Vaccine against porcine circo virus type 2

Abstract

The present invention pertains to a vaccine comprising a non-replicating immunogen of porcine circo virus type 2 for use in prophylactically treating an animal that has circulating antibodies directed against porcine circo virus type 2, against an infection with pathogenic porcine circo virus type 2 by a single dose administration of the vaccine into the dermis of the animal.

Claims

1. A method of prophylactically treating an animal against an infection with pathogenic porcine circo virus type 2 comprising administering a single dose of a vaccine comprising recombinantly expressed ORF2 protein of porcine circo virus type 2 and a Lawsonia intracellularis antigen into the dermis of an animal that has circulating antibodies directed against porcine circo virus type 2; wherein the vaccine is administered with a needle-less vaccination device.

2. The method of claim 1, wherein the single dose administration comprises less than 0.5 ml of the vaccine.

3. The method of claim 2, wherein the single dose administration comprises less than 0.2 ml of the vaccine.

4. The method of claim 1, wherein the vaccine comprises an adjuvant comprising mineral oil, wherein the amount of mineral oil in the vaccine is between 1 and 10% v/v.

5. The method of claim 3, wherein the vaccine comprises an adjuvant comprising mineral oil, wherein the amount of mineral oil in the vaccine is between 1 and 10% v/v.

6. The method of claim 5, wherein the amount of mineral oil is between 2.5 and 5% v/v.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 depicts the serology in a DOI study.

(2) FIGS. 2A-2C show the viral load in serum, feces, and in organs, respectively.

(3) FIG. 3 shows the average body temperatures in days, post vaccination.

(4) FIG. 4 depicts the average total PCV2 Ig Ab results.

(5) FIG. 5 shows the average PCV2 IgM Ab results.

(6) FIG. 6 depicts the antibody titers in a duration study.

(7) FIG. 7 shows the viral load in the organs.

(8) FIG. 8 depicts the viral load in the organs.

DEFINITIONS

(9) Non-replicating immunogen of a virus is any substance or compound corresponding to the virus, other than the replicating (live) virus as a whole, against which an immunological response is to be elicited, such that the corresponding virulent virus or one or more of its virulence factors will be recognized by the host's immune system as a result of this immune response and is ultimately neutralized. Typical examples of non-replicating immunogens are killed whole viruses and subunits of these viruses such as capsids and surface expressed proteins, for example recombinantly expressed. A vaccine comprising non-replicating immunogen of a virus differs not only from a live virus vaccine, but also from a DNA vaccine in which the DNA is not an immunogen as such, but after incorporation into host cells, expresses the corresponding immunogen.

(10) Prophylactic treatment against an infection with a virus is aiding in preventing or ameliorating an infection resulting from a post treatment challenge with a pathogenic virus, in particular to reduce viral load in the host after such challenge.

(11) Prophylactic treatment against disease resulting from an infection with a virus is aiding in preventing or ameliorating one or more clinical manifestations resulting from a post treatment infection with the virus.

(12) Administration of a vaccine into the dermis means a sufficient amount of the vaccine is deposited in dermis, leading to an immunological response significantly different (in particular: when using the Wilcoxon rank sum test in a test set up as outlined in Example 3, the p value should be less than 0.10, preferably less than 0.05) from an intramuscular administration with the same vaccine and volume thereof. Several devices are commercially available for intradermal vaccination, for example the IDAL vaccinator (MSD Animal Health), the Pulse 50 MicroDose (Pulse Needle Free Systems), or other devices as described in Vaccine, 2012 Jan. 11; 30(3):523-38 (see in particular Table 1, page 525: An overview of different devices for liquid and solid formulation administration)

(13) Single dose administration of a vaccine for use in prophylactically treatment means that in order to obtain protective immunity, the vaccination does not need to be boosted with a second administration. In a two-shot regime, the first (prime) vaccination is typically boosted within 6 weeks from the first administration, commonly within 3 or even 2 weeks from the first administration, and only after the second (boost) administration protective immunity may be obtained.

Embodiments of the Invention

(14) In a first embodiment the single dose administration comprises less than 0.5 ml of the vaccine, preferably 0.2 ml of the vaccine. A dosage below 0.5 ml appears to be suitable for intradermal vaccination. At such a low dosage the dermis remains at least to a sufficient extent intact and available for receiving the vaccine, whereas at a substantially higher dosage, there is a risk of rupturing the dermis, leaving the dermis unavailable for receiving the vaccine.

