COMBINATION VACCINE FOR INTRADERMAL ADMINISTRATION

Abstract

The present invention relates to the field of veterinary vaccinology, namely to combination vaccines for swine. In particular the invention relates to a combination vaccine for protection against a pathogenic infection with porcine circo vims type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhyo) comprising non-replicating immunogen of PCV2 and non-replicating immunogen of Mhyo. The vaccine is characterized in that it is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica. In another embodiment, the invention relates to a combination vaccine for protection against a pathogenic infection with PCV2 and Mhyo by intradermal administration.

Claims

1. A combination vaccine comprising a non-replicating immunogen of porcine circo virus type 2 and a non-replicating immunogen of Mycoplasma hyopneumoniae, characterized in that the combination vaccine is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica.

2. The combination vaccine according to claim 1, characterized in that it comprises squalane in an amount of 1 to 15% w/v.

3. The combination vaccine according to claim 1, characterized in that it comprises vitamin E-acetate in an amount of 2 to 20% w/v.

4. The combination vaccine according to claim 1, characterized in that it comprises an emulsifier with an HLB value of 8 to 20.

5. The combination vaccine according to claim 4, characterized in that the emulsifier is polysorbate 80.

6. The combination vaccine according to claim 4, characterized in that the emulsifier is present in an amount of 0.5 to 10% w/v.

7. The combination vaccine according to claim 1, characterized in that it comprises silica in an amount of 0.02 to 2% w/v.

8. The combination vaccine according to claim 1, characterised in that the non-replicating immunogen of porcine circo virus type 2 is recombinantly expressed protein encoded by the ORF2 gene of porcine circo virus type 2.

9. The combination vaccine according to claim 1, characterised in that the non-replicating immunogen of Mycoplasma hyopneumoniae comprises killed whole Mycoplasma hyopneumoniae.

10-12. (canceled)

13. A method of prophylactically treating an animal against an infection with porcine circo virus type 2 and an infection with Mycoplasma hyopneumoniae by administering into the dermis of the animal the combination vaccine according to claim 1.

14. An adjuvant composition for formulating a non-live vaccine, characterized in that the composition is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica.

Description

EXAMPLES

Example 1: Preparation of the Combination Vaccine

[0116] The combination vaccine according to the invention was prepared as follows:

[0117] This oily emulsion in 2× concentration was prepared according to the following subsequent process steps: [0118] required amounts of vitamin E-acetate and squalane were weighed off, and combined in a beaker, [0119] the vitamin E-acetate/squalane mixture was homogenised by low-energy mixing (magnetic stirrer), at room temperature, [0120] the required amount of Polysorbate 80 was weighed off, and added to the homogenised vitamin E-acetate/squalane mixture, [0121] the combined mixture was homogenised again, by low-energy mixing at room temperature, [0122] the homogenised mixture was sterilised by filtration through an 0.2 micrometre filter (Pall, Ultipor™ N66), [0123] the required amount of (heat sterilised) silica was weighed off and added to the homogenised mixture, after which the combined mixture was homogenised again, by low-energy mixing at room temperature, [0124] the mixture was heated to 65-75° C., [0125] the water for injection (sterilised) was heated to 65-75° C., [0126] the heated oil-phase and the water were pre-mixed using high energy mixing by Ultra Turrax with N18 rod, for 5-15 minutes; the temperature decreased from 65 to 55° C. [0127] the pre-mix was given 3 passages through a Microfluidizer™ at 800 bar; temperature was kept below 50° C. with a cooling spiral.

[0128] Of the final oily emulsion, completeness and level of homogenisation were checked by light-microscopy. Further pH (7.34), and osmolality (221 mOsm/kg) were also checked. Particle size measurements revealed: D100=300 nm; D99=250 nm; D90=200 nm, and D50=130 nm.

[0129] The aqueous phase (in 2× concentration) was prepared by taking the required amount of each of the non-replicating immunogens: Mhyo: 6% v/v of a 10× concentrated inactivated culture and PCV: 50 μg ORF2.

