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Definitions

• the present invention relates to a vaccine against porcine circovirus (PCV-2) and a method for the manufacture of such a vaccine, for protecting piglets against PCV infection.
• PCV-2 porcine circovirus
• PCV-2 is thought to be linked to the post-weaning multisystemic wasting syndrome (PMWS) observed in young pigs.
• PMWS post-weaning multisystemic wasting syndrome
• PCV1 and PCV-2 are small (17 nm) icosahedral non-enveloped viruses containing a circular single stranded DNA genome.
• the length of the PCV-2 genome is about 1768 bp.
• PCV-2 isolates originating from different regions in the world seem to be closely related to each other and display 95 to 99% nucleotide sequence identities (Fenaux et al., J. Clin. Micorbiol., 38(7), 2494-2503, 2000).
• ORF-2 of PCV encodes the putative capsid protein of the virus.
• the ORF 2 of PCV 2 encodes a protein of 233 amino acids.
• the ORF 2 of all PCV-2 isolates share 91-100% nucleotide sequence identity and 90-100% deduced amino acid sequence identity. Between the ORF 2 genes of PCV 1 and PCV-2 there exists only 65 to 67% nucleotide identity and 63 to 68% amino acid sequence identity (Fenaux et al., supra).
• PDNS diporcine dermatitis and nephropathy syndrome
• a vaccine could be based on recombinant antigens derived from PCV-2.
• PCV-2 proteins have already been expressed in various expression systems. For example, Liu et al. (Protein Expression and Purification, 21, 115-120 (2001) expressed a fusion protein of the entire protein encoded by ORF-2 of PCV-2 linked to a MBP His tag, in E. coli . Kim et al. (J. Vet. Sci, 3(1), 19-23, 2002) expressed ORF 1 and 2 of PCV 2 in a baculovirus expression system. Blanchard et al. (Vaccine, 21, 4565-4575, 2003) expressed ORF 1 and ORF 2 in baculovirus based system in insect cells as well.
• the insect cells which had produced the PCV-2 proteins were lysed and formulated into a vaccine which was used to vaccinate specific pathogen free (SPF) piglets.
• the piglets received either one of the proteins in a prime boost regimen where the subunit vaccine followed a DNA vaccination or, in another group of piglets, the piglets received the ORF 1 and ORF 2 protein in two injections.
• SPF pathogen free
• the piglets need to obtain their priming vaccination already in the first week(s) of age so they can receive the booster vaccination round the time of weaning and have obtained full protection against PCV-2 infection just after weaning.
• Piglets are likely to have maternally derived antibodies (MDA) against PCV-2.
• MDA maternally derived antibodies
• a distribution of MDA titers in piglets used in experiments with a vaccine according to the invention is given in the Examples). It is however, well known that the presence of maternally derived antibodies will interfere with vaccination.
• Piglets may have different titers of MDA. Very high passive MDA titers may protect the piglets against PCV-2 infection (Merial: “PCV-2 Diseases: From research back to the field strain”, 18 th IPVS, Hamburg Germany, June 2004, page 99-101).
• the MDA titer may be too low to provide protection against PCV-2 infection, while still high enough to interfere with vaccination with, for example, a conventional inactivated PCV-2 vaccine.
• an inactivated vaccine may contain less antigen due to the fact that the virus can not be propagated to high titers in cell culture (or complicated and time consuming concentration procedures should be introduced in vaccine production).
• a vaccine according to the invention has been found to provide adequate protection against PCV-2 infection.
• a vaccine has been provided that can be used in a method to protect piglets, even piglets which are MDA positive against PCV-2, against infection with PCV-2, and thus against PCV-2 related diseases, most notably PMWS and PDNS.
• the present invention provides a vaccine against PCV-2 comprising at least 20 microgram/dose of ORF-2 protein of porcine circovirus type 2 (PCV-2).
• a vaccine containing at least 20 microgram (ug) of ORF-2 protein of PCV-2 per dose is capable of eliciting a protective immune response against PCV-2 infection (and thus against PCV-2 related diseases like PMWS and PDNS) even in the face of MDA.
• the vaccine contains at least 50 ug per dose, and most preferably 80 ug per dose.
• Vaccines according to the present invention with an antigenic mass up to 275 ug per dose could even be prepared, and such vaccines still did not elicit local reactions at the injection site.
• a vaccine according to the invention may contain a recombinant ORF-2 protein, wherein said recombinant protein is preferably produced by way of expression from a baculovirus expression vector in insect cells, said baculovirus expression vector containing the PCV-2 ORF-2 gene sequence under control of a suitable promoter.
• baculo expression system results in the production of high yields of viral antigen, which moreover show a good antigenicity.
• the use of the baculo expression system thus eliminates the need for complicated and time consuming procedures to concentrate the antigen to a suitable level when it cannot be produced at a high concentration, for example in a virus infected cell culture.
• the most commonly used baculo expression vector is Autographa californica often used with an insect cell culture of SF-9, SF-21 or High five insect cells.
