Vaccines for use against asymptomatic Lawsonia infections in pigs

Abstract

To provide an improved vaccine that can be used to reduce the shedding of Lawsonia intracellularis bacteria by an asymptomatic infected pig.SOLUTION: The present invention pertains to a pharmaceutical composition comprising non live Lawsonia intracellularis antigen and a pharmaceutically acceptable carrier.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the use of Lawsonia intracellularis in pigs. Lawsonia intracellularis associated with asymptomatic infection with To reduce shedding of Lawsonia intracellularis bacteria The present invention relates to a vaccine for use in the method. [Background technology]

[0002] Proliferative enteropathy, also known as ileitis, is caused by the obligate intracellular bacterium Lawsonia intracellularis. Caused by Lawsonia intracellularis is a common intestinal disease of postweaned pigs worldwide. The characteristic lesions are the intestinal crypts of the ileum. These cells normally contain primary cytoplasmic At autopsy, histological lesions were observed, especially in enterocytes, ranging from 1.5 to 2.5 μm The presence of vibrio-like bacteria of length 10 mm was considered to be Lawsonia positive. These bacteria can also be found in the mucous membranes between the crypts and in the mesenteric lymph nodes. They are often found in macrophages located in the lamina propria. The clearance of bacteria from enterocytes is related to This leads to the resolution of the associated proliferative lesions, indicating a direct local effect of bacteria on the crypts ( McOrist et al., “Developed and resolving lesions s in porcine proliferative enteropathy:p ossible pathogenic mechanisms,Journal of Comparative Pathology,115,1996,pp35-4 5) Lawsonia intracellularis in these lesions The presence of C. tracellularis may be indicative of animals that exhibit only asymptomatic infection (diarrhea or abnormal As in animals that do not show feces, animals that show disease (i.e. diarrhea or abnormal red / black color) ) using PCR in both Clinical cases usually present in the grow-finishing stage and in some older finishing pigs. In this case, an acute hemorrhagic form has been documented.

[0003] Lawsonia intracellularis s) The relationship between seropositive pigs and the proportion of pigs showing clinical or asymptomatic infection has been previously studied. (Van der Heijden, Prevalence of expo sure and infection of Lawsonia intracell ularis among slaughter-age pigs.,Res Vet Sci, December 2004, 77(3), pp197-202). Some pigs may be carrier pigs, i.e. infected but without clinical signs of ileitis. In Europe, animals considered to be disease-free have a high bacterial load in the intestine at the time of slaughter. They appear to have asymptomatic infection as evidenced by persistence. Lawsonia intracellularis in The explanation for the positive results for the presence of B. aris bacteria is that these populations Lawsonia intracellularis (Lawsonia intracellularis) Only in certain circumstances can the law be transmitted to the Nia infections result in clinical problems, including cases of acute hemorrhagic enteropathy.

[0004] Vaccination against Lawsonia infection is rare in clinically asymptomatic populations. However, asymptomatic infections have a negative effect on average daily weight gain (ADWG) in pigs. Some grow-finish farms have been using immunizations to combat localized infections in the intestine. To achieve this, the only commercially available ileitis vaccine on the market, namely Enterisol® ) Ileitis (available from Boehringer Ingelheim) The vaccine contains a live attenuated bacterium, Lawsonia intracellularis. In fact, at present, It is believed that local infection can only be countered by inducing local immunity. This is different for animals that develop clinical disease. These animals are not immune to the systemic effects of non-live vaccines. It has been shown that vaccination can successfully protect against No. 144088, assigned to Intervet International BV). Theory While not being bound by the present invention, the reason for this is that these animals may be exposed to infection in the system. This is because the patient has severe gastrointestinal lesions that cause infection, which explains why the systemic immune response is unable to stop the infection. However, it is thought that the mechanism of action of the serotonin receptor agonist, which is thought to explain why the serotonin receptor agonist is useful for stopping microintestinal infections. If there is only a corresponding asymptomatic infection, IgA and cellular immunity are required to contain the infection. A local immune response, including immunity, is thought to be required. A vaccine containing European Enterisol® Ileitis Sympathomimetic in Serona Published at the Research and Development Forum in Mike Roo, known as Enterisol® Ileitis This is confirmed in the paper by F. In Table 1.1 on page 2, the systemically administered killed vaccine is classified as "liquid / whole Although it can induce "systemic immunity," it cannot induce both "mucosal immunity" and "cellular immunity." The latter two have been shown to be effective in preventing localized infections of the intestine involving intracellular pathogens. It is generally known that the al Immunology, seventh edition, 1991, p. 206. The secretory immune system protects the external mucosal surfaces”, p. 209 “Defense is by cell-mediated immunity" and page 210 "Ac activated macrophages kill intracellular p In a 2005 paper by Mike Roof, "Killed Bacteria Early studies looking at the phosphoprototype have shown that killed vaccines can protect against intestinal colonization. It was confirmed that the vaccine did not induce a protective response (page 2, left column, 2 whole paragraphs).