[0130] Next, both concentrated compositions (oily emulsion with adjuvants, and aqueous phase with immunogens) were combined in an approximate 50:50 volume ratio, by low-energy mixing at room temperature.

[0131] The following vaccine adjuvant compositions are prepared as oil-in-water (o/w) emulsions (all percentages are % w/v) using the procedure described above. To some compositions aluminum hydroxide was added in the specified amount (as double concentrate) together with the Polysorbate 80 and squalane when preparing the oily emulsion:

TABLE-US-00001 TABLE 1 Reference Reference Composition Composition Composition Composition 1 2 3 4 Polysorbate 80 3.24% 3.24% 3.24% 3.24% (d = 1.08 g/ml) Squalane 6.75% 6.75% 6.75% 6.75% (d = 0.81 g/ml) Vitamin 7.94% 7.94% 7.94% 7.94% E-acetate (d = 0.95 g/ml) Aerosil 380  0.2%  0.2% — — Water for 28.78%  28.78%  31.97%  28.98%  injection Alhydrogel — 9.83% 1.73% 9.83% (Al(OH).sub.3 3%) PBS** + Balance Balance Balance Balance Immunogen *Phosphate Buffered Saline

Example 2: Efficacy of PCV2/Mhyo ID Formulations in Pigs Against Mhyo Challenge Infection

[0132] The effectiveness of vaccination with Mhyo intradermal (ID) formulations (0.2 ml, vaccinated at the right side of the neck with the IDAL® vaccine) against Mhyo challenge is tested in Specific Pathogen-Free (SPF) piglets (ToJaPigs). The formulations have been prepared with various treatments of Mhyo immunogen and are formulated at 5 PCVU/ml (approx. 25% w/v of an inactivated Mhyo culture). Animals were vaccinated at 3 weeks of age according to the following scheme (Table 2). Four weeks after vaccination all animals were infected with Mhyo. All animals were challenged on two consecutive days at 7 weeks of age, i.e. 4 weeks post vaccination, with 10 ml intra-tracheal Mhyo strain 98 with 10.sup.9 and 10.sup.9 CCU/ml respectively. Three weeks post challenge, the animals were sacrificed and the extent of Mhyo-induced consolidated pneumonia was scored according to Goodwin (maximum score: 55).

TABLE-US-00002 TABLE 2 Group Immunogens (N) PCV2 Mhyo Adjuvant 1 (15) X X Reference Composition 3 2 (15) X X X-Solve12* + Aerosil380 3 (15) — — Non-vaccinated control group *Proprietary adjuvant of Porcilis .sup.® PCV ID (MSD Animal health)

[0133] Results:

[0134] Effectiveness of vaccination is determined by lung lesion scores (LLS, averaged), which were recorded for each pig and compared to the non-vaccinated control group.

TABLE-US-00003 TABLE 3 Group Lung Lesion Score (LLS) 1 13.2 2 11.5 3 15.4

[0135] No significant reduction in lung lesion score was obtained with any of the vaccines tested in groups 1 and 2 compared to the control group. Instead, lung lesion core was similar as in the non-vaccinated control group.

[0136] Thus, neither the combination of the adjuvants allohydrogel, squalane, vitamin E acetate and silica in combination with the immunogens of PCV2 and Mhyo nor the combination of the commercially available PCV vaccine Porcilis® PCV ID and silica as adjuvant was suitable for safe and effective intradermal administration.

Example 3: Efficacy of a PRRS Vaccine Reconstitituted in PCV2/Mhyo ID Formulations in Pigs

[0137] The objective of this study was to compare the safety and serological efficacy of different PCV2 and/or Mhyo vaccines containing Aerosil200 or Aerosil380 reconstituted with Porcilis® PRRS, when administered intradermally (ID) in the neck of 5 weeks-old piglets. Piglets were allotted to the treatment groups indicated below (Table 4). The piglets were vaccinated intradermally when they were approximately five weeks old. Piglets from groups 4 and 5 were vaccinated with Porcillis PRRS with 10.sup.4.5 TCID.sub.50 virus reconstituted in different PCV2-Mhyo vaccine formulations as described below

