PL246286B1 - Method of producing protein complexes with bacterial lipopolysaccharides, their use, vaccine containing an immunogenic LPS/protein complex and method of producing the vaccine - Google Patents

Description

Description of the invention

The invention relates to a method of producing complexes of bacterial lipopolysaccharides (LPS) with proteins with immunogenic properties and their use for the formulation of antibacterial vaccines. LPS complexes with a carrier protein are used as immunogens, i.e. molecules capable of inducing an immune response in vertebrate organisms in order to produce an immune response aimed at protecting against pathogens or to obtain antibodies specific to the immunogen used. Conjugates of proteins with LPS or sugar fragments obtained from it are used in antibacterial vaccines and for obtaining sera containing antibodies specific to sugar antigens and obtaining monoclonal antibodies specific to these antigens. The invention also relates to a vaccine containing an immunogenic complex and a method of producing the vaccine.

Gram-negative bacterial infections constitute a significant percentage of bacterial infections and hospital infections. Mortality is significant despite the use of antibiotics. The problem is getting worse with the increase in the number of multidrug-resistant strains, which are responsible for particularly severe cases of bacteremia, often ending in sepsis and death of the patient. Sepsis therapy is one of the important medical issues that is still far from being resolved. Currently, a number of therapeutic strategies are used and studied, the most important of which are: removal of bacterial endotoxins from the bloodstream, therapies supporting vital functions of key organs, inhibition of excessive activation of the immune system, neutralization of endotoxins with antibodies. Another approach is to generate immunity to pathogens and endotoxins using appropriate vaccines. The target for vaccines is traditionally protein antigens, but due to the fact that the main component of the outer membrane of the cell wall of gram-negative bacteria is bacterial lipopolysaccharide, a number of studies have been undertaken to use LPS as a target for vaccines.

Lipopolysaccharides are a group of macromolecules composed of a lipid component, usually called lipid A, and a sugar part with a diverse structure and structure. LPS also contains low-molecular-weight substituents that affect the biological and physicochemical properties of LPS. These include, for example, phosphate groups, diphosphates, phosphoethanolamine, methyl-phosphoramidate groups, amino acids, glycerol and its derivatives, and others. LPS containing a sugar part composed of a small number of sugar residues is often referred to as LOS (lipooligosaccharide). The sugar part of LPS is typically composed of two parts: a core oligosaccharide and a polysaccharide also called the O antigen. LPS strongly interacts with immune system cells, responsible for the toxic effects observed during gram-negative bacteremia and sepsis. Lipid A is responsible for most of the biological activities responsible for LPS toxicity. The LPS molecule is amphiphilic due to its structure, in water it forms complex micellar systems, which may have different structures and sizes. LPS can be dissolved in some solvents, e.g. dimethyl sulfoxide (DMSO) after appropriate preparation US patent 8,426,221. In an acidic environment and at elevated temperature, LPS decomposes with the release of lipid A, the sugar part, which can be purified and used to create vaccines.

The LPS construction diagram is shown below:

Lipid A

Inner Core Outer Core Antigen O

The concept of obtaining immunity to Gram-negative pathogens by inducing antibodies directed against the O-specific antigen of lipopolysaccharide was proposed by Robbins et al., 1992.

Due to the poor immunogenicity of LPS and the lack of T cell activation, obtaining a good response is associated with the necessity of creating conjugates of O-antigens with a carrier protein, similarly to the vaccines already used in humans against /7. influenza, pneumococcus, meningococcus, which contain conjugates of capsular polysaccharides with protein. Oligosaccharides obtained by enzymatic hydrolysis of LPS from Salmonella thyphimurium conjugated with a carrier protein induced a strong response to sugar antigens in mice and rabbits (Swenson and Lindberg, 1981; Swenson et al., 1979).

Subsequent work focused on glycoconjugate vaccines based on O-specific antigens, which provided effective protection in animal models. Currently, a number of vaccines against Gram-negative pathogens based on the above concept (S. paratyphiA, E. coliO157:H7, Pseudomonas aeruginosa, Shigella) have passed or are in clinical trials (Konadu et al., 2000, Levine et al., 2007; Ahmed et al., 2006).

The disadvantage of vaccines based on the O-specific antigen is the limited reactivity of the obtained antibodies - limited to strains of a given serotype.

Gram-negative bacterial infections are also a major problem for animal farmers, especially in poultry, pig and cattle production. The main species of gram-negative bacteria responsible for losses in animal production are E. coli, Salmonella, Listeria, Campylobacter. Currently, the global animal vaccine market reached a value of USD 5.81 billion in 2016 and is estimated to grow to USD 7.68 billion in 2021. The compound annual growth rate (CAGR) in 2016-2021 will be 5.7%.

Veterinary vaccines are mostly killed or attenuated bacterial cells of the strains most often causing infections. For example, the Coliprotect F4/F18 vaccine produced by (Prevtec Microbia GmbH) consists of two strains of live E. coli bacteria (O8:K87 and O141:K94) that do not produce toxins that cause disease. Others contain bacterial proteins that are virulence factors such as adhesins and toxoids (Porcilis Porcoli Diluvac Forte produced by Intervet International B.V.; Nobilis E.Coli produced by: Intervet International B.V.). Conjugate veterinary vaccines are currently in the laboratory testing phase, and LPS-based vaccines are not used in veterinary medicine. The disadvantage of current vaccines is their serotype specificity limited to the strains used for immunization. The use of LPS as an antigen allows the production of vaccines with both narrow and broader specificity.

The O antigen is the most variable part of LPS, unique to a given serotype, and therefore one of the key antigens for serotyping bacteria. Core oligosaccharides have a much more conserved structure, e.g. within the E. coli species, all serotypes have one of five types of core oligosaccharide, while in Salmonella bacteria, only one type of core oligosaccharide has been identified so far. LPS is a major component of the outer membrane of the cell wall of gram-negative bacteria, and is therefore one of the major antigens on the cell surface and a potential target for vaccines.

