ES2666671T3 - Vaccine for protection against Lawsonia intracellularis - Google Patents

Abstract

A non-living composition containing a carbohydrate, the carbohydrate also being found in the live cells of Lawsonia intracellularis in association with the outer cell membrane of these cells, for use as a vaccine for protection against a Lawsonia intracellularis infection, by means of systemic administration of the vaccine, wherein the composition containing the carbohydrate is the material resulting from the inactivation of Lawsonia intracellularis bacteria and where the composition containing the carbohydrate contains whole cells of inactivated Lawsonia intracellularis bacteria.

Description

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DESCRIPTION

Vaccine for protection against Lawsonia intracellularis

The present invention relates to a vaccine for protection against an infection by Lawsonia intracellularis, being a vaccine, in this sense, a composition that provides at least a decrease in a negative influence of Lawsonia intracellularis infection, said influence being negative , for example, tissue damage and / or clinical signs such as a decrease in weight gain, diarrhea, etc.

Proliferative enteropathy (also called enteritis or ileitis) in many animals, particularly in pigs, presents with a clinical sign and a pathological syndrome with mucosal hyperplasia of immature crypt epithelial cells in the terminal ileum. Other areas of the intestine that may be affected include the jejunum, the cecum and the colon. Piglets and young adult pigs are mainly affected with a typical clinical manifestation of rapid weight loss and dehydration. Natural clinical disease in pigs occurs worldwide. The disease is consistently associated with the presence of a curved intracellular bacterium, currently known as Lawsonia intracellularis.

In general, oral vaccination against Lawsonia intracellularis has proven to be an economically effective measure for the control of ileitis and to allow greater exploitation of the genetic growth potential of the pig (Porcine Proliferative Enteropathy Technical manual 3.0, July 2006; available at Boehringer Ingelheim). Additionally, oral vaccination instead of parenteral will reduce the transmission of haematological infections such as PRRS through multipurpose needles and the reduction of reactions at the injection site and needles retained in corpses. It will reduce animal and human stress, time, cost and effort compared to individual vaccination (McOrist: "Ileitis - One Pathogen, Several Diseases" at the IPVS Ileitis Symposium in Hamburg, June 28, 2004).

It is generally understood that the advantage of a methodology with a live attenuated vaccine is that the efficacy of immunity is usually relatively good, since the host's immune system is exposed to all the antigenic properties of the organism in a more "natural" way. ". Specifically, for intracellular bacterial agents such as Lawsonia intracellularis, it is believed that the methodology with an attenuated live bacteria vaccine offers the best protection available for vaccinated animals, due to an immune response based on complete and appropriate T lymphocytes. This contrasts with the between variable and poor immunity associated with the types of subunit vaccine or bacteria inactive for intracellular bacteria. This is also specifically true for obligate intracellular bacteria such as Lawsonia intracellularis or Chlamydia species, which cause pathogenic infections in the mucosa. Some studies indicate that the complete live attenuated forms of the intracellular bacteria in question are best administered in the target mucosa, which are necessary in the form of complete living bacteria to produce a fully protective immune response in the target mucosa, but also that they are immunologically superior compared to the use of partial bacterial components.

It has become a general understanding that a vaccine against Lawsonia intracellularis must be administered orally (see, among others, the Technical Manual 3.0 mentioned above). This is based on the fact that the basis of the body's resistance to an ileitis is local immunity in the intestine, which is the product of cellular immunity and local defense through antibodies, especially IgA. According to current knowledge, serum antibodies (IgG) do not provide any protection, simply because they do not reach the intestinal lumen. Some studies have shown that oral vaccination produces cellular immunity, as well as local production of IgA in the intestine (Murtaugh, in Agrar- und Veterinar-Akademie, Nutztierpraxis Aktuell, Ausgabe 9, June 2004; and Hyland et al. in Veterinary Immunology and Immunopathology 102 (2004) 329-338). In contrast, intramuscular administration did not result in protection. In addition, together with the general understanding that a successful vaccine against an intracellular bacterium must induce cellular immunity, as well as the production of local antibodies, the skilled professional knows that only a very low percentage of orally digested antigens are actually absorbed. by enterocytes, and that the incorporation of Lawsonia intracellularis in the cell is an active process initiated by the bacteria. Consequently, a vaccine of inactivated bacteria would provide the intestine with an insufficient immunogenic antigen (Haesebrouck et al. In Veterinary Microbiology 100 (2004) 255-268). This is why it is believed that only vaccines with live attenuated bacteria induce sufficient cellular protection in intestinal cells (see Technical Manual 3.0 mentioned above). There is currently only one vaccine on the market to protect against Lawsonia intracellularis, namely Enterisol ® Ileitis, marketed by Boehringer Ingelheim. This vaccine is, in fact, a live bacteria vaccine for oral administration.