TABLE-US-00004 TABLE 4 Group Immunogens (N) PCV2 Mhyo Other components 4 (10) X X Porcilis .sup.® PRRS + Aerosil200 5 (10) X X Porcilis .sup.® PRRS + Aerosil380 6 (10) X — Porcilis .sup.® PCV ID + Porcilis .sup.® PRRS; non-mixed (positive control) 7 (10) — — Non-vaccinated control group [0138] Piglets from group 4 were vaccinated intradermally with a single dose (0.2 ml) of vaccine formulated with PCV2 (10000 AU/ml; approx. 80 μg/ml;)+M. Hyo (10 PCVU/ml) and the adjuvant Aerosil200. [0139] Piglets from group 5 were vaccinated intradermally with a single dose (0.2 ml) of vaccine formulated with PCV2 (10000 AU/ml)+M. Hyo (10 PCVU/ml) and the adjuvant Aerosil380. [0140] Piglets from group 6 were vaccinated intradermally with a single dose (0.2 ml each) of vaccine Porcilis® PCV ID+Porcilis® PRRS, non-mixed. [0141] Piglets from group 7 were not vaccinated (negative control group). All animals were examined for injection site reactions.

[0142] All piglets were observed daily after vaccination for clinical signs. Temperatures were taken and serum samples were collected from all animals. Samples were tested for antibodies against PCV2, M. hyopneumoniae and PRRSV.

[0143] Results:

[0144] Following vaccination, the body temperatures of all groups were comparable. At the day of vaccination all animals had comparable PCV2 antibody titers. PCV2 antibody titers remained at a constant level in all groups until the end of the study. The antibody titers of the control group 7 decreased over time. At the day of vaccination all animals were negative for PRRS antibodies. Following all groups showed 0-20% PRRS responders. The control group 7 remained negative. At the start of the study all animals were serologically negative for M. hyopneumoniae. All groups showed 0-20% responders at SD21. At SD28 most groups showed positive animals. The control group 7 showed no M. hyopneumoniae positive response. Results of IgM antibody response (predictive for vaccine take) against PCV2 and Mhyo are shown in Tables 5 and 6.

TABLE-US-00005 TABLE 5 Percentage of PCV2 specific IgM positive animals following vaccination Group SD0 SD14 SD21 4 0 80 40 5 20 50 80 6 0 100 100 7 20 20 20

TABLE-US-00006 TABLE 6 Percentage of Mhyo serological antibody response positive animals following vaccination Group SD0 SD14 SD21 SD28 4 0 0 20 20 5 0 0 0 10 6 0 0 0 10 7 0 0 0 0

[0145] From this study, it can be concluded that no vaccinated group showed an acceptable antibody response against PCV2 or Mhyo. Thus, neither the adjuvant Aerosil A380 nor Aerosil A200 when reconstituted with Porcilis® PRRS and PCV2 and Mhyo immunogen was suitable for safe and effective intradermal administration in pigs.

Example 4: Efficacy of a PRRS Vaccine Reconstituted in Mhyo ID Formulations in Pigs Against Mhyo Challenge Infection

[0146] Effectiveness of vaccination with Mhyo intradermal (ID) formulations mixed with PRRS (A212D, 10.sup.5.1 TCID50/dose) against Mhyo challenge infection is tested in SPF piglets. The formulations have been prepared with various adjuvants. Animals were vaccinated ID (0.2 ml) in the right side of the neck at 3 weeks of age according to the following scheme (Table 7). Three weeks after vaccination all animals were infected with Mhyo.

TABLE-US-00007 TABLE 7 Group Immunogens (N) PRRS Mhyo Vaccine adjuvant 8 (12) X X Reference Composition 3 9 (12) — X X-Solve (Porcilis .sup.® MHyo ID once), positive control for Mhyo 10 (12)  X — Porcilis .sup.® PRRS, positive control for PRRS

[0147] All animals were infected on two consecutive days at 6 weeks of age with 10 ml intra-tracheal Mhyo strain 98 with 9 and 8 CCU, respectively.

