Combination vaccine for swine

Abstract

The present invention relates to a combination vaccine for swine, comprising non-replicating antigen from porcine circovirus type 2 (PCV2), and live porcine reproductive and respiratory syndrome virus (PRRSV); the combination vaccine is formulated as an oil-in-water emulsion, and is adjuvated with squalane and vitamin E-acetate. This combination vaccine was found to be immunologically effective against all pathogens: PCV2, and PRRSV.

Claims

1. A combination vaccine comprising non-replicating antigen from porcine circovirus type 2 (PCV2) and live porcine reproductive and respiratory syndrome virus (PRRSV), wherein the vaccine is an oil-in-water emulsion comprising squalane and vitamin E-acetate; wherein the oil-in-water emulsion is a submicron emulsion.

2. The combination vaccine of claim 1, comprising squalane in an amount of between about 1 and about 9% w/v.

3. The combination vaccine of claim 1, comprising vitamin E-acetate in an amount of between about 1 and about 10% w/v.

4. The combination vaccine of claim 1, also comprising non-replicating antigen from Mycoplasma hyopneumoniae (Mhyo).

5. The combination vaccine of claim 1, also comprising non-replicating antigen from Lawsonia intracellularis (Lawsonia).

6. A kit of parts comprising at least two containers: one container comprising non-replicating antigen from PCV2 in an oil-in-water emulsion comprising squalane and vitamin E-acetate; and one container comprising live PRRSV in freeze-dried form; wherein the oil-in-water emulsion is a submicron emulsion.

7. A method for the preparation of the combination vaccine of claim 1, comprising the steps of: preparing an aqueous phase comprising non-replicating antigen from PCV2 and live PRRSV, and admixing said aqueous phase with an oily emulsion comprising squalane and vitamin E-acetate.

8. A method for the preparation of the combination vaccine of claim 1, comprising the steps of: preparing live PRRSV in a freeze-dried form, preparing an aqueous phase comprising non-replicating antigen from PCV2, admixing said aqueous phase with an oily emulsion comprising squalane and vitamin E-acetate, and reconstituting said freeze-dried live PRRSV with said admixture of aqueous phase and oily emulsion.

9. A method for the preparation of combination vaccine of claim 1, comprising the steps of: preparing an admixture of an aqueous phase comprising non-replicating antigen from PCV2, and of an oily emulsion comprising squalane and vitamin E-acetate, and reconstituting live PRRSV in a freeze-dried form with said admixture.

10. A method for vaccinating a swine against PCV2 and PRRSV, comprising administering to said swine the combination vaccine of claim 1.

11. A combination vaccine comprising non-replicating antigen from porcine circovirus type 2 (PCV2) and live porcine reproductive and respiratory syndrome virus (PRRSV), wherein the vaccine is an oil-in-water emulsion comprising squalane and vitamin E-acetate; wherein the amount of vitamin E-acetate is about 1 to about 10% w/v.

12. The combination vaccine of claim 11, wherein the amount of squalane is about 1 to about 9% w/v.

13. The combination vaccine of claim 11, further comprising non-replicating antigen from Mycoplasma hyopneumoniae (Mhyo).

14. The combination vaccine of claim 11, further comprising non-replicating antigen from Lawsonia intracellularis (Lawsonia).

Description

EXAMPLES

(1) 1. Preparation of the Combination Vaccine

(2) The combination vaccine according to the invention was prepared as follows:

(3) The oily emulsion in 2× concentration contains per 100 g: Polysorbate 80 (Tween 80): 3.24 g; Squalane: 6.75 g; DL-alpha tocopherol acetate: 7.94 g; water for injection: 82.07 g.

(4) This oily emulsion was prepared according to the following subsequent process steps: required amounts of Tween 80 and squalane were weighed off, and combined in a beaker the Tween 80/squalane mixture was homogenised by low-energy mixing (magnetic stirrer), at room temperature, the required amount of DL-alpha tocopherol acetate was weighed off, and added to the homogenised Tween 80/squalane mixture the combined mixture was homogenised again, by low-energy mixing at room temperature, the mixture was heated to 65-75° C. the water for injection was heated to 65-75° C. 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. the pre-mix was given 3 passages through a Microfluidiser at 800 bar; temperature was kept below 50° C. with a cooling spiral. the microfluidised oily emulsion was sterilised by filtration through an 0.2 micrometre filter (Pall, Ultipor™ N66); the filter had been preheated to 55-75° C. via its double wall.

