Swine vaccine

Abstract

The invention pertains to a vaccine comprising in combination non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans and live attenuated PRRS virus, and a pharmaceutically acceptable carrier, for use in a method for prophylactic treatment of a swine against an infection with Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans and PRRS virus, wherein the vaccine is administered in a single dose with regard to the treatment against an infection with PRRS virus.

Claims

1. A method for prophylactically treating a swine that is seronegative for PRRS virus against an infection with Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans, and PRRS virus comprising administering a vaccine comprising a pharmaceutically acceptable carrier and a non-replicating immunogen of an Erysipelothrix rhusiopathiae, a non-replicating immunogen of a porcine parvo virus, a non-replicating immunogen of a Leptospira interrogans, and a live attenuated PRRS virus; wherein the vaccine is administered in a single dose with regard to the treatment against an infection with PRRS virus to said swine that is seronegative for PRRS virus.

2. The method of claim 1, wherein the non-replicating immunogen of the Erysipelothrix rhusiopathiae, the non-replicating immunogen of the porcine parvo virus, and the non-replicating immunogen of the Leptospira interrogans are inactivated pathogens of the Erysipelothrix rhusiopathiae, the porcine parvo virus, and the Leptospira interrogans, respectively.

3. The method of claim 1, wherein the Leptospira interrogans pathogen comprises bacteria of the serogroup Pomona.

4. The method of claim 3, further comprising a bacterium selected from the serogroup consisting of a Tarassovi, an Australis, a Grippotyphosa, an Icterohaemorrhagiae, and a Canicola.

5. The method of claim 4, wherein the bacterium of the Australis is from a serovar Bratislava.

6. The method of claim 1, wherein the vaccine is administered parenterally.

7. The method of claim 1, wherein the vaccine is administered intramuscularly.

8. A kit for a swine that is seronegative for PRRS virus comprising a combination vaccine and a set of instructions; wherein the combination vaccine comprises a first vaccine comprising a pharmaceutically acceptable carrier and a non-replicating immunogen of an Erysipelothrix rhusiopathiae, a non-replicating immunogen of a porcine parvo virus, and a non-replicating immunogen of a Leptospira interrogans and a second vaccine comprising a freeze-dried live attenuated PRRS virus; and wherein the set of instructions state that: (i) the second vaccine is to be mixed with the first vaccine to form the combination vaccine; and (ii) the combination vaccine is to be administered to said swine that is seronegative for PRRS virus in a single dose with regard to the treatment against an infection with PRRS virus.

9. The kit of claim 8, wherein the non-replicating immunogen of the Erysipelothrix rhusiopathiae, the non-replicating immunogen of the porcine parvo virus, and the non-replicating immunogen of the Leptospira interrogans are inactivated pathogens of the Erysipelothrix rhusiopathiae, the porcine parvo virus, and the Leptospira interrogans, respectively.

Description

EMBODIMENTS OF THE INVENTION

(1) In an embodiment, the non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans are inactivated pathogens of Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans respectively. For example by simply killing the pathogens, a simple way is provided to have (all) immunogens available in a non-replicating form. Although any subunit vaccine might also be suitable for use in the present vaccine, by having the inactivated pathogens available, the relevant immunogens are present per se, and in a way (similar to the way wherein) they are present in the live, naturally occurring pathogen.

(2) In a further embodiment, the Leptospira interrogans pathogen comprises bacteria of the serogroup Pomona, which is the most important swine leptospira pathogen. Optionally bacteria of at least one the serogroups Tarassovi, Australis, Grippotyphosa, lcterohaemorrhagiae and Canicola, are also present, wherein the bacteria of the serogroup Australis in particular are bacteria of the serovar Bratislava.

(3) As indicated here above, also in a further embodiment of the vaccine for use in a method for prophylactic treatment of a swine against an infection with Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans and PRRS virus, the non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans are inactivated (e.g. killed) pathogens. In particular, the Leptospira interrogans pathogen comprises bacteria of the serogroup Pomona, and optionally bacteria of at least one the serogroups Tarassovi, Australis, Grippotyphosa, lcterohaemorrhagiae and Canicola, wherein the bacteria of the serogroup Australis in particular are bacteria of the serovar Bratislava.

(4) In still another embodiment the vaccine is administered parenterally, i.e. administered elsewhere to the body than the mouth and alimentary canal. The vaccine can for example be administered intramuscularly.