(15) In a next embodiment the vaccine is administered with a needle-less vaccination device, using a jet of the vaccine to reach the dermis through the skin of the animal. Vaccination into the dermis is in this embodiment provided by a needle-less vaccination device using a liquid jet of the vaccine (a high pressurized fluid stream), typically using a very low volume of vaccine in the range of 0.05 to 0.2 ml. This further increases the safety of the vaccine and method of administration.

(16) In another embodiment the non-replicating immunogen is recombinantly expressed ORF2 protein of porcine circo virus type 2, for example expressed by baculo virus as known in the art, or in a PCV1-PCV2 chimeric virus. This recombinant protein has proven to be suitable for application in the present invention. In particular, the ORF2 protein can be expressed in a baculo virus expression system such as described in WO2007/028823, WO 2007/094893 or WO2008/076915.

(17) In still another embodiment the animal at the time of administration has circulating antibodies at a titer of 7 log 2 (7 units of a 2 log) or above (established according to the method as described here beneath under Example 1, Serology). It was found that even at this medium to high antibody titer, the present invention is still effective.

(18) In an embodiment the single dose of the vaccine comprises less than 5000 AU of the immunogen (the minimum amount being an amount still leading to an effective immune response). It was found that at this dose, lower than the dose in the existing vaccine PORCILIS PCV (a vaccine comprising porcine circovirus type 2 ORF2 subunit antigen, licensed with a two shot vaccination regime to reach a DOI of 22 weeks), one can still obtain a DOI of more than 23 weeks after only one vaccination. In an embodiment the single dose of the vaccine comprises about 2000 AU of the immunogen. This amount appears to be sufficient to obtain the advantages of the present invention.

(19) In another embodiment the vaccine comprises non-live immunogen of Mycoplasma hyopneumoniae to treat the animal against an infection with pathogenic Mycoplasma hyopneumoniae bacteria, and/or the vaccine comprises non-live immunogen of Lawsonia intracellularis to treat the animal against an infection with pathogenic Lawsonia intracellularis bacteria.

(20) In still another embodiment the vaccine comprises an adjuvant comprising mineral oil, characterised in that the amount of mineral oil in the vaccine is between 1 and 10% v/v (volume of oil over total volume of the vaccine). It is commonly known that mineral oil is a very good potentiator of the immune response (also referred to as adjuvant). However, in pigs it often leads to severe local reactions (swellings having a mean diameter of more than 5 cm), in particular if administered intradermally. It was surprisingly found that with the current intradermal route of vaccination, such severe local reactions may not occur at all, and the mean diameter of the swellings may even stay below 3 cm which is found to be acceptable. In a further embodiment the amount of mineral oil is between 2.5 and 5% v/v. It was found that even at this very low concentration of mineral oil, the immune response was effective.

(21) In embodiments of the method according to the invention an animal receives a single dose administration of a vaccine comprising non-replicating immunogen of porcine circo virus type 2 into the dermis, and concurrently (that is within one day, preferably within a few hours, minutes or seconds) receives a single dose administration of a vaccine comprising non-live immunogen of Mycoplasma hyopneumoniae (Mhyo) into the dermis to treat a porcine circo virus type 2 infection and an Mycoplasma hyopneumoniae infection at the same time. Several non-live Mycoplasma hyopneumoniae immunogens to be used in this or any of the other embodiments of the invention are known in the art such as bacterins (Swine Health and Production. 1998; 6(3):107-112), recombinantly expressed Mhyo antigens (Infection and Immunity, June 1997 vol. 65 no. 6 2502-2507), the soluble fraction of an Mhyo culture (WO 2013/152083) etc.

(22) In another embodiment an animal receives a single dose administration of a vaccine comprising non-replicating immunogen of porcine circo virus type 2 into the dermis, and concurrently receives a single dose administration of a vaccine comprising non-live immunogen of Lawsonia intracellularis into the dermis to treat a porcine circo virus type 2 infection and an Lawsonia intracellularis infection at the same time. With regard to Lawsonia intracellularis immunogens to be used in this or any of the other embodiments of the invention, several types of non-live immunogen are known in the art, and are for example described in WO2009/144088 (killed whole cell immunogen), WO2005/070958 (sub-units) and WO97/20050 (killed whole cell immunogen). Combination with PCV and Mhyo antigens as such is described in WO2009/144088.

Examples

(23) Example 1 is an experiment to show that a single dose intradermal vaccination can provide twenty three weeks of immunity against an infection with porcine circo virus type 2.

(24) Example 2 is another experiment with a PCV2 ID once vaccination approach showing that vaccination is safe and leads to protective titers.