It is an objective of the present invention to provide an alternative vaccine to protect against a Lawsonia intracellularis infection. To this end, the use of a composition containing an inactivated carbohydrate has been contemplated, the carbohydrate also being found in the live cells of Lawsonia intracellularis in association with the outer cell membrane of these cells, for the preparation of a vaccine for protection against an infection by Lawsonia intracellularis, the vaccine being in a form suitable for systemic administration, in which the composition containing the carbohydrate is the material resulting from the destruction of bacteria

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Lawsonia intracellularis, and in which the composition containing the carbohydrate contains whole cells of destroyed Lawsonia intracellularis bacteria. Surprisingly, in the face of the persistent general understanding of how to combat Lawsonia intracellularis, it was found that through the use of an inactivated composition containing a carbohydrate as an antigen in a vaccine, protection against Lawsonia intracellularis can be induced that is comparable or even enhanced with with respect to the protection provided by using the Enterisol ® Ileitis bacteria vaccine (administered according to the corresponding instructions), when the antigen is administered systemically, that is, in a way that reaches the body's circulatory system (which comprises the system cardiovascular and lymphatic), thus affecting the body as a whole rather than a specific place such as the gastrointestinal tract. Systemic administration can be carried out, for example, by the administration of antigens in muscle tissue (intramuscular), in the dermis (intradermal), below the skin (subcutaneous), below the mucosa (submucosa), in the veins (intravenous) etc. Apart from the very good protection obtainable, an important advantage of the present attenuated vaccine is that it is inherently safe when compared to a live bacteria vaccine.

In general, the carbohydrate-containing composition can be used for the preparation of a vaccine by using methods known in the art that basically comprise mixing the composition containing the antigenic carbohydrate (or a composition derived therefrom, such as a dilution or concentrate of the original composition, or an extract, one or more purified components, etc.) with a pharmaceutically acceptable carrier, for example, a liquid carrier (optionally buffered) such as water or a solid carrier such as those used usually to obtain lyophilized vaccines. As such, processing may take place in an industrial environment, but the antigens could also be mixed with the other constituents of the vaccine in situ (i.e., in a veterinary practice, on a farm, etc.), for example (immediately) before actual administration to an animal. In the vaccine, the antigens could be present in an immunologically effective amount, that is, in an amount capable of stimulating the immune system of the target animal sufficiently to reduce at least the negative effects of post-vaccination exposure with natural microorganisms. . Optionally, other substances such as adjuvants, stabilizers, viscosity modifiers or other components may be added, depending on the intended use or the required properties of the vaccine. Many forms are suitable for systemic vaccination, in particular liquid formulations (with dissolved, emulsified or suspended antigens), but also solid formulations such as implants, or an intermediate form such as in the form of a solid carrier for the suspended antigen. in a liquid Systemic vaccination, in particular parenteral vaccination (i.e., that which is not through the food channel), and suitable (physical) forms of vaccines for systemic vaccination have been known for more than 200 years.

It is appreciated that the subunits of Lawsonia intracellularis cells have been determined as antigens in a vaccine for protection against this bacterium. However, these are mainly recombinant proteins, and so far none of them have proven capable of providing good protection. Inactivated bacteria (which inherently contain the carbohydrate that is also found in the live cells of Lawsonia intracellularis in association with the outer cell membrane) have also been suggested as antigens in vaccines against Lawsonia intracellularis, but no vaccine based on inactivated whole cells has been really tested and determined as providing good protection. Apart from this, systemic administration has not been used together with these inactivated bacteria, due to the general understanding that there is no reasonable expectation of success for systemic administration of antigens to combat locally (i.e., in the intestine) Lawsonia intracellularis.

In this regard, it is noted that WO 97/20050 (Daratech PTY Ltd) mentions the use of inactivated bacteria of Lawsonia intracellularis to immunize pigs. However, systemic administration is not mentioned. Based on current knowledge that vaccination is only effective after oral administration, it is usually understood that the oral route was the route of administration chosen for the experiments described in the Daratech application. Another patent application that mentions inactivated bacteria is WO 2005/011731 (Boehringer Ingelheim). However, only the use of a live bacteria vaccine administered orally is actually reported. It is not shown that an inactivated bacteria vaccine can be effective, much less that the inactivated bacteria vaccine can be administered systemically. EP 843 818 (Boehringer Ingelheim) describes the intramuscular administration of a vaccine of inactivated bacteria (paragraph [0115] together with paragraph [0119]). Paragraph [0115] states that the bacteria were destroyed by storage at 4 ° C at normal atmospheric conditions. However, as is well known, under such conditions, the Lawsonia intracellularis bacteria survives. Therefore, this document does not teach the subject matter of the present invention. It is also indicated that a composition containing the carbohydrate, in which the carbohydrate is also found in the living cells of Lawsonia intracellularis in association with the outer cell membrane of these cells, is known from Kroll et al. (Clinical and Diagnostic Laboratory Immunology, June 2005, 693-699). However, this composition is used for diagnosis. It has not been tested as a protective antigen for the reasons stated above.