(5) Of the final oily emulsion (in 2× concentrate), 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.

(6) The aqueous phase (in 2× concentration) was prepared by taking the required amount of each of the non-replicating antigens: Mhyo: 6% v/v of a 10× concentrated inactivated culture; Lawsonia: 2×10{circumflex over ( )}9 inactivated cells; and PCV: 50 μg ORF2.

(7) Next, both concentrated compositions (oily emulsion with adjuvants, and aqueous phase with antigens) were combined in 50:50 volume ratio, by low-energy mixing at room temperature.

(8) This vaccine mixture was used to resuspend ampules of Porcilis PRRS, with the required volume to reach a full dose of PRRSV (10{circumflex over ( )}5 TCID50) per 2 ml of the combination vaccine.

(9) The final combination vaccine contained: 3.375% w/v squalane; 3.97% w/v vitamin E-acetate, and 1.62% w/v Tween 80, and had a density of 0.9913 g/ml. Products were stored at 2-8° C.

(10) 2. Test for Virucidal Effect

(11) The oily emulsion of the invention was incubated with a sample of live PRRSV, to determine if any virucidal effect would occur.

(12) In short: an ampule of Porcilis PRRSV was reconstituted in PBS to a final volume of 7 ml, to reach a titre of 6 Log 10 TCID50/ml. A 50 μl sample of this virus suspension was combined with 450 μl of a combination vaccine without live PRRSV, prepared as in Example 1, and comprising: squalane, vitamin E-acetate, and polysorbate 80, microfluidised in water, with non-replicating antigens of Mhyo, Lawsonia and PCV2. A control sample of PRRSV was mixed with 450 μl PBS. Both samples were incubated for 1 hour at room temperature. Next the incubated samples were titrated to determine the remaining titre of PRRSV. Titration was done on 1 day old monolayers of MA104 cells. 10 rows of starting wells received 25 μl of incubated virus sample, this was diluted 1:10 through 7 subsequent wells. 2 columns of untreated cells served as negative controls. This was done in duplo. Next the plates were incubated for 3 days at 37° C. in 5% CO2 atmosphere. Finally PRRSV viral replication was detected by Immuno-fluorescence using an anti-PRRSV monoclonal antibody and a fluorescently labelled detection antibody. Titres were calculated using the Spearman-Kaerber algorithm.

(13) TABLE-US-00001 Titre.sup.(1) (Log10 Sample TCID50/ml) combination vaccine (0.9x) 4.6 control 4.8 .sup.(1)Titre is the average of two determinations

(14) With a spread in titration values found of ±0.2 Log 10 TCID50, the results demonstrated that samples of live PRRSV incubated in an 0.9× concentrated combination vaccine according to the invention, did not experience a significant reduction of titre.

(15) 3. Vaccination-Challenge Experiment

(16) 3.1. Introduction

(17) A combination vaccine without live PRRSV, prepared as in Example 1, and comprising: squalane, vitamin E-acetate, and polysorbate 80, microfluidised in water, with non-replicating antigens of Mhyo, Lawsonia and PCV2, was tested in animals. Vaccination was given as a one shot dose, by intramuscular route, at 3 weeks of age. Mhyo efficacy was tested by challenge infection, at 4 weeks post vaccination. Several other adjuvants were compared.

(18) 3.2. Study Design

(19) 84 SPF piglets were used for this study. 6 groups of 12 animals were vaccinated once intra-muscularly at the age of three weeks (+/− three days). One group of 12 pigs was left unvaccinated and served as challenge-control group. Prior to vaccination and two days after vaccination rectal temperatures were measured. Furthermore, for the SVEA group, injection sites were palpated weekly for local reactions. Four weeks after vaccination all animals were infected with a virulent Mhyo strain. Three weeks post-challenge all animals were euthanized and investigated post-mortem for lung lesions. From all animals, blood samples were taken: prior to vaccination, before challenge and at post-mortem.