(25) Example 3 is a direct comparison between intradermal and intramuscular vaccination.

(26) Example 4 describes experiments with combined intradermal vaccination.

(27) Example 5 describes an experiment with combination vaccines, various antigen dosages and various adjuvants.

(28) FIG. 1 Serology in a DOI study

(29) FIG. 2 Viral load in serum, feces and organs

(30) FIG. 3 Average body temperatures

(31) FIG. 4 Average total PCV2 Ig Ab results

(32) FIG. 5 Average PCV2 IgM Ab results

(33) FIG. 6 Antibody titers in a duration study

(34) FIG. 7 Viral load in organs

(35) FIG. 8 Viral load in organs

Example 1

(36) Experimental Design

(37) Progeny of 10 sows with antibodies against PCV2 were used for this study. Piglets were divided across litters into 2 groups of 15 animal animals. At 3 weeks of age, the piglets of group 1 were vaccinated intradermally on the right side of the neck with 0.2 ml of a vaccine comprising recombinantly expressed ORF2 protein of porcine circo virus type 2 (see WO 2007/028823 for the provision of the protein), using the commercially available intradermal vaccination device IDAL (available from MSD Animal Health, Boxmeer, The Netherlands), while group 2 was left unvaccinated and served as a control group. All study animals were observed daily for clinical signs. Blood samples of all animals were taken at time of vaccination, 9, 17, 19 and 21 weeks later. Twenty-three weeks following vaccination each animal was challenge infected using a wild-type PCV2 challenge virus strain applied intranasally.

(38) Serum samples and fecal swabs were taken one day before challenge and one, two and three weeks after challenge and were examined for PCV2 viral nucleic acid by quantitative PCR. In addition serum samples were examined for PCV2 antibodies. Three weeks following challenge, all animals were necropsied and inguinal lymph node, tonsil and lung were sampled for determination of PCV2 viral antigen and nucleic acid.

(39) The vaccine used was given as an oil-in-water emulsion, comprising 5% v/v of the mineral oil MARCOL 52 (Exxon), 0.30% w/v vitamin E acetate and 0.32% Polysorbate 80 (TWEEN 80; Sigma Aldrich), water for injection and 2000 AU of PCV2 protein per 0.2 ml. The AU units are calculated based on an AlphaLISA test of PerkinElmer. For this test the wells of a polystyrene microtitre-plate are filled with serial dilutions of test sample containing PCV2 ORF2 antigen alongside serial dilutions of a reference standard. These dilutions are incubated with acceptor-beads (coated with monoclonal antibody directed against PCV2 ORF2), and biotin-labeled secondary antibody which is also directed against PCV2 ORF2. The amount of bound secondary antibody is then quantified by incubation with streptavidin coated donor-beads and chemiluminescent detection. The reference standard is such that the commercially available vaccine Porcilis PCV is set to contain 5000 AU per (2 ml) dose.

(40) Experimental Procedure

(41) Daily Observation

(42) All pigs were observed daily for clinical signs of disease. Observations consisted of systemic reactions including loss of appetite, tendency to lie down, listlessness or drowsiness, shivering, bristling, oedema (especially around the eyes), vomiting, diarrhoea and dyspnoea.

(43) Sampling of Blood

(44) Blood samples were collected before vaccination, 9, 17, 19 and 21 weeks later. Blood samples were collected one day before challenge and 7, 13 and 19 days after challenge. This was done from all pigs individually.

(45) Fecal Swabbing

(46) Fecal swabs were taken from all animals, using one dry swab per animal, one day before challenge, 7, 13 and 18 days post challenge. Swabs were taken using standard procedures, into medium containing antibiotics. Suspensions of swabbed material in medium was clarified by centrifugation, aliquotted and stored at 18 C. until further use.

(47) Serology

(48) All serum samples were examined for antibodies against PCV2, using standard ELISA procedures. In brief, serially diluted serum samples were incubated on microtiter plates coated with baculovirus expressed PCV2 ORF2 antigen. After removing the sera, all wells were incubated with a fixed amount of biotin-labeled PCV2-specific monoclonal antibody. Bound MoAb was then incubated with peroxidase-conjugated streptavidin followed by chromophoric detection. Titers were expressed as log 2 titers.

(49) Postmortem Examination

(50) At the end of the experiment all animals were euthanized by bleeding following stunning. During necropsy the animal was opened and the viscera are inspected in-situ, paying particular attention to the following organs: lungs, inguinal and mesenteric lymph nodes, tonsils, thymus, spleen, liver and kidneys. Following this, samples from tonsil, lung (lobus accessories), and inguinal lymph node were removed for quick freezing and later analysis by quantitative PCR (qPCR).