According to the invention, the composition containing the carbohydrate is the material resulting from the destruction of the bacteria of Lawsonia intracellularis. It has been found that a very convenient way of providing the carbohydrate for use according to the present invention is simply to inactivate Lawsonia intracellularis cells and use the resulting material as a carbohydrate source.

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The material as a whole could be used, for example, a whole cell suspension or a cell lysate of Lawsonia intracellularis. This method could be carried out using relatively simple techniques known in the art.

According to the invention, the carbohydrate-containing composition contains whole cells of inactivated bacteria of Lawsonia intracellularis. It has been shown that this is the most convenient way to provide the carbohydrate in the form of an antigen in the vaccine. Moreover, the efficacy of the vaccine is even further increased, possibly because this way of offering the antigen to the immune system of the target animal better simulates the natural carbohydrate environment.

In one embodiment, the vaccine comprises an oil-in-water adjuvant containing oil droplets of a submicron size. In general, an adjuvant is a non-specific immunostimulating agent. In principle, any substance that is capable of favoring or amplifying a particular process in the cascade of immunological events, ultimately resulting in a better immune response (i.e., the integrated body response to an antigen, in particular lymphocyte mediated , and which normally involves the recognition of antigens by specific antibodies or by previously sensitized lymphocytes), can be defined as an adjuvant. It has been shown that the use of an oil-in-water adjuvant containing oil droplets of a submicron size provides very good protection against Lawsonia intracellularis. In fact, the application of oil adjuvants in water as such is common in relation to inactivated antigens. However, it is generally known that the best immunostimulatory properties are obtained when the oil droplets have a large diameter. In particular, oil droplets with a diameter below 1 micrometer are used in particular when it is believed that safety is an important issue. In that case, small droplets could be used, since it is known that they cause less tissue damage, clinical signs, etc. However, in the case of obtaining protection for a disorder related to the intestine through systemic vaccination (as is the case in the present invention), large droplets would be chosen, since it would be expected that the immune response has to be triggered. in a meaningful way. On the contrary, we found that using small droplets of oil in the composition provided very good results with respect to protection against Lawsonia intracellularis.

In a more preferred embodiment, the helper comprises droplets of a biodegradable oil and droplets of a mineral oil, the droplets of the biodegradable oil having an average size that differs from the average size of the droplets of the mineral oil. It has been shown that the use of a mixture of biodegradable oil and mineral oil provides very good results in relation to efficacy and safety. In addition to this, the stability of the composition is very high, which is an important economic advantage. The stability has been shown to be very good, in particular when the average size (weighted by volume) of any of the droplets of the biodegradable oil or the droplets of the mineral is below 500 nm (preferably around 400 nm).

In one embodiment, the vaccine additionally comprises Mycoplasma hyopneumoniae and porcine circovirus antigens. To date, combination vaccines of Lawsonia intracellularis have been suggested in the prior art. However, the effectiveness of not many of these combinations has been really tested. The reason for this is that it is generally understood that the combination of antigens with Lawsonia intracellularis antigens can only result in successful protection if Lawsonia antigens are provided in the form of live (attenuated) cells. In this regard, we refer to WO 2005/011731, which also suggests all types of combination vaccines based on Lawsonia intracellularis. However, with respect to the description and structure of the claims of the patent application, the beneficiary (Boehringer Ingelheim) seems to be convinced that combination vaccines can only be expected to have a reasonable probability of success when Lawsonia antigens They are present in the form of living cells. The same is true for WO2006 / 099561, also granted to Boehringer Ingelheim. In fact, based on general usual knowledge, this thought is obvious.

The invention will be further explained using the following examples.

Example 1 describes a method for obtaining a substantially protein-free composition containing carbohydrate and a vaccine that is made by using this composition.

Example 2 describes an experiment in which a second vaccine according to the present invention is compared with the currently marketed vaccine and an experimental vaccine comprising Lawsonia intracellularis subunit proteins.

Example 3 describes an experiment in which two different vaccines according to the present invention are compared with the vaccine currently marketed.

Example 4 describes an experiment in which the effect of the dose of a vaccine according to the invention is established.

Example 1

In this example, outside the scope of the invention, a method for obtaining a carbohydrate composition substantially free of proteins associated with the outer cell membrane of the cells of the invention is described.