Vaccines that use isolated, purified antigens are called subunit vaccines, their advantage is the strong induction of a response to the selected antigen, which in the case of such a complex immunogen as the entire bacterial cell is not always possible due to antigenic competition between different molecules on its surface. Other advantages include a strictly defined, repeatable composition, a smaller number of side reactions, and targeting the response to antigens that provide protection.

Lipopolysaccharides, like polysaccharides and oligosaccharides, are weak immunogens, and therefore constitute haptens, which only after conjugation with a protein carrier acquire the ability to induce IgG antibodies and immunological memory necessary for obtaining good protection. The formation of LPS protein conjugates requires partial decomposition of LPS in order to release the water-soluble sugar part. As a result of acid hydrolysis of lipopolysaccharide, it is cleaved at the junction of the inner part of the core oligosaccharide, usually containing 3-deoxy-D-manno-octulosonic acid (KDO) molecules, with lipid A. The released sugar part of LPS is then purified and conjugated to the carrier protein. There are several methods of conjugating oligosaccharides and polysaccharides to proteins known to those skilled in the art. Most often, conjugation takes place in two steps: activation of the saccharide and conjugation to the protein. Coupling by reductive amination between terminal ketose or aldehyde groups of the saccharide and amino groups of proteins in the presence of a reducing agent, usually sodium cyanoborohydride, is often used. Regardless of the method used, the key step is hydrolysis of LPS to release the sugar moiety. Despite the relatively mild reaction conditions, the inner core part composed of KDO is degraded, and a significant portion of non-sugar substituents, which are important for the antigenic properties of LPS as elements of epitopes, are released and lost.

The method described in the invention allows obtaining a complex in a way that does not cause degradation and other chemical modifications of LPS, while fully preserving its antigenic features. The invention describes a method of conjugating LPS with proteins, in which there is no need for partial degradation of LPS, conjugation does not require the formation of a covalent bond between LPS and protein, which can also lead to modification and loss of some epitopes. Conjugation consists in the formation of a non-covalent complex between LPS and protein in a multi-stage process. In the first stage, LPS is subjected to a procedure of removing metal cations bound to it and exchanging them for protons, which results in an acidic form of LPS, highly soluble in DMSO, in an analogous process a protein is obtained, in which all ionically bound cations are converted into protons. Then LPS and protein are dissolved in DMSO and both solutions are mixed together, obtaining a mixture of LPS protein in DMSO in the desired weight ratio. The DMSO is then gradually diluted with water and the DMSO is removed by dialysis, ultrafiltration or gel filtration. The complex can then be used to formulate an immunogen or vaccine with any adjuvant.

The invention is characterized in that the resulting complex is capable of inducing an immune response with characteristics typical of TH2 type antigens, i.e. with antibody class switching, resulting in the synthesis of IgG, IgA antibodies, while the primary response is characterized by the production of IgM class antibodies and immunological memory, contains a complete LPS molecule, does not cause toxic effects in vaccinated animals in doses used for immunization. Advantageously, the lipid A contained in the complex, which is a component of LPS, has immunostimulating properties, ensuring a strong immune response.

The process of obtaining the vaccine consists of the following steps:

a) bacterial cell cultures;

b) LPS isolation;

c) converting LPS into a DMSO-soluble form;

d) forming the complex by mixing LPS and protein solutions in DMSO and changing the solvent to water;

e) lyophilization or formulation with an adjuvant.

The obtained complex can be administered by injection without an adjuvant or in a formulation with an additional adjuvant providing a deposition effect, e.g. mineral oil. The results of the experiments indicate that the obtained complexes are capable of inducing a high titer of IgG antibodies specific to the LPS used for vaccination and antibodies specific to the carrier protein, which indicates appropriate activation of T lymphocytes. Due to the very similar physicochemical properties of LPS, the method is universal and allows the preparation of an immunogen from LPS isolated from any strain of gram-negative bacteria. In response to the immunogen, antibodies directed against the used carrier protein are also formed, thanks to which the progress of immunization can be monitored in the case of a residual level of antibodies to the LPS of a given bacterium as a result of environmental exposure and antibody transfer from the parent organism. This is important in the case of veterinary vaccines, where a so-called marker is required.

At the same time, the lack of toxicity of the immunogens used in the vaccination tests of the Balb/C strain mice was found. The LPS dose was about 1.1 mg/kg of body weight. This is a dose 5 times lower than the lethal dose for pigs (5 mg/kg of body weight). The vaccine preparations were also tested on cell lines, where their toxicity was also not demonstrated.

The subject of the invention is a method for producing bacterial lipopolysaccharide (LPS)/protein immune complexes, characterized in that it comprises the following steps:

a) LPS is isolated from bacteria using any method and then converted into a form that is highly soluble in DMSO by removing metal cations associated with LPS and exchanging them for protons, converting the bacterial lipopolysaccharide into an acidic form;

b) a protein is obtained that is highly soluble in DMSO, with all ionically bound cations being converted into protons;

c) dissolving the LPS and protein in DMSO and mixing the two solutions to obtain a mixture of LPS and protein in DMSO in a specified weight ratio; and then

d) DMSO is gradually diluted and removed, without causing degradation and chemical modification of LPS, maintaining the complete LPS molecule and its antigenic properties, and conjugation of LPS with the protein occurs in a non-covalent manner.

Preferably, the method is characterized in that the LPS is derived from any strain of Gram-negative bacteria such as Escherichia, Hafnia, Salmonella and Campylobacter.

Preferably, the method is characterized in that the protein used in the complex may be any protein with immunogenic properties that is soluble in DMSO.

Preferably, the method is characterized in that the protein used in the complex is casein or bovine serum albumin.

Preferably, the method is characterized in that the DMSO is diluted with water.

More preferably, the method is characterized in that the DMSO is removed by dialysis, ultrafiltration or gel filtration.

Another subject of the invention is a lipopolysaccharide/protein complex produced by the above method for use in inducing an immune response.

Preferably, the lipopolysaccharide/protein complex is for use in the production of an immunogen or an antibacterial veterinary vaccine.