• SF-9 and SF-21 are ovarian cell lines from Spodoptera frugiperda
• High five cells are derived from egg cells from Trichoplusia ni .
• the PCV-ORF-2 gene should be placed under the control of a suitable promoter.
• Most commonly used promoters in the baculovirus expression system are the promoters for the polyhedrin gene and the promoter for the p10 gene, meaning that the ORF-2 PCV-2 gene sequence is inserted in an insertion site in either the polyhedron locus or the p10 locus in the baculovirus genome.
• baculovirus expression system is given in “Baculovirus Expression vectors” by D. R. O'Reilly, L. K. Miller, and V. A. Luckow (1992, W.H. Freeman & Co, New York). Furthermore baculovirus derived expression vectors and complete expression systems are commercially available from many different companies.
• a vaccine according to the invention may further comprise a suitable adjuvant.
• suitable adjuvant systems are known in the art, for example commonly used oil in water adjuvant systems. Any suitable oil may be used, for example a mineral oil known in the art for use in adjuvantia.
• the oil phase may also contain a suitable mixture of different oils, either mineral or non-mineral. Suitable adjuvantia may also comprise vitamin E, optionally mixed with one or more oils.
• the water phase of an oil in water adjuvanted vaccine will contain the antigenic material. Suitable formulations will usually comprise from about 25-60% oil phase (40-75% water phase). Examples of suitable formulations may comprise 30% water phase and 70% oil phase or 50% of each.
• a vaccine according to the invention may be administered via any suitable route known in the art such as intramuscularly, intradermally or subcutaneously, whereby intramuscular administration is preferred.
• the present invention further provides a method for the manufacture of a vaccine intended for the protection of young piglets, which are PCV-2 MDA positive, against PCV-2 infection, wherein said vaccine is provided with at least 20 ug/dose of ORF-2 protein of porcine circovirus type 2 (PCV-2).
• a vaccine (prepared by a method) according to the invention can be used in a method to protect young piglets against PCV-2 infection.
• a vaccine according to the invention can even be used in a method for the protection of young piglets, which are positive for maternally derived antibodies (MDA) against PCV-2, against infection with PCV-2.
• MDA maternally derived antibodies
• a vaccine according to the invention can protect piglets, even when they have a relatively high titer of MDA against PCV-2.
• a distribution of MDA titers in young piglets encountered in the field at various farms across Europe are reflected in table 1, and the protection provided by a vaccine according to the invention is reflected in table 2.
• a vaccine according to the invention can even provide adequate protection against PCV-2 infection to piglets that have MDA titers falling in cluster 2′ as defined in the Examples (Table 2). Piglets falling in this cluster have MDA titers between 8 and 12 log2 which is a high MDA titer.
• a vaccine according to the invention can therefore even be used in a method for the protection of young piglets, which have an MDA titer against PCV-2 up to 10 log2, or even 12log2 (as measured with a method as indicated in the Examples).
• the vaccine is preferably administered in a 2 shot vaccination regimen, whereby the first shot (priming vaccination) is given to the piglets in the first to fourth week of age, preferably prior to weaning, for example in the first week of age.
• the second shot (boosting vaccination) can be given about 3 weeks later. In this way the piglets will have obtained full protection against PCV-2 infection just after weaning which is when the piglets are most susceptible to PCV-2 infection and thus become susceptible to PMWS and PDNS.
• Antibody titers against PCV-2 can be determined in the following manner:
• a monolayer of PK15 cells was formed in 96-well tissue culture plate. At 80% confluency the cells were infected with a field isolate of PCV-2 and further incubated for 2 days at 37° C. in a CO 2 incubator. After this period the cells were fixed in Ethanol and stored at 2-8° C. until use. Plates are used for tests when approximately 20% of the cells were infected.
• PCV-2 specific antibody titre of a given serum serial dilutions are made and incubated on the ethanol fixated cells. After 1 hour of incubation at 37° C. the plates are washed with tap water and the bound antibodies detected by incubation with FITC-labeled Rabbit anti-Swine IgG. The titre of a given serum is expressed as the reciprocal of the highest dilution where a PCV-2 specific antibody response can still be observed.
• Sera were collected from 232 piglets from various countries across Europe.
• Cluster 1 piglets with titres smaller than 8, cluster 2; piglets with titres from 8 to 12 and cluster 3; piglets with titres of 13 and higher.
• cluster 3 the maternally derived antibody titres are that high that it is expected that the piglets will be protected during the critical period of age (Merial: “PCV-2 Diseases: From research back to the field strain”, 18 th IPVS, Hamburg Germany, June 2004, page 99-101).
• cluster 1 the maternally derived antibody titres are that low that most of these piglets can be easily vaccinated.
• cluster 2 the antibody titres are of such a magnitude that a conventional vaccination approach will probably fail to immunize the majority of this group. Since more than halve of the piglets seem to fall into this cluster it will be of the uttermost importance to be able to protect the piglets in this cluster if one wants to eliminate PMWS from a farm.