(20) TABLE-US-00002 TABLE 1 Schedule of treatment of Example 3 Group Vaccine-adjuvant 1 Amphigen 2 SVEA 3 SVEA + Al(OH)3 4 MF59 + DDA 5 SP oil (Metastim) 6 Vaxliant S5 7 no vaccine
3.3. Adjuvants Tested

(21) A number of vaccine formulations were tested, which differed only in respect of the type of formulation and adjuvant used; antigen content was the same. The following adjuvants were tested: Amphigen: oil-in-water of mineral oil with lecithin SVEA: microfluidised oil-in-water emulsion with squalane, vitamin E-acetate and Tween 80 SVEA+Al(OH)3: SVEA with 0.2% w/v aluminiumhydroxide (same as in Porcilis PCV Mhyo) MF59+ DDA: MF59 is an oil-in-water emulsion with squalene, polysorbate 80 and Span 85; DDA is a cationic lipid: dimethyldioctadecylammonium. SP oil: pluronic, squalane and Tween, as described in WO 2013/152086. Vaxliant™ S5: proprietary adjuvant of unknown composition
3.4. Methods and Materials
Challenge:

(22) Challenge material was Mhyo, virulent field strain, fresh 3 day culture in FRIIS medium with porcine serum. 10 ml culture containing 9 CCU, was administered intra-tracheally per animal, on two consecutive days. All animals were under regular veterinary supervision

(23) Vaccination:

(24) Vaccination was at three weeks, while animals were still with their sow. Dose was 3 ml, given intramuscularly, at right side of the neck. Weaning was at 4 weeks of age. One week prior to challenge pigs were transferred to challenge facilities.

(25) Serology:

(26) Blood samples (from vena jugularis) were taken just before vaccination (T=0), just before challenge (T=4) and at post-mortem (T=7). Samples were kept at ambient temperature, till serum was derived. Presence of relevant antibodies in serum samples for PCV2 (via Elisa), or for Lawsonia, was determined according to standard procedures.

(27) Palpation

(28) Injection sites of the SVEA group were inspected for local reactions: just before vaccination, four hours after vaccination, daily for two days and weekly for five weeks after vaccination. Animals that still showed local reactions five weeks after vaccination were palpated individually weekly until local reactions disappeared.

(29) Rectal Temperatures and Clinical Observations

(30) Rectal temperatures were measured and clinical observations (0=Normal; 1=less active; 2=vomiting; 3=lies down) were done one day before, and just before vaccination, four hours and one and two days after vaccination.

(31) Post Mortem Examination

(32) At the end of this experiment, 3 weeks after challenge, all pigs were euthanized. Injection sites were investigated for local reactions in individual animals. Percentage lung lesion score was recorded for each pig individually according to Goodwin & Whittlestone score.

(33) 3.5. Results

(34) TABLE-US-00003 TABLE 2 Results of Example 3 PCV2 Ab titre.sup.(2) Lawsonia Ab titre at x weeks p.v. at x weeks p.v. Group Vaccine adjuvant Mhyo LLS.sup.(1) 0 4 7 0 4 7 1 Amphigen 1.5 8.2 8.1 <6.7 <4.2 <5.7 <6.8 2 SVEA 0.75 8.2 9.3 8.9 <4.1 7.3 9.1 3 SVEA + Al(OH)3 2 8.1 8.7 <7.6 <4.2 7.6 9.6 4 MF59 + DDA 0.5 <7.9 7.7 <6.4 <4.2 <4.7 <4.6 5 SP oil (Metastim) 0 8.6 8.0 <6.9 <4.2 6.7 8.4 6 Vaxliant S5 0 7.5 <7.1 <6.3 <4.0 <4.6 <6.1 7 no vaccine 3.5 8.0 <6.4 <4.8 <4.1 <4.1 <3.9 .sup.(1)Mhyo LLS = median lung lesion score
Results of Palpations and Temperature:

(35) The group monitored by palpation of vaccination site and checked for temperature was the group receiving vaccine adjuvated with SVEA (group 2). These showed that no detectable local swelling, and no rise in temperature was observed throughout the monitoring period.