Another subject of the invention is a method for producing a veterinary vaccine containing an LPS/protein immune complex comprising the following steps: culturing bacterial cells, isolating LPS, characterized in that LPS is converted into a DMSO-soluble form by removing metal cations bound to LPS and exchanging them for protons, obtaining a DMSO-soluble protein in which all ionically bound cations are exchanged for protons, dissolving LPS and the protein in DMSO and then mixing the obtained solutions to form a non-covalent complex exhibiting immunogenicity, wherein the diluent used to remove DMSO from the preparation is water.

Preferably, LPS is isolated by the phenol-water extraction method.

Preferably, DMSO is removed by dialysis, ultrafiltration or gel filtration.

Preferably, the obtained preparation is freeze-dried or formulated with any adjuvant.

Still another subject of the invention is a veterinary vaccine containing the complex produced by the method described above.

Preferably the vaccine is in lyophilized form.

Preferably, the vaccine is formulated with an oil adjuvant such as Freund's incomplete adjuvant.

Preferably, the carrier protein of the complex is a marker that allows tracking of the progress of immunization in a subject.

Advantageously, the vaccine demonstrates a broad spectrum of activity against all LPS versions (serotypes) found in Escherichia coli, Salmonella and Hafnia alvei strains.

Advantageously, the vaccine stimulates a specific humoral response in the individual, causing an increase in anti-LPS IgG antibodies.

Preferably, the subjects to be immunized may be birds and mammals, such as chickens and pigs.

Preferably, immunization is by intramuscular or intraperitoneal administration.

Preferably, the vaccine does not cause toxic effects in vaccinated animals.

Preferably, the vaccine comprises a complex of LPS with the protein in PBS and optionally any other adjuvant.

The technical problem solved by the invention is to obtain an innovative vaccine, which has not been used in veterinary medicine so far. The invention is distinguished by the fact that the described technology can easily obtain immunogenic LPS protein complexes. The presence of the protein in the complex provides a TH2 type response, which is desirable in the case of vaccines. At the same time, it is not necessary to chemically couple LPS to the protein to obtain the complex, as is the case with known vaccines. Surprisingly, the response to LPS after immunization with such a complex is very strong and exceeds the response to LPS in the case of immunization with killed bacterial cells. Vaccines containing whole, unmodified LPS demonstrate a broader spectrum of action. This is an improvement over vaccines based on the O antigen, whose reactivity of the obtained antibodies is limited to strains of a given serotype. In the case of the vaccines disclosed in the present invention, the reactivity of antibodies obtained by immunization with the LPS/protein complexes used in these vaccines is specific to all versions of LPS found in E. coli strains, which ensures that the vaccine is effective against all serotypes of E. coli found in the environment. Broad reactivity is achieved by using a mixture of rough lipopolysaccharides representing all 5 core types known in E. coli in the complexes. Similarly, in the case of Salmonella, which has 1 core type, the use of rough LPS ensures reactivity against all serotypes of Salmonella.

The accompanying figures show: Fig. 1 shows a standard curve for Salmonella R60 LPS in the range 0-100 µg LPS, Fig. 2 shows the level of anti-Salmonella LPS IgG antibodies in chicken serum, and Fig. 3 shows the level of anti-carrier protein IgG antibodies in chicken serum.

The invention has been presented in detail in the following examples, but these do not limit its scope.

Examples of execution

Example 1. LPS isolation

Lipopolysaccharides from bacterial strains: Hafnia alvei 1216, Hafnia alvei 1286, Eschericha coli: F470, F576, F653, F2513, W3100 and Eschericha coli K12 C600 (K-12) Salmonella minnesota R60, Campylobacter jejuni were isolated by the phenol-water extraction method according to the publication by Westphal and Jann, New York: Academic Press Inc.; 1965(5):83-91.

Dry bacterial mass was suspended in water in the ratio of 5 g of bacterial mass per 100 ml of water, then the suspension was mixed with an equal volume of 90% phenol heated to 65°C and stirred at this temperature in a water bath for 15 min. After cooling, the mixture was centrifuged to separate the phases (5000 χ g, 20 min), the water phase was collected, the phenolic phase was supplemented with clean water to the previous volume and re-extracted at 65°C. The material was centrifuged as before, the water phase was collected and the extraction process was repeated with another portion of water. The combined water phases were dialyzed in a dialysis bag (cut-off mass 12 kDa) until the phenol odor was lost. The dialysate was subjected to centrifugation (5000 g, 10 min) to remove precipitated particles, then LPS was pelleted by ultracentrifugation (100,000 χ g, 2 h). The LPS pellet was suspended in 0.2 M NaCl (25 mg LPS/ml), nucleases (RNase and DNase) were added in the amount of 50 μg/ml, the mixture was incubated for 2 h at 37°C. Then LPS was subjected to 2-time ultracentrifugation in water to remove fragments of nucleic acids and nucleotides. The purity of the preparation (no nucleic acids) was controlled spectrophotometrically, the absence of a peak at a wavelength of 260 and 280 nm indicated the absence of contamination with proteins and nucleic acids.

Example 2. Preparation of DMSO-soluble LPS mg LPS was dissolved in 2 ml of 2% SDS with EDTA (50 μl of 0.5 M r-ru), centrifuged to remove insoluble impurities (16,000 χ g for 5 min) and precipitated with 4 volumes of ethanol. The material was centrifuged (16,000 χ g for 30 min), the supernatant was collected and discarded, the pellet was suspended in ethanol (95%) and centrifuged (16,000 χ g for 30 min), the step was repeated. The pellet was dissolved in 2 ml of water and passed through a column filled with Dowex 50Wx8 ion exchanger in acidic form. The column was washed with water, fractions exhibiting acidic pH were collected, combined and lyophilized. The procedure was applied to LPS from E. coli: F470, F576, F653, F2513, W3100 and E. coli K12 C600.

