description

EXAMPLES

Example 1: Assembly of Different PCV2 ORF2 Expressing Recombinant Constructs

(1) For the expression of PCV2 ORF2 proteins in insect cells, recombinant baculoviruses were generated, using standard procedures. In short:

(2) The wildtype PCV2 ORF2 genes used in these experiments were obtained from different sources: the PCV2a parental virus was from a vaccine strain from USA; PCV2b was from French isolate Imp1011, GenBank acc. nr. AF055394; PCV2d was from China, strain BDH, GenBank acc.nr: HM038017.

(3) The ORF2 gene was subcloned by amplification using PCR, and insertion into a cloning plasmid. Next, some of the ORF2 genes were mutated, either to encode a Proline at amino acid position 131, by using PCR-directed mutation. Alternatively, or additionally, ORF2 genes were codon-optimised to resemble the codon usage table of the AcMNPV polyhedrin gene. The mutated sequences used herein for PCV2b are presented in the attached sequence listing, as described.

(4) Synthetic constructs of mutated ORF2 gene inserts were ordered from a commercial supplier (BaseClear, Netherlands; or Genscript, Piscataway, N.J., USA).

(5) For use with the Bac-to-Bac system these were subcloned into cloning vector pFastBac1, and for use with the ProEasy system in transfervector pVL1393. In both instances the insert was behind the polyhedrin promoter. Generation and selection of recombinant baculovirus was performed according to the manufacturer's instructions. Some of the PCV2a recombinant baculovirus constructs had been constructed using the classical selection technique of transfection and isolation of recombinants by plating of infected insect cells under soft agar, and plaque picking.

(6) All recombinant baculoviruses were amplified on Sf9 cells, harvested, stored refrigerated or frozen, and were titrated. Virus stocks used for further experiments were typically between 7.5 and 8.5 Log 10 TCID50/ml.

(7) A selection of the recombinant baculoviruses used for the expression of PCV2 ORF2 protein in insect cells is listed in the below schedule: NB: All inserts were inserted behind the polyhedrin promoter. Codon optimisation (when applied) was towards the codon use of the AcMNPV polyhedrin gene.

(8) TABLE-US-00001 Virus PCV2 Baculovirus Codon Amino acid number genotype construct optimalisation at 131 1 2a plaque picked None Proline 2 2a plaque picked Yes Proline 3 2a Bac-to-Bac None Proline 4 2a Bac-to-Bac Yes Proline 5 2a ProEasy Yes Proline 6 2b Bac-to-Bac None Threonine 7 2b Bac-to-Bac Yes Threonine 8 2b Bac-to-Bac None Proline 9 2b ProEasy Yes Proline 10 2d ProEasy Yes Proline

Example 2: Expression in Insect Cells

(9) The different recombinant baculovirus constructs, as described above, were used to infect insect cells, and express several variants of the PCV2 ORF2 protein. The protein produced was visualised and analysed to assess the effect of the various mutations made, by comparing to unmodified protein, or to protein of another genotype of PCV2. Some recombinant baculoviruses were scaled-up, to produce protein for the formulation of PCV2 subunit vaccine for testing in pigs.

(10) 2.1 Insect Cell Cultures

(11) Standard infection and expression experiments were done at small scale, using Sf9 insect cells, which were taken from a spinner culture of clean cells that were regularly split to maintain exponential growth. Typical infection rates were between 0.01 and 0.1 moi, and culture durations were between 3 and 5 days. Cultures were monitored regularly by light microscopy to check the health of uninfected cells, or to monitor the progress of viral replication in infected cultures.

(12) Culture vessels used were either T25 or T75 flasks (Falcon) with monolayer cultures of 5 or 15 ml respectively. Alternatively 100 ml suspension cultures were run in spinner flasks (Corning). Flasks were incubated at 27° C., and the culture medium used was Sf-900™ II (Thermo Fisher scientific). No serum was added, but a mixture of antibiotics: 50 μg/ml gentamycin and 0.25‰ natamycin.