[0005] Live Enterisol Ileitis Vaccine Reduces Infection-Associated Weight Gain The claim that the vaccine reduces the loss of income is actually approved and used for that purpose. The disadvantage of Chin is that it requires oral administration rather than the routine method of vaccinating pigs. that antibiotic use must be suspended in animals for six days. (otherwise the bacteria in the vaccine would be killed). Another drawback is that this vaccine However, it has not been approved for reducing bacterial shedding. The data used to determine the efficacy of serotonin-dependent chemoreceptor peptides did not show a reduction in bacterial shedding. (Eds., Vaccine 33, 2015, 156-162) has Ileitis provides no protection against shedding of Lawsonia bacteria after infection This was confirmed by vaccinating all animals and preventing weight gain problems and clinical signs. Although the incidence of Lawsonia intracellularis in animal populations is significantly reduced, This may indicate that infection with P. onia intracellularis may persist. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2009 / 144088 [Non-patent literature]

[0007] [Non-Patent Document 1] McOrist et al., Journal of Comparative Pathology, 115, 1996, pp35-45 [Non-Patent Document 2] Van der Heijden,Res Vet Sci,December 2004,77(3),pp197-202 [Non-Patent Document 3] Mike Roof, European Enterisol® Ileitis Symposium, October 13-15, 2005, Barcelona, ​​Spain [Non-Patent Document 4] Ivan Roitt, Essential Immunology, seventh edition, 1991, pages 206, 209 and 210 [Non-Patent Document 5] Riber et al., Vaccine 33, 2015, 156-162 Summary of the Invention [Problem to be solved by the invention]

[0008] Asymptomatic infection of pigs with Lawsonia intracellularis To provide an improved vaccine that can be used to reduce shedding of the bacterium acelluaris. It is an object of the present invention to reduce the adverse effects on ADWG associated with asymptomatic infection. It is a further object to provide improved vaccines which further [Means for solving the problem]

[0009] In order to meet the primary objectives of the present invention, The pharmaceutical composition is a pharmaceutical composition comprising the non-viable bacterium Lawsonia intracellularis. (Lawsonia intracellularis) antigens and pharma- ceutical acceptable carriers and is suitable for systemic administration.

[0010] Surprisingly, systemically administered non-live Lawsonia antigen (WO 2009 / 144 Different types of non-live Lawsonia antigens, such as those described in No. 088, react with IgA and cytoplasmic antigens. It is generally understood that the immune system is unable to induce cellular immunity, much less a local immune response in the intestine. Although such compositions have no effect on the asymptomatic infection of pigs, Lawsonia intracellularis in It has been found that it is possible to reduce and even substantially or completely prevent emissions of The reasons for this are unclear and are not consistent with the prior art for live vaccines. The advantages of the novel compositions are clear: they can be administered systemically, e.g. intramuscularly or intradermally. It can be administered without interrupting antibiotic use because it contains only non-viable Lawsonia antigens. . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] definition A vaccine is a composition that provides protection against post-vaccination infection with a pathogenic microorganism, i.e. In the inoculated host, the microorganism typically blocks itself, e.g., via antibodies. A composition that prevents or reduces infection or damage resulting from infection by a microorganism. Vaccination therefore has the potential to prevent infection or at least reduce the level of infection. and / or prevent or at least reduce the level of damage resulting from the infection. This reduces the

