TWI361695B - Pcv-2 vaccine - Google Patents

Description

1361695 (1) Description of the Invention [Technical Field of the Invention] The present invention relates to a vaccine against porcine circovirus (PC V-2) and a method of manufacturing the same to protect piglets against PCV infection. PCV-2 is thought to be associated with post-weaning multisystemic obliteration (PMWS) observed in piglets. The disease first appeared in Canada in 199.

Its clinical signs and pathology were published in 1997 ((: 13|^^31· Proc. Am. Assoc. Swine. Pract > 1 997:499-5 0 1 > Harding et al., Proc. Am. Assoc. Swine. Pract, 1 997:503 ), including: progressive consumption, difficulty breathing, urge to breathe and occasional jaundice

Nayar et al., Can. Vet. J. Volume 38, June 1997 porcine circovirus was detected in pigs with clinical signs of PMWS, and it was concluded that the species was not considered to be natural to PK-15 cells. The PCV outside the occupant's PCV is related to PMWS. Later literature (Hamel et al., J. Virol., 72 (6), 5262-5267 * 1 998; Meehan et al;, J. gen.

Virol, 79, 2171-2 179, 1998) confirmed these findings and proposed (Meehan et al., supra) to refer to these pathogenic PCVs as PCV-2, whereas the original PK-15 cell culture isolates ( Tischer et al., Nature 295, 64-66, 1 9 82 ) should be referred to as PC V-1. PCV-1 and PCV-2 are small (17 nm) icosahedral coatless viruses containing a circular single-stranded DNA genome. The length of the PC V-2 genome is approximately 1768 bp. PCV-2 isolates from different regions of the world appear to be closely related to each other and exhibit 95 to 99% nucleotide sequence identity (-4- 8 (2) (2) 1361695 F enaux et al., J Clin. Micorbiol·, 38 (7), 2494-2503, 2 000 ). ORF-2 of PCV encodes the capsid protein of the inferred virus. ORF-2 of PCV-2 encodes a protein consisting of 233 amino acids. All 卩(^-2 isolate 01^-2 shared 91-100% nucleotide sequence identity and 90-1 00% of the amino acid sequence consensus. PCV-1 and PCV-2 There is only 65 to 67% nucleotide identity between the ORF-2 gene and 63 to 68% amino acid sequence identity (?611311\6131., 51^13