[0148] Results:

[0149] Effectiveness of vaccination is determined by lung lesion scores (LLS), which were recorded for each pig and compared to the non-vaccinated control group.

TABLE-US-00008 TABLE 8 Lung Lesion Score (LLS) Group Score % reduction 8 9.0 22 9 3.0 74 10 11.5 —

[0150] It could be shown that the intradermal administration of a combination of Mhyo and PRRS immunogens with alhydrogel, squalane and vitamin E-acetate as adjuvant composition as in Reference Composition 3 gives a very low and thus unacceptable lung lesion score of 22% reduction versus 74% in the positive control group using a commercial Mhyo vaccine. Thus, the combination of the immunogens with the adjuvants alhydrogel, squalane and vitamin E-acetate was not suitable for safe and effective intradermal administration.

Example 5: Efficacy of PCV2-Mhyo ID Formulations in SPF Pigs Against Mhyo Challenge Infection

[0151] Groups of 12 pigs were intradermally vaccinated according to the following scheme (Table 9) at the age of three weeks (+/−three days), group 17 was not vaccinated and serves as Mhyo challenge control. Four weeks after vaccination all animals were infected with a virulent Mhyo strain. Three weeks post-challenge all animals were post-mortem investigated for lung lesions. Prior to vaccination, before challenge and at post-mortem blood samples were taken.

TABLE-US-00009 TABLE 9 Group Mhyo PCV Volume (N) Adjuvant/product immunogen immunogen & Route 11 (12) Composition 1 Strain 11 PCV2 0.2 ml 12 (12) Composition 1 Strain J ID, 13 (12) Composition 2 Strain 11 neck 14 (12) Reference Composition 4 Strain 11 right 15 (12) ISA28 + micro-fortasol* Strain 11 side (Reference) 16 (12) Porcilis .sup.® Mhyo Strain 11 — ID Once** 17 (12) —*** — — — *Montanide .sup.® ISA 28 of Seppic to which microfluidized vitamin E acetate was added **Positive control group for Mhyo ***Non-vaccinated control group

[0152] Experimental Procedures

[0153] Serology

[0154] Blood samples (vena jugularis) were taken just before vaccination (T=0, three weeks of age), just before challenge (T=4, 7 weeks of age) and at post-mortem (T=7, 10 weeks of age). Samples were transported at ambient temperature. From the blood samples serum was derived in duplicate. The presence of the relevant antibodies in all serum samples was determined in Elisa tests for M hyo and PCV according to standard procedures.

[0155] Clinical Observations and Rectal Temperature

[0156] Clinical observations were done and rectal temperatures were measured just before vaccination, four hours and one and two days after vaccination. Observations and temperature were noted.

[0157] Post Mortem Examination

[0158] At the end of this experiment, 4 weeks after challenge, the pigs were sedated by means of electrical stunning and subsequent euthanized by bleeding to death. Lung lesions were scored according to Goodwin.

[0159] Results:

[0160] No injection site reactions that were unacceptable were observed. No unacceptable temperatures were observed.

[0161] Effectiveness of vaccination is determined by lung lesion scores (LLS), which were recorded for each pig and compared to the non-vaccinated control group. Results are shown in Table 10:

TABLE-US-00010 TABLE 10 Lung Lesion Score (LLS) Group Score % reduction 11 8.7 47% 12 6.0 63% 13 2.8 83% 14 13.0 20% 15 20.0 −23%  16 4.0 75% 17 16.3 —

[0162] The above data of lung lesion scores show that groups 11, 12 (inventive compositions) and 13 (Reference Composition 2) gave acceptable scores, since the reduction compared to the non-vaccinated control group (group 17) was above 45% (which was used as cut-off value for substantial effectiveness). Instead, groups 14 and 15 gave unacceptable scores, as the LLS in group 15 was even higher than in the non-vaccinated control group, and the LLS in group 14 was only slightly reduced compared to the negative control group by 20%.