(51) Quantitative PCR

(52) Quantitative PCR (qPCR) was performed on all sera and fecal swabs, and on 10% tissue homogenates of tonsil, lung and inguinal lymph nodes. In brief, DNA was extracted from the samples using a commercial kit. PCV2 genomic DNA in each sample was quantified by polymerase chain reaction (PCR), using primers and a dually labeled hydrolysis probe specific for PCV2. The cycle number where specific fluorescence exceeded the threshold was correlated with the cycle numbers for a set of samples containing known amounts of a PCV2-containing plasmid. Results were expressed as log 10 copies/l of reaction mixture (log 10 c/l).

(53) Results

(54) At the start of the experiment all animals were found to be healthy. In the control group one animal was found dead at 6 weeks post vaccination (wpv). Two vaccinated animals had slight local problems, viz. a slight motional dysfunction (stiffness in one leg). Given the low problem, these animals were not treated. None of the vaccinated animals showed any signs of disease or systemic reactions such as hyperthermia, reduced feed intake, anaphylactic shock or vomiting.

(55) The results of the serology are given in FIG. 1. It is clear that the vaccinated animals keep an anti-PCV2 titer that seems to level out to about 4.0 log 2, whereas in the control animals the titer decreases below the detection limit. After challenge (23 wpv, at an age of 26 weeks), titers slightly rise in the vaccinated group. In the control group titers rise to the same level.

(56) The qPCR results are shown in FIGS. 2A, 2B and 2C (dpc=days post challenge). It appears that the vaccinated animals, 23 weeks after vaccination were protected from challenge infection with PCV2, as shown by the significant reduction of PCV2 nucleic acid in serum, lymphoid organs and lung. Furthermore, the vaccine was capable to reduce the viral shedding as demonstrated by a significant reduction of the viral load in fecal swabs against PCV2 of at least 23 weeks. This was done in field animals, having circulating anti-body titers against PCV2 of approximately 7 log 2, which is considered a medium level.

Example 2

(57) Experimental Design

(58) A total of 46 piglets from one farrowing batch were allotted to 4 treatment groups: two vaccinated groups of 13 piglets each and two control groups of 10 piglets. Group one was vaccinated as indicated above under Example 1 when the piglets were approximately two weeks old, group two was vaccinated when the piglets were approximately three weeks old. The piglets were intradermally vaccinated in the right side of the neck with a single dose of vaccine. Groups 3 (control group 2 week old animals) and 4 (control group 3 week old animals) were not vaccinated. Serum samples were collected from all animals on the day of vaccination, 2, 3 and 4 weeks after vaccination. Temperatures were taken one day before vaccination, at the day of vaccination and four hours later and at 1, 2, 3, 4 days post vaccination.

(59) Experimental Procedure

(60) Before vaccination, the piglets were observed for general health. Body temperatures were taken of all piglets, on day T=1, day T=0 at 0 and 4 hours after vaccination, and on day T=1, 2, 3, 4 post vaccination.

(61) Blood samples were collected on the day of vaccination and 2, 3 and 4 weeks later. This was done from all pigs individually according to standard procedures. The blood samples were collected without the addition of anti-coagulant. Serum was prepared from the clotted blood samples and aliquots were filled and stored at 20 C. until analysis.

(62) Total PCV2 Ig antibody and PCV2 IgM antibody ELISA were tested as indicated here above under Example 1 (Serology), except that in the case of IgM antibody ELISA, the plates were coated with IgM antibody and thereafter incubated with PCV2 ORF2 antigen, before incubation with a fixed amount of biotin-labeled PCV2-specific monoclonal antibody.

(63) Results

(64) At the start of the experiment all animals were found to be healthy. Average results of the body temperatures are shown in FIG. 3. No difference could be seen in the average increase in body temperature between either the two and three week old animals (maximum average increase was between 0.0-0.3 C.). Also, the maximum increase in body temperature of individual animals in group 1 and in group 2 was comparable to the maximum temperature increase of individual animals in the two control groups.

(65) Total PCV2 Ig Antibody ELISA

(66) Average results of the total PCV2 Ig antibody response are summarized in FIG. 4. At the time of vaccination, piglets vaccinated at 2 weeks of age had higher (most likely maternally derived) PCV2 antibody titers than piglets vaccinated at 3 weeks of age. The vaccinated animals showed an increase in titer considerably higher than the control animals.