(5) As stated here above, the invention also pertains to a method to constitute the combination vaccine wherein the method comprises mixing a first composition comprising live attenuated PRRS virus with a second composition comprising the immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus, and Leptospira interrogans in the pharmaceutically acceptable carrier. In a further embodiment the first composition comprises freeze-dried PRRS virus, for example in a stabiliser such as known from Porcilis® PRRS. The mixing takes preferably place at most 24 hours before the vaccine is administered to the animal, or at most 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or ½ an hour before administration is due to take place.

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

EXAMPLES

Experiment 1: The Combination of Non-Live Immunogens of Ery, Parvo and Lepto

(7) In a first experiment, the safety and efficacy of the combination of non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans was assessed. For such a combination of non-live antigens, in particular safety is an important aspect. This is because of the relatively high amount of antigens that is typically used in such a (non-live) vaccine, with the risk of an endotoxic shock, in particular when the immunogens comprise inactivated pathogens of gram-negative bacteria (which bacteria the cells walls comprise endotoxic lipopolysaccharides).

(8) The study was designed as a historically controlled field study. It was conducted on a farm with a history of increased abortion rates, associated with leptospira infection. High antibody titres against serogroup Pomona were found in a number of animals indicating a recent Leptospira serogroup Pomona infection. The farm was negative for PRRS virus infection.

(9) At the start of the study, all breeding sows and replacement gilts on the farm were vaccinated in the neck muscle twice, with an interval of 4 weeks, with a vaccine containing non-replicating immunogens of Erysipelothrix rhusiopathiae (formalin inactivated bacteria, strain M2), porcine parvo virus (BPL inactivated virus, strain 014) and Leptospira interrogans (BPL inactivated bacteria of the serogroups Pomona, Tarassovi, Australis (serovar Bratislava), Grippotyphosa, lcterohaemorrhagiae and Canicola) in an aqueous dl-α-tocopherol-acetate based adjuvant. This vaccine composition is denoted as “Ery/parvo/lepto”. The animals were re-vaccinated with Ery/parvo/lepto in the second week of every following lactation. New replacement gilts were vaccinated using the same schedule.

(10) The reproductive performance of the pigs in the study was monitored. The farrowing results and relevant breeding data were collected to determine if vaccination had any effect on the incidence of abortion. The results obtained during the study period were compared to the historical data. Since parvovirus and Ery also have a significant negative impact on reproduction (parvo may kill the foetuses and Ery may cause abortion), a substantial improvement in this respect is a good indication that the vaccine is also effective against these pathogens.

(11) No adverse events were reported during the study, indicating that the vaccine was safe. Regarding efficacy, the percentage abortions between the study period and the pre-study period were compared, taking into consideration the effect of season. After vaccination a rapid improvement on abortions was manifested, from 12.6% pre-vaccination, down to 0.5% after vaccination had started. This is a decrease in abortion rate of 96%. The total number of abortions on the farm decreased from 55 to 6. The trend of having a low number of abortions at the farm was stable after the vaccination program started and in the following season. The frequency of abortions remained low, viz. 0.6%. The safety and efficacy of the vaccine was confirmed in another study reported in Porcine Health management in November 2015 (Porcine Health Management 2015 1:16/DOI: 10.1186/s40813-015-0011-0). This study focused on viraemia, renal infection and urinary shedding.

(12) It may thus be concluded that the vaccine comprising in combination non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans is safe and efficacious against all of these pathogens. Adding a live PRRS virus to this vaccine, which (inherently) contains a relatively low amount of antigen when compared to the inactivated pathogens of ery/parvo/lepto, it is not expected that there is a negative effect on the efficacy of the ery/parvo/lepto immunogens. However, safety might be compromised and the effect of the inactivated immunogens on the live PRRS virus is not known and needs to be assessed. This was done in another experiment, of which the results are indicated here below.

Experiment 2: The Effect of the EPL Combination Vaccine on Live PRRS Vaccine

(13) The objective of this study was to evaluate the vaccine potential in six-week-old SPF piglets of a live attenuated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 vaccine (Porcilis PRRS) reconstitution in a vaccine comprising in combination non replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans.