[0163] Thus, it could be shown that the inventive vaccine composition including the adjuvant combinations of squalane, vitamin E-acetate and silica gave acceptable lung lesion scores showing effective vaccination.

Example 6: Efficacy of PCV2-Mhyo ID Formulations in SPF Pigs Against PCV Challenge Infection

[0164] Piglets were allotted to five treatment groups of 10 piglets each and vaccinated intradermally when they are approximately three weeks old. Piglets from group 1 through 3 were vaccinated with the vaccines described below. Group 4 was vaccinated with Porcilis® PCV ID and Porcilis® M Hyo ID ONCE as positive control group. Group 5 was not vaccinated (negative control). At three weeks post vaccination (6 weeks of age) all animals were challenged using 5.0 log10 TCID50/mL of wild-type PCV2b challenge virus strain 112/11 applied intranasally (3 ml per nostril). The treatment scheme is shown below in Table 11. Three weeks post challenge, all animals were necropsied and inguinal lymph node, mesenteric lymph node, tonsil and lung were sampled for the detection of PCV2.

[0165] All piglets were observed daily after vaccination for clinical signs. Serum samples were collected on the day of vaccination and on SD14, SD20, SD35 and SD42 (during necropsy). Samples were tested for antibodies against PCV2 viral nucleic acid by qPCR. Fecal swabs were collected on SD20, 35 and SD41 from all animals and were examined for PCV2 viral nucleic acid by qPCR.

TABLE-US-00011 TABLE 11 Group Adjuvant/ Mhyo PCV Volume & (N) product immunogen immunogen Route 18 (10  Composition 1 Strain 11 PCV2 0.2 ml ID, neck 19 (10) Composition 2 right side 20 (10) ISA28* + micro-fortasol 21 (10) Porcilic .sup.® 0.2 ml ID R1 PCV ID + (PCV) + 0.2 Porcilis .sup.® ml ID R2 (Mhyo) M Hyo ID Once** 22 (10) —*** — — — *Montanide .sup.® ISA 28 of Seppic to which microfluidized mineral oil was added **Positive control group for Myho and PCV2 ***Non-vaccinated control group

[0166] Treatment

[0167] Dosage and Administration

[0168] Vaccinations were done by the intradermal route (0.2 ml), on the right side of the neck. Group 21 was vaccinated twice on the right side. Challenge was done by the intranasal route, 6 ml, 3 ml per nostril using a MAD applicator.

[0169] Test System

[0170] Only healthy animals were used. In order to exclude unhealthy animals, they were examined before vaccination (general physical appearance and absence of clinical abnormalities or disease). Just before vaccination, all animals will be individually numbered with eartags. All pigs were observed daily for clinical signs of disease. Observations will consist of systemic reactions such as loss of appetite, reluctance to move, tendency to lie down, listlessness or drowsiness, shivering, bristling, edema (especially around the eyes), vomiting and diarrhea and dyspnea.

[0171] Experimental Procedures

[0172] Sampling of Blood

[0173] Blood samples were collected from all animals on the day of vaccination, one day before challenge, two weeks later and at the day of necropsy. A minimum of 4 ml and a maximum of 8 ml blood was taken per animal. This was done from all pigs individually according to standard procedures. The blood samples were collected without the addition of anti-coagulant.

[0174] Fecal Swabs

[0175] Fecal swabs were taken one day before challenge infection, at 2wpc and one day before necropsy. Swabs were taken into medium containing antibiotics.

[0176] Post Mortem Examination

[0177] At 3 weeks post challenge the animals were transported to the necropsy room. They were anaesthetised using an electrocution apparatus and exsanguinated in accordance with standard procedures. The carcasses of the animals were destroyed according to standard procedures. 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. Subsequently, samples from tonsil, lung, mesenteric lymph node and inguinal lymph node were removed and divided in two parts, one part for freezing and analysis by PCV2 qPCR and one part for fixation followed by (immuno)histochemical analysis.