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Lawsonia intracellularis and a vaccine that can be made using this composition. In general, a carbohydrate is an organic compound that contains carbon, hydrogen and oxygen, usually in a ratio of 1: 2: 1. Some examples of carbohydrates are sugars (saccharides), starches, cellulose and gums. They usually serve as the main source of energy in the animals' diet. Lawsonia intracellularis is a gram-negative bacterium, which therefore contains an outer membrane that is not only made up of phospholipids and proteins, but also contains carbohydrates, in particular polysaccharides (usually polysaccharides such as lipopolysaccharides, lipooligosaccharides, or even non-lipopolysaccharides).

FRACTION OF CARBOHYDRATES FOR THE PREPARATION OF THE VACCINE

Twenty milliliters of buffered water (0.04 M PBS, phosphate buffered saline) containing Lawsonia intracellularis cells at a concentration of 3.7E8 (= 3.7 x 108) cells / ml were collected. The cells were lysed by keeping them at 100 ° C for 10 minutes. Proteinase K (10 mg / ml) in 0.04 M PBS was added at a final concentration of 1.7 mg / ml. This mixture was incubated at 60 ° C for 60 minutes in order to degrade all proteins and keep carbohydrates intact. Subsequently, the mixture was incubated at 100 ° C for 10 minutes to inactivate proteinase K. The resulting material, which is a carbohydrate-containing composition, in particular that contains carbohydrates as present in the live bacteria of Lawsonia intracellularis in association With its outer cell membrane (see next paragraph), it was stored at 2-8 ° C until later use. The composition was formulated in Diluvac strong adjuvant. This adjuvant (see also EP 0 382 271) comprises 7.5 percent by weight of vitamin E acetate droplets with a volume-weighted average size of approximately 400 nm, suspended in water and stabilized with a 0 , 5 weight percent Tween 80 (sorbitan polyoxyethylene monooleate). Each milliliter of vaccine contained the material that had been extracted from 1.2E8 Lawsonia intracellularis cells.

IMMUNOPRECIPITATION OF LAWSONIA CARBOHYDRATE ANTIGENS

Two batches of the monoclonal antibodies (AcMc) created against Lawsonia intracellularis whole cells were precipitated with Na2SO4 saturated at room temperature according to the usual procedures. The precipitate was pelleted by centrifugation (10,000 g for 10 minutes). The sediment was washed with 20% Na2SO4 and resuspended in 0.04 M PBS. Tylosyl activated Dynal microspheres (DynaBeads, DK) were pre-washed with 0.1 M NaPO4 (at pH 7.4), according to the manual of the maker. From each batch of the AcMc, 140 pg were taken and pre-washed microspheres were added to 2E8, and incubated overnight at 37 ° C. The microspheres were pelleted by centrifugation, and unbound AcMc were removed by aspiration of the supernatant. Spectrophotometric measurements showed that between 20 and 35% of the added AcMc had bound to the microspheres.

Ultrasound was applied to two batches of 1 ml of Lawsonia intracellularis cells (3.7E8 / ml) in 0.04 M PBS for 1 minute. The resulting cell lysates were added to the Tylosyl-monoclonal activated microsphere complexes and incubated overnight at 4 ° C. Tylosyl-monoclonal activated microsphere complexes were washed three times with 0.1 M NaPO4 (at pH 7.4). The bound compounds were eluted by washing the microspheres with 0.5 ml of 8 M urea in 0.04 M PBS (E1); 0.5 ml of 10 mM Glycine at pH 2.5 (E2); and 0.5 ml of 50 mM HCl (E3), in a sequential manner. After elution, E2 and E3 were neutralized with 100 pl and with 200 pl of 1 M Tris / HCI (at pH 8.0).

Samples were taken from each stage and loaded into SDS-PAGE gels. The gels were stained using Commassie bright blue (CBB) and stained with silver or transferred. The transferred ones were developed using the same AcMc mentioned above. Inspection of gels and transferred showed that the AcMc recognized bands with an apparent molecular weight of 21 and 24 kDa that were not observed in CBB gels, but were visible in gels stained with silver. It was also established that the fraction of the cells that bound the AcMc was resistant to proteinase K. Therefore, based on these results it can be concluded that this fraction contains carbohydrates (namely: all protein is lysed, and the factions of DNA to which ultrasound was applied did not show such a clear band in a silver stain), and that carbohydrates are in association with (that is, being part of, or attached to) the outer cell membrane of Lawsonia intracellularis ( namely: the AcMc created against this fraction also recognized the whole cells of Lawsonia intracellularis). Given the fact that Lawsonia intracellularis is a gram-negative bacterium, it is believed that the carbohydrate composition comprises polysaccharide (s).

Example 2

This experiment was carried out to test a convenient way to formulate the carbohydrate antigen in a vaccine, namely, through an inactivated whole cell (also known as bacterin). As controls, the commercially available Enterisol® ileitis vaccine and an experimental subunit vaccine comprising protein subunits were used. Following this, unvaccinated animals were used as control.