[0012] Non-live antigens of wild-type bacteria are expressed by the corresponding pathogenic bacteria or one or more of its virulence factors. are recognized and at least partially neutralized by the host's immune system as a result of the immune response of the host. Any substance or compound, other than a live bacterium itself, that is intended to induce an immunological response, such as Typical examples of non-viable antigens of wild-type bacteria are killed whole cells, surface-expressed proteins, etc. a bacterial subunit and a toxin, the latter two of which may be recombinantly expressed or Lawsonia intracellularis may not be present. Regarding C. acellularis, several types of non-live antigens are known in the art. For example, International Publication No. 2009 / 144088 (killed whole cells) and International Publication No. No. 05 / 070958 (subunit) and WO 97 / 20050 (killed whole cell It is known to

[0013] A pharma- ceutical acceptable carrier is a medium that is biocompatible, i.e., capable of acting remarkably in a subject after administration. A vehicle that does not induce any adverse reactions and that can effectively deliver the antigen to the immune system of the target animal after administration of the vaccine. Such a carrier can be water and / or some other biocompatible solvent. It may be a liquid containing glycerol, such as those commonly used to obtain freeze-dried vaccines. It can also be a solid (sugar and / or protein based).

[0014] Lawsonia intracellularis Asymptomatic infections are mostly or completely asymptomatic (without signs or symptoms), especially Asymptomatically infected animals do not show diarrhea or fecal abnormalities. Lawsonia intracellularis Symptomatic carrier animals may have reduced weight gain. The presence of asymptomatic infection may be due to fecal or Microbiological culture of the intestine (the latter after slaughter) or DNA techniques such as the polymerase chain reaction It is identified by:

[0015] Systemic administration of an antigen means that the antigen is delivered to the body's circulatory system, which includes the cardiovascular system and the lymphatic system. ), thus affecting the whole body rather than a specific site such as the gastrointestinal tract. Systemic administration means, for example, administration into muscle tissue (intramuscular), into the skin (intradermal), Administering antigens under the skin (subcutaneous), under the mucous membranes (submucosal), and into a vein (intravenous) This can be done by:

[0016] The finishing stage for pigs is when the pigs are around 10 weeks old (weight approx. 25-30 kg) to around 28 weeks old (weight approx. The finishing period is the period when the weight of the animal is between 110 and 130 kg. This is part of the period between approximately 3 weeks of age and 28 weeks of age, the age at which most pigs are slaughtered.

[0017] Compositions that include killed whole bacterial cells as antigens include antigens that are derived from the killing of live whole bacterial cells. In the clear sense, this means that bacterial cells are at least partially ruptured or or killed whole cell extracts or homogenates are actually contained in "killed whole cell vaccines" It is not excluded that killed whole-cell Lawsonia intracellularis can be used as an antigen. (Lawsonia intracellularis) is described, for example, in International Publication No. 2009 This is known from WO 97 / 20050 and WO 144088.

[0018] Embodiment In a first embodiment, the method is for preventing the shedding of bacteria. Cutin is sensitive to more than 90% of animals, even up to 99%, despite being administered systemically. It may even be possible to induce a local immune response in the intestine and thus prevent infection. The live Enteritis vaccine should be adjusted to prevent asymptomatic infection in Even though ol® Ileitis simply reduces the excretion of Lawsonia bacteria, This is quite surprising given the fact that it cannot.

[0019] In a further embodiment, the method further comprises the step of: Daily intake of pigs associated with asymptomatic infection with Sonia intracellularis The aim of this vaccine is to reduce the adverse effect on average weight gain (ADWG) of the vaccine. could have a positive effect on ADWG in asymptomatically affected pigs. Even if the beneficial difference in weight gain is small, this is of great importance to any pig producer. This is an important parameter.

[0020] In another embodiment, the vaccine is administered only once. The second dose of the vaccine is generally administered 2 to 4 weeks after the first dose. This may be done after a period of time to improve the level of immune response.