PDNS (porcine dermatitis and kidney disease) is another major problem for pig farmers, which occurs at the same time as PMWS. PDNS is characterized by red/brown ring skin lesions with bleeding, usually found in the ears, ribs, legs and hips. A review of PCV-2 related syndromes and diseases is found in Chae. C (2005) Vet. J. 169 3 26-3 3 6 . [Prior Art] There is a need for a vaccine that protects piglets against PCV-2 related diseases such as PMWS and PMWS. However, there are currently no vaccines available for anti-PCV-2 related diseases. Traditionally, individuals have considered traditional vaccines for pigs based on the deactivated whole PC V-2 virus. However, the situation with PCV-2 is more complicated because PCV-2 does not replicate high titers in cell culture. An alternative vaccine can be derived from the recombinant antigen of PCV-2 as a substrate. PCV-2 proteins have been shown in different expression systems. For example: Liu et al. (Protein Expression and Purification, 2 1 , 1 1 5-120 -5 - 1361695 *- (3) (200 1 ) will be encoded by PCV-2's ORF-2 (which is linked to the MBP His tag) The fusion protein of the entire protein is expressed in E. coli. Kim et al. (J. Vet. Sci, 3 (1), 19-23, 2002) expressed ORFs 1 and 2 of PCV-2 in the baculovirus expression system. Blanchard (Vaccine, 21 ., 4565-45 75, 2003) et al. showed ORF 1 and 〇RF 2 in a baculovirus-based system in insect cells. Insects that have produced PC V-2 protein The cells are lysed and prepared into a vaccine for immunizing piglets that do not contain a special pathogen (SPF). In the first-additional method, the piglet is inoculated with either of the two proteins, of which the inoculation unit The DNA vaccine is vaccinated after the vaccine, or, in another group of piglets, the piglets receive the 0RF1 and ORF2 proteins in the second injection. However, all experiments were performed on SPF pigs without pathogens, therefore, Any antibody derived from the mother against PCV-2. From PCV-2 bow to PMWS and PDNS available in four It is observed in piglets as large as about 15 weeks old. It seems that until the weaning, the piglets can safely be free of PCV-2 related diseases, and the piglets only have the chance of clinical symptoms after weaning. Therefore, in order to To protect piglets with vaccines, ideally, piglets must be protected from the time of weaning, because when PCV-2 related diseases are unpredictable, in order to achieve this goal by secondary vaccination, piglets need to be in the first place. The first vaccination is carried out at Chow, so that it can be vaccinated at about the time of weaning, and it is completely protected against PC V-2 infection after weaning. The pig seems to have a maternal-derived anti-PC V- Antibody 2 (MDA) -6- 8 1361695 *- (4). (The distribution of MDA titers of piglets used in the experiments of the vaccine of the present invention is listed in the examples). However, the art is well known: when there is Maternal-derived antibodies will interfere with vaccination in the presence of piglets. Piglets can have different titers of MDA. Very high passiveness _ MDA titer protects piglets against PCV-2 infection (Merial: 'PCV-2 Diseases: From research back to the Field strain" 1 18,h IPVS > Hamburg Germany, June 2004, page 99-101 ). However, piglets with lower MDA titers do not protect piglets when they reach the relevant age (ie, after weaning) Fight against PCV-2 infection. For those piglets that appear to be the main encounter in the area, the MDA titer may be too low to provide protection against PC V-2 infection, but still high enough to interfere with the vaccine (eg, traditional deactivation) PCV-2 vaccine) Inoculation. In particular, since the virus cannot proliferate to a high titer in cell culture, the deactivated vaccine may contain less antigen (or a complex and time consuming concentration procedure should be introduced into the vaccine manufacturing method). The vaccine of the present invention can specifically provide protection against PCV-2 infection in piglets of this population. SUMMARY OF THE INVENTION By the present invention, a vaccine for use in a method for protecting piglets can be provided, and even those resistant to PCV-2 MDA-positive, anti-PCV-2 infection and thereby against PCV-2-related diseases can be provided. People pay attention to the protection of piglets for PMWS and PDNS). The present invention provides an anti-PCV-2 vaccine comprising at least 20 micrograms per dose of a second type of swine ring disease 1361695 • (5) virulence (PCV-2) of the 0RF-2 protein. It has now been found that a vaccine containing at least 20 micrograms (/zg) of ORF-2 protein of PCV-2 per dose can elicit a protective immune response against PCV-2 infection even in the presence of MDA (to thereby combat PCV -2 related diseases, like PMWS and PDNS). Suitably, the vaccine contains at least 50 micrograms per dose, most suitably at least 80 micrograms per dose. The vaccine of this technology can even be prepared with a vaccine of up to 275 micrograms per dose, and such vaccines will not cause local reactions at the injection site. Of course, even more micrograms of antigen can be added to the vaccine dose of the vaccine of the present invention, but if the vaccine with a higher dose fails to improve the protective effect obtained, the increased antigen load is only an expensive expense rather than a necessity. . In addition, avoiding an increase in antigen dose may eventually result in an unacceptable local response at the injection site. The method for measuring the amount of antigen is described in the experimental part of the present application. The vaccine of the present invention may comprise a recombinant ORF-2 protein, wherein the heavy Group protein is preferably produced by expression of a baculovirus expression vector in an insect cell, the baculovirus expression vector containing the appropriate promoter Controlled PCV-2 ORF-2 gene sequence. While other suitable expression systems known in the art can also be used in the methods of preparing the vaccines of the present invention, it has been found that the use of a baculovirus expression system produces high yields of viral antigens and can exhibit good antigenicity. When high concentrations of antigen are not produced (e. g., in virus-infected cell culture), the use of a baculovirus expression system eliminates the need for a complex and time consuming procedure to concentrate the antigen to a suitable level. -8 - 1361695 V (6) The most commonly used baculovirus expression vector is Spodoptera litura, which is usually used with SF-9, SF-21 or High five insect cells. SF-9 and SF-21 are egg cells derived from the ovary cell line 'High five cell line of Spodoptera frugiperda. The PC V-0RF-2 gene should be under the control of a suitable promoter. The most commonly used promoter in the baculovirus expression system is the promoter for the polyhedrin gene and the promoter for the pl〇 gene, which means that the 0RF-2 PCV-2 gene sequence is inserted into the polymorphic gene of the baculovirus genome. The insertion site in the locus or pl〇 gene locus. Other suitable promoters (homologous or heterologous) known in the art can also be used. Detailed descriptions of all aspects of the baculovirus manifestation system are listed in DR O'Reilly, LKMiller and VALuckow, "88. 111〇乂11:115£\卩1655; 011'\^(^015" (1992, \^.1'1·