(13) When most of the cells showed cytopathogenic effect (cpe), the cultures were harvested: T-flask cultures required tapping firmly to loosen the cells. Culture-harvests were then centrifuged, and culture supernatant was discarded as the ORF2 protein produced was mostly contained within the insect cells. Depending on the intended further use the cell-pellet could then be resuspended in a required liquid at a desired concentration.

(14) The small scale cultures were not routinely inactivated; samples were either used in containment facilities, or were treated with denaturing electrophoresis sample buffer, and then considered inactivated.

(15) Large scale cultures followed essentially the same outline, with adaptations for volume and equipment. In experimental setting, intermediate large scale insect cell suspension cultures were run at 2 to 10 litre volume, in industrial fermenters with automated process controls. Clean Sf9 insect cells were obtained from a pre-culture. Moi was between 0.01 and 0.1, and culturing was for 5-7 days. When the infection had progressed sufficiently, the cells were left to settle by gravity. Then the top 90% of the culture volume was removed, the cells were harvested in 1/10th of the original culture volume, and were lysed by sonification. At medium scale sonification was performed batch-wise, on ice, using a tip sonifier (Vibra Cell™, Sonics, CT, USA). At larger scales (100 l volumes or more), sonication was done by passage through a flow-through sonification cell (Sonobloc™, Bandelin)

(16) Next the sonicate was inactivated using BEI. This was then neutralised using Na-thiosulphate. Then the inactivated harvest was clarified by centrifugation and the supernatant containing the ORF2 protein, was kept as the watery phase of the bulk antigen. This was stored at 2-8° C. for quality control and further processing. In these products the pharmaceutically acceptable carrier for the antigen is thus spent medium from the insect cell culture. When needed the bulk antigen could be concentrated, of could be dialysed against PBS. Alternatively the antigen could be diluted with fresh insect cell culture medium, or with PBS.

(17) 2.2 ELISA

(18) 2.2.1 Outline of ELISA Insect cell cultures were infected with recombinant baculoviruses number 6 (Threonine at 131), or virus number 8 (T131P substitution), to test the effect of the T131P substitution in PCV2b ORF2 protein. Both are constructs in Bac-2-Bac format, and did not have codon-optimisation. Of virus 6, two isolates were tested.

(19) T175 flask cultures of Sf9 cells were infected, incubated, and harvested by centrifugation of the whole culture. The cell pellets were taken up into 10 ml water-for-injection, which effectively lysed all the cells. The cell-lysates were then tested for their antigenic mass in a sandwich ELISA, briefly as follows:

(20) The wells of a polystyrene micro-titration plate were coated with a monoclonal antibody directed against PCV2 ORF2a. Serial dilutions of lysate samples were incubated alongside a series of dilutions of a known reference standard of PCV2a ORF2. Next the plates were incubated with a fixed amount of a secondary antibody also directed against PCV2 ORF2a, which was conjugated with biotin. Finally the amount of bound conjugate was then quantified by incubation with peroxidase-conjugated streptavidin, followed by chromophoric detection by automatic plate reader.

(21) The amount of antigen in the lysates was calculated against the reference standard, of which the amount of antigen was arbitrarily set at 100 antigenic units (AU)/ml.

(22) 2.2.2 Results and Conclusions of ELISA

(23) The amount of ORF2 protein detected in the lysates was as follows: virus 6 infected cells (two variants): 5.5 and 7.4 AU/ml, virus 8 infected cells: 68 AU/ml.

(24) A lysate prepared in pure water from the cells infected with the recombinant virus expressing PCV2b ORF2 protein without mutation at 131, contained only very little ORF2 protein as detected by ELISA. However such a lysate from cells infected with recombinant baculovirus expressing ORF2 with the T131P substitution, contained about 10 times more protein.

