Vaccines containing swine pathogens for associated non-mixed use

Abstract

The present invention pertains to a combination of a first vaccine comprising non-replicating immunogen of porcine circo virus type 2 (PCV2) and non-replicating immunogen of Mycoplasma hyopneumoniae, and a second vaccine comprising live attenuated porcine reproductive and respiratory syndrome (PRRS) virus, for use in prophylactically treating an animal against an infection with porcine circovirus type 2, an infection with Mycoplasma hyopneumoniae and an infection with PRRS virus, by associated non-mixed administration of the first vaccine and the second vaccine to the animal. The invention also pertains to a kit-of-parts comprising the first and second vaccine and to a method of protecting an animal against such infections using these vaccines.

Claims

1. A method of prophylactically treating an animal against a porcine circo virus type 2 (PCV2) infection, a Mycoplasma hyopneumoniae infection, and a porcine reproductive and respiratory syndrome (PRRS) virus infection by concurrent associated non-mixed administration of a first vaccine and a second vaccine to the animal, wherein the first vaccine comprises a non-replicating immunogen of porcine circo virus type 2 and a non-replicating immunogen of Mycoplasma hyopneumoniae, and the second vaccine comprises a live attenuated porcine reproductive and respiratory syndrome virus (PRRS) vaccine and wherein the associated non-mixed administration occurs simultaneously and the first vaccine and the second vaccine are administered at separate application sites of the animal and wherein the associated non-mixed administration of the first vaccine and the second vaccine confers improved protection to the animal against the PCV2 infection when compared to the administration of the first vaccine alone in the absence of the concurrent non-mixed administration of the live attenuated PRRS vaccine.

2. The method of prophylactically treating the animal of claim 1, wherein the second vaccine is administered into the dermis of the animal.

3. The method of prophylactically treating the animal of claim 1, wherein the first vaccine further comprises a non-replicating immunogen of Lawsonia intracellularis which reconstitutes the first vaccine.

4. The method of prophylactically treating the animal of claim 3, wherein the non-replicating immunogen of Lawsonia intracellularis is added to the first vaccine within 24 hours before the associated non-mixed administration of the first vaccine.

5. The method of prophylactically treating the animal of claim 4, wherein the non-replicating immunogen of Lawsonia intracellularis comprises freeze-dried killed whole cells of Lawsonia intracellularis.

6. The method of prophylactically treating the animal of claim 1, wherein the first vaccine and the second vaccine are administered by a single dose.

7. The method of prophylactically treating the animal of claim 1, wherein the non-replicating immunogen of PCV2 is a recombinantly expressed protein encoded by the ORF2 gene of the PCV2.

8. The method of prophylactically treating the animal of claim 7, wherein the non-replicating immunogen of PCV2 is baculovirus expressed protein of the PCV2.

9. The method of prophylactically treating the animal of claim 1, wherein the non-replicating immunogen of Mycoplasma hyopneumoniae comprises killed whole Mycoplasma hyopneumoniae.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 depicts the titer of PRRSV versus the number of days post challenge for various vaccination experiments.

(2) FIG. 2 depicts the number of PCV2 virus copies in various organs after different treatments.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

(3) A vaccine is a pharmaceutical composition that is safe to administer to a subject animal, and is able to induce protective immunity in that animal against a pathogenic micro-organism, i.e. to induce a successful prophylactic treatment as defined here below.

(4) Non-replicating immunogen of a pathogen is any substance or compound corresponding to the pathogen, other than the live replicating pathogen as a whole (either in wild type of attenuated form), against which pathogen an immunological response is to be elicited, such that the corresponding virulent pathogen 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 are ultimately at least partly neutralized. Typical examples of non-replicating immunogens are killed whole pathogens (which term includes these pathogens in lysed form) and subunits of these pathogens such as capsid proteins, surface expressed molecules (for example recombinantly expressed proteins or lipopolysaccharides) and excreted molecules such as toxins.

(5) A live attenuated pathogen is a viable, replication competent form of the pathogen having reduced virulence. The process of attenuation takes an infectious pathogen and alters it so that it becomes harmless or less virulent, typically by either multiple passages of the pathogen through cell systems or by genetically modifying the pathogen.