(36) 3.6. Conclusions

(37) Table 2 demonstrates that broad efficacy for a combination vaccine is hard to achieve; several of the adjuvants tested, did induce for one or even two of the pathogens an immunity that could be assigned as protective; this either in terms of low Mhyo lung lesion score, or as sufficiently high specific antibody titre for PCV2 and Lawsonia. However only for the SVEA adjuvanted vaccine, was protection sufficient to good for all three pathogens. None of the other adjuvants came to that level of broad efficacy.

(38) The data from Example 2, on the lack of virucidal effect of SVEA adjuvated combination vaccine for live PRRSV, predict that a combination vaccine with SVEA adjuvant will also be effective against PRRSV. Together this demonstrates that a combination vaccine according to the invention is immunologically effective against each of the pathogens Mhyo, Lawsonia, PCV2, and PRRSV. Further it is safe for swine.
4. Four-Way Vaccination-Challenge Experiments Further vaccination-challenge experiments were performed, whereby pigs were vaccinated with the four-way combination vaccine according to the invention, and in 4 separate experiments this vaccine was tested for efficacy of all the 4 components: Mhyo, Lawsonia, PCV and PRRSV. Specifically, a 3-way combination vaccine was prepared as described in Examples 1 and 3, containing a sub-micron emulsion of squalane, vitamin E acetate, and Tween 8, mixed 1:1 with an aqueous phase comprising inactivated antigens of Mhyo and Lawsonia, and recombinant expression product of PCV2-ORF2. Subsequently, and shortly before vaccination of pigs, this 3-way vaccine was used to dissolve an ampule of freeze-dried commercial PRRS vaccine, using the required volume to reach a full dose of PRRSV of 10{circumflex over ( )}5 TCID50, per 2 ml animal dose of the 4-way combination vaccine. Vaccination was given as a one-shot dose, by intramuscular route, to pigs at 3 weeks of age.

(39) Subsequent analysis of the vaccine efficacy of each of the four vaccine antigens was done in separate experiments, by challenge infection of the vaccinates and controls, to allow focus on the specific symptoms of infection and disease for these different conditions. However, no differences in outcome of vaccination-efficacy against any of these challenge infections was expected as compared to the results described in Example 3 above; it was highly unlikely that there would be any different effect of the use of a 3-way as compared to a 4-way vaccine in respect of the protection against the inactivated antigens. In other words, the presence of PRRSV virus in the 4-way combination vaccine could not be expected to affect the efficacy of that combination vaccine against any one of the Mhyo, Lawsonia, or PCV challenge infections. Further, no effect was expected on the viability and efficacy of the live PRRSV component, as the previous experiments had already indicated that there was no significant effect of the 3-way vaccine in SVEA adjuvant on the viability of PRRS virus. Consequently, as the PRRS virus was not killed or its infectivity damaged by mixing into the 3-way vaccine, there was no reason why it would not be able to induce effective protection.

(40) These expectations were indeed confirmed by the results of the 4 challenge experiments described below: the 4-way combination vaccine according to the invention was found to induce effective immune protection against infection and signs of disease induced by a challenge infection with a pathogen from each of its 4 components. Also, there was no negative effect or interference from its combination.

(41) 4.1. M. Hyopneumoniae Efficacy

(42) The 4-way combination vaccine was prepared as described above, using Porcilis PRRS freeze dried vaccine.

(43) Experimental Outline

(44) 24 SPF piglets were used for this study. 1 group of 12 animals was vaccinated once intra-muscularly at the age of about three weeks. One group of 12 pigs was left unvaccinated and served as challenge-control group. Four weeks after vaccination all animals were challenge-infected with a virulent Mhyo strain. Three weeks post-challenge all animals were euthanized and investigated post-mortem for lung lesions. From all animals, blood samples were taken: prior to vaccination, before challenge and at post-mortem.