(67) PCV2 IgM Antibody ELISA

(68) Average results of the PCV2 IgM antibody response are shown in FIG. 5. At the time of vaccination all animals were negative for IgM antibodies. Following vaccination the three week old animals had a considerable faster and higher IgM antibody response than the two week old animals. The control animals remained negative throughout the study.

(69) Based on these results it may be concluded that the one dose intradermal vaccination of piglets at 2 and 3 weeks of age resulted in an acceptable safety profile and a good serological response. Comparable results were obtained with another experiment (data not shown) where the starting level of circulating anti-body titers was even higher, viz. up to 9.4 log 2, which is considered to be at the high end of a medium range.

Example 3

(70) Experimental Design

(71) A total of 40 piglets were allotted to four treatment groups of 10 piglets each. Piglets from groups 1 and 2 were intradermally vaccinated with a single dose of vaccine as indicated hereabove under Example 1. The difference between group 1 and 2 was that the IDAL vaccinator of group 2 had a slightly higher pressure build-up (125% of the value of the IDAL vaccinator used for group 1). Piglets from group 3 were intramuscular vaccinated with a single dose of the same vaccine, in the same amount at the same place (in the neck), and piglets from group 4 were left untreated. Serum samples were collected from all animals on the day of vaccination, three and five weeks after vaccination.

(72) Experimental Procedure

(73) Before vaccination, the piglets were observed for general health, according to standard procedures. Sampling of blood and serology of total anti-PCV2 antibodies and PCV2 ORF2 specific IgM antibodies was done according to the procedure as indicated here above under Example 2.

(74) Results

(75) At the start of the study all animals were found to be healthy. Results of the serology are summarised in Table 1 (titers expressed as log 2). At the time of vaccination mean antibody titers were relatively high. Following vaccination, none of the animals showed an increase in PCV2 Ab titer. At 3 and 5 weeks post vaccination, in the ID groups higher mean PCV2 antibody titers than in the IM group could be observed.

(76) TABLE-US-00001 TABLE 1 Average antibody results Groups Titer 0 wpv Titer 3 wpv Titer 5 wpv 1 8.2 7.3 6.2 2 8.3 7.0 5.9 3 8.6 6.5 5.1 4 8.4 6.2 4.3

(77) Results of the anti PCV2 IgM serology are summarised in Table 2 (titers expressed as log 2). At three weeks post vaccination anti PCV2 IgM antibody titers of the two ID groups were considerably higher than of the IM group and the control group.

(78) TABLE-US-00002 TABLE 2 Average anti PCV2 IgM antibody results Group IgM titer 3 wpv 1 12.7 2 8.9 3 3.4 4 1.0

(79) When applying the Wilcoxon rank sum test, the p values for the difference in IgM response for each of the ID groups (1 and 2) versus the IM group (3) was below 0.05, viz. 0.0001 and 0.0329 respectively.

Example 4

(80) Progeny of several sows with antibodies against PCV2 were available for this study. Piglets were divided across litters into 3 groups of 18 animals. At 3 weeks of age, the piglets of group 1 and 2 were vaccinated intradermally as indicated here above under Example 1. The animals in group 2 were vaccinated intradermally at the same time with the commercially available vaccine Porcilis M Hyo ID Once (containing an Mhyo bacterin) according to manufacturer's instructions at the other side of the neck. Animals of group 3 (control group) remained untreated. All study animals were observed daily. Serum samples were taken at the time of vaccination and every other week until animals were sent to slaughter (23-25 weeks of age). These samples were examined for PCV2 antibodies.

(81) Experimental procedures were as indicated here above under Example 1. The resulting serology is shown in FIG. 6. From this figure it becomes clear that the anti-PCV2 titers remain well above the level of 4 log 2 as established with the experiments as described under Example 1 and found to be protective. There is no indication of negative interference between the vaccines.

(82) This experiment was repeated to check protection against virulent Mycoplasma hyopneumoniae. For this repeat experiment sixty piglets were used. Forty animals were vaccinated at the age of 18-24 days with the Mhyo vaccine and twenty of these animals were also vaccinated with the PCV vaccine. Twenty animals were not vaccinated and served as challenge controls. Three weeks after vaccination all animals were infected with a virulent M. hyopneumoniae strain and three weeks post-challenge all animals were post-mortem investigated for lung lesions. Lung lesion scores (LLS) were compared between the groups.