(6) Prophylactic treatment against an infection with a pathogen is aiding in preventing, ameliorating or curing an infection with that pathogen or a disorder arising from that infection, resulting from a post treatment challenge with the pathogenic pathogen, in particular to reduce its load in the host after such challenge and optionally to aid in preventing or ameliorating one or more clinical manifestations resulting from the post treatment infection with the pathogen.

(7) Associated non-mixed administration of vaccines, also referred to as concurrent administration, is the administration of these vaccines separately, thus not mixed before administration, to the target animal, but within a time frame such that immunological interference may occur, typically within 24 hours. Examples of associated non-mixed use are the simultaneous administration at separate application sites in the target animal, and the administration at the same or separate application sites but at different times, typically separated by 1-6 hours.

(8) Simultaneous administration of vaccines means the administration at exactly the same time or at least within a time frame of 1 hour, preferably within 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 minutes or even a time frame of 1 minute.

(9) Single dose administration of a vaccine for use in prophylactically treatment means that in order to arrive at protective immunity, the vaccination does not need to be boosted with a second administration of the vaccine. 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, i.e. a successful prophylactic treatment as defined here above, may be obtained.

EMBODIMENTS OF THE INVENTION

(10) In an embodiment of the combination of a first and a second vaccine for use according to the invention, the associated non-mixed administration occurs simultaneously. This embodiment has the advantage that an animal needs to be handled only once for applying both the vaccines.

(11) In another embodiment of the present combination of vaccines, the second vaccine is administered into the dermis of the animal. Such administration of at least the PRRS vaccine has shown to be safe and efficacious while at the same time being less stressful for the animal when compared to intramuscular administration. Regarding administration into the dermis (also referred to as intradermal administration), although such administration is often carried out using a needle-less vaccination device such as the IDAL® vaccinator (available from MSD Animal Health, Boxmeer, The Netherlands), “intradermal” administration per se should not be equated with “needle-less” administration. The World health Organization in its Aug. 27, 2009 paper titled “Intradermal Delivery of Vaccines; A review of the literature and the potential for development for use in low- and middle-income countries” indeed clearly indicates that “needle-less” vaccination does not necessarily mean “intradermal” vaccination (see Table 1, Page 3 of the review). Only when a needle-less device is “configured for intradermal vaccination”, then a vaccine may indeed be delivered (at least partly) into the dermis. Otherwise the vaccine may be delivered subcutaneous or intramuscularly in its entirety.

(12) In yet another embodiment of the combination of the first and a second, the first vaccine comprises non-replicating immunogen of Lawsonia intracellularis. In this embodiment the novel combination of vaccines is capable of providing protection against four major swine pathogens by using just one vaccination protocol. The immunogen of Lawsonia intracellularis in an embodiment is combined with the immunogen of PCV2 and Mycoplasma hyopneumoniae within 24 hours before administration, preferably within 6 hours before administration. Combining the antigens right before administration provides more freedom to choose the excipients since long-term stability, although known for many pharmaceutical compositions, even for combination vaccines including PCV2 ORF2 antigen (for example Porcilis® PCV M Hyo, available from MSD Animal Health), as such is known, might still not be straightforward to achieve, at least not for any and all pharmaceutically acceptable carrier compositions. In a further embodiment the immunogen of Lawsonia intracellularis is added to the vaccine in the form of a composition comprising freeze-dried killed whole cells of Lawsonia intracellularis.

(13) In still another embodiment of the novel combination of a first and a second vaccine, the first and second vaccine are administered by a single dose. It was found that a single dose administration led to an effective vaccination against all pathogens. This provides for a very convenient and economical way to protect animals against these pathogens.

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

(15) In again another embodiment, the non-replicating immunogen of Mycoplasma hyopneumoniae comprise killed whole Mycoplasma hyopneumoniae. Such Mhyo antigen is relatively easy to produce and has a good track record of efficacy in the everyday swine industry practice.

(16) The invention will now be explained in more detailed, using the following examples.

EXAMPLES

(17) Study 1: Safety and Protection Against PRRSv Challenge by the Associated Non-Mixed Use of Porcilis® PCV MHyo with Lawsonia Immunogen Reconstituted Therein, and Porcilis® PRRS.