[0021] In another embodiment, the vaccine is administered when the pig is between 3 and 10 days of age. Vaccinating pigs at a young age provides early protection, i.e. immediately after weaning. It can be provided.

[0022] In yet another embodiment, the non-viable fungus Lawsonia intracellularis The antigen is derived from the live bacterium Lawsonia intracellularis ( In Lawsonia intracellularis, the outer cells of these bacteria These carbohydrates are specific for anti-Lawsonia intracellularis. Lawsonia intracellularis: Enabling immune response , has been shown to increase the vasopressin-releasing hormone (see WO 2009 / 144088).

[0023] In yet another embodiment, the non-viable fungus Lawsonia intracellularis (Lawson The Lawsonia intracellularis antigen is a killed Lawsonia intracellularis bacterium. Antibodies purified from a composition containing Lawsonia intracellularis Purification may be used to reduce any site reactions, if any. , non-specific bacterial material can be removed from the actual antigens.

[0024] In a practical embodiment, the non-viable fungus Lawsonia intracellularis (Lawsoni The antigen is derived from killed whole-cell Lawsonia intracellularis. The squirrel is Lawsonia intracellularis.

[0025] [Example] Experiment 1 Experiment 1 Design This study was carried out in older finishing pigs and breeding gilts in a Dutch herd. Lawsonia intracellularis s)(LI) infection. The experiment followed a randomized, controlled, blinded design.

[0026] A randomized study of 158 piglets aged 3-10 days (mostly aged 3-5 days) was conducted within litters. Piglets in the test group were given inactivated lotion in an oil-in-water emulsion. Sonyabacterin vaccine (see WO 2009 / 144088, Example 3) The piglets were vaccinated at enrolment and again 3 weeks later. The control piglets were vaccinated with placebo on the same days. In the test animals, the mice received an injection of IL-1 (antigen-free emulsion). Antibiotic therapy (e.g., tylosin, lincomycin, tiamulin, tetracycline, Ikrin) was not approved.

[0027] After weaning, i.e., during the growing and finishing phases, test and control piglets were kept together. In addition, during the growing and fattening periods, non-experimental pigs were housed in the same pen but in a different location from the experimental animals. It was housed in a pen.

[0028] Local and post-vaccination studies were performed 4 hours, 1 day, 3 days, 1 week, and 2 weeks after each vaccination. Piglets were examined periodically for systemic reactions.

[0029] All pigs were screened for 1 week or more during the expected period of exposure to filed infection. Fecal samples were collected at 2-week intervals. Fecal samples from 10 animals per treatment group were pooled. The presence of LI bacteria was then confirmed by qPCR (hereinafter also referred to as "PCR"). If a pooled fecal sample showed the presence of bacteria, the original sample from the pool was analyzed. Individual fecal samples were tested as soon as the first sample was positive. At the time of first vaccination, 10, 16, and 21–23 weeks after first vaccination, all The animals were weighed.

[0030] Results of Experiment 1 During the experiment, Lawsonia infection was observed in the stool samples, as evidenced by PCR data. All but one animal in the control group showed any clinical signs. As no animals were present, this group was not susceptible to asymptomatic infection resulting from field exposure to (wild-type) bacteria. He was infected.

[0031] Fecal bacterial load The average PCR results are shown in Table 1, and the number of positive animals and the average concentration of LI-DNA are shown in Table 2. vinegar.

[0032] From week 13 onwards, LI-derived DNA was found in many control animals. The percentage of control animals testing positive ranged from 0 at 12 weeks after the first vaccination to 13 weeks after vaccination. This gradually increases through 2-8% at ~15 weeks to around 18% at 16-18 weeks, after which After the 20th week, this dropped to 0%. [Table 1]

[0033] In the test (vaccine) group, one PCR-positive fecal sample was obtained per animal on one collection day. Only fecal samples were found (1 positive out of 72, i.e. 1.4%). Of the 75 cats, 31 had at least one PCR-positive fecal sample (41.3%). The differences between the groups were statistically significant (Fisher's exact test: p value < 0.00). 1). [Table 2]