Example 3. Preparation of E. coli LPS soluble in DMSO mg of LPS from E. coli K12 C600 was dissolved in 4 ml of 2% SDS, then passed through a column packed with Chelex 100, the column was washed with water, the fraction containing LPS was collected (the presence of LPS was detected by a colorimetric test phenol, sulfuric acid). LPS was precipitated with 4 volumes of ethanol and recovered by centrifugation (16,000 g for 30 min), the supernatant was collected and discarded, the pellet was suspended in ethanol (95%) and centrifuged (16,000 g for 30 min), the step was repeated. The pellet was dissolved in 2 ml of water and passed through a column packed with Dowex 50Wx8 ion exchanger in acid form. The column was washed with water, LPS fractions exhibiting acidic pH were collected, combined and lyophilized. This procedure was also used to obtain acidic forms of LPS: Hafnia alvei 1216, Hafnia alvei 1286.

Example 4. Preparation of Salmonella minnesota LPS soluble in DMSO mg of Salmonella minnesota R60 LPS was suspended in water (10 ml) and 1 ml of 0.5 M EDTA pH 8.0 was added. The material was transferred to a dialysis bag (12 kDa cut-off mass) and dialyzed into water to remove EDTA. The material after dialysis was collected in a Falcon tube (50 ml), 2 ml of Dowex 50Wx8 suspension in acid form was added, incubated for 1 hour on a serological rotator and then centrifuged on a swing-out rotor (5000 χ g for 5 min), the aqueous phase was collected and lyophilized.

The above methods from examples 2-4 presenting the preparation of LPS from bacterial cells are alternative and equivalent methods, the basis of their application is the removal of divalent metal ions from LPS and then its transformation into an acidic form, which is a necessary step of the procedure. These methods can be used interchangeably for any lipopolysaccharides isolated from different bacteria. The choice of technique does not depend on the origin and structure of the lipopolysaccharide.

Example 5. Preparation of Salmonella R60 LPS/cow's milk casein complex mg LPS soluble in DMSO was dissolved in 2 ml DMSO. 10 mg cow's milk casein was dissolved in 2 ml DMSO, the suspension was lightly sonicated until a clear solution was obtained. The solutions were mixed and incubated overnight with stirring at room temperature. The mixture was then slowly diluted with water with constant stirring. The rate of water addition was adjusted to avoid an increase in the temperature of the mixture by more than 10°C. After reaching a 1:1 ratio, the mixture was transferred to a dialysis bag and dialyzed against water until the DMSO was removed.

The material was transferred to a centrifuge tube and centrifuged to recover the LPS/protein complex (16,000 x g, for 20 min). The complex was collected and dissolved in PBS. The LPS content in both fractions, the supernatant and the complex, was then determined by a colorimetric method using phenol and sulfuric acid as described in DuBois M. et al. "Colorimetric Method for Determination of Sugars and Related Substances" Anal. Chem., 1956, 28 (3), pp. 350-356.

For this purpose, a standard curve was prepared for Salmonella R60 LPS in the range of 0-100 μg LPS (shown in Fig. 1) and the LPS content in both fractions was determined, showing that 66.2% of LPS was in the fraction containing the complex.

Example 6. Preparation of Salmonella R60/casein and H. alvei 1216/casein, H. alvei 1286/casein LPS complexes from cow's milk

Each of the samples, 10 mg of LPS in DMSO-soluble form, was dissolved in 1 ml of DMSO and mixed with 1 ml of DMSO containing dissolved cow's milk casein. The solutions were incubated overnight with stirring at room temperature. Then the mixtures were slowly diluted with water under constant stirring, the rate of water addition was set to avoid the temperature of the mixture increasing by more than 10°C. After reaching a 1:1 ratio, the mixtures were transferred to dialysis bags and dialyzed into water until DMSO was removed. The material was transferred to a centrifuge tube and centrifuged to recover the LPS/protein complex (16,000 x g for 20 min). The complexes were collected and dissolved in PBS, the presence of LPS was confirmed by colorimetric method.

Example 7. Preparation of E. coli LPS complexes: F470, F576, F653, F2513 and K12 with cow's milk casein

Samples of 50 mg of E. coli LPS: F470, F576, F653, F2513 and K12 were dissolved in 5 ml of DMSO and mixed with 5 ml of DMSO containing 50 mg of cow's milk casein. The solutions were incubated overnight with stirring at room temperature. Then the mixtures were slowly diluted with water with constant stirring, the rate of water addition was set to avoid the temperature of the mixture to increase by more than 10°C. After reaching a 1:1 ratio, the mixtures were transferred to dialysis bags and dialyzed into water until DMSO was removed. The material was transferred to a centrifuge tube and centrifuged to recover the LPS/protein complex (16,000 g for 20 min). The complexes were collected and dissolved in PBS, the presence of LPS was confirmed by colorimetric method.

Example 8. Prepared Bovine Serum Albumin (BSA) Proteins Soluble in DMSO 100 mg BSA was dissolved in 10 ml of water, 1 ml of 0.5 M EDTA pH 8.0 was added and dialyzed in a dialysis bag to remove EDTA. Then the protein solution was passed through a column packed with Dowex 50Wx8 bed, the column was washed with water, the fractions containing the protein were collected and lyophilized.

Example 9. Prepared Salmonella LPS/BSA complex mg BSA obtained in Example 8 was dissolved in 2 ml DMSO, separately 10 mg Salmonella R60 LPS was dissolved in 1 ml DMSO. Both solutions were mixed and incubated overnight with stirring at room temperature. Then the mixture was gradually diluted with water in a 1:1 ratio with constant stirring, the rate of water addition was set so as to avoid an increase in the temperature of the mixture by more than 10°C. The diluted mixture was transferred to a dialysis bag and dialyzed into water until DMSO was removed and then into PBS.

Example 10. Immunization of mice with Hafnia alvei 1216/cow's milk casein complex

Balb/c mice were immunized with a Hafnia alvei 1216/casein complex in PBS in an amount corresponding to 20 μg of LPS per dose. The complex was administered without additional adjuvant intraperitoneally. Animals were immunized 3 times at 3-week intervals. Control groups were given LPS alone or casein alone (20 μg per mouse). Blood samples were collected 10 days after the last vaccination,

PL 246286 Β1 serum was prepared and analyzed by ELISA for reactivity with the antigen used for immunization. The titer of the sera to Hafnia alvei 1216 LPS is presented in the table below.