EXPERIMENTAL DESIGN OF EXAMPLE 2

An inactivated whole cell vaccine was developed as follows. Live cells of Lawsonia intracellularis derived from pig intestines with PPE were collected. Cells were inactivated with 0.01% BPL (beta-

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proprioctone). The resulting material, which is inherently a non-living composition that contains the carbohydrate within the meaning of the present invention (in particular, since it contains carbohydrates as they are present in the live bacteria of Lawsonia intracellularis in association with its outer cell membrane), Diluvac (adjuvant) was formulated in a strong adjuvant at a concentration of approximately 2.8 x 108 cells per ml of vaccine.

The subunit vaccine contained recombinant P1 / 2 and P4 as known from EP 1219711 (the 19/21 and 37 kDa proteins, respectively), and the recombinant proteins expressed by the genes 5074, 4320 and 5464 as reported. described in WO2005 / 070958. Proteins were formulated in Diluvac strong adjuvant. The vaccine contained approximately 50 pgrams of each protein per milliliter.

Forty six-week-old SPF pigs were used. The pigs were assigned to 4 groups of ten pigs each. Group 1 was vaccinated once orally (at T = 0) with 2 ml of "Enterisol ® ileitis" of live bacteria (Boehringer Ingelheim) according to the manufacturer's instructions. Groups 2 and 3 were vaccinated twice intramuscularly (in T = 0 and in T = 4 weeks) with 2 ml of the Lawsonia inactivated whole cell vaccine and with the recombinant subunit combination vaccine as described above. respectively. Group 4 was left as a control without vaccinating. At T = 6 weeks all pigs were exposed orally to a mucosal homogenate infected with Lawsonia intracellularis. Subsequently, all pigs were observed daily to evaluate the clinical signs of porcine proliferative enteropathy (PPE). At regular intervals before and after exposure, blood serum (for serology) and feces (for PCR) samples were collected from pigs. At T = 9 weeks all pigs were slaughtered and they underwent a necropsy. Histological samples of the ileum were collected and analyzed under a microscope.

The exposure inoculum was prepared from infected mucosa: 500 grams of infected mucosa (scraped from infected intestines) were mixed with 500 ml of physiological saline. This mixture was homogenized in an omnimixer for one minute at maximum speed on ice. All pigs were exposed orally to 20 ml of the inoculum of exposure at T = 6 weeks.

At T = 0, 4, 6, 7, 8 and 9 weeks, a stool sample (in quantities of grams) and a blood serum sample from each pig were taken, and stored frozen until the test. Stool samples were tested in a quantitative PCR test (Q-PCR) and expressed as the logarithm of the amount found in picograms (pg). Serum samples were tested in the IFT test usually applied (immunofluorescent antibody test to detect antibodies against whole cells of Lawsonia intracellularis cells in the serum). For the histological score, the relevant ileum sample was taken, fixed with 4% buffered formalin, included routinely and cut into sheets. These sheets were stained with Hematoxylin-Eosin (staining with HE) and with an immunohistochemical staining using anti-Lawsonia intracellularis monoclonal antibodies (IHQ staining). The slides were analyzed under a microscope. Histology scores are as follows:

Staining with HE:

no anomalies were detected score = 0

doubtful injury score = ^

minor injuries score = 1

moderate lesions score = 2

serious injuries score = 3

IHQ staining:

there are no evident L. intracelluaris bacteria doubtful presence of bacteria

presence of individual bacteria / low amounts of bacteria on the slide presence of a moderate amount of bacteria on the slide presence of large amounts of bacteria on the slide

score = 0 score = ^ score = 1 score = 2 score = 3

All data were recorded individually for each pig. The score per group was calculated as the average of the positive animals for the different parameters after exposure. The non-parametric Mann-Whitney U test was used to assess statistical significance (bilateral test and significance level set at 0.05).

RESULTS OF EXAMPLE 2

Serology

Prior to the first vaccination, all pigs were seronegative when antibody titers were tested on the IFT. After vaccination with the whole cell bacterin (group 2), pigs developed high antibody titres in the IFT, while controls and pigs vaccinated with the subunit vaccine remained negative until exposure (Table 1). Two of the pigs vaccinated with Enterisol® (group 1) developed moderate titres in the IFT, while all other pigs in this group

They remained seronegative. After exposure, all pigs developed high antibody titers in the IFT. The average results are represented in Table 1 (with the dilution used, 1.0 was the detection level of the lower side).

5 Table 1. Average antibody titres in the IFT (2 log) in the serum of pigs after vaccination and

exposition

 Group

 T = 0 weeks T = 4 weeks T = 6 weeks T = 9 weeks

 one

 <1.0 1.1 1.7> 11.4

 2

 <1.0 3.7> 11.8> 12.0

 3

 <1.0 <1.0 <1.0> 11.6

 4

 <1.0 <1.0 <1.0> 12.0

Real-time PCR of stool samples

10 Before exposure all stool samples were negative. After exposure, positive reactions were found in all groups. Group 1 (p = 0.02), group 2 (p = 0.01) and group 3 (p = 0.03) had a significantly lower release level compared to the control. Table 2 provides a post-exposure summary.