Freeman & Co, New York). Furthermore, performance vectors derived from baculovirus and complete performance systems are commercially available from a number of different companies. The vaccine according to the invention may further comprise a suitable adjuvant. A variety of adjuvant systems are known in the art, such as the conventional oil-in-water adjuvant system. Any ® suitable oil can be used, for example, mineral oils known in the art for assisting. The oil phase may also contain a suitable mixture of different oils (mineral or non-mineral). Suitable adjuvants may also contain vitamin E optionally mixed with one or more oils. The aqueous phase of the vaccine supplemented with oil-in-water will contain the antigenic material. Suitable blends typically comprise from about 25 to about 60% oil phase (40 to 75% aqueous phase). Examples of suitable blends may comprise 30% aqueous phase and 70% oil phase or 50% each. The vaccine of the invention may be administered by any suitable route known in the art, for example: intramuscular, intradermal or Subcutaneously, it is preferred to administer -9-8 via the intramuscular route. (7) 1361695 The present invention also provides a method for producing a vaccine against PCV-2 MDA positivity against PCV-2 infection, which is intended to protect young pigs, wherein the vaccine An ORF-2 protein having a second type porcine circovirus (PCV-2) of at least 20 micrograms per dose. The vaccine of the present invention (prepared by the method of the present invention) can be used in a method for protecting young pigs against PCV-2 infection. • The vaccine of the present invention can even be used in a method for protecting young pigs, wherein the young pig is a mother-derived anti-PCV-2 antibody (MDA) positive, anti-PCV-2 infection. It has now been found that the vaccine of the present invention protects piglets even when the piglet has a very high titer of MDA against PCV-2. Table 1 reflects the distribution of MDA titers in young pigs encountered in different farms across Europe, and the protective effects provided by the vaccines of the present invention are reflected in Table 2. It shows that the vaccine of the present invention can even provide appropriate protection against PCV-2 infection in young pigs whose MDA titer is defined as "Group 2" as defined in the examples (Table 2). Piglets classified in this group have an MDA titer between 8 and 12 l〇g2 (this high MDA titer). Thus, the vaccine according to the invention can even be used in a method for protecting young pigs having an MDA titer against PCV-2 of up to 101 g2, or even 121 gPa (measured by the method indicated in the examples). From Table 1, it can be found that about 55% of the piglets collected from different farms across Europe belong to Group 2 (of course, belong to Group 1, with lower -10- 8 1361695 *- (8) MDA titration Piglets containing 32% of the population can also be protected by the vaccine of the invention). Thus, the conclusion is that the vaccine of the present invention provides protection for the vast majority of piglets encountered in the field, including those with high MDA titers. In order to provide appropriate protection, it is advisable to administer the vaccine with a two-injection vaccine regimen, so that when the piglet is from the first week to the fourth week (before weaning, for example, during the first week) One injection (first vaccine). The second ® injection (additional vaccine) can be given after about three weeks. In this way, piglets can achieve complete protection against PCV-2 infection after weaning, while weaning is the most sensitive time for piglets to PCV-2 infection, and thus becomes sensitive to PMWS and PDNS. [Examples] Example 1: Determination of PCV-2 specific antibody titer (mother-derived) Method The antibody titer against PCV 2 infection can be determined in the following manner: Monolayer PK1 5 cells are formed in a 96-well tissue culture dish. At 80% confluency, cells were infected with PCV-2 field isolates and continued for 2 days in a C2 incubator at 37 °C. After this period of time, the cells were fixed in ethanol and stored at 2-8 ° C until use. When about 20% of the cells in the culture plate were infected, the plate was used for testing. To determine PCV-2-specific antibodies to specific sera, serial dilutions were made and incubated on ethanol-fixed cells. After incubating for 1 hour at 37 ° C, the plates were washed with tap water and incubated with FITC-labeled rabbit anti-porcine IgG-1361695 ·- (9) to detect bound antibodies. The titer of a particular serum is expressed as the reciprocal of the highest dilution in which the PCV-2 specific antibody response is still observed. 0 The parenterally derived anti-PCV-2 antibody titer listed in Table 1 for pre-weaning piglets. Typical distribution. The serogroup was collected from 23 piglets from different countries across Europe.