(83) The LLS for the groups vaccinated with Porcilis M Hyo ID Once were significantly lower than those of the control group (p<0.05, Dunn's test). There was no significant difference between the groups that had been vaccinated with Porcilis M Hyo ID Once alone or in association with the PCV vaccine. It may thus be concluded that the combined vaccination has no negative effect on the immunity obtained with Porcilis M Hyo ID Once.

Example 5

(84) In total eight vaccines were formulated containing PCV2 (250 to 6000 AU/0.2 ml), M hyo (at the same level as in Porcilis Mhyo ID Once) and Lawsonia antigen (see WO 20089/127684, example 2 for the killed whole cells antigens: at a level of approximately 110.sup.9 cells per 0.2 ml). The vaccines contained different adjuvants. Some vaccines used the existing adjuvant Diluvac Forte (MSD Animal Health, Boxmeer, The Netherlands; called DF). Others used the adjuvant formulation as described here above under Example 1 (called X-solve 12), or adjuvants formulated with the same constituents as X-solve 12, but at half of the concentrations (called X-solve 6), or 2% times the concentrations as in X-solve 12 (called X-solve 30). The resulting vaccines were as follows (the Mhyo and Lawsonia antigens are not recited; content per dose):

(85) Group 1: 2000 AU PCV2/X-solve 30

(86) Group 2: 250 AU PCV2/X-solve 12

(87) Group 3: 500 AU PCV2/X-solve 12

(88) Group 4: 2000 AU PCV2/X-solve 12

(89) Group 5: 2000 AU PCV2/X-solve 6

(90) Group 6: 500 AU PCV2/DF

(91) Group 7: 2000 AU PCV2/DF

(92) Group 8: 6000 AU PCV2/DF

(93) The progeny of 8 sows were used for this study. The piglets had moderate (medium level) maternally derived antibodies (MDA) against PCV2 (average: 6.7 log 2). At three/four weeks of age the piglets from groups one through eight were vaccinated intradermally with a single dose, using an IDAL vaccinator. Piglets from group nine remained unvaccinated. At seven weeks post vaccination all animals were transported to the challenge facilities. One day later all animals were challenge infected with a challenge strain of PCV2. All piglets were observed daily for clinical signs.

(94) Local reactions were monitored by palpation, starting on the day of vaccination and every two days after vaccination until twenty days post vaccination.

(95) Blood samples were collected from all animals on the day of vaccination and three weeks later, one day before challenge, 1 and 2 weeks later and at the time of necropsy. Serum samples taken from each animal were tested for antibodies against PCV2 and M hyo. During necropsy, mesenteric and inguinal lymph node, tonsil and lung were sampled for quantification of PCV2 nucleic acid.

(96) Experimental procedures were the same as described here above under Example 2. PCV2 IgM antibody response at time of vaccination was below detection level for all groups (below 2.0 log 2). At 3 weeks post vaccination the PVC antibody titer was the highest for the 2000 AU PCV2/X-solve 30 vaccine, viz. 20 log 2. The X-solve 12 groups, comprising 250-, 500- and 2000 AU PCV2 antigen per dose had a titer of 9, 16 and 19 log 2 respectively. The group that received the 2000 AU/X-solve 6 vaccine had a titer of 15 log 2. The DF groups comprising the 500-, 2000- and 6000 AU of PCV2 antigen per dose had a titer of 8, 14 and 16 log 2 respectively. The controls had a titer below detection level.

(97) In this study, systemic and local reactions were assessed. No systemic reactions attributable to vaccination were observed. As far as local effects are concerned, no more than three vaccinated animals (having received the X-solve 12 500 and 2000 AU, or DF 6000 AU PCV2 per dose vaccine respectively) had slight motility problems, the same number as in the control animals. Therefor these reactions may reasonably be regarded as unrelated to the vaccination. With regard to other local reactions, many animals (between about 60-100%) vaccinated with X-solve showed local reactions, the average size of the swellings was less than 3 cm, viz. between 1-2 cm, and the swellings disappeared within 2-6 days. Using DF, only about 30% of the animals showed local swellings, the mean size being less than 0.5 cm, and they disappeared within a day.

(98) In FIGS. 7 and 8 the viral load in the organs (averaged) is depicted for the various vaccines. It appears that all vaccines are able to substantially (in these cases at least 3 logs) reduce the viral load in the relevant organs.

(99) In this study it appeared that all adjuvants used were safe, induced an anti PCV2 IgM response and were able to treat an animal against an infection with pathogenic porcine circo virus type 2. No negative interference between the different antigens was found.