(14) The study was performed with forty PRRSV antibody negative piglets, evenly distributed over 2 groups. Groups 1 and 2 were vaccinated once intramuscularly (IM) with 2 ml Porcilis PRRS diluted to 4.5 log.sub.10 TCID.sub.50 per dose (this dose represents a dose slightly above minimal dose of 4.0 log.sub.10 TCID.sub.50 for Porcilis® PRRS). Group 1 received the PRRS vaccine diluted in an experimental Ery/Parvo/Lepto combination vaccine as indicated here above in experiment 1. The vaccines were mixed at a temperature of 25° C. and left for at least 1 hour before actual administration to the animals. Group 2 received the live attenuated virus diluted in Diluvac Forte, and thus de facto received the regular vaccine Porcilis® PRRS slightly above its minimal dose. This group in fact served as a positive control.

(15) The following parameters were measured: local and systemic reactions, PRRSV viremia and PRRSV serology. This way, it can be established whether the vaccine strain is able to normally replicate in the host animal, which is a requisite for induction of an effective immune response. From the day of vaccination onwards until the end of the experiment (28 days post vaccination, dpv), all piglets were observed daily for clinical signs. Blood samples were collected just before vaccination, and 2 and 4 weeks post vaccination. Serum samples obtained at time points after vaccination were used to determine PRRSV-specific viremia and serological responses.

(16) Veterinary examination showed that all piglets were healthy on the day of vaccination. No adverse events which could be related to the vaccination were found. One piglet in group 2 was found dead on the evening post blood sampling at 14 days post vaccination. Necropsy was performed and cause of death was most likely a hematoma in the neck, due to blood sampling.

(17) The PRRS virus serology results are indicated in Table 1. The PRRS virus viraemia results are indicated in Table 2.

(18) TABLE-US-00001 TABLE 1 PRRSV serology results: percentage of positive animals, 0, 14 and 28 dpv Group 0 dpv 14 dpv 28 dpv 1 0 80 100 2 (positive control) 10 100 100

(19) TABLE-US-00002 TABLE 2 PRRSV viraemia results: percentage of positive animals, 14 and 28 dpv Group 14 dpv 28 dpv 1 70 100 2 (positive control) 100 100

(20) It appeared that the serology as well as the viraemia results for PPRS virus of the combination vaccine was comparable to that of the positive control (Porcilis® PRRS as a stand alone vaccination), both at the minimum protective dose as well as the higher dose. These data thus indicate that the PRRS virus is able to survive incubation in a vaccine comprising in combination non replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans. Based on these data, it is understood that a (two-way) combination vaccine comprising non-replicating immunogens of Erysipelothrix rhusiopathiae and live attenuated PRRS virus will also be safe and effective. To this vaccine, non-replicating immunogens of porcine parvo virus may be added to provide also protection against an infection with porcine parvo virus. Such a three-way vaccine is also understood to be safe and effective based on the present results. This all provides a number of different vaccines which enable to help in providing the following vaccination schemes for swine, specifically directed at enhancing reproductive performance of these animals: a basic vaccination, for example a prime-boost vaccination, of an animal before its first gestation (typically between 20 and 30 weeks of age) with a combination vaccine comprising non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans, wherein, if a prime-boost scheme is used, either the prime or boost vaccine is combined with live attenuated PRRS virus in line with the present invention (the PRRS component of the vaccine in any case being used as a single dose vaccine). A prime-boost vaccination could for example take place at 20 and 24 weeks age. This basic vaccination could then be repeated yearly against ery/parvo/lepto/prrs infection with a single dose of the combination vaccine according to the present invention, or with a combination vaccine wherein, depending on the circumstances, one or more of the antigens are not present (for example no leptospira antigens if a farm is not expected to be infected with any leptospira bacteria). Regarding PRRS, depending on the PRRS infection pressure on the farm, vaccination could be repeated every 6 months (or even every 3 months) if deemed necessary. Such a re-vaccination at a shorter interval could be done with a combination vaccine comprising next to the live attenuated PRRS virus, non-replicating immunogens of Erysipelothrix rhusiopathiae. For leptospira and parvo virus such a short term re-vaccination is typically not regarded as necessary since leptospira and parvo vaccines may have a duration of immunity of about a year in swine.

(21) In conclusion, it can be said that the safety as well as the efficacy of the novel combination vaccine is sufficient. Additional serology data (in particular for porcine parvo virus and leptospira bacteria) and/or challenge data (in particular for PRRS virus and Erysipelothrix rhusiopathiae) may be used to confirm the efficacy of the new combination vaccine for each of the corresponding pathogens.