[0178] Processing of Samples

[0179] Serum was prepared from the clotted blood samples and aliquots (e.g. 2×0.8 ml) were filled. The samples were NOT heat-inactivated. Fecal samples were prepared from swabs and aliquots (e.g. 2×0.8 ml) were filled. Until use the samples were stored at ≤−15° C. The period between sampling and storage did not exceed 36 (serum) or 48 (swabs) hours.

[0180] PCV2 Antibody ELISA

[0181] Sera were tested for antibodies against PCV2 according to standard 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 (MoAb). Bound MoAb is then incubated with peroxidase-conjugated streptavidin followed by chromophoric detection. Titers were defined as the reciprocal of the interpolated serum dilution with an extinction value equal to the 50% maximum extinction for the test. Results were expressed as log.sub.2 titers. Titers below 2.0 log.sub.2 were considered negative. For negative samples, a value of 1.0 log.sub.2 was used for calculation purposes.

[0182] Quantitative PCR

[0183] Quantitative PCR (qPCR), on PCV2 nucleic acid, was performed on all sera, fecal swabs, and on 10% tissue homogenates of tonsil, lung, mesenteric lymph nodes and inguinal lymph nodes according to standard procedures. 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 probe specific for PCV2-ORF2. The cycle number where specific fluorescence exceeds the threshold is correlated with the cycle numbers for a set of samples containing known amounts of a PCV2-ORF2-containing plasmid. Results were expressed as log.sub.10 copies/0 of extracted DNA (log.sub.10 c/μl). Values lower than 1.00 log.sub.10 c/μl are considered negative, were taken as 0.00 log.sub.10 c/μl for calculation purposes.

[0184] Immunohistology (IHC)

[0185] Tonsil and lymph node samples were prepared for histological examination. Samples were fixed in 10% formalin, paraffin embedded, and immunohistochemistry was performed for detection of PCV2 antigen on slides. An anti-PCV2 rabbit serum was used as primary antibody and Envision+ (DAKO, Denmark) was used as detection system, according to the manufacturer's instructions. The slides were counterstained with hematoxylin. Microscopic examination was done. For tonsil and lymph nodes, characteristic brown staining was scored on an ordinal scale as follows: [0186] 0 No specific positive staining cells observed [0187] 1 scattered (single) positive staining cells present in (less than), 10% of follicles [0188] 2 positive staining (single) cells observed in 10-50% of follicles, or focal collections of >15 positive staining cells in <10% of the follicles. [0189] 3 specific staining in >50% of follicles

[0190] The results were recorded as the total score, being the sum of scores of the individual tissues.

[0191] Results:

[0192] Pcv2 Serology:

[0193] At start of measurement at SD1, log 2 titers of all groups was between 4 and 5. The positive control (group 21) gave a log 2 titer of 10 between SD34 and SD41. In the same week, the log 2 titers in the groups 18, 19 and 20 was between 8 and 10. Non-vaccinated negative control group 22 led to a log 2 titer below 4.

[0194] PCV2 Viral Load in Serum (qPCR Serum)

[0195] Between SD0 (start of measurement) and SD19, no viral load could be detected in all groups (log 10 c/μl=0).

[0196] At SD 34 viral load in the positive control (group 21) increased to about 1.0 log 10 c/μl and further increased to about 1.6 at SD41.

[0197] At SD 34 viral load in the non-vaccinated negative control (group 22) increased to about 4.1 log 10 c/μl and slightly decreased at SD41 to about 3.6.

[0198] The viral load in groups 18, 19 and 20 was between 1.3 and 1.8 log10 c/μl at SD 34 and between 1.6 and 2.1 at SD41.

[0199] PCV2 Viral Load in Fecal Swabs (qPCR Fecal Swab)

[0200] At SD19 (start of measurement), no viral load could be detected in all groups (log10 c/μl=0). At SD 34 viral load in the positive control (group 21) increased to about 1.5 log10 c/μl and further increased to about 2.2 at SD41.

[0201] The viral load in the non-vaccinated negative control (group 22) increased to about 3.6 log10 c/μl at SD34 and remained basically constant up to SD41.

