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

Infections with gram-negative bacteria account for a significant proportion of bacterial infections and hospital-acquired infections. Mortality is considerable despite the use of antibiotics. The problem worsens with an increase in the number of multidrug-resistant strains, which are responsible for particularly severe cases of bacteraemia, often ending in sepsis and patient death. Sepsis therapy is one of significant medical issues, which is still far from being solved. A number of therapeutic strategies are currently being used and studied, with the most important ones including: the removal of bacterial endotoxins from the bloodstream, therapies supporting vital functions of key organs, inhibition of excessive activation of the immune system, neutralisation of endotoxins with antibodies. Another approach is to develop immunity to pathogens and endotoxins with suitable vaccines. Traditionally, vaccines target protein antigens, however, due to the fact that the main component of the outer membrane of the cell wall of gram-negative bacteria is a bacterial lipopolysaccharide, a number of studies have been undertaken to use LPS as a target for vaccines.

Lipopolysaccharides are a group of macromolecules built from a lipid component usually called lipid A and a sugar portion with a varying composition and structure. LPS also comprises low molecular weight substituents affecting biological and physicochemical properties of LPS. These are, for example, phosphate groups, diphosphates, phosphoethanolamine, methyl- phosphoramide groups, amino acids, glycerol and its derivatives and others. LPS comprising a sugar portion built from a small number of sugar residues is often referred to as LOS (lipooligosaccharide). A sugar portion of LPS is typically composed of two portions: a core oligosaccharide and a polysaccharide also called an O-antigen. LPS strongly interacts with the cells of the immune system, being responsible for the toxic effects observed during gram-negative bacteraemia and sepsis. Lipid A is responsible for most of the biological activities responsible for LPS toxicity. Due to its composition, the LPS molecule is amphiphilic, and in water it forms complex micellar systems which may have different structure and size. LPS can be dissolved in some solvents, e.g., dimethyl sulfoxide (DMSO), after appropriate preparation - patent US 8,426,221. In an acidic environment and at elevated temperatures, LPS breaks down into lipid A and a sugar portion, which can be purified and used to form vaccines.

The LPS composition model is shown below:

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

Due to weak immunogenicity of LPS and the lack of T-cell activation, obtaining a good response is associated with the need to create conjugates of O-antigens with the carrier protein, as in the case of the vaccines already used in humans against H. influenza, pneumococcus, meningococcus and comprising conjugates of capsular polysaccharides with the protein. The oligosaccharides obtained by enzymatic hydrolysis of LPS from Salmonella thyphimurium conjugated to the carrier protein elicited a strong response to sugar antigens in mice and rabbits (Swenson and Lindberg, 1981; Swenson et al., 1979).

Subsequent works 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. paratyphi A, E. coli 0157:1-17, Pseudomonas aeruginosa, Shigella) have been investigated or are being investigated in clinical trials (Konadu et al., 2000, Levine et al., 2007; Ahmed et al., 2006).

A drawback of O-specific antigen-based vaccines is limited reactivity of the antibodies obtained, i.e., limited to the strains of a given serotype.

Infections with gram-negative bacteria are also a major problem for animal breeders, especially in the production of poultry, pigs and cattle. The main species of gram-negative bacteria responsible for losses in animal production include E. coli, Salmonella, Listeria, Campylobacter. Currently, the global market of animal vaccines in 2016 reached the value of USD 5.81 billion and its value is estimated to increase to USD 7.68 billion in 2021. The compound annual growth rate (CAGR) for 2016-2021 will be 5.7%.

Veterinary vaccines are mostly killed or attenuated bacterial cells of the strains which most often cause infections. For example, the Coliprotect F4/F18 vaccine produced by (Prevtec Microbia GmbH) consists of two live E. coli bacteria strains (08:K87 and 0141:K94), which do not produce toxins causing diseases. Others contain bacterial proteins constituting 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 either. A drawback of current vaccines is their serotype specificity limited to the strains used for immunization. The use of LPS as an antigen allows to obtain vaccines with both narrow and broader specificity.

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

Vaccines in which isolated, purified antigens are used are referred to as subunit vaccines, their advantage is a strong induction of a response to a selected antigen, which in the case of a complex immunogen such as a whole bacterial cell is not always possible due to antigenic competition between various molecules on its surface. Other advantages include a well-defined, repeatable constitution, fewer side reactions, and a targeted response to antigens which provides protection.

Lipopolysaccharides, like polysaccharides and oligosaccharides, are weak immunogens and are therefore haptens, which only after coupling to a protein carrier obtain the ability to induce IgG antibodies and immunological memory necessary to obtain good protection. The formation of LPS protein conjugates requires partial decomposition of LPS to release the water-soluble sugar portion. Following acid hydrolysis, the lipopolysaccharide is cleaved at the site of connection of the inner portion of the core oligosaccharide typically containing 3-deoxy-D-manno-octulosonic acid (KDO) molecules with lipid A. The released LPS sugar portion is then purified and coupled to the carrier protein. There are a number of ways of coupling oligo- and polysaccharides to proteins known to those skilled in the art. Most often, coupling takes place in two stages: activation of saccharide and conjugation with the protein. Coupling by reductive amination between terminal ketose or aldehyde groups of saccharide and amine 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 LPS hydrolysis to release the sugar portion. Despite relatively mild reaction conditions, the internal portion of the core built from KDO is degraded, and a significant portion of non-sugar substituents, which are important for LPS antigenic properties as epitope elements, is also released and lost.