Experiment 3: Serology Results of the EPL/PRRS Combination Vaccine

(22) The objective of this experiment was to confirm the serological response of associated mixed use of a Ery+Parvo+Lepto vaccine (Porcilis® Ery+Parvo+Lepto) with a live PRRS vaccine (Porcilis® PRRS) compared to the separate vaccines. Porcilis® PRRS normally is administered once whereas the basic vaccination of Porcilis® Ery+Parvo+Lepto (EPL) consists of two vaccinations with a 4-week interval. Thus Porcilis® PRRS can be co-administered during EPL primary vaccination or during the EPL booster vaccination. Both mixing options have been tested in this study.

(23) For this study sixty-four 20 week-old pigs (sero-negative for all vaccine components) were used and allotted to 4 groups of 16 animals each. Group 1 was vaccinated with Porcilis EPL+Porcilis PRRS at 20 w of age and with Porcilis EPL at 24 w of age. Group 2 was vaccinated with Porcilis EPL at 20 w of age and with Porcilis EPL+Porcilis PRRS at 24 w of age. Group 3 was vaccinated with Porcilis EPL at 20 w of age and at 24 w of age. Group 4 was vaccinated with Porcilis PRRS at 20 w of age. During one 20 w after vaccination blood was sampled regularly and the sera were tested in the respective serological tests.

(24) The test was valid if the animals are seronegative at the start of the study and if at least 10 animals in each control group remains sero-negative for the component omitted from the vaccinations i.e. group 4 negative for Ery, parvo and Leptospira and group 3 negative for PRRS. These criteria were fulfilled. The overall results per antigen are described here below.

(25) PRRS Serology (Using the PRRS X3 Test, IDEXX)

(26) At the start of the study most animals were seronegative. The responses after the associated mixed use vaccinations (group 1 and 2) were at least as good as for Porcilis PRRS alone (group 4), whereas the Porcilis Ery+parvo+Lepto alone group 3 remained at a low level. Therefore, it can be concluded that the associated mixed use at 20 w or at 24 w of age had no negative effect on the PRRS component of Porcilis PRRS.

(27) Ery Serology (Using the CIVTEST Suis SE/MR, Hipra)

(28) At the start of the study most animals were seronegative. The responses after the associated mixed use vaccinations (group 1 and 2) were at least as good as for Porcilis Ery+Parvo+Lepto alone (group 3), whereas the Porcilis PRRS alone group 4 remained at a low level. Therefore, it can be concluded that the associated mixed use at 20 w or at 24 w of age had no negative effect on the Ery component of Porcilis Ery+Parvo+Lepto.

(29) Parvo Serology (Using an in House HI Test and the INgezim PPV Test, Ingenasa)

(30) Although the HI test is often used to detect post-infection Parvo antibodies it is less suitable for post-vaccination serological studies because after vaccination many non-responders are found. Therefore, this antigen was tested in the HI test as well as in a more sensitive commercially available ELISA.

(31) At the start of the study most animals were seronegative in both tests. The responses after the associated mixed use vaccinations (group 1 and 2) were at least as good as for Porcilis Ery+Parvo+Lepto alone (group 3), whereas the Porcilis PRRS alone group 4 remained at a low level. Therefore it can be concluded that the associated mixed use at 20 w or at 24 w of age had no negative effect on the Parvo component of Porcilis Ery+Parvo+Lepto.

(32) Lepto Serology (Using in House Serogroup Specific ELISA's)

(33) The MAT (micro agglutination) test is generally used to detect post-infection Leptospira antibodies. However, this test mainly measures IgM antibodies which are shortly lived and predominantly induced after primary vaccination, whereas mainly IgG antibodies are induced after a booster vaccination. In addition, some serotypes do hardly respond in the MAT test which also makes this test less suitable for serological studies. Because IgG has been implicated in protection and because the MAT test is less suitable, serotype specific antibody ELISA's were in-house developed and validated. These serotype specific inhibition ELISA's were used to measure serotype specific IgG responses in this study.

(34) At the start of the study all animals were seronegative. The responses after the associated mixed use vaccinations (group 1 and 2) were at least as good as Porcilis Ery+Parvo+Lepto alone (group 3), whereas the Porcilis PRRS alone group 4 remained at a low level. Therefore, it can be concluded that the associated mixed use at 20 w or at 24 w of age had no negative effect on any of the Lepto components of Porcilis Ery+Parvo+Lepto. In addition, the decline in antibody titre over time gave similar profiles suggesting that also the duration of immunity is not influenced.