(25) These ELISA results illustrated the big difference in the amount of ORF2 protein that could be readily isolated from infected insect cells, with or without the substitution of ORF2 amino acid number 131. On the one hand this related to the ease with which the protein was released in a non-denaturing lysate. On the other this also reflected the difference in overall production of PCV2b ORF2 protein in insect cells, without or without mutation of the amino acid at position 131.

(26) 2.3 Immunofluorescence Assays

(27) 2.3.1 Outline of IFT Assays

(28) For Immunofluorescence tests (IFTs) clean insect cells were seeded in the wells of a 96-well microtitration plate, typically at 2.5×10{circumflex over ( )}4 cells per well, in 100 μl of medium. After attachment the cells were infected with the particular recombinant baculovirus to be investigated. Typically dilution ranges of the virus were inoculated to allow observation at different moi's. The plates were analysed after 4-5 days of incubation, when the insect cells had become properly infected but were not yet lysed. The medium was removed, the cells were fixed with cold ethanol, and stained with appropriate antibodies according to standard procedures. A FITC-conjugated second antibody was used for the visualisation. The insect cells were counter-stained with Evans blue to enhance the contrast with the background.

(29) For these IFT studies the primary antibody used was a swine polyclonal anti-PCV2a antiserum, this was sufficiently potent to also stain ORF2 protein of PCV2b and PCV2d genotype.

(30) 2.3.2 Results of IFT Assays

(31) When comparing the IFT results observed for the different ORF2 protein expression products, several observations were made: The recombinant viruses 1-5 as described above, all expressing PCV2a ORF2 protein, invariably all caused a cytoplasmic staining pattern whereby essentially the whole of the insect cell was showed a bright colouration. However insect cells infected with recombinant baculovirus number 6, expressing wildtype PCV2b ORF2 protein, displayed an essentially different pattern, where the staining was located exclusively around the nucleus of the insect cells. No cytoplasmic staining was observed. This IFT pattern was largely the same for insect cells infected with virus number 7, which is also expressing PCV2b ORF2 protein but from a codon-optimised gene. Only difference was that a small amount (approximately 10% of the insect cells) showed cytoplasmic staining, while the majority of the cells still displayed nuclear staining. Surprisingly, insect cells infected with virus number 8 gave an IFT pattern where the fluorescence pattern was the reverse of that for recombinant virus number 7: most of the insect cells displayed cytoplasmic staining, with some cells still showing nuclear staining. Virus number 8 carried a PCV2b ORF2 gene wherein the codon encoding amino acid number 131 had been mutated from a Threonine to a Proline (T131P). Finally, insect cells infected with virus number 9 (PCV2b ORF2 gene with codon optimisation and carrying the T131P substitution, in a clean construct) all exclusively showed cytoplasmic staining. Remarkably, insect cells infected with recombinant baculovirus number 10 (encoding PCV2d ORF2 protein with the T131P substitution, codon-optimisation, and in a clean construct) all still displayed a nuclear staining pattern.
2.3.3 Conclusions from IFT Assays

(32) It was concluded that the effective expression of ORF2 protein from PCV2b in insect cells, required the substitution of the amino acid at position number 131 by Proline, displaying a cytoplasmic staining pattern, in an IFT with an anti-PCV2a antibody. Without such mutation, the PCV2b ORF2 protein displayed a staining exclusively at or around the nucleus of the insect cell expressing that protein.

(33) The mutation of the ORF2 gene by codon optimisation only, gave a slight shifting of the IFT staining pattern, probably as a result of the increased efficiency of the expression.

(34) The nuclear staining pattern could be further reversed when the recombinant baculovirus was not based on a construct containing residual elements from the cloning vector (such as in a recombinant produced using the Bac-to-Bac system), but was a ‘clean’ baculovirus construct, i.e. not contain further genetic elements from the recombination process in its genome. Such clean recombinant baculoviruses can be obtained by using classical homologous recombination and plaque purification, or more conveniently by using a commercial kit such as the ProEasy system.