(10) 1361695 Table 1 Distribution of antibody titers derived from maternal population in a population containing 232 young pigs in a population with population-derived PCV-2 specific antibody titer (l〇g2) Percentage of piglets per category Percentage of piglets per group ^ 4 1 . 3 5 9.9 32 6 9. 1 7 12.1 8 9.5 9 19.4 10 11.2 55 11 9.9 12 4.7 13 5.2 14 3.0 15 2.6 13 16 1.3 ^ 1 7 0.8

Three groups can be distinguished in Table 1: Group I; piglets with titers less than 8, Group 2; piglets with titers from 8 to 12 and Group 3; with 13 and higher The titer of the piglet. In group 3, the antibody titer produced from the parent-derived-13 - -- (11) 1361695 is so high that it is expected that the piglet will be protected at a critical age (Merial: 'PCV-2 Diseases: From research back to The field strain"> 18'b IPVS * Hamburg Germany * June 2004, page 99-101). In Group 1, the antibody titers derived from the mother were so low that most of these piglets were easily vaccinated. However, in Group 2, this range of antibody titers made it impossible for traditional vaccination methods to successfully immunize most piglets. Since • more than half of the pigs seem to belong to this group, it is most important to protect this group of pigs if they want to exclude P M W S from this farm. It is well known in the art that vaccination can be aided by adjuvant or high antigen content when antibodies derived from the mother are present. Which adjuvant or antigen content can destroy the parent-derived antibody against a particular pathogen is currently unknown. Therefore, in this experiment, we have attempted to define a method for protecting the lowest antigen amount of Group 2 piglets against PCV-2 infection. ® Example 2. Construction of Recombinant Baculovirus Expressing PCV-2 0RF-2 Lung Tissue from Piger Pig (Feeder Pig) showing clinical and histopathological signs of PMWS using PCV-free porcine testis (ST) cells The PCV-2 virus was isolated. The virus was propagated on PK-15 cells without PCV by subculture for 5 passages.