[0034] Weight gain Body weight and average daily weight gain are summarized in Tables 3 and 4 below. A relevant parameter often used to measure the efficacy of Lawsonia vaccines is However, this parameter varies depending on several different conditions (secondary infection, climate, feeding, etc.). The ADWG difference of 25 grams per day is nonspecific because it is affected by The pre-calculated sample size to obtain a statistically significant value (p<0.05) was The number of mice should be about 300. For reasons of efficiency, the groups used in practice were much smaller. In this pilot study, group sizes were only 78 and 80 animals. , even though the difference in ADWG was about 25 grams per day, the resulting p-values ​​were 0 in both cases. It was estimated to be greater than .05. [Table 3]

[0035] Comparison of mean daily weight gain during the finishing phase by treatment group (see below) showed, as expected, No statistically significant difference was obtained (p value 0.2042). When vaccinated, the daily weight gain during the finishing phase was on average 1 day faster than the control group. The 90% confidence interval ranged from 6.4 grams per day lower to 1.5 grams per day higher. The average daily intake ranged from 49.2 grams per day to as high as 21.4 grams per day. The mean difference in ADWG between the two groups was a substantial difference. The least squares mean (LSM) for weight gain was 881 grams per day in the vaccinated group. and 860 grams per day in the control group. These LSMs were compared using ANCOVA. However, the small difference in the covariate "weight at vaccination" between groups was not significant. The averages are adjusted for the number of cases and therefore differ slightly from the usual averages from Table 3. [Table 4]

[0036] The p-value for the treatment effect on overall mean daily weight gain was 0.2804. When adjusted for baseline values, the vaccinated group had a 12. The mean daily weight gain was 5 g / day higher. The mean growth rate was 6.6 g / day lower in the vaccinated group than in the control group, ranging from 31.5 g / day lower. / day ranged from high to low growth.

[0037] Experiment 2 Design of Experiment 2 The objective of this experiment was to determine whether the pigs were fed the same diet as those used in the first experiment in finishing pigs under field conditions. By assessing the efficacy of the vaccine, but when used as a one-shot vaccine This study was a randomized, blinded, saline-controlled trial of clinical efficacy. Approximately 750 3- to 4-week-old piglets were randomly assigned within the same litter to a test or control group. The test group piglets were given a single intramuscular injection of 2 mL of vaccine, and the control group piglets were given a single intramuscular injection of 2 mL of vaccine. mL of saline was injected.

[0038] At 10 (±1) weeks of age, 648 test piglets, 324 from each treatment group, were transported to the finishing farm. After that, the control pigs were sent to the same unit in separate pens from the vaccinated pigs (unit The animals were housed in eight pens (8–11 animals per treatment group) until sacrifice (at 25 weeks of age).

[0039] The first parameters evaluated were different stamina scores at enrolment, after transfer to the finishing farm and immediately before slaughter. The second parameter evaluated was the average daily weight gain (ADWG) of the pigs between weighings. Mehta measured Lawsonia excretion at 15 and 20 weeks of age after transfer to the finishing farm and immediately before slaughter. It was.

[0040] Results of Experiment 2 During this second experiment, Lawsonia infection was confirmed by PCR data in feces. Only one animal in the control group showed minimal clinical signs (determined after necropsy). It was confirmed that this was the result of Lawsonia infection. The group was confirmed to have asymptomatic infection resulting from field exposure to the (wild-type) bacteria. obtain.

[0041] The effect on ADWG is shown in Table 5. As mentioned above, the average body weight per day during the growing period The gain was 319 grams per day in the test group and 307 grams per day in the control group. The difference in average daily body weight gain (ADWG) during the rearing period was 0.01% between the control and test groups. There was a significant difference between the ADWG and the sham-treated group (ANOVA: p=0.0203). The difference between the control and test groups was 7 grams per day. The difference in ram is commercially significant (leading to a difference in final weight of approximately 1.4 kg) , but was not statistically significant. [Table 5]

[0042] Regarding Lawsonia excretion, all fecal samples contained Lawsonia intracerebellum during the sixth week of the experiment. The results were negative for Lawsonia intracellularis. At week 11, seven samples (19%) from the control group were positive, and one sample (10%) from the test group were positive. At week 16, seven samples (19%) from the control group and one from the test group were positive. One sample from the group was positive. At week 21, all animals were negative again. During the study The number of pens with PCR Lawsonia-positive fecal samples (1 or 2 consecutive weeks) was compared with the control group. The incidence rate was significantly lower in the study group compared with the control group (Fisher's exact test, p<0.001). ).