(35) Thus: a reversion of the IFT nuclear staining pattern observed in insect cells expressing PCV2b ORF2 protein, can be obtained by mutation of the triplet encoding the ORF2 amino acid number 131 to encode Proline. Further optimisation of cytoplasmic ORF2 protein expression can be reached by codon-optimising the encoding gene, and by using a clean recombinant baculovirus construct.

(36) 2.4 Quantification by Gel-Electrophoresis

(37) 2.4.1 Outline of Gel-Electrophoresis Assays

(38) To allow a quantification of the expression level of the different ORF2 proteins, samples from infected insect cell cultures were run on SDS-PAGE, alongside lanes with known quantities of bovine serum albumin (BSA) as marker protein. After staining, the gels were scanned and analysed. By using a strongly denaturing sample buffer this experiment revealed the total expression capacity of insect cells infected with the various recombinant baculovirus constructs

(39) The different recombinant baculoviruses to be tested were cultured in T75 flasks as described, harvested, centrifuged, and the cell pellets were taken up in 7.5 ml PBS. Samples of the supernatant and of the resuspended cells were then taken up into standard denaturing Laemmli SDS-PAGE sample buffer (containing bromophenol blue indicator and beta-mercaptoethanol).

(40) Samples were run on standard poly-acrylamide gels (precast Criterion TGX™, Any kD (15%), Bio-Rad), in standard Tris-Glycine-SDS running buffer. After the electrophoresis, the gels were stained with Instant Blue™ (Expedeon). Finally the gels were digitised and analysed with a Bio-Rad GS-900 calibrated densitometer using Image Lab™ Software, version 5.2.1, to assign an amount of protein to individual bands on the gel.

(41) 2.4.2 Results of Gel-Electrophoresis Assays

(42) In FIGS. 1 and 2 some representative images are presented of gel such as used in these experiments: FIG. 1: samples from cultures of insect cells infected with viruses no. 3, 6, or 7; and FIG. 2: samples from cultures with viruses no. 8 or 9. Per type of virus, the first lane contains a sample of culture supernatant, the next three lanes contain samples of the 2× concentrated cells, with 30, 20, and 10 μml from left to right. Left- and right-most lanes: molecular weight markers 10-250 kDa (Precision Plus Protein™ Standards, Bio-Rad), the band sizes are indicated in the Figures.

(43) Each gel also contained a dilution range of BSA (Albumin Standard high-quality, Thermo Fisher Scientific), that was used as a 100 ng/μl solution. The amounts used were: FIG. 1: in lanes 14-17: 0.25, 0.5, 1, and 2 μg BSA; FIG. 2: in lanes 11-15: 0.25, 0.5, 1, 2, and 3 μg BSA.

(44) The analyses by densitometer focussed on the bands of PCV2 ORF2 protein, at 26 kDa. All samples were analysed on gels two times, the calculated yield results are averages of the Duplo's.

(45) The yields of PCV2 ORF2 protein are expressed in micrograms of protein per ml of the original T75 culture (15 ml volume). The yields were found to be as follows:

(46) TABLE-US-00002 ORF2 Virus PCV2 Baculovirus Codon Amino acid yield no. genotype construct optimalisation at 131 (μg/ml) 3 2a Bac-to-Bac None Proline 32 6 2b Bac-to-Bac None Threonine 26 7 2b Bac-to-Bac Yes Threonine 36 8 2b Bac-to-Bac None Proline 58 9 2b ProEasy Yes Proline 76