(45) Details of the Experiment

(46) Challenge material was from an Mhyo virulent field strain, as fresh 3 day culture in FRIIS medium with porcine serum. 10 ml culture containing 10 and 9 CCU, respectively, was administered intra-tracheally per animal, on two consecutive days. All animals were under regular veterinary supervision

(47) Vaccination was at three weeks of age, while animals were still with their sow. The dose was 2 ml, given intramuscularly, at the right side of the neck. Weaning was at 4 weeks of age. One week prior to challenge pigs were transferred to challenge facilities.

(48) Blood samples (from vena jugularis) were taken just before vaccination (T=0 weeks), just before challenge (T=4 weeks) and at post-mortem (T=7 weeks). Samples were kept at ambient temperature, till serum was derived. Presence of relevant antibodies in serum samples for PCV2, or for Lawsonia, was determined by ELISA according to standard procedures.

(49) Data Analysis:

(50) At the end of this experiment, 3 weeks after challenge, all pigs were euthanized. Percentage lung lesion score was recorded for each pig individually according to the Goodwin & Whittlestone score (supra).

(51) Results

(52) TABLE-US-00004 TABLE 3 Results of Mhyo and serology data of Example 4.1 PCV2 Ab titre Lawsonia Ab titre at 7 weeks p.v. at 7 weeks p.v. Group Vaccination Mhyo LLS .sup.(1) 0 4 7 0 4 7 1 4-way in SVEA 8.0 <4.8 10.6 9.9 <3.9 <6.6 9.2 2 no vaccine 12.7 5.4 <4.8 <4.3 <3.9 <3.9 <3.9 .sup.(1) Mhyo LLS = median of lung lesion scores

Conclusion

(53) Vaccination of pigs with the 4 way combination vaccine according to the invention, was effective in protecting against infection and signs of disease induced by an Mhyo challenge infection. Also, there was a good development of protection against Lawsonia and PCV2, as measured by serology.

(54) 4.2. PCV2 Efficacy

(55) The 4-way combination vaccine was prepared as described above, using Porcilis PRRS freeze dried vaccine.

(56) Experimental Outline & Details of the Experiment

(57) Piglets were allotted to treatment groups of 10 piglets each. The piglets were vaccinated intradermally or intramuscularly when they were approximately five weeks old: one group was vaccinated intramuscularly with the 4-way combination vaccine in SVEA adjuvant: a single 2 ml dose of vaccine formulated with PCV2-Orf2 (2500 AU/ml), Mhyo (1.0 PCVU/ml), Lawsonia (5000 AU/ml), and PRRSV at 10.sup.5TCID.sub.50/dose. Time between dissolving the PRRS vaccine and vaccination was 1 hour. group 2 was the positive control, and this was vaccinated using the commercial vaccines Porcilis PCV ID and Porcillis PRRS ID, both by intra-dermal route, but non-mixed and given at different sites on the back of the pigs. piglets in the third group were not vaccinated (negative control group), but were challenged.

(58) At three weeks post vaccination (8 weeks of age, 3 weeks post vaccination, sample date [SD]22 days) all animals were challenged using 5.0 log.sub.10TCID.sub.50/mL of wild-type PCV2b challenge virus, strain 112/11, which was inoculated intranasally, at 3 ml per nostril.

(59) 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, bt qPCR, and by immunhistochemistry.

(60) All piglets were observed daily after vaccination for clinical signs. Temperatures were taken at SD-1, SD0, SD0+4 hours and SD1. Serum samples were collected from all animals, these were tested for antibodies against PCV2, PRRSV and Lawsonia.

(61) Serum, tissue, and fecal- and nasal swab samples were collected from all animals, and were examined for PCV nucleic acid by qPCR.

(62) Data Analysis & Results:

(63) Temperature readings showed no significant results.

(64) Combined serological results are represented in Table 4 below.

(65) PCV2 Serology:

(66) All piglets from the two vaccinated groups reached 100% seroconversion for anti-PCV2 antibodies already at sample date 22 (3 weeks p.v.). Actual titres increased some more after that, and reached a plateau from SD35. Unvaccinated controls only seroconverted after challenge, but never reached more than 20% seroconversion.