The antibody titer against Hafnia alvei LPS in mouse serum is shown in Table 1.

Table 1

titer (serum dilution at which the OD450 reading is 0.2 above the background level (IU/ml) before vaccination after 1 vaccination after 2nd vaccination after 3rd vaccination mouse 1 148 17442 34183 49149 mouse 2 304 11576 5757 10548 mouse 3 222 3124 6157 3961 mouse 4 219 14629 19108 97137 mouse 5 256 1301608 523436 1665934

Example 11. Immunogenicity study of the Salmonella R60 LPS/casein complex from cow's milk in chickens.

Laying hens were immunized twice in the breast muscle with the Salmonella R60/casein LPS complex in the amount of 200 pg of the complex in PBS without additional adjuvant. Blood samples were taken before immunization. The interval between vaccinations was 3 weeks. Blood samples were taken again 14 days after the second vaccination. Sera were analyzed by ELISA by testing a series of serum dilutions, the titer was calculated based on the obtained curves, defined as the highest dilution at which the signal is > 0.2 units above the background level. The titer of the sera is presented in the table below.

The IgG antibody titer in the chicken sera in response to the Salmonella R60/casein LPS complex is presented in Table 2.

Table 2

IgG titer before vaccination IgG titer 2 weeks after second vaccination Hen 1 1715 2696 Hen 2 898 1107 Hen 3 2234 2521 Hen 4 1148 2429 Hen 5 1392 5517 mean 1477 2854

Example 12. Testing the effectiveness of the vaccine in chickens experimentally infected with Salmonella bacteria.

In the study, 6-week-old hens were immunized with the Salmonella minnesota/casema LPS complex using an oil adjuvant (incomplete Freund's adjuvant). The hens were divided into groups: control, group A (unvaccinated) and two test groups vaccinated with different amounts of the complex: group B - 50 pg of the complex, group C - 150 pg of the complex. The animals were immunized with two doses at an interval of 4 weeks. After the second vaccination, the animals were subjected to an experimental infection with a field isolate of Salmonella. The infection was performed twice on the 28th and 62nd day after the second vaccination. Approximately one week after the last infection, the animals were euthanized and the following organs were collected for microbiological examination: liver, bursa of Fabricius, spleen, blood samples and cloacal swabs.

PL 246286 Β1

The results of the organ analysis are summarized in Table 3 below. Results of microbiological examination of the organs of immunized hens. (-) - no presence of Salmonella bacteria in the organ, (+) - presence of Salmonella bacteria in the organ.

Table 3

Table 3 Collection of material on the day of completion of experiments Sample collection before experimental infection Collection of material after introducing the chickens into the hen house liver spleen bag Fab. Cloaka Cloaca Cloaca Control Group A Group B 1 - - - - 2 - - ' - - - 3 - - - 4 + - - - - 5 + - - - - 6 - - 7 + + - - - 8 + + + - - 9 + - - - 10 11 + + ---:----------- -------------------------------------- - 12 13 - --—............. .........................—.............— 15 - - - - - 16 17 + - - ...................————— ..........................—. 18 - - : - - - 19 - - - 20 + + - - —................... — - - - 22 ~23 —.....r- ·-....... ----------------------- ........................................- 24 + - + - - 25 —— + - - 27 - - ' - - - 28 - - - 29 - - - 30 + + +

Cloacal swabs taken on day 1, 56 and 98 did not show the presence of Salmonella in this organ. The presence of Salmonella was also tested in organs taken from birds after slaughter (day 98). In the case of liver of unvaccinated hens, the presence of bacteria was confirmed in 60% of hens, in the groups of vaccinated hens

PL 246286 Β1

20%. Similarly, in the case of testing for Salmonella in the spleen - 30% in unvaccinated hens and 10% in groups of vaccinated hens.

In order to check the immunogenicity of the prepared preparations, the level of IgG antibodies in the serum of chickens after vaccination was analyzed using the ELISA method (Fig. 2 - Salmonella LPS and Fig. 3 - carrier protein).

A significant increase in antibodies against Salmonella LPS and carrier protein was confirmed.

Example 13. Testing the effectiveness of the vaccine on pigs

The study aimed to investigate the activity of the developed vaccine against colibacillosis and confirm its effectiveness in preventing this disease in piglets. The study used a total of 9 sows and 27 piglets that were descendants of these sows (3 from each litter). The experiment consisted of 3 experimental groups of 3 sows in each group.

In order to obtain animal material for the study, 26 DanBred hybrid gilts were purchased from a Danish breeding farm. DanBred Hybrid is the first cross between DanBred Landrace and DanBred Yorkshire. It is an optimal mother breed that ensures high production efficiency by combining the best features.

Study groups:

Group D - sows vaccinated twice with the experimental vaccine and their piglets infected with an infectious dose of E. coli strain F4 (K88) producing Stx2e toxin (1 x ¹⁰⁹ CFU/1 ml in a volume of 5 ml/piglet).

Gr. K1 - unvaccinated sows, infected piglets identically to piglets from group D.

Gr. K2 - unvaccinated sows and piglets, sows and piglets from this group were not infected intentionally, infections in this group occurred spontaneously, as is the case on farms.

Vaccination was performed with a preparation (300 pg per dose) consisting of an equilibrium mixture of complexes prepared according to Example 5 containing a total of 150 pg of LPS (total LPS) per dose. The complex was administered with an oil adjuvant (incomplete Freund's adjuvant).

Composition of the immunization mixture (single dose):

0.5 ml of LPS/casein complex mixture in PBS containing a total of 150 pg LPS;

0.5 ml of incomplete Freund's adjuvant.

Pregnant sows were vaccinated intramuscularly at 3-week intervals (8th and 11th weeks of gestation).

Piglets were infected on the 7th day of life. Then, clinical observations, temperature measurement, health control were carried out and production results were assessed based on body weight gain. Piglets were euthanized on the 21st day of life, material for histopathological examination was collected: samples of jejunum, duodenum, ileum and colon. The same samples were the material for microbiological examination.

Antibody levels were analyzed in serum of vaccinated sows, piglets and colostrum.