15 Table 2. Mean PCR results in stool samples (log pg) after vaccination and exposure

 Group

 T = 6 weeks T = 7 weeks T = 8 weeks T = 9 weeks Total after exposure

 one

 0 1.3 3.6 1.8 6.3

 2

 0 0.8 2.8 1.9 5.5

 3

 0 0.5 3.8 2.0 5.9

 4

 0 0.8 4.9 4.9 10.0

Histology Scores

Group 2 had the lowest histology score of HE (p = 0.05), IHQ score (p = 0.08) and 20 total histology score (p = 0.08). The other groups had higher scores and were not significantly different from the control group. See table 3.

Table 3. Average histology score for ileus.

 Group

 HE score IHQ score Total score

 one

 1.8 1.5 3.3

 2

 1.3 1.5 2.7

 3

 1.8 1.6 3.4

 4

 2.4 2.3 4.7

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CONCLUSIONS REGARDING EXAMPLE 2

From the results it can be concluded that the systemic administration of a Lawsonia intracellularis non-living whole cell vaccine that inherently contains the carbohydrate as it is also found in association with the outer membrane of Lawsonia intracellularis living cells, induced at least one partial protection All parameters studied and histology scores were significantly or practically significantly better compared to controls.

Example 3

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This experiment was carried out to test a vaccine comprising a composition containing the carbohydrate as an antigen. A second vaccine to be tested also contained inactivated whole cells of Lawsonia intracellularis, Mycoplasma hyopneumoniae and porcine circovirus antigens (the "combi" vaccine). As a control, the commercially available Enterisol® ileitis vaccine was used. Along with this, the unvaccinated animals were used as the second control.

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EXPERIMENTAL DESIGN OF EXAMPLE 3

The vaccine based on a composition containing the substantially protein-free carbohydrate was obtained as described in Example 1.

The experimental combi vaccine contained inactivated whole cells of Lawsonia intracellularis as an antigen (see Example 2 for the method used to provide the inactivated bacteria) in an amount of 1.7 x 108 cells / ml. Next to this it contained inactivated PCV-2 antigen (20 pgrams of the protein encoded by PCV 2 ORF 2 per ml; the protein being expressed in a baculovirus expression system as is commonly known in the art, for example, according to It is described in WO 2007/028823) and inactivated Mycoplasma hyopneumoniae antigen (the same antigen at the same dose, as is known from the commercially available vaccine Porcilis Mhyo®, available from Intervet, Boxmeer, The Netherlands). The antigens were formulated in a double "X" emulsion adjuvant. This adjuvant is a mixture of 5 parts by volume of adjuvant "A" and 1 part by volume of adjuvant "B". Adjuvant "A" consists of droplets of mineral oil with an approximate average size (volume-weighted) of approximately 1 pm, stabilized with Tween 80 in water. Adjuvant "A" comprises 25% by weight of the mineral oil and 1% by weight of Tween. The rest is water. Adjuvant "B" consists of biodegradable vitamin E acetate droplets with an approximate average size (volume weighted) of 400 nm, also stabilized with Tween 80. Adjuvant "B" comprises 15% by weight of vitamin E acetate and 6% by weight Tween 80. The rest is water.

Sixty-four 3-day-old SPF piglets were used. The pigs were assigned to four groups of 14 piglets and a group of 8 piglets (Group 4). Group 1 was vaccinated intramuscularly at 3 days of age with 2 ml of the combination vaccine, followed by a second vaccination at 25 days of age. Group 2 was vaccinated intramuscularly once with 2 ml of the combined vaccine at 25 days of age. Group 3 was vaccinated orally with 2 ml of Enterisol® ileitis (Boehringer Ingelheim) at 25 days of age as prescribed. Group 4 was vaccinated intramuscularly at 3 and 25 days of age with 2 ml of the protein-free carbohydrate vaccine. Group 5 was left unvaccinated as an exposure control group. At 46 days of age, all pigs were exposed orally to an infected mucosa homogenate. Subsequently, all pigs were observed daily to evaluate the clinical signs of porcine proliferative enteropathy (PPE). At regular intervals before and after exposure, blood serum samples and stool samples were collected from pigs for serology and PCR, respectively. At 68 days of age all pigs were slaughtered and a post-mortem analysis was performed. The ileus was analyzed histologically.

The other aspects of the experimental design were the same as those described in Example 2, unless otherwise indicated.