(67) PCV2 IHC and qPCR:

(68) Immunohistological screening and scoring was performed for signs of PCV in lymphnodes and tonsils. Results showed that in both vaccinated groups the scores were on average 0.3, while in the unvaccinated-challenged control group IHC scores were on average 1.6. qPCR results showed that in both vaccinated groups pigs had very little PCV2 present in serum, nasal- or fecal swabs, or tissue samples (lymph nodes, tonsils, and lungs). However, the control group became strongly positive for PCV2 nucleic acids after challenge.
Lawsonia Serology

(69) Lawsonia serology for group 1 showed an increase in titer from SD22, while in the control group the Lawsonia titer showed a steady decrease.

(70) PRRSV Serology

(71) Serological results for PRRSV are typically expressed as an SP (Sample to Positive) ratio. When this ratio is above 0.4, a sample is considered positive for PRRSV seroconversion. The section of Table 4 for PRRSV represents these SP values. They show that animals in the positive control group 2 (PRRSV vaccine by id route) were strongly positive for PRRSV-seroconversion; nevertheless, animals in group 1 (4-way combination vaccine by im route) also reached good seroconversion rates. Unvaccinated controls hardly showed any seroconversion for PRRSV, as no SP ratios above 0.05 were found.

(72) TABLE-US-00005 TABLE 4 Combined serological results of Example 4.2 Serology per group SD0 SD22 SD28 SD35 SD43 PCV 1. 4-way vaccine, im 3.7 7.2 7.7 10.0 10.4 2. Porcillis PCV ID + 3.7 7.0 8.1 9.5 9.9 Porcilis PRRS ID 3. unvaccinated controls 4.2 2.3 2.1 3.5 5.7 Lawsonia 1. 4-way vaccine, im 4.7 4.3 5.0 5.7 6.3 3. unvaccinated controls 3.3 3.3 3.3 2.9 2.9 PRRSV (SP ratios) 1. 4-way vaccine, im 0.0 0.9 1.3 1.3 1.4 2. Porcillis PCV ID + 0.0 1.6 1.8 2.1 2.3 Porcilis PRRS ID 3. unvaccinated controls 0.0 0.0 0.0 0.0 0.0

Conclusions

(73) Vaccination of pigs with the 4-way combination vaccine according to the invention, was effective in protecting against infection and signs of disease induced by a PCV2 challenge infection. Also, there was a good development of protection against Lawsonia and PRRSV, as measured by serology.

(74) 4.3. Lawsonia Efficacy

(75) The 4-way combination vaccine was prepared as described above, using Prime Pac PRRS freeze dried vaccine.

(76) Experimental Outline

(77) Three groups of three week old pigs were vaccinated with the 4-way combination vaccine, and were given a challenge infection with virulent Lawsonia bacteria 5 weeks later. One group received the full 4-way vaccine, one other group received a control vaccine comprising all the same ingredients, except the Lawsonia antigen. The third group received the control vaccine, but no challenge as these were necropsied at the time of the challenge to confirm the absence of Lawsonia infection in the herd prior to challenge. The groups were compared to determine the efficacy of a single dose of the combination-vaccine against disease caused by the Lawsonia challenge: ileitis, colonization of the ileum by Lawsonia, shedding, and effect on weight gain.

(78) All pigs were evaluated every other day for local and systemic reactions for 21 days following the vaccination, or until resolution. Fecal samples were collected to confirm the absence of field infection by Lawsonia prior to the challenge. Blood samples were collected throughout the study to evaluate the antibody responses to vaccination and challenge, also fecal samples were collected for Lawsonia qPCR, and body weight data were recorded. At three weeks post challenge (11 weeks of age) all remaining animals were necropsied and scores were determined of gross lesions of the ileum, and mucosal scrapings were collected for Lawsonia quantitative PCR (qPCR). Also a section of the ileum was collected for immunohistochemistry (IHC) and histopathology.
Details of the Experiment

(79) Pigs were of mixed sex, and were a mixed American Yorkshire-Landrace-Duroc breed. Water and age-appropriate feed were provided at libitum.