Research results

The most frequently reported symptom of infection was diarrhea, observed with varying intensity in all study groups.

Table 4 below presents clinical observations of piglets subjected to experimental infection. Piglets were infected on the 7th day of life with a bacterial suspension at a dose of 1 χ 10 ⁶ CFU/1 ml in a volume of 5 ml/piglet. The percentage of piglets with specific symptoms and the percentage of deaths were calculated as a percentage of the number of initial piglets, i.e. on the day before the recorded symptom. Abbreviations: A - body stiffness and apathy, lack of appetite; B - diarrhea; C - weight loss; K - lameness; U - death of the animal (death).

Table 4

Group Initial number of piglets symptom code Days after experimental infection* 1 2 3 4 5 G 7 8 9 10 11 12 13 1+ n(%) n (%) n(%) (%) O (%) n<%) n(%) n(%) n (%) n(%) O (%) n(%) O (%) n(%) KI 35 A B K U 0(0.00) 16(45.71) 7 (20.00) 0(0.00) 2(551) 0 (0.00) 5 (15.13)1 4(12.12) 0 (0.00) 0 (0.00) 0 (0.00) 6|18.18) 4(12.12) 0 (0.00) 2 (6.06) 0(0.00) 11 (33.48) 6 (19.35) 0(0.00) 1(3.12) 0 (0.00) 9(30.00) 6 (20.00) 0 (0.00) 0 (0.00) 0(0.00) 8(26.66) 4(13.33) 0 (0.00) 4(12.9) 0(0.00) 4(15.38) 4(13.38) 0(0.00) 1 (3.84) 0 (0.00) 3 (12.00) 0 (0.00) 0 (0.00) 0 (0.00) 0(0.00) 0(0.00) 1 (4.00) 0(0.00) 0(0.00) 0(0.00) 0 (0.00) 1 (4.00) 0 (0.00) 0 (0.00) 0(0.00) 0(0.00) 1 (4.00) 0(0.00) 0(0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 1 (4.00) 0(0.00) 0(0.00) 0(0.00) 0(0.00) 0(0.00) 0 (0.00) 0 (0.00) 1 (4.16) 0 (0.00) 0 (0.00) K2 37 B C K u 0(0.00) 25 (67.56) 26(70.27) 0(0.00) 0(0.00) 0 (0.00) 22 (59.45) 23 (62.16) 0 (0.00) 0 (0.00) 0 (0.00) 14(37.83) 24 (64.86) 0 (0.00) l (2.70) 0(0.00) 5(13.88) 16 (44.44) 0(0.00) 0(0.00) 0 (0.00) 1 (2.77) 8(22.22) 0 (0.00) 3 (8.33) 0(0.00) 0 (0.00) 7(21.21) 0 (0.00) 0(0.00) 0(0.00) 0(0.00) 7(21.21) 0(0.00) 0(0.00) 0 (0.00) 0 (0.00) 5(15.15) 0 (0.00) 0 (0.00) 0(0.00) 0(0.00) 3(9/09) 0(0.00) 1 (3/03) 0 (0.00) 0 (0.00) 2(6.25) 0 (0.00) 0 (0.00) 0(0.00) 0(0.00) 1(3.12) 0(0.00) 0(0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0(0.00) 0(0.00) 1(3.12) 0(0.00) 0(0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) D 34 B c K u 0(0.00) 23(67.64) 15(44.11) 0(0.00) 0(0.00) 1 (2.94) 14 (41.17) 10 (29.41) 0 (0.00) 1 (2.94) 1 (3/03) 6,118.18) 10 (30^0) 0 (0.00) 0 (0.00) 3 (9.09) 4 (12.12) 4(12.12) 0(0.00) 2 (6.06)' 0 (0.00) 1 (3.03) 3 (9.09) 0 (0.00) 0 (0.00) 0(0.00) 1 (3.03) 3 (9.09) 0 (0.00) 0(0.00) 0(0.00) 2 (6.06) 2 (6.06) 0(0.00) 1 (3.03) 0 (0.00) 0 (0.00) 2 (6.25) 0 (0.00) 0 (0.00) 0(0.00) 0(0.00) 1 (3.12) 0(0.00) 0(0.00) 0 (0.00) 0 (0.00) 1(3.12) 0 (0.00) 0 (0.00) 0(0.00) 0(0.00) 1(3.12) 0(0.00) 0(0.00) 0 (0.00) 1(3.12) 2 (6.24) 1(3.12) 0 (0.00) 0(0.00) 1 (3/12) 2(6.24) 1(3.12) 0(0.00) 0 (0.00) 1(3.12) 2 (6.24) 1(3.12) 0 (0.00)

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Mortality of piglets after 7 days of life (i.e. after experimental infection) was the lowest in the case of animals that were offspring of vaccinated sows (total 5.88% excluding piglets that died as a result of manipulation during blood collection), and the highest in the case of piglets infected experimentally and offspring of unvaccinated sows (34.28%). Mortality of piglets that were offspring of unvaccinated and uninfected sows during the 14-day observation period was (13.51%). This means a reduction of deaths by about 28% compared to piglets subjected to experimental infection.

Further beneficial effects of the vaccine could be observed by examining other parameters, such as: body weight and rate of weight gain, antibody levels, and the number of bacteria in the gastrointestinal tract.

Table 5 presents the body weight of experimental piglets in the individual stages of the study (kg). AB - a = 0.01 ab - a = 0.05; * mean values of traits in group D were assumed as 100%.

Table 5

Detail Statistic measure Group KI K2 D Body weight at 7 days of life X 2.02 ^1.83b 2.16 ^a SD 0.285 0.207 0.154 %* 93.5 85.9 100 Body weight at 14 days of age X ^{2.56B 2.84b 3.58A} SD 0.460 0.479 0.545 %* 71.5 79.3 100 Body weight at 21 days of life X ^3.66B 3.89 ^4.53A SD 0.667 0.614 0.629 %* 80.7 85.8 100

AB - a = 0.01 ab - a = 0.05; ^average values of features in group D were assumed as 100%

As can be seen from the data presented in Table 5, piglets from group D, being the offspring of vaccinated sows, were characterized by a significantly (a = 0.05) and highly significantly (a = 0.01) higher body weight in comparison to piglets from the other groups in all study periods.