The PC V-2 virus product was purified from the supernatant of infected PK-15 cells, and DNA was isolated from the preparation. According to the published sequence, the primer containing the BamHl restriction site was used (forward primer: CGG GAT CCG TTT TCA GCT ATG ACG TAT, reverse bow | object: CGG GAT CCT -14- ·- (12) 1361695 TTA TCA CTT CGT AAT GGT T) PCR was performed to amplify the ORF-2 gene. The resulting amplicon contains the complete 〇RF-2 gene plus the adjacent BamHl restriction. After gel electrophoresis, the amplicon was excised and purified. Then, the purified PC V-2 0RF-2 fragment was decomposed with BamHl and ligated into P A c AS 3 decomposed by BamHl (V1 ak et · a 1 · (1 9 9 0) V ir ο 1 ogy 179 312-320). This plastid contains the pl〇 promoter upstream of the insertion site, so that the foreign gene can be expressed under the control of the pl〇 promoter. The TOP 10F' bacteria (Invitrogen, Carlsbad, USA) were transformed with the ligation mixture, and the selected plants containing the correct constructs were selected according to the sequence of the selected strains. The positive selection strain was amplified and the DNA of the transferred plastid was retested by sequencing. Prior to transfection, B. cerevisiae was decomposed with Bsu361 (AcNPV, described in Martens et. al. (1 995) J. Virol. Methods 52 15-19). The Bsu361 position in this virus is a unique restriction of ® in the plO gene locus. Then, CellFectine (Life Technologies, Inc., Gettysburg, USA) was used to transfect P. californica (Sf9) cells with transfer plastids and AcNPV baculovirus DNA decomposed by Bsu361. Transfection supernatants were harvested on day 3 post-transfection and plaque purification was performed on Sf9 cells. The plaque was amplified and analyzed for the presence of PC V in the virus produced by analyzing the sequence of the isolated viral DNA and performing immunofluorescence analysis on Sf9 cells using anti-PC V-2 rabbit and pig serum. -2 ORF-2 gene insertion. -15- 1361695 • (13) Prepare seeds of the recombinant baculovirus BacPCV-2-ORF-2 called "Masterseed". The PCV-2 0RF-2 insert in the fifth generation of the major seeds on major seeds and Sf9 cells was tested by analyzing the sequence of the isolated DNA and performing immunofluorescence analysis on Sf9 cells. stability. Titration is performed to measure the amount of infectious virus in the viral preparation. Titration was performed on Sf9 cells, and PCV-2 0RF-2 specific immunofluorescence analysis was performed by observing baculovirus-specific CPE and / ® or using multiple rabbit anti-PCV-2 immune sera. The results show that this method produces a plaque-purified major seed of recombinant AcNPV baculovirus BacPCV-2 0RF-2. By sequencing and immunofluorescence analysis to test the main seed and the main column of Sf9 cells, the fifth generation can be judged that the construct can stably display PCV on Sf9 cells under the control of plO promoter. - ORF-2 protein" • Example 3. Production method of PCV-2 antigen In order to obtain the maximum amount of performance product, preliminary experiments were carried out to optimize the conditions for obtaining recombinant PCV-2 0RF-2 protein. All experiments were carried out at 28 ° C using Spodoptera frugiperda 21 (Sf2 1) cells cultured in suspension. Infection was carried out using BacPCV-2-ORF·2 virus from the 4th generation level of the main seed. In order to optimize the production conditions, the cell density at the time of infection was 1.4 x 106 cells/ml, the multiplicity of infection (MOI) was 〇.〇1, and culture was continued for 6 days after infection. The resulting mixture is referred to as a product of the performance product. In a single experiment over a period of one year, under the optimal conditions -16 - 8 (14) 1361695 5 performances. Since the antigen system is located inside the cell, all of the cells containing the cells and the supernatant are subjected to ultrasonic treatment until at least 90% of the cells are destroyed. • Thereafter, each batch of the ultrasonically treated final product was continuously stirred with 33 mM bisacetimimine (BEI) at 37 ° C, pH 7.5 for 72 hours to deactivate the viable recombinant virus. After deactivation, 1.6 times the molar excess of sodium thiosulfate was added to neutralize the BEI. • After neutralization, centrifuge at 600g for 1 低 minutes to remove cell debris and polyhedra. The supernatant produced is referred to as a deactivated virus suspension. Check if the finished product is sterile to check if the deactivation is complete. The deactivation was tested for completeness by transplanting the deactivated virus suspension on Sf9 cells for 2 weeks and visually examining the presence of no baculovirus-specific CPE. The results showed that the baculovirus titer obtained by the method was 8.5 l〇g1GTCID5()/ml, which was completely deactivated after BEI treatment. Example 4. Method for determining the amount of PCV-2 antigen The sample of the cell culture suspension of the deactivated suspension and the mother transfer virus before and after low speed centrifugation according to Laemmli (Laemmli, UK (1970) Nature 227, 680-685) Denatured SDS-polyacrylamide gel electrophoresis was performed. A 4-12% gradient gel stained with Coomassie Brilliant Blue was used. When the western blot analysis was completed, the gel-derived protein was transferred to a nylon membrane by electroporation to block the skim milk in PBS, -17- 8 ·- (15) 1361695 and resistant Multiple strains of porcine serum dilution of PC V-2 on-site isolates 1 μL (#1) of this suspension was sprayed on the gel in a similar manner to measure the resulting deactivated virus suspension The antigen content, and serial dilutions of bovine serum albumin (St. Louis, Sigma, USA, catalog number A-2 15 3) were simultaneously swam on the same gel for reference. The camera was used to capture angiography and using GeneTools (SynGene, 3rd edition) for computer analysis to compare the density of the BSA reference to the density of the sample containing PCV-2 to quantify the deactivated virus. The ORF-2 gene product in suspension. When the deactivated intact product before and after low-speed centrifugation was condensed on SDS-PAGE, the precision plus markers (Precision Plus markers) (Bio-Rad, Hercules, USA) were electrophoretically separated to compare with each other before centrifugation. The material produces two main bands of approximately 30 and 26.8 kDa surface molecular weight (MW) of equal density, while the centrifuged material contains only lower bands. When the ® parent transfer virus is swimming next to the recombinant virus, the mother transfer virus contains only the higher of the two bands, which indicates that the lower band is the ORF-2 of PCV, and the higher band is centrifuged. After removing the polyhedron. The characteristics of the lower 26.8 kDa band were further confirmed by Western blot analysis, which showed that the band reacted with multiple pig sera against PCV-2 live virus, and the 30 kDa band did not. The performance level of PCV-2 ORF-2 was determined in 5 individual experiments, and in each case, the amount was much higher than the detection side limit of the test, specifically, the amount was 40 to 550 micrograms (//g). ) /ml (ml) to deactivate the virus suspension (16) 1361695. Example 5. Effect of the amount of PCV-2 0RF-2 on the vaccine adoption of MDA-positive young pigs A vaccine with different PCV-2 0RF-2 antigen content was used and used to produce different levels of self-derived anti-PC V-2 antibodies. Young pigs (MDA) are vaccinated. Secondary vaccination was given and spaced 3 weeks apart from each other. Serum responses against the antigen were measured 5-6 weeks after the first ® vaccination. From these data, the effect of antigen content on the adoption of vaccines in the presence of MD A can be calculated. A plurality of different antigen dilutions are prepared and mixed with an oil-in-water adjuvant (as commonly used in the art) at a ratio of 1:1 (vol/vol). Then, between the 1st and 4th week of the young pigs, a litter of pigs is divided into groups and treated with a vaccine containing different amounts of PCV-2 0RF-2 protein via the intramuscular route or without vaccination. Repeat the vaccination after 3 weeks. The following groups were made: 1 14 piglets (group 1) were inoculated with 1-14 micrograms of PCV-2 0RF-2 protein/dose, and 85 piglets were inoculated with 20 and 80 micrograms of PCV-2 ORF-2 protein/dose. (Group 2). Blood was taken at the first vaccination and after 5-6 weeks. Serum was prepared and examined for PCV-2 antibodies by immunofluorescence analysis. For this test, a PK1 5 cell monolayer in a 96-well tissue culture dish was infected with the on-site isolate of PCV-2. After 2 days of culture, when about 20-3 0% of the cells were infected, the monolayer was fixed in ethanol and stored at 2-8 °C until use. For the determination of titer, serial dilutions of test serum were incubated on cells - 19-.-(17) 1361695, incubated at 37 ° C for 1 hour, and after washing the plates, they were compared with FITC-labeled rabbit anti-pigs. IgG (Tolder, Netherlands, Nordic) was incubated together to detect bound antibodies. The measured titer is the reciprocal of the highest dilution in which PCV-2 specific fluorescence is still observable. The decline in antibody titer between the first and second blood collections was determined for all animals. If the antibody titer does not decrease or increase during this period, the vaccine is considered to have been adopted by the animal concerned. However, when the PCV-2 specific antibody titer was reduced, then ® considered that the vaccination was not successful and the pig did not adopt the vaccine. The minimum amount of antigen required to vaccinate a sufficient number of piglets can be determined by correlating different vaccine doses to be titrated with maternal-derived antibodies at the time of vaccination. The results of this analysis are listed in Table 2 〇 • 20· 8 (18) 1361695 Table 2. Percentage of vaccines taken at different MDA titers and antigen concentrations