(47) Several observations could be made: For virus infections with viruses numbers 8 and 9, the two constructs of PCV2b ORF2 with T131P substitution, the gel lanes (see FIG. 2) looked slightly darker than for the other viruses tested, even though for all cultures compared by SDS-PAGE the moi (0.1), culture time (4 days), and other conditions were all the same. This could indicate that the infection may not have progressed as far as for the other viruses, resulting in more cell-material in the sample. Perhaps the Proline substitution caused a slight attenuation of the recombinant baculovirus. However protein-expression levels appeared unaffected and were better than for PCV2b ORF2 without this mutation. None of the samples of supernatant showed an amount of ORF2 protein that could be visualised by the CBB staining that was used. Consequently, under the conditions as applied in these experiments, effectively all of the expressed ORF2 protein was contained in the insect cells. The expression level of unmutated PCV2b ORF2 protein (virus 6) was lower than that of PCV2a ORF2 (virus 3), with 26 versus 32 μg/ml. It is reminded that these samples display the total of all protein produced, because of the aggressive denaturation employed (boiling in SDS and beta-mercaptoethanol). This indicates that the total protein expression from the unmutated PCV2b ORF2 gene is actually lower over-all in insect cells. NB: This difference in yield is much worse in practice, when harvesting is without (strong) denaturation. In that case lysates from insect cells expressing unmutated PCV2b ORF2 hardly showed any detectable ORF2 protein at all. The yield of unmutated PCV2b ORF2 protein could be slightly increased by the use of a codon optimised ORF2 gene: compare the yields of virus-cultures 6 and 7, with total yields of 26 and 36 μg/ml respectively. However the most significant increase was achieved by the introduction of the T131P substitution in the PCV2b ORF protein, see the yields of virus-cultures 6 and 8, with 26 versus 56 μg/ml. The T131P substitution was thus able to cause more than a doubling in total yield of ORF2 protein, even in these small scale cultures without any optimisation. Even better yields of PCV2b ORF2 protein could be reached when all mutations were combined: T131P substitution, codon optimisation and using a clean baculovirus construct: comparing the yields of virus-cultures 6 and 9, shows a tripling of protein yield, from 26 to 76 μg/ml.

(48) A similar analysis of protein yield was also performed on samples that had been produced in intermediate large scale cultures of 2 litres. Harvests were obtained in 7.7× concentration compared to the total culture volume. The ORF2 protein from these cultures was also used to produce the experimental PCV2 vaccines described hereafter.

(49) ORF2 protein yields obtained were as follows, indicated in μg/ml of the original 2 litre culture volume:

(50) TABLE-US-00003 ORF2 Virus PCV2 Baculovirus Codon Amino acid yield no. genotype construct optimalisation at 131 (μg/ml) 5 2a ProEasy Yes Proline 44.2 9 2b ProEasy Yes Proline 103.9

(51) The culture conditions in the fermenters were clearly more optimal then in the small scale culture sin T-flasks. Not only because of the automated control of temperature, pH, and dissolved oxygen, but also because these were suspension cultures, which allow higher cell-densities per ml of culture.

(52) The recombinant baculoviruses used were also constructed in the optimal way in the sense that they had a codon-optimised ORF2 gene, in a clean baculovirus construct (produced using the ProEasy system).

(53) This resulted in a protein yield for insect cells infected with virus no. 9 of over 100 μg/ml of culture, while in T-flasks this was 76 μg/ml.

(54) Remarkably the yield of protein from insect cells expressing mutant PCV2b ORF2 protein with the substitution of amino acid number 131 by Proline, was substantially higher than that of unmodified PCV2a ORF2 protein, even though the same constructs and culturing conditions were used. In particular because the ORF2 gene of the PCV2a used here also had a Proline at amino acid position 131 from its native sequence. It is not immediately apparent what might cause this difference, though it is evidently highly favourable for the production of PCV2b ORF2 protein for the manufacture of subunit vaccines against PCV2.

(55) 2.4.3 Conclusions from Gel-Electrophoresis Assays

(56) Although not all effects observed could be explained at this time, the analysis by gel-electrophoresis made it explicitly clear that expression in insect cells of PCV2b ORF2 protein without any adaptation, at best yielded protein amounts lower than for the expression of PCV2a ORF2. This difference is even worse when the cells are harvested using non-denaturing conditions.