(35) In conclusion, the antibody responses after associated mixed use of Porcilis PRRS and Porcilis Ery+Parvo+Lepto were at least as high as after vaccination with either vaccine alone and therefore, it can be concluded that the take of the vaccines and the level of immunity are not negatively influenced by the associated mixed use. Likewise, the decline in antibody titre over time showed similar profiles indicating that the duration of immunity for all vaccine components also is not negatively influenced by the association.

Experiment 4: PRRS Efficacy at a Minimum Dose in a Combination Vaccine with Ery+Parvo+Lepto

(36) The objective of this experiment was to evaluate the efficacy of a live attenuated PRRSV Type 2 vaccine (Prime Pac® PRRS) reconstituted at the minimal dose of 4.0 log 10 TCID50/animal in an EPL vaccine.

(37) Sixty-six 5-week-old piglets seronegative for PRRSV were included in this study. The piglets were vaccinated with a minimal dose of Prime Pac® PRRS reconstituted in either an EPL formulation (group 1; one hour of waiting time at 25° C. between reconstitution and vaccination) or in Diluvac Forte (group 2), intramuscularly in the right side of the neck. Piglets in group 3 were vaccinated intramuscularly in the right side of the neck with 2 ml of the same EPL formulation and served as PRRSV-challenge controls. At 4 weeks post vaccination the piglets were challenged with a dose of 5.0 log 10 TCID50 of a virulent PRRSV Type 2 strain, Nebraska-1, by the intranasal (IN) route, 1 ml per nostril. Blood samples from the piglets were taken at day of vaccination, day of challenge, 5, 7, 10, 14 and 28 days post challenge. Rectal temperatures were measured on 1 day before challenge, just before challenge, 4 hours post challenge, and thereafter daily from 1 until 10 days post challenge. Bodyweight were measured on the day before challenge, 9 and 25 days post challenge. On 10 days post challenge 11 pigs per group were euthanized and observed for lung lesions. On 28 days post challenge the remaining piglets were euthanized.

(38) Body Temperature and Body Weight

(39) With regard to body temperature, in each of the vaccine groups the temperature was lower than in the control group 3. It was also found that there is no difference between the mixed use group 1 and the Prime Pac® PRRS only group (group 2). With regard to the body weight, the vaccinated animals grew faster than the unvaccinated animals in group 3. It was also found that the animals in the mixed use group 1 grew at a similar rate as the animals in the Prime Pac® PRRS only group (group 2).

(40) Lung Lesions Scores

(41) Half of the animals, in total 11 animals per group, were euthanized at 10 days post challenge to observe their lungs for PRRS related pneumonia. Table 3 shows per group the mean estimated percentage of the lung affected with visible pneumonia. From the table it can be concluded that in general the PRRSV challenge strain caused on average hardly any lung lesions. Nevertheless, at 10 days post vaccination, the vaccinated animals showed fewer lung lesions than the controls. Overall, not much difference was seen between the mixed use group and the Prime Pac® PRRS only group.

(42) TABLE-US-00003 TABLE 3 Mean lung lesion scores per group, 10 dpc Group 10 dpc PrimePac + EPL 0 PrimePac 0.2 Control 0.85
Serological Response

(43) On the day of vaccination all animals were seronegative for PRRSV, demonstrating that PRRSV negative animals were used for this study. Animals in the unvaccinated control group remained seronegative until the day of challenge (i.e. 4 weeks post vaccination). In contrast, on the day of challenge an antibody response was measured for the vaccinated groups, indicating that the animals in both the mixed use group 1 and the Prime Pac® PRRS only group (group 2) were successfully primed by PRRS vaccination. At later time points post challenge, i.e. 10 and 28 dpc, an increase in the height of the antibody level was observed, most likely caused by the challenge infection.

(44) PRRS Viremia

(45) The mean virus titer per group is represented in table 4. The table clearly shows that the vaccinated animals (group 1 and 2) have on average a reduced virus load in their serum as compared to the control animals (group 3). There is little difference in height of the viremia between each of the vaccinated groups.

(46) TABLE-US-00004 TABLE 4 PRRS titer dpc, log10 TCID50/ml Group 0 5 7 10 14 28 1 0 0.6 0.5 0.7 0.6 0 2 0 0.1 0.2 0.2 0.3 0 3 0 2.0 1.8 2.2 2.1 0

(47) In conclusion, it has been established that even in the lowest dose (i.e. the lower limit according to the label) the commercial vaccine Prime Pac® PRRS reconstituted in an EPL formulation adequately seroconverted animals, and protection against pathogenic PRRSV is obtained.