(80) The full 4-way vaccine comprised per 2 ml animal dose: 2.0 RP of Mhyo antigen; 6000 Elisa units of Lawsonia antigen/ml; 5.7 μg/ml of PCV2 Orf2 antigen; and 10{circumflex over ( )}5 TCID50 of PRRSV.

(81) Just prior to challenge 2 pigs vaccinated with control vaccine were necropsied, to verify absence of any Lawsonia infection prior to challenge.

(82) Lawsonia challenge was given by oral route, at 5 weeks post vaccination (about 8 weeks of age), with an inoculum of 4.6 10 Log TCID50 of live virulent Lawsonia per animal.

(83) Lesion scoring of the ileum was done on a 2.5 cm section of the ileum, excised and collected in 10% buffered formalin, which was used for histopathological examination, and for the presence of Lawsonia by immunohistochemistry. The rest of the ileum was opened and the mucosal surface was visually examined for the presence of Lawsonia-associated lesions of ileitis (also called Porcine Proliferative Enteritis or PPE) including mucosal proliferation with thickening of the intestinal wall, edema, hyperemia, congestion, necrosis and hemorrhaging. Gross lesions were given a score ranging from 0 (normal mucosa) to 5 (severe PPE with hemorrhaging and/or necrosis). Also ileum scrapings were collected and frozen until analysis.
Data Analysis:

(84) The score for Ileitis was based on microscopic lesion score of ileitis by histopathology, and the gross lesion severity score of the ileum.

(85) Lawsonia infection (colonization) was determined based on microscopic IHC score. qPCR was used to score for the presence of Lawsonia in rectal swabs and intestinal-mucosa scrapings, and for shedding in feces.

(86) Results

(87) Fecal Shedding:

(88) No significant differences were found between the groups at 14 days post challenge (dpc). However fecal shedding results at 20 dpc showed that the vaccinated pigs were shedding significantly less Lawsonia than the placebo group at this time (p=0.0134), indicating earlier recovery by the vaccinates.

(89) Colonisation:

(90) qPCR of ileal scrapings collected at 20 dpc showed significantly more colonization by Lawsonia in the group receiving the placebo vaccine (p=0.0094).

(91) Daily Weight Gain:

(92) Following challenge, daily weight gain was also significantly improved in the 4-way vaccinated pigs, as compared to group receiving the placebo vaccine (p=0.0337).

Conclusion

(93) Vaccination of pigs with the 4-way combination vaccine according to the invention, was effective in protecting against infection and signs of disease induced by a Lawsonia challenge infection.

(94) 4.4. PRRSV Efficacy

(95) The 4-way combination vaccine was prepared as described above, and comprised PRRSV from reconstitution of an ampule of PrimePac™ PRRS vaccine, shortly before vaccination.

(96) Experimental Outline

(97) The objective of this study was to evaluate the immunogenicity of the PRRS fraction of the combination vaccine according to the invention, by vaccination of three week old pigs, and challenge with virulent PRRSV 4.5 weeks later. Two groups of 25 healthy pigs each, negative for anti-PRRSV antibodies and PCV2 viremia, were vaccinated at three weeks of age by the intramuscular route with a 2 ml dose of the 4-way combination vaccine, or with a placebo (3-way) vaccine not comprising PRRSV. All antigens were present at estimated field dose levels. At approximately 7.5 weeks of age, the pigs were challenged intranasally with PRRS virus strain NADC-20. Throughout the post-challenge period, pigs were evaluated for clinical observations and clinical scores, and blood and nasal swab samples were collected. At 14 days post-challenge, all pigs were euthanized and necropsied. Gross lesions of the lung were scored and lung sections and lymph nodes collected for histopathology and immunohistochemistry (IHC). The results were evaluated to determine the efficacy of a single dose of combination vaccine for the reduction of respiratory disease, viremia and/or shedding of PRRSV following a challenge infection. Body weights were recorded at the time of vaccination, at challenge, and at end of test.
Details of the Experiment

(98) PRRSV challenge was administered intranasally with 2 mL per nare of diluted challenge material. The challenge virus was prepared shortly before administration and kept on ice before and during use. The total challenge dose was approximately 4.2 log.sub.10TCID.sub.50 per animal.