(57) The lack in yield can however be more than made up by substituting the amino acid at position 131 in PCV2b ORF2 by Proline, which at least doubles the yield as compared to that of unmutated PCV2b ORF2 protein.

(58) Even further increases in yield can be reached by expressing the 131P mutation from a codon-optimised ORF2 gene, and using a clean baculovirus construct. Still further improvements in the yields can be reached in large scale suspension cultures.

(59) Together these findings enable for the first time the commercial and large-scale production of ORF2 protein from PCV2b genotype, by a recombinant expression system.

Example 3: Vaccination-Challenge Experiments in Pigs

(60) 3.1 Introduction

(61) 3.1.1 Objective

(62) This study was performed to compare and evaluate the efficacy of porcine vaccines based on insect-cell expressed mutant PCV2 ORF2 proteins according to the invention. To provide a thorough test of their protective capacity, the ORF2 proteins of different genotypes were used not only against a homologous, but also against a heterologous PCV2 challenge-infection. Also the vaccine doses used contained relatively low amounts of ORF2 protein.

(63) 3.1.2 Study Outline

(64) For this study 140 piglets were used, allotted to 2 sets of 7 treatment groups with 10 animals each, one set for each of the two challenges. The 140 animals were derived from 14 sows. The piglets had detectable levels of anti-PCV2 ORF2 antibodies ranging from low to very high titres; the average MDA titre for all animals was 5.8 Log 2 by antibody-ELISA

(65) The piglets were vaccinated intramuscularly in the neck with 2 ml each, when they were approximately three weeks old. The vaccines used comprised different amounts of PCV2 ORF2 protein produced via the baculovirus-insect cell expression system, from one of three genotypes: PCV2a, 2b or 2d. The recombinant baculoviruses used were numbers 5, 9, and 10, as described above, having a Proline at amino acid position 131, expressed from an encoding ORF2 gene that was codon optimised towards the AcMNPV polyhedrin gene, and using a clean baculovirus construct. The vaccines were formulated as oil-in-water emulsions with Emunade™ adjuvant. The vaccine additionally contained antigen from Mycoplasma hyopneumoniae (J strain), to resemble the commercial vaccine: Porcilis™ PCV M Hyo. With regard to the PCV2 ORF2 antigen, the vaccine tested comprised either a quarter or a sixteenth of a standard full dose per animal.

(66) For both sets, the division of the vaccine treatments over the groups was as indicated in the schedule below.

(67) NB: Animals in group 7 of both sets were not vaccinated, to serve as challenge controls.

(68) TABLE-US-00004 Group 1 2 3 4 5 6 7 Vaccine dose 20 5 20 5 20 5 No (μg ORF2 prot./animal) vaccine ORF2 protein genotype 2a 2b 2d

(69) Two weeks post vaccination all animals were transported to the challenge facilities. At 3 weeks post vaccination (at 6 weeks of age), all animals received a challenge infection with virulent PCV2, either from 2b or from 2d genotype. Challenge was by way of intranasal administration of 6 ml (3 ml per nostril) of PCV2 in PBS, at 5 Log 10 TCID50/ml; using for set 1: a PCV2b challenge virus (Dutch isolate, 2003), and for set 2: a PCV2d challenge virus (German isolate, 2015). The challenge viruses had been produced on PCV-free PK15 cells, and had been checked for bacterial and fungal sterility.

(70) Three weeks post challenge (at 9 weeks of age), all animals were euthanized and examined: the viscera were inspected in-situ, paying particular attention to: lungs, inguinal and mesenteric lymph nodes, tonsils, thymus, spleen, liver and kidneys. Next, samples from tonsil, lung, mesenteric lymph node and inguinal lymph node were removed and fixed for detection of PCV2 challenge virus, by immunohistochemistry (INC).