The growth rate analysis shows that piglets from vaccinated sows show significantly higher growth than piglets from other groups.

Table 6 shows the weight gain rate of piglets from immunized sows (D) and control sows (K1 and K2).

Table 6

rate of growth group % more in group D measurement day 7........; KI 119 K2 1451 D 194 in stack, to KI 63 in a stack, to K2 14 114 j 150 and 151 21 123 1481 173

The protective effect correlates with high antibody titer in serum and colostrum of immunized sows. Piglets acquired passive immunity thanks to antibodies transferred in colostrum, which is reflected in the level of antibodies in serum of piglets that are offspring of immunized sows.

Table 7 shows the level of IgG antibodies in the colostrum of immunized sows (D) and control sows (K1 and K2).

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Table 7

colostrum D KI K2 ) 109653 8408 10754 29282 7121 14023 142077 6073 7863 median and 109653 7121 10754 mean ; 93670 7201 10880 ratio gr D to KI and K2for: median: t 15 10 average: and 13 9

Table 8 shows the level of IgG antibodies in the serum of sows in immunized (D) and control groups (K1 and K2) at different stages of the experiment.

Table 8

download 1 dungeon D KI | K2 download2 dungeons D KI K2 download3 I 0 dungeons KI and K2 4231 3447 I 5552 _ _ . 2891 ; 3492 ' 3953 2725 ^; 5976 3721 : 49097 3083 6095 [ 15036 3205: 2798 20080 3746: 6348 ------------------- ' 28825 and 9733 15276 2800: 4892 ___4566] _ 2253 : 3195 ................-—i------------------------------------------------........—1 median 2891 3492 j 3953 mean 3283 4305 Ϊ 4742 median 20080 3205Γ 6095 mean 28071 3345] 5080 median 15276 average 17945 3683J 3195 3683' 3447 ratio of gr.D to KI and K2 for: medianH ___1.2] 0.7 mean: 1.3 0.7 ................. ratio of group D to KI and K2 for: median:_· _ \ 6.3___ 3.3 mean: 8.4] 5.5 —......— ratio of group D to Ki and median: I _ mean: K2for: | 4.9: 5.2

Table 9 shows the level of IgG antibodies in the serum of piglets from immunized sows (D) and control groups (K1 and K2) at different stages of the experiment.

Table 9

download 1 D piglets KI K2 3949 769 2101 6475: 5751: 2718 8092 951’ 975 14011: 916: 895 7891 726 576 25976; 705: 603 1651 2276 3870 median | 7592 843 1651 mean ] 11066 1636 and 1741 ratio gr. Odo KI and K2 for: . median: : 9.5' 4.8 average: 6,8. 6.4

download 2 D piglets KI K2 1003· 649: 803 973 and 1800: 867 1792 450 586 4923: 492; 653 1864: 409 989 5984: 378: 702 1199 681 1328 median j 1828i 471: 803 mean 2757 696 _: 868 ratio gr. D to KI and K2 for: and median: ί 3.9 2,3 average: and 4.0: 3,2

Piglets from immunized sows also showed a reduction in the colonization of the E. coli strain used for infection, primarily in the jejunum and colon.

Table 10 presents the results of microbiological examination of the gastrointestinal tract sections of piglets from the offspring of immunized sows (D) and control sows (K1 and K2).

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Table 10

number of bacteria % reduction and KI ’ K2 D and in the stack, to KI in a stack, to K2 in the jejunum ; 3097744.44; 279222.22; 86455.55] 97.21 69.04 in the colon 491277.77 292625; 73400: 85.06 74.92

To sum up, the conducted experiment concluded that:

The preparation used stimulates a specific humoral response in sows, causing an increase in IgG class anti-LPS E. coli antibodies. Piglets that are offspring of immunized sows acquire passive immunity through the transfer of antibodies via colostrum, which results in their presence in the piglet serum. Piglets that are offspring of immunized sows tolerated the experimental infection better, which is reflected in faster body weight gains and a reduction in the number of bacteria in the tested sections of the digestive tract and a smaller number of deaths compared to the control groups.

Example 14. ELISA test procedure for determining antibody levels in serum and colostrum samples.

1) A 384-well plate was coated with LPS solution (equilibrium mixture of E. coli LPS including all 5 core types: R1, R2, R3, R4, K12). LPS mixture concentration - 5 pg/ml in coating buffer, 35 µl per well and incubated overnight.

Coating buffer:

Proportions: 8 ml 0.2 M Na2CO3 + 17 ml 0.2 M NaHCO3 - 32 ml Na2CO3 and 68 ml NaHCO3 mixed

2) The entire buffer was then manually removed from the plate.

3) The plate was washed three times with TBS-T solution (TBS with 0.1% TWEEN 20) using a Tekan washer.

4) Then, serum diluted in TBS buffer with 1% BSA was applied to the plate in a series of dilutions, 35 µl per well, the first serum dilution - 100x, each subsequent one: 2x.

5) The plate was incubated at 37°C for 1 h.

6) All serum was then manually removed from the plate.

7) The plate was washed three times with TBS-T solution (TBS with 0.1% TWEEN 20) using the Tekan washer using the ELISA_384_TL program.

8) Anti-IgG antibody conjugate with HRP diluted in TBS buffer with 1% BSA was applied to the plate, 35 µl per well.

For pigs: goat anti-porcine IgG antibodies from Jackson ImmunoResearch at a 5000x dilution.

9) The plate was incubated at 37°C for 1 h.

10) After one hour, all conjugate or antibody solution was removed from the plate.

11) The plate was washed six times with TBS-T solution (TBS with 0.1% TWEEN 20) using a Tekan washer using the ELISA_384_TL program.

12) 35 µl/well of TMB substrate solution in citrate buffer (205 mM citric acid in H2O + KOH to pH 4.0) was added and the reaction was developed for 20 min.