(18) This study was performed to evaluate the safety and efficacy of Porcilis® PRRS in associated non-mixed use with freeze-dried killed whole cells of Lawsonia intracellularis (also referred to as “Lawsonia freeze-dried”) reconstituted in Porcilis® PCV M Hyo. The study was performed with sixty PRRSV antibody negative piglets, evenly distributed over 5 groups of 12 piglets each. Four groups were vaccinated at the age of 3 weeks with either 2 mL of Porcilis® PRRS intramuscularly (IM; groups 1 and 3) or 0.2 mL of Porcilis® PRRS intradermally (ID; groups 2 and 4). Piglets from groups 1 and 2 were vaccinated with Lawsonia freeze-dried reconstituted in Porcilis® PCV M Hyo (2 mL; IM) within a couple of minutes after receiving the PRRS vaccine. Piglets in group 5 (also 12 piglets) were not vaccinated and served as non-vaccinated challenge controls. Four weeks after vaccination all piglets were challenged by the intranasal (IN) route using a heterologous virulent PRRSV Type 1 strain (Isolate 2).

(19) After challenge infection, the following parameters were measured: clinical signs, rectal temperatures, and PRRSV viremia and serology.

(20) No clinical signs related to PRRSV challenge were observed in any of the groups. Rectal temperatures were measured for 12 days starting on one day before challenge until 10 days post challenge. No significant differences were observed between any of vaccinated groups. The rectal temperatures of all PRRS vaccinated groups were lower compared to the control group at several time points post challenge. There was neither a statistical significant difference between IM or ID vaccinated groups, nor between groups treated with Porcilis® PRRS alone or Porcilis PRRS in combination with Lawsonia freeze-dried reconstituted in Porcilis® PCV M Hyo.

(21) A significant reduction of PRRSV viremia compared to the non-vaccinated controls was shown if piglets were vaccinated with Porcilis® PRRS and if piglets were vaccinated with Porcilis® PRRS combined with vaccination of Lawsonia freeze-dried reconstituted in Porcilis® PCV M Hyo (FIG. 1). No significant difference was detected between single and non-mixed associated use of Porcilis® PRRS administered via the IM or ID route.

(22) All vaccinated groups showed an anti-PRRSV antibody titer 4 weeks post vaccination at the day of challenge, while the control group did not. Four weeks post challenge all groups show an anti-PRRSV antibody titer and the titer in the vaccinated groups is higher than on the day of challenge. No statistical significant difference was detected between single and non-mixed associated use of Porcilis® PRRS administered via the IM or ID route.

(23) In conclusion, the concurrent administration of Lawsonia freeze-dried reconstituted in Porcilis® PCV M Hyo had no negative effect on Porcilis® PRRS vaccination via either the IM or ID route. It was unexpected that the associated use of the triple Lawsonia-PCV-Mhyo vaccine had no negative effect at all on the efficacy of the PRRS vaccine (see study 5).

(24) Study 2: PCV2 Efficacy Study of the Associated Non-Mixed Use of Porcilis® PCV MHyo with Lawsonia immunogen reconstituted therein, and Porcilis® PRRS.

(25) A total of 45 piglets with low to moderate maternally derived PCV2 antibody titers (<5.5 log.sub.2), no PCV2 viral load and no PRRSV antibody titers were allotted to 3 treatment groups: 3 groups of 15 piglets each. The piglets were vaccinated once at 3 weeks of age. The piglets of group 1 and 2 were vaccinated with Lawsonia FD dissolved in Porcilis PCV M Hyo, both as a single dose (intramuscular route; IM) and the piglets from group 1 were also vaccinated at about the same time with Porcilis PRRS (via both the intramuscular and intradermal route; IM and ID). The animals from group 3 remained unvaccinated (control).

(26) At 5 weeks of age (2 weeks post vaccination), each animal was challenge infected using wild-type PCV2 challenge virus applied intranasally. 21 days following challenge, all animals were necropsied and inguinal lymph node, tonsil and lung were sampled for the detection of PCV2.