(71) Other analyses were also performed, such as serology on blood samples, and qPCR on tissue and swab samples. However the IHC data gave the most explicit results.

(72) 3.2 Materials and Methods

(73) 3.2.1 Pcv2 Orf2 Proteins:

(74) The PCV2 ORF2 gene sequences of PCV2a, 2b and 2d were produced in intermediate large scale cultures as described in section 2.1 above. Their quantification by gel-electrophoresis is described in section 2.3.2 above.

(75) 3.2.2 Test Animals

(76) Test animals were negative for PCV2 viral load, and had moderate levels of anti-PCV2 antibodies. The test would have been rejected if any of the animals had developed clinical signs of PCV2 infection prior to challenge.

(77) Only healthy animals were included in the trial. All piglets were observed daily for general health, and for clinical or systemic signs of disease, such as loss of appetite, reluctance to move, tendency to lie down, listlessness or drowsiness, shivering, bristling, oedema (especially around the eyes), vomiting and diarrhoea and dyspnoea.

(78) Piglets were marked individually by ear-tags, with piglets of the 14 litters being equally divided over the test groups. After weaning tap water was available ad libitum and feeding was done according to standard procedures.

(79) The vaccination was given at the farrowing farm. After transport to the challenging facilities one week acclimatisation was included.

(80) 3.2.3 Laboratory Experimental Procedures

(81) 3.2.3.1 Immunohistochemistry

(82) Tissue samples were prepared for histological examination by fixing in 10% formalin and embedding in paraffin. Microscopy slides were prepared. These were incubated with a rabbit polyclonal anti-PCV2 serum as primary antibody, and visualised with peroxidase staining (Envision+™, DAKO). The slides were counterstained with hematoxylin. In the microscopic examination of tonsils and lymph nodes, characteristic brown staining was given a score depending on extent of colouring: score 0: lymphoid follicles showed no specific positive staining; score 1: less than 10% of the lymphoid follicles contained up to 15 cells with specific positive staining; score 2: 10-50% of the lymphoid follicles contained up to 15 cells with specific positive staining, or less than 10% of the lymphoid follicles contained more than 15 cells with specific positive staining; score 3: >50% of the lymphoid follicles contained cells with specific positive staining.

(83) The sum of the scores of the individual tissues were recorded as the total IHC score per animal, and these were combined for all animals of a group.

(84) 3.2.3.2 Vaccine Formulations

(85) The test vaccines were formulated as oil-in-water emulsions with Emunade™, this is a dual adjuvant with mineral oil dispersed in an aqueous phase containing the vaccine antigens as well as Aluminium-hydroxide. The preparation, with 9% Mhyo antigen, was according to the procedure as disclosed in WO 2016/091998.

(86) Also the vaccine was prepared in line with EP 1.926.496, so that 25% of a full dose (2 ml/animal) comprised approximately 20 μg of ORF2 protein, and a 6.25% dose comprised 5 μg ORF2 protein/animal dose.

(87) 3.3 Results & Conclusions

(88) By way of immunohistochemistry, the tissue samples of tonsil, mesenteric- and inguinal lymph nodes collected post-mortem, were analysed to assess the amount of detectable challenge virus. A graphical overview of these results is presented in FIGS. 3 and 4: FIG. 3 represents the results for the animals in groups 1-7 of set 1, receiving a challenge infection with virulent PCV2b virus; FIG. 4 represents the IHC data for all animals of set 2, in which the animals were challenged with virulent PCV2d virus.

(89) The vertical axis presents the score of IHC intensity according to the scale described above. The IHC results can be used to determine the efficacy of the vaccines based on PCV2 ORF2 protein, by comparing the level of reduction of PCV2 infection in vaccinated versus unvaccinated groups.