TMB (tetrabutylammonium borohydride -21.1 mg, tetramethylbenzidine -98.54 mg, N,N-dimethylacetamide -10 ml).

13) The reaction was stopped by adding 35 µl of 1 Μ H3PO4 solution per well.

14) The plate was read in a microplate reader at a wavelength of 450 nm

Publications:

1. Robbins, J. B., Chu, C., and Schneerson, R. (1992). Hypothesis for vaccine development: protective immunity to enteric diseases caused by nontyphoidal salmonellae and shigellae may be conferred by serum IgG antibodies to the O-specific polysaccharides of their lipopolysaccharides. Clin. Infect. Dis. 15, 346-361.

2. Svenson, S.B. and Lindberg, A.A. (1981). Artificial Salmonella vaccines: Salmonella typhimurium O-antigen-specific oligosaccharide-protein conjugates elicit protective antibodies in rabbits and mice. Infect. Immun. 32, 490-496.

3. Svenson, S.B., Nurminen, M., and Lindberg, A.A. (1979). Artificial Salmonella vaccines: O-antigenic oligosaccharide-protein conjugates induce protection against infection with Salmonella typhimurium. Infect. Immun. 25, 863-872.

4. Konadu, E.Y., Lin, F.Y., Ho, V.A., Thuy, N.T., Van Bay, P., Thanh, T.C., Khiem, H.B., Trach, D.D., Karpas, A.B., Li, J., et al. (2000). Phase 1 and phase 2 studies of Salmonella enterica serovar paratyphi A O-specific polysaccharide-tetanustoxoid conjugates in adults, teenagers, and 2- to 4-year-old children in Vietnam. Infect. Immun. 68, 1529-1534.

5. Levine, M.M., Kotloff K.L., Barry, E.M., Pasetti, M.F., and Sztein, M.B. (2007). Clinical trials of Shigella vaccines: two steps forward and one step back on a long, hard road. Nat. Rev. Microbiol. 5, 540-553.

6. Ahmed, A., Li, J., Shiloach, Y., Robbins, J.B., and Szu, S.C. (2006). Safety and immunogenicity of Escherichia coli O157 O-specific polysaccharide conjugate vaccine in 2-5-year-old children. J. Infect. Dis. 193, 515-521.

7. Westphal O, Jann K. Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. In: Whistler RL, Wolfan ML, editors. Methods in Carbohydrate chemistry. New York Academic Press lnc.:1965(5):83-91

Claims (22)

Patent Claims 1. A method for producing bacterial lipopolysaccharide (LPS)/protein immune complexes, characterized in that it comprises the following steps: a) LPS is isolated from bacteria using any method and then converted into a form that is highly soluble in DMSO by removing metal cations associated with LPS and exchanging them for protons, converting the bacterial lipopolysaccharide into an acidic form; b) a protein is obtained that is highly soluble in DMSO, with all ionically bound cations being converted into protons; c) dissolving the LPS and protein in DMSO and mixing the two solutions to obtain a mixture of LPS and protein in DMSO in a specified weight ratio; and then d) DMSO is gradually diluted and removed, without causing degradation or chemical modification of LPS, maintaining the complete LPS molecule and its antigenic properties, and conjugation of LPS with the protein occurs in a non-covalent manner. 2. The method according to claim 1, characterized in that the LPS is derived from any strain of Gram-negative bacteria, such as Escherichia, Hafnia, Salmonella and Campylobacter. 3. The method according to claim 1, characterized in that the protein used in the complex may be any protein with immunogenic properties, soluble in DMSO. 4. The method according to claim 3, characterized in that the protein used in the complex is casein or bovine serum albumin. 5. The method according to claim 1, characterized in that the DMSO is diluted with water. 6. The method according to claim 1, characterized in that the DMSO is removed by dialysis, ultrafiltration or gel filtration. 7. A lipopolysaccharide/protein complex produced by the method of any one of claims 1 to 6 for use in inducing an immune response. 8. The lipopolysaccharide/protein complex of claim 7 for use in the production of an immunogen or an antibacterial veterinary vaccine. 9. A method of producing a veterinary vaccine comprising an LPS/protein immune complex comprising the following steps: culturing bacterial cells and isolating LPS, characterized in that LPS is converted into a DMSO-soluble form by removing metal cations bound to LPS and exchanging them for protons, obtaining a DMSO-soluble protein in which all ionically bound cations are exchanged for protons, dissolving LPS and the protein in DMSO and then mixing the resulting solutions to form a non-covalent complex exhibiting immunogenicity, wherein the diluent used to remove DMSO from the preparation is water. 10. The method according to claim 9, characterized in that the LPS is isolated by the phenol-water extraction method. 11. The method according to claim 9, characterized in that the DMSO is removed by dialysis, ultrafiltration or gel filtration. 12. The method according to claim 9, characterized in that the obtained preparation is subjected to lyophilization or formulated with any adjuvant. 13. A veterinary vaccine comprising a complex produced by the method of any one of claims 1 to 6. 14. The vaccine according to claim 13, characterized in that it is in lyophilized form. 15. The vaccine of claim 13, characterized in that it is formulated with an oil adjuvant such as incomplete Freund's adjuvant. 16. The vaccine according to claim 13, characterized in that the carrier protein of the complex is a marker allowing monitoring of the progress of immunization in an individual. 17. The vaccine according to claim 13, characterized in that it has a broad spectrum of activity against all versions of LPS (serotypes) found in strains of Escherichia coli, Salmonella and Hafnia alvei. 18. The vaccine according to claim 13, characterized in that it stimulates a specific humoral response in an individual causing an increase in anti-LPS IgG antibodies. 19. The vaccine according to claim 13, characterized in that the subjects to be immunized may be birds and mammals, such as chickens and pigs. 20. The vaccine according to claim 13, characterized in that the immunization is performed by intramuscular or intraperitoneal administration. 21. The vaccine according to any one of the preceding claims 13 to 20, characterized in that it does not cause toxic effects in the vaccinated animals. 22. The vaccine of claim 13, characterized in that it comprises a complex of LPS with the protein in PBS and optionally any other adjuvant.