Piglet vaccination with PCV-2 specific antibody titer (l〇g2) derived from maternal example 1; group; 1-丨4 μg/dose group 2; 20-80 μg/dose per category Number of piglets/number of piglets adopting vaccines Percentage of vaccines adopted per group Number of piglets per category/Number of piglets adopting vaccines Percentage of vaccines adopted per group S 4 3/3 1/1 5 0/0 90 % 3/3 100% 6 15/13 5/5 7 .2/2 3/3 8 12/8 4/4 9 12/5 17% 11/10 10 6/0 13/10 76% 11 21/0 15/10 12 26/0 7/4 13 15/0 6/1 14 2/0 11/0 15 0/0 0% 4/0 4% 16 0/0 1/0 ^ 17 0/0 1/0 Total Π4/31 27% 85/51 60% Total protected* 114/48 42% 85/73 86% *The total number of protected piglets is the number of piglets in which the titer is less than 13 and the vaccine is used plus The number of piglets that have a titer of 13 or higher. ·- (19) 1361695 In this table, "adoption of vaccine means that the titer of PCV-2 specific antibody produced by the piglet one week after the additional vaccine is equal to or higher than the PC V produced by the first vaccination. 2 Vaccination of titers of specific antibodies. In all such cases, the vaccine is shown to be active against PCV-2, and in this case, piglets are considered to be protected against PCV-2 infection. However, the titer produced one week after the vaccine was added was less than the titer produced after the first vaccination and did not cause an immune response, and the observed antibody derived from the mother naturally decreased. This situation makes these animals susceptible to PCV-2 infection. This table shows that when using a vaccine dose equal to or less than Μ micrograms, 90% of the animals in Group 1 (MDA titer S7) will be vaccinated. Transformation, therefore, can be considered protected. However, in Group 2 (MDA titer > 7 and < 13), only 17% of the animals were seroconverted when vaccinated with a vaccine dose of less than or equal to 14 micrograms. Protected. This group Seventeen animals have titers of 13 or higher and, therefore, have been protected by native-derived PCV-2 specific antibodies derived from the parent. Therefore, individuals have concluded that all 114 piglets in this group Only 48 (42%) were only protected; 17 piglets with antibody titers derived from maternal, plus 31 piglets seroconverted in groups 1 and 2 at 20 μg/ Significantly more animals were protected in the dose- or higher-dose population; all animals in Group 1 and 76% of animals in Group 2 were protected against seroconversion of PC V-2, plus After 13 or more MDA titers, 88% of piglets in this population were found to be protected. -22- 8 (20) 1361695 Since animals are harvested when about 80% or more of the animals are protected Group protection, it can be concluded that the antigenic dose of the vaccine against PC V-2 must contain at least 20 micrograms or more of antigen to effectively protect the herd against the consequences of PCV-2 infection.