(90) Several observations could be made: The unvaccinated animals showed considerably higher IHC scores than the vaccinated groups, indicating that the dose of the challenge was high enough, and that the challenge infection was effective, even in the context of moderate levels of anti-PCV2 maternal antibodies in the piglets. As can be seen: the vaccine doses comprising 5 or 20 μg ORF2 protein gave strong protection against replication of challenge virus, reaching reduction of IHC scores of up to 90%. Therefore these PCV2 vaccines were highly efficacious. Somewhat lesser protection was observed for the lowest dose of PCV2a ORF2 protein against PCV2d challenge. This indicates that PCV2 vaccines based on insect-cell expressed mutant ORF2 protein from PCV2b or from PCV2d according to the present invention are very effective as vaccine against PCV2 infection, even at a single dose, and even in the context of maternally derived antibodies. At equal doses, these vaccines based on mutant ORF2 protein from PCV2b or from PCV2d even appeared to be more effective than the traditional vaccine based on PCV2a ORF2 protein. While homologous protection was mostly better than heterologous protection, there was a clear level of cross-protection against the PCV2b and PCV2d challenge viruses, by all the three vaccine genotypes tested: the PCV2a and PCV2d ORF2 protein vaccines were comparable in protective effect at their lowest dose against PCV2b challenge, while PCV2a ORF2 protein was better at the higher dose. However vaccine based on mutant PCV2b ORF2 protein was better than PCV2a ORF2 protein against PCV2d challenge at both doses.

BRIEF DESCRIPTION OF DRAWINGS

(91) FIG. 1:

(92) Scan of SDS-PAGE gel with samples from cultures of insect cells expressing different PCV2 ORF proteins via infection with recombinant baculoviruses.

(93) Lane numbers and an indication of the lane content is depicted above the gel image. Lanes 1 and 18: Molecular weight markers; indications of molecular weights in kDa are indicated to the left of the image Lanes 2-5: cells infected with virus 3; Lane 2: supernatant; Lanes 3-5 cell-pellet, respectively 30, 20, and 10 μl Lanes 6-9: cells infected with virus 6; Lane 6: supernatant; Lanes 7-9 cell-pellet, respectively 30, 20, and 10 μl Lanes 10-13: cells infected with virus 7; Lane 10: supernatant; Lanes 11-13 cell-pellet, respectively 30, 20, and 10 μl Lanes 14-17: reference samples of BSA protein, respectively: 0.25, 0.5, 1, and 2 μg.

(94) FIG. 2:

(95) Scan of SDS-PAGE gel with samples from cultures of insect cells expressing different PCV2 ORF proteins via infection with recombinant baculoviruses.

(96) Lane numbers and an indication of the lane content is depicted above the gel image. Lanes 1 and 16: Molecular weight markers; indications of molecular weights in kDa are indicated to the left of the image Lanes 2-5: cells infected with virus 8; Lane 2: supernatant; Lanes 3-5 cell-pellet, respectively 30, 20, and 10 μl Lanes 6-9: cells infected with virus 9; Lane 6: supernatant; Lanes 7-9 cell-pellet, respectively 30, 20, and 10 μl Lane 10: empty Lanes 11-15: reference samples of BSA protein, respectively: 0.25, 0.5, 1, 2, and 3 μg.

(97) FIGS. 3 and 4:

(98) Graphical representations of the results from immunohistochemistry on different tissues of pigs that received a challenge infected with PCV2, after receiving a vaccine prepared from insect cell expressed PCV2 ORF2 protein. Tissue results are presented for tonsil, mesenteric lymph nodes (‘Mes. In’) and inguinal lymph nodes (‘Ing. In’)

(99) The various vaccines applied (or not) are indicated on the horizontal axis; the vertical axis presents the arbitrary score of IHC intensity according to a particular scoring grade.

(100) FIG. 3 represents the IHC results for the animals in groups 1-7 of set 1, receiving a challenge infection with virulent PCV2b virus;

(101) FIG. 4 represents the IHC results for the animals in groups 1-7 of set 2, in which the animals were challenged with virulent PCV2d virus.