(27) Serum samples were taken at the time of vaccination, one day before challenge, at 2 and 3 weeks after challenge. Samples were examined for PCV2 antibodies and viral nucleic acid. Faecal and nasal swabs taken one day before challenge, at 2 and 3 weeks after challenge were examined for viral nucleic acid.

(28) Serology showed that both vaccinated groups developed anti-PCV2 antibodies to a comparable level (6 log 2) at the time of challenge, whereas the antibodies in the control group had declined significantly. After challenge, antibodies in the vaccinated groups rose to about 13 log 2 measured 3 weeks post challenge. In the control group the level was 5 log 2 at that time. After challenge, viral nucleic acid could be detected in the control animals in the serum, nasal and faecal swabs. The level was substantially lower in the vaccinated animals. In the faecal samples, the animals vaccinated with both vaccines simultaneously had no detectable levels of viral nucleic acid, suggesting that this group was best protected against the PCV2 challenge. This surprising finding of a possible improved protection was confirmed by the qPCR measurements in the tissue, as can be seen in FIG. 2 (representing the PCV2 copies found in Tonsils, Lung and Inguinal lymph node respectively going from left to right). The lowest values for viral load were consistently found in the animals that received both vaccines (left column), even lower than for the animals that received the commercial PCV vaccine Porcilis® PCV M Hyo (middle columns), which were still considerably lower than control (right columns).

(29) Study 3: Efficacy of Freeze-Dried Lawsonia Reconstituted in Porcilis® PCV M Hyo Used Concurrently with Porcilis® PRRS in Pigs, Against Lawsonia Challenge.

(30) For this study seventy-five 3-week-old piglets, divided over three groups, were used.

(31) Group 1 was vaccinated with freeze-dried Lawsonia antigen reconstituted in Porcilis® PCV M Hyo (2 ml IM) administered at the same time (within 1 hour) with Porcilis® PRRS (2 ml IM+0.2 ml ID), group 2 was vaccinated with freeze-dried Lawsonia antigen reconstituted in Porcilis PCV M Hyo (2 ml IM) and group 3 was left as unvaccinated controls. At 7 w of age (4 weeks after vaccination) all pigs were challenged orally with homogenized Lawsonia infected intestinal mucosa. After challenge the pigs were daily observed for clinical signs. At regular times before and after challenge serum blood (for serology) and faeces (for qPCR) samples were collected. Three weeks after challenge the pigs were euthanized and post-mortem examined. The intestines were checked macroscopically for Lawsonia intracellularis infection and ileum samples were collected for qPCR and (immuno-)histological scoring.

(32) On day of vaccination most pigs were either seronegative to Lawsonia whereas the others had a low to moderate antibody titer. After the vaccination the titer of groups 1 and 2 showed a similar increase in the antibody titre, whereas the controls showed a slight decrease and remained at a low level. On the day of vaccination the pigs had low to moderate maternally derived PCV antibody titers. After the vaccination the PCV antibody titer of groups 1 and 2 showed a similar increase whereas the controls showed a decrease of maternal antibodies. On day of vaccination the pigs were seronegative to Mhyo and PRRSv. After vaccination groups 1 and 2 showed a similar antibody response to Mhyo whereas the control group 3 remained seronegative until the end of the trial. Group 1 responded to the PRRS vaccination whereas groups 2 and 3 remained seronegative until challenge.

(33) The results for the different parameters after Lawsonia challenge are summarized in Table 1 below (PCR data in log.sub.10 pg DNA/μl on day 21 post challenge).

(34) TABLE-US-00001 TABLE 1 PCR on PCR on Macroscopic Immunohistological Group faeces mucosa ileum scores ileum scores 1 0.00 0.22 40 0.6 2 0.60 0.41 84 0.8 3 1.72 1.15 161 5.0

(35) From the results it can be concluded that Lawsonia freeze-dried antigen dissolved in Porcilis® PCV M Hyo administered concurrently with Porcilis® PRRS (group 1), induced significant protection against Lawsonia infection 4 w after vaccination. This was demonstrated by a significant reduction in shedding, infection (qPCR ileum mucosa), macroscopic ileum lesion scores as well as microscopic ileum lesion scores. No negative influence of Porcilis® PRRS on the Lawsonia efficacy was observed. On the contrary, the associated mixed use group tended to be better for the above mentioned parameters.