Claims (1)

1361695 匕日 Revision I ^ in»··· ιι. ——I _ I· · · Annex 3A: No. 094131364, the scope of the patent application is amended. The amendment of the Republic of China on August 9, 100. The scope of application for patent 1. The ORF-2 protein of the second type porcine circovirus (PCV-2) is used in the manufacture of a vaccine for protecting a piglet-positive antibody (MDA)-positive piglet from PCV-2 infection, the vaccine comprising at least 20 Micrograms/dose of this ORF-2 protein. 2. Use of the ORF-2 protein of PCV-2 for the manufacture of a vaccine for protection of MDA-positive piglets from PC V-2 infection, the vaccine comprising at least 50 μg/dose of the ORF-2 protein. 3. The use of claim 1 or 2, wherein the ORF-2 protein has been produced by means of a baculovirus expression vector in insect cells, and wherein the baculovirus expression vector comprises a suitable promoter Controlled PCV-2 ORF-2 gene sequence. 4. The use of claim 3, wherein the promoter is a p 10 promoter. 5. A pharmaceutical composition for use as a vaccine for protection of MDA-positive piglets from PCV-2 infection, comprising at least 20 micrograms per dose of ORF-2 protein of PCV-2 and a pharmaceutically acceptable carrier. 6. A pharmaceutical composition for use as a vaccine for protecting MDA-positive piglets from PCV-2 infection, comprising at least 50 micrograms/dose of ORF-2 protein of PCV-2 and a pharmaceutically acceptable carrier. 7. The pharmaceutical composition of claim 5 or 6, wherein the composition comprises a suitable adjuvant. 1361695 8. The pharmaceutical composition of claim 7, wherein the adjuvant is an oil-in-water emulsifier. 9. The pharmaceutical composition of claim 7, wherein the adjuvant comprises vitamin E.