(99) The full 4-way vaccine comprised per 2 ml animal dose: 2.0 RP of Mhyo antigen; 7000 Elisa units of Lawsonia antigen/ml; 5.7 μg/ml of PCV2 Orf2 antigen; and 10{circumflex over ( )}5 TCID50 of PRRSV.

(100) Data Analysis

(101) Clinical observations of respiratory distress and lethargy were recorded immediately before and subsequently after the challenge. Clinical scores were indicated ranging from 0: normal, to 3: severe dyspnea and/or tachypnea and/or prominent abdominal breathing when stressed, whereby stress was induced by briefly inducing rapid movement of the pig.

(102) Lung scoring was applied according to Halbur et. al. (1995, Vet. Pathol., vol. 32, p. 648-660 [Appendix 7, Ref. 1]). Macroscopic lung lesions were given a score to estimate the percentage of the lung affected by pneumonia. Each lung lobe was assigned a number of points to reflect the approximate volume percentage of the entire lung represented by that lobe. Ten possible points (5 for dorsal, 5 for ventral) were assigned each to the right anterior lobe, right middle lobe, anterior part of the left anterior lobe, and caudal part of the left anterior lobe. The accessory lobe was assigned 5 possible points, and 27.5 possible points (15 for dorsal and 12.5 for ventral) were assigned to each of the right and left caudal lobes to reach a total of 100 possible points. Gross lung lesion scores were recorded as the number of points that reflect the approximate volume percentage of that lobe affected by PRRS associated pneumonia. The total lung lesion score for each lung was calculated as the sum of the gross lung lesion scores of all lobes.

(103) Lung samples were taken from each pig for histopathological examination, specifically: the tip of the left middle lobe, a piece of the accessory lobe, and the anterior part of the right caudal lobe. In addition, sections were taken from mediastinal and bronchial lymph nodes. The tissues were fixed in 10% neutral buffered formalin for histopathology and IHC.

(104) Further collections were serum samples, for detection of anti-PRRSV antibodies; nasal swabs, for detection of PRRSV nucleic acid by a quantitative RT-PCR; and tissue samples, for microscopic analysis and IHC.

(105) PRRSV samples were titrated on the MARC145 cell line. For each replicate, serial 10-fold dilutions were added to ten wells in a 96-well plate containing pre-formed cell monolayers, and the plates were incubated with 5% CO2 at 35-39° C. for five days. Next the plates were fixed and stained with an anti-PRRSV fluorescent antibody, and scored by IFT.

(106) Outcome variables for vaccination efficacy were evaluated by analysis of post-challenge period clinical observations and clinical scores, body temperature, body weights, nasal shedding and viremia as determined by PCR, as well as macroscopic and microscopic analysis of lung lesions at 14 days post-challenge. Respiratory disease was assessed by macroscopic lung lesion scores Secondary variables of efficacy by tissue analysis by histopathology and IHC, maximum amount of viremia, maximum amount of shedding, weight gain following challenge, clinical observations following challenge, and respiratory clinical scores following challenge.
Results

(107) The median of the lung lesion scores, in % of the lung involved, were: 20% for the placebo vaccine group, versus 7% for the 4-way vaccine group (p=0.0014).

(108) The histopathology of the three collected lung samples was scored on a scale of 0 (normal) to 4 (severe interstitial pneumonia). The maximal scores were: 3 for the placebo vaccine group, versus 2 for the 4-way vaccine group (p=0.0010).

(109) The lung sample immunohistochemistry was scored on a scale of 0 (no PRRSV-antigen positive cells) to 4 (>100 positive cells per tissue section). The maximum value of the scores of the three collected lung samples were 2 for the placebo vaccine group, versus 1 for the 4-way vaccine group (p=0.0006).

Conclusion

(110) Vaccination of pigs with the 4-way combination vaccine according to the invention, was effective in protecting against infection and signs of disease induced by a PRRSV challenge infection.