[0043] Experiment 3 Design of Experiment 3 The primary objective of this study was to evaluate the efficacy and safety of immunization against bacterial shedding after one or two vaccination schemes. The aim of the study was to further evaluate the efficacy of the vaccine used in the previous experiment under field conditions. The study was conducted on a randomized and randomized controlled trial on a family farm with Lawsonia infection in its population. The study was conducted according to a blinded design. Healthy 3- to 10-day-old suckling piglets were divided into two test groups within the same litter. were randomly assigned to one of the control groups (320 per group) and one test group (2x 1 mL) of the vaccine was administered to piglets at 3 to 10 days of age, and 21 days later, the vaccine was administered to The other test group (1x2mL) of piglets received 2mL of vaccine at 24-31 days of age. The control piglets were vaccinated with 1 mL of saline at 3–10 and 21 days of age. Fecal samples for Lawsonia PCR were collected at weeks 6, 8, 10, and 12. and then weekly until the pigs were slaughtered at 23 weeks of age.

[0044] Results of Experiment 3 During this second experiment, Lawsonia infection was confirmed by PCR data in feces. No animals showed clinical signs of Lawsonia infection. Therefore, this group was suffering from asymptomatic infection resulting from field exposure to the (wild-type) bacteria. .

[0045] Regarding Lawsonia excretion, a large difference in the occurrence of positive fecal samples was observed between farrowing batches in the control group. In the first farrowing batch, the positive control samples were later in the finishing phase (18th and 19th weeks). In the second batch, positive samples were found only in week 21, and in the third batch, One positive sample was found in the 21st week. However, the fourth batch from the 17th week onwards Positive samples were detected in the 14th week, while positive samples were already found in the 5th batch. In summary, 50% of controls yielded one or more positive samples after week 14, Two specimens (6%) from the 2x1mL group and four specimens (12%) from the 1x2mL group were positive. The number of animals with PCR Lawsonia-positive fecal samples (1 or 2 consecutive weeks) during the experiment was Compared with the control group, the 1x2mL group (Fisher's exact test, p<0.0001) and and 2x1mL group (Fisher's exact test, p=0.0002). .

Claims

1. 1. A method for reducing shedding of Lawsonia intracellularis bacteria associated with asymptomatic infection following vaccination with Lawsonia intracellularis in pigs, comprising: said vaccination comprising systemically administering to said pig a pharmaceutical composition comprising a non-live Lawsonia intracellularis antigen and a pharma- ceutical acceptable carrier; wherein the pharmaceutical composition is administered prophylactically prior to infection with Lawsonia intracellularis; and wherein the presence of said asymptomatic infection is by the presence of Lawsonia intracellularis DNA in the faeces of pigs 13-20 weeks after the first vaccination if the first vaccination was at 3-10 days of age; or If the first vaccination was at 3-4 weeks of age, confirmation by the presence of Lawsonia intracellularis DNA in the pig feces 11-20 weeks after the first vaccination. method.

2. 2. The method of claim 1, wherein the pharmaceutical composition is for preventing excretion of the bacteria.

3. 3. The method of claim 1 or 2, wherein the pharmaceutical composition is administered only once.

4. 4. The method of any one of claims 1 to 3, wherein the non-viable Lawsonia intracellularis antigens comprise carbohydrates that, in live Lawsonia intracellularis, are associated with the outer cell membrane of these bacteria.

5. The method according to any one of claims 1 to 4, characterized in that the non-live Lawsonia intracellularis antigen is an antigen purified from a composition comprising killed Lawsonia intracellularis.

6. 6. The method of any of claims 1 to 5, wherein the non-live Lawsonia intracellularis antigen is killed whole cell Lawsonia intracellularis.

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