RESULTS OF EXAMPLE 3

Serology

Before the first vaccination, all pigs were seronegative for antibody titers in the IFT against Lawsonia. After vaccination with the combi vaccine (groups 1 and 2) and with the protein-free carbohydrate vaccine (group 4), many pigs developed antibody titers in the IFT, while controls and pigs vaccinated with Enterisol remained negative until The exhibition. After exposure, all pigs (except two in the Enterisol group) developed antibody titers in the IFT. For a summary of the average values obtained, see table 4 (due to the higher dilution when compared to example 2, the detection level was 4.0).

Table 4. IFT antibody mean titers against Lawsonia (2 log) of pig serum after ___________________ vaccination and exposure ___________________

 Group

 T = 3 days T = 25 days T = 46 days T = 67 days

 one

 <4.0 <4.0 7.9 10.3

 2

 <4.0 <4.0 4.8 9.8

 3

 <4.0 <4.0 <4.0 8.5

 4

 <4.0 <4.0 6.9 10.6

 5

 <4.0 <4.0 <4.0 9.0

With respect to Mhyo, at the beginning of the experiment, as well as the day of revaccination (with 25 days of age), all pigs were seronegative for Mhyo. After revaccination, group 1 developed higher antibody titers against Mhyo, comparable to those obtained with the commercially available vaccine.

With respect to PCV, at 3 days of age the piglets had high titers of antibodies against the PCV of maternal diversion. On the day of revaccination (with 25 days of age) the vaccinated (group 1) had a degree

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similar in comparison with group 2 and with the control group. The title of the PCV at 25 days of age was slightly lower compared to the title at 3 days of age. After vaccination at 25 days of age, the titles of group 1 (2 vaccinations on day 3 and 25) and group 2 (one vaccination on day 25) remained at a high level, while the control piglets showed a normal decrease in maternal bypass antibodies. The PCV titres obtained are comparable to the titles obtainable with commercially available vaccines.

Real-time PCR of stool samples

Three weeks after exposure, pigs in groups 1, 2 and 4 had less Lawsonia (DNA) in their feces compared to groups 3 and 5. Only the differences between groups 1 and 3 (Enterisol) and groups 4 and 3 were statistically significant (p <0.05, Mann-Whitney U test). For the average results, see table 5.

Table 5. Mean PCR results in stool samples (log pg) after vaccination and exposure

 Group

 Middle value

 one

 1.0

 2

 1.2

 3

 2.0

 4

 0.6

 5

 1.8

Histology Scores

The histology scores of groups 1 and 4 were significantly lower compared to those of groups 3 and 5 (p <0.05, bilateral Mann-Whitney U test (see table 6). pigs with a confirmed PPE was 2/13 in group 1, 6/12 in group 2, 12/14 in group 3, 2/7 in group 4 and 12/14 in the group of control 5. Groups 1 and 4 had a significantly lower incidence of PPE compared to groups 3 and 5 (p <0.05, exact Fischer bilateral test).

Table 6. Average histology score for ileus.

 Group

 HE score IHQ score Total score

 one

 0.4 0.6 1.0

 2

 0.7 0.7 1.4

 3

 1.6 1.4 3.0

 4

 0.4 0.4 0.8

 5

 1.9 1.5 3.4

CONCLUSION OF EXAMPLE 3

From the results it can be concluded that the systemic administration of Lawsonia whole cell bacterin combined with PCV and Mhyo antigen, as well as the vaccine comprising (substantially protein-free) carbohydrate, administered at 3 days of age and at 25 days of age, both induce partial protection against experimental infection with Lawsonia intracellularis. It is particularly surprising that the vaccine is effective when the first administration takes place before weaning (under 21-25 days). It can be seen that in Examples 2 and 3, vaccines, as regards Lawsonia antigens, contain per ml antigenic material derived from more than 1E8 Lawsonia intracellularis cells. Given the fact that these vaccines, despite the use of mild adjuvants (namely, adjuvants containing small droplets and no, or little, mineral oil), provide good protection against ileitis, in particular When compared to the commercially available Enterisol® Ileitis vaccine, the dose of the antigens could be decreased. This could be done by administering less vaccine (reduced to, for example, 0.2 ml, suitable for an application, for example, intradermally), or by reducing the antigenic content of the vaccine. Based on analogs in vaccine technology, it is believed that with an antigenic dose (by vaccination) derived from, or containing, 1 E7 cells, in particular 2.5E7 cells or greater, still comparable or even comparable results can be obtained better than those obtained with the current commercially available vaccine. Given the fact that the combination vaccine provided Mhyo and PCV antibody titres at a level comparable to the levels obtainable with commercially available individual vaccines, it is understood that the combination vaccine also provides protection against Mycoplasma hyopneumoniae and the porcine circovirus

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

Example 4

This experiment was carried out to establish the effect of the dose of a vaccine according to the invention. Also, in this experiment, unvaccinated animals were used as control.