(36) Study 4: Efficacy of Associated Non-Mixed Use of Porcilis® PCV M Hyo with Lawsonia Freeze-Dried Reconstituted Therein, with Porcilis® PRRS in SPF Piglets at Three Weeks of Age Against M. Hyopneumoniae Challenge Infection 4 Weeks after Vaccination.

(37) Hundred pigs from a M. hyopneumoniae and PRRS virus free herd, 3 weeks of age, were used for this study in groups of 25 animals each. One group was vaccinated IM with Porcilis® PCV M Hyo+Lawsonia freeze-dried and concurrently with Porcilis® PRRS (IM+ID; see here above); one group was vaccinated with Porcilis® PCV M Hyo and at about the same time (within 1 hour) with Porcilis® PRRS (IM+ID) and one group with Porcilis® PCV M Hyo; the fourth group was not vaccinated and served as challenge control. Four weeks after vaccination all animals were infected with a virulent M. hyopneumoniae strain and three weeks later all animals were post-mortem investigated for lung lesions.

(38) All animals were serologically negative for M. hyopneumoniae at vaccination and non-vaccinated animals remained serologically negative until challenge infection. Vaccinated groups showed similar serological responses against M. hyopneumoniae four weeks post vaccination and three weeks post challenge infection.

(39) A significant reduction in lesion score was observed for all vaccinated groups compared to non-vaccinated animals. Porcilis® PCV M Hyo+Lawsonia freeze-dried, vaccinated concurrently with Porcilis® PRRS had a reduction of 90%, Porcilis® PCV M Hyo vaccinated concurrently with Porcilis® PRRS had a reduction of 95%. Porcilis® PCV M Hyo induced 100% reduction in median M. hyopneumoniae lesions. The differences between the vaccinated groups were not significant.

(40) It can be concluded that associated non-mixed use of Porcilis® PCV M Hyo alone or in combination with freeze-dried Lawsonia antigen and Porcilis® PRRS is efficacious in reducing M. hyopneumoniae-induced lung lesions.

(41) Study 5: The Effect of the Use of an Inactivated PCV Vaccine on the Efficacy of a Live PRRS Vaccine.

(42) In this experiment the effect of mixing a first vaccine containing non-replicating immunogen of porcine circo virus type 2 (CircoFLEX, Boehringer Ingelheim) and a second vaccine comprising live attenuated porcine reproductive and respiratory syndrome virus (Ingelvac PRRS, Boehringer Ingelheim) was examined. Since the survival of the live PRRS virus is the main critical parameter when mixing these two vaccines, the survival of the PRRS virus after an incubation of 1, 2 and 4 hours was examined.

(43) On day −1, MA104 cells were seeded at 10.sup.5 cells/ml and put in wells at 225 μl per well. These cells were kept at 37° C. in air with a 5% carbon dioxide content. On day 0 Ingelvac PRRS was prediluted in PBS to a titer of 5.3 log 10 TCID50/ml. This mixture was either diluted with PBS (control) or diluted with CircoFLEX (two different batches) to a final titer of 5.0 log 10 TCID50/ml. The final mixtures were incubated at room temperature for 1, 2 and 4 hours to mimic practical circumstances in a stable when vaccinating with a freshly mixed combination vaccine. After this, 25 μl of each of the incubated mixtures was incubated in the MA 104 cells for 7 days at 37° C. and 5% carbon dioxide to grow the PRRS virus. At day 7 the PRRS virus was read out. The data are given here below in Table 2.

(44) TABLE-US-00002 TABLE 2 PRRS titer in 10 log TCID50/ml after incubation with PCV 1 h 2 h 4 h Sample Start incubation incubation incubation PRRS control 5.0 4.90 4.80 4.64 PRRS + PCV batch 1 5.0 3.90 3.90 3.15 PRRS + PCV batch 2 5.0 3.85 3.55 3.55

(45) As can be seen from the table, the PRRS virus loses viability by the short incubation with the PCV vaccine at room temperature. Vaccine efficacy will be negatively influenced by a loss of vaccine titer.