[0202] The viral load in groups 18, 19 and 20 was in the range of the positive control group (between about 1.5 and 1.8 at SD34 and between about 1.8 and 2.2 at SD41).

[0203] Safety

[0204] No injection site reactions that were unacceptable were observed. Maximum average temperature increase was 0.4° C. at T=0+4 h. Thus, it could be shown that the inventive vaccine composition including the adjuvant combinations of squalane, vitamin E-acetate and silica gave acceptable results as regards vaccination efficacy without causing unacceptable site reactions.

Example 7: Use of Different Pharma Grade Silica's to Constitute the Combination Vaccine

[0205] In this example various different types of silica's, all pharma grade (colloidal) amorphous silica's, are shown to be useful to produce a combination vaccine according to the invention. The method for preparing the vaccine was largely in line with Example 1, although for some vaccines the silica was added only after passage through the Microfluidizer™. This had no substantial impact on the final composition, all silica's were roughly distributed in the final composition with an average particle size of about 120 nm. However, when adding the silica after the passage through the Microfluidizer™, a very small percentage of the total volume of silica was present as large agglomerates (solid particles) above 10 μm (up to 200 μm). This however has no negative impact on the performance of the vaccine. The data for the various compositions are given in Table 12.

TABLE-US-00012 TABLE 12 Osmolality Microscopic Mastersizer mOsmol/ Silica view in μm Appearance pH kg Aerosil Solubilisate 0.10 = 0.08 Homo- 7.41 214 200 with particles 0.50 = 0.12 geneous, up to 3 μm 0.90 = 0.17 nearly white 0.99 = 0.22 Aerosil Solubilisate 0.10 = 0.08 Homo- 7.42 189 200 with 0.50 = 0.12 geneous, particles up 0.90 = 0.22 nearly white 3 μm, also  0.99 = 66.96 solid particles up to 50 μm Aerosil Solubilisate 0.10 = 0.08 Homo- 7.38 212 200vv with particles 0.50 = 0.12 geneous, up to 3 μm 0.90 = 0.17 nearly white 0.99 = 0.23 Aerosil Solubilisate 0.10 = 0.08 Homo- 7.42 186 200vv with 0.50 = 0.12 geneous, particles up  0.90 = 13.10 nearly white 3 μm, also 0.99 = solid particles 143.79 up to 200 μm Aeroperl Solubilisate 0.10 = 0.08 Homo- 7.40 211 300 with particles 0.50 = 0.12 geneous, up to 3 μm 0.90 = 0.17 nearly white 0.99 = 0.22 Aeroperl Solubilisate 0.10 = 0.08 Homo- 7.42 187 300 with 0.50 = 0.12 geneous, particles up 0.90 = 0.18 nearly white 3 μm, also  0.99 = 63.65 solid particles upto 100 μm Aerosil Solubilisate 0.10 = 0.08 Homo- 7.40 211 380F with particles 0.50 = 0.12 geneous, up to 3 μm 0.90 = 0.17 nearly white 0.99 = 0.22 Aerosil Solubilisate 0.10 = 0.08 Homo- 7.41 186 380F with 0.50 = 0.13 geneous, particles up  0.90 = 25.44 nearly white 3 μm, also  0.99 = 85.02 solid particles upto 100 μm

[0206] Other silica's suitable for use in the present invention are Aerosil® 90, Aerosil® 130, Aerosil® 150, Aerosil® 200F, Aerosil® 255, Aerosil® OX 50, Aerosil® TT600 and Aeroperl® 300/30.

CONCLUSIONS

[0207] The Examples 1 to 7 show that of the tested compositions only the novel adjuvant combination of squalane, vitamin E-acetate and silica gave acceptable results as regards vaccination efficacy against PCV2 and Mhyo infections without causing unacceptable site reactions. Hence, using this adjuvant composition, a combination vaccine can be provided against PCV2 and Mhyo using non-replicating immunogen of porcine circo virus type 2 and Mycoplasma hyopneumoniae for safe and effective intradermal administration.