EXPERIMENTAL DESIGN OF EXAMPLE 4

Vaccines were made with inactivated whole cells as indicated in Example 2. The antigenic material was formulated in Diluvac strong adjuvant at a concentration of approximately 2.0 x 108 cells per ml vaccine, respectively 5.0 x 107 and 1.25 x 107 cells per ml of vaccine. Sixty 3-day-old SPF piglets were used. The pigs were assigned to four groups of 15 pigs each. The piglets of groups 1, 2 and 3 were vaccinated intramuscularly (in the neck) at 3 days of age and at 25 days of age with 2 ml of the vaccine each time. Group 4 was left as a control without vaccinating. At 46 days of age all pigs were orally exposed to Lawsonia bacteria as indicated in example 2. At 67 days of age all pigs were slaughtered and analyzed. The tests were performed as indicated in example 2. Next to this a PCR of mucosa samples is performed. To do this, a sample of the ileum of each animal was taken, when applicable from an area that showed thickening.

RESULTS OF EXAMPLE 4

Weight gain

From 14 days onwards, significant differences appeared in the total weight gain between the groups. Group 1 showed an average gain in total weight of approximately 5350 grams. In group 2 this was 5150 grams. Group 3 showed a weight gain of approximately 4250 grams, while group 4 showed a weight gain of 4550 grams.

Real-time PCR of stool samples

Three weeks after exposure, positive reactions were found in all groups. Group 1 and group 2 had a significantly lower level of release compared to the control. Table 7 provides a post-exposure summary of the number of infected animals (as determined by PCR).

Table 7. PCR result of stool samples after vaccination and exposure

 Group

 Number of infected animals after exposure

 one

 1/15

 2

 2/15

 3

 7/15

 4

 8/15

Real-time PCR of mucosal samples

Three weeks after exposure, positive reactions were found in all groups. Group 1 and Group 2 had a significantly lower level of release compared to the control. A summary post-exposure summary of the number of infected animals is provided in Table 8 (as determined by PCR).

Table 8. PCR result of mucosal samples after vaccination and exposure

 Group

 Number of infected animals after exposure

 one

 0/15

 2

 2/15

 3

 5/15

 4

 6/14 (no samples of pig n ° 8)

Histology Scores

The total histology score and the number of animals that were confirmed to have PPE are shown in Table 9.

Table 9. Average histology score for ileus.

 Group

 Total score Number of animals with PPE

 one

 0.3 0/15

 2

 0.8 2/14

 3

 1.1 4/15

 4

 1.9 7/15

CONCLUSION OF EXAMPLE 4

5 Contrary to expectations, the results indicate that there is a very sudden decrease in the protective effect around the lowest dose used in these experiments. Although a dose of antigenic material derived from 2.5 x 107 cells still provided a protective effect comparable to that of the commercially available vaccine, the fact that the reduction between a dose that is only 0.6 log greater is so significant (barely observed no effect on weight gain, the number of infected animals and the mucosal PCR; however, there is still a decrease in the number of animals recognized with PPE), which provided the perception that, in general, a lower Practical effective dose of the antigen can result in: an amount of antigens smaller than that obtained from, or containing, 1 x 107 cells, which in practice, under current market circumstances, will not result in economically relevant results . The reason for the existence of this apparent cut-off value is not 100% clear. A more gradual decrease in protection is usually expected 15 when the dose is reduced. It could be that to combat a local infection in the mucosa of the intestine through an immune response of systemic bypass a minimum amount of antigens is required.

Along with the above, the surprising effect observed in Example 3, namely that a vaccine based on a carbohydrate antigen and administered systemically is effective when the first administration takes place before weaning (under 21-25 days) , is confirmed in this experiment with the use of another adjuvant. Therefore, it can be reasonably understood that this characteristic is generic for all vaccines comprising a carbohydrate antigen.

Claims (3)

1. A non-living composition containing a carbohydrate, the carbohydrate also being found in the live cells of Lawsonia intracellularis in association with the outer cell membrane of these cells, for use as a vaccine for protection against a Lawsonia intracellularis infection , by systemic administration of the vaccine, wherein the composition containing the carbohydrate is the material resulting from the inactivation of Lawsonia intracellularis bacteria and where the composition containing the carbohydrate contains whole cells of inactivated Lawsonia intracellularis bacteria. A non-living composition containing a carbohydrate for use according to claim 1, characterized by that the vaccine comprises an oil-in-water adjuvant containing oil droplets of submicron size. 3. A non-living composition containing a carbohydrate for use according to claim 2, characterized in that the adjuvant comprises droplets of a biodegradable oil and droplets of a mineral oil, the droplets having of the biodegradable oil an average size that differs from the average size of the mineral oil droplets. 4. A non-living composition containing a carbohydrate for use according to any of the preceding claims, characterized in that the vaccine additionally comprises Mycoplasma hyopneumoniae antigens 20 and porcine circovirus.