• PCV-2 virus was isolated from lung tissue of a feeder pig showing clinical and histopathological signs of PMWS using PCV-free Swine Testis (ST) cells. The virus was propagated through five passages on PCV-free PK15 cells.
• DNA was isolated from a preparation of PCV-2 virus purified from infected PK15 cell supernatant. PCR was done to amplify the ORF-2 gene based on published sequences, using primers containing BamH1 restriction sites (forward primer CGG GAT CCG TTT TCA GCT ATG ACG TAT, reverse primer: CGG GAT CCT TTA TCA CTT CGT AAT GGT T). The resulting amplicon encompasses the complete ORF-2 gene plus flanking BamH1 restriction sites.
• the amplicon was excised and purified.
• the purified PCV-2 ORF-2 fragment was then digested with BamH1, and ligated into BamH1-digested pAcAS3 (Viak et. al. (1990) Virology 179 312-320).
• This plasmid contains the p10 promoter upstream of the insertion site, allowing for expression of foreign genes under control of the p10 promoter.
• TOP 10F′ bacteria (Invitrogen, Carlsbad, USA) were transformed with the ligation mixture, and clones which contained the correct construct were selected based on their sequence. A positive clone was expanded and the transfer plasmid DNA was again retested using sequencing.
• Sf9 Spodoptera frugiperda
• transfer plasmid and Bsu36I-digested AcNPV baculovirus DNA using CellFectine (Life Technologies, Gaithersburg, USA).
• the supernatant of the transfection was harvested at 3 days post transfection and plaque purifications were performed on Sf9 cells.
• Plaques were expanded and the resulting virus was screened for PCV-2 ORF-2 gene insertion by sequencing of isolated viral DNA, and immunofluorescence on Sf9 cells using anti-PCV-2 rabbit and pig sera.
• a seed of recombinant baculovirus BacPCV-2-ORF-2 was prepared called “Masterseed”.
• the Masterseed and the 5 th passage of the Masterseed on Sf9 cells were tested for stable insertion of the PCV-2 ORF-2 gene by sequencing of isolated DNA and immunofluorescence on Sf9 cells.
• Titrations were done to measure the amount of infectious virus in the virus preparations. Titrations were done on Sf9 cells, and were read by observing baculovirus specific CPE and/or PCV-2 ORF-2 specific immunofluorescence using polyclonal rabbit anti-PCV-2 immune serum.
• the total harvest containing both cells and supernatant was subjected to sonication to the extent that at least 90% of the cells were disrupted. Thereafter the live recombinant virus in batches of sonicated harvest was inactivated with 33 mM Binary Ethylenimine (BEI) at 37° C. for 72 hours under continuous stirring, at a pH of 7.5. After inactivation, the BEI was neutralized by the addition of a 1.6 fold molar excess of Sodium Thiosulphate.
• BEI Binary Ethylenimine
• inactivated virus suspension After neutralization, cell debris and polyhedra were removed by low-speed centrifugation at 600 g for 10 min. The resulting supernatant was named inactivated virus suspension. Harvests were checked for sterility and for completeness of inactivation. Completeness of inactivation was tested by passaging inactivated virus suspension on Sf9 cells for 2 weeks and visual inspection for the absence of baculovirus specific CPE.
• Baculovirus titres of was 8.5 log 10 TCID 50 /ml were obtained which were completely inactivated after treatment with BEI.
• PCV-2 ORF-2 Expression levels of the PCV-2 ORF-2 were determined in 5 separate experiments, and in each instance, the amount was well above the detection limit of the test, specifically ranging from 40 to 550 microgram/milliliter (ug/ml) of inactivated virus suspension.
• Vaccines of different PCV-2 ORF-2 antigen content were formulated and used to vaccinate young piglets with varying levels of maternally derived antibodies (MDA) against PCV-2. Two vaccinations were given, 3 weeks apart. The seroresponse against the antigen was measured at 56 weeks after the first vaccination. From these data, the influence of the antigen content on the take of the vaccine in the face of MDA was calculated.
• MDA maternally derived antibodies
• Titres were determined as the reciprocal of the highest dilution where a PCV-2 specific fluorescence could still be observed. For all animals, the decline of the antibody titer between the first and second bleeding was determined. If in this period the antibody titre had not declined or was increased it was regarded that in the animal concerned the vaccine had taken. However when the PCV-2 specific antibody titre was decreased it was regarded that vaccination had not succeeded and the vaccine didn't take.
• vaccine take means that the vaccination of a piglet resulted in a PCV-2 specific antibody titre at 1 week post booster vaccination that is equal or higher than the PCV-2 specific titre at primary vaccination.
• the vaccine mounted an active serum response against PCV-2 and in which case piglets can be regarded as being protected against a FCV-2 infection.
• the titre at 1 week post booster was smaller than at primary vaccination the vaccine was unable to induce an immune response and the natural decline of maternally derived antibodies was observed which, in time, will render these animals susceptible for a PCV-2 infection.
• the antigenic mass of a vaccine directed against PCV-2 must at least contain 20 ug of antigen or more to be able to efficiently protect a herd against the consequences of a PCV-2 infection.

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