The method described in the invention makes it possible to obtain the complex in the manner which does not cause degradation and other chemical modifications of the LPS, while fully preserving its antigenic characteristics. The invention describes a method of coupling LPS to proteins in which there is no need for partial degradation of LPS, the coupling does not require the formation of a covalent connection between LPS and the protein, which may also lead to modification and loss of some epitopes. The coupling involves the formation of a non-covalent complex between LPS and the protein in a multi-stage process. In the first step, LPS is subjected to the procedure of removing metal cations bound to it and exchanging them for protons whereby an acidic form of LPS is obtained, which is well soluble in DMSO, in an analogical process a protein is obtained in which all the ionically bound cations are exchanged for protons. The LPS and the protein are then dissolved in DMSO and both solutions are mixed together to obtain a mixture of the LPS and the protein in DMSO in a desired weight ratio. Subsequently, DMSO is gradually diluted with water and DMSO removed in the process of dialysis, ultrafiltration, or gel filtration. The complex can then be used to formulate an immunogen or a 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 antibodies and immunological memory, contains a complete LPS molecule, does not cause toxic effects in vaccinated animals at the doses used for immunization. Preferably, the lipid A component of LPS contained in the complex has immunostimulating properties providing a strong immune response.

The method for obtaining the vaccine consists of the following steps: a) culturing bacterial cells; b) isolating LPS; c) converting LPS into a DMSO-soluble form; d) producing the complex by mixing LPS and protein solutions in DMSO and changing the solvent to water; e) lyophilizing or formulating with an adjuvant.

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

At the same time, no toxicity of the immunogens used in the Balb/C mouse vaccination tests was reported. The LPS dose was approx l.lmg/kg body weight. This is 5 times lower than the lethal dose for pigs (5mg/kg body weight). Vaccine preparations were also tested on cell lines, where their toxicity was also not demonstrated.

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

(a) isolating from bacteria LPS, well soluble in DMSO by removal of metal cations bound to the LPS and their exchange for protons to convert the bacterial lipopolysaccharide into an acidic form;

(b) obtaining a protein, well soluble in DMSO, wherein all ionically bound cations are exchanged for protons;

(c) dissolving the LPS and the protein in DMSO and mixing both solutions together to obtain a mixture of the LPS and the protein in DMSO in a specific weight ratio; following which

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

Preferably, the method is characterized in that 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 can be any protein with immunogenic properties and 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 DMSO is diluted with water.

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

A further object of the invention is the use of the lipopolysaccharide/protein complex produced by the above method to induce an immune response.

Preferably, the use is for the production of an immunogen or an antibacterial veterinary vaccine.

A further object of the invention is a method for producing a veterinary vaccine containing the LPS/protein immune complex comprising the steps of: culturing bacterial cells, isolating the LPS, converting the LPS into a DMSO-soluble form by the removal of metal cations bound to the LPS and their exchange for protons, obtaining a DMSO-soluble protein wherein all ionically bound cations are exchanged for protons, characterized in that the LPS and the protein are dissolved in DMSO and then the resulting solutions are mixed to form a non-covalent complex exhibiting immunogenicity, wherein the diluent used to remove DMSO from the preparation is water.

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

Preferably, DMSO is removed in the process of dialysis, ultrafiltration, or gel filtration.

Preferably, the preparation obtained is lyophilized or formulated with any adjuvant.

A still further object of the invention is a veterinary vaccine comprising the complex produced by the method described above.

Preferably, the vaccine is in a lyophilized form.

Preferably, the vaccine is in the form of formulation with an oil adjuvant such as an incomplete Freund's adjuvant.

Preferably, the carrier protein of the complex is a marker which allows to follow the progress of immunization in a subject.

Preferably, the vaccine exhibits a broad spectrum of action against all LPS versions (serotypes) found in Escherichia coli, Salmonella, Hafnia alvei and Campylobacter jejuni strains.

Preferably, the vaccine stimulates a specific humoral response in a subject to result in an increase in anti-LPS IgG antibodies.

Preferably, the immunized subjects may be birds and mammals such as chickens, mice and pigs. Preferably, immunization takes place by intramuscular or intraperitoneal administration. Preferably, the vaccine causes no toxic effects in vaccinated animals.

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

The technical problem solved by the invention is the obtaining of an innovative vaccine which has not been previously used in veterinary medicine. The invention is distinguished in that with the technology described LPS/protein immunogenic complexes can be easily obtained. The presence of protein in the complex provides a TH2-type response, which is desirable in the case of vaccines. At the same time, there is no need to chemically couple LPS to the protein to obtain the complex, as is the case with known vaccines. Unexpectedly, the response to LPS after immunization with such a complex is very strong and outweighs the response to LPS in the case of immunization with killed bacterial cells. The vaccines containing the whole, unmodified LPS exhibit a broader spectrum of action. This is an improvement as compared to O-antigen-based vaccines, where the reactivity of the antibodies obtained is limited to the strains of a given serotype. In the case of the vaccines disclosed in the invention, the reactivity of the antibodies obtained by immunization with the LPS/protein complexes used in these vaccines is specific for all LPS versions found in E.coli strains, which ensures the effectiveness of the vaccine for all E.coli serotypes found in the environment. Broad reactivity is obtained by using in the complexes a mixture of rough lipopolysaccharides representing all 5 core types known in E.coli. Similarly, for Salmonella, which has 1 core type, the use of rough LPS provides reactivity to all Salmonella serotypes.

The enclosed figures depict the following: Figure 1 shows a standard curve for Salmonella R60 LPS in the range of 0-100 pg LPS, Figure 2 shows the level of anti-Salmonella LPS IgG antibodies in hen serum, and Figure 3 shows the level of anti-carrier protein IgG antibodies in hen serum.

The invention is presented in more detail in embodiments, which, however, do not limit the scope thereof.

Embodiments

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 NCTC 11168 were isolated by phenol-water extraction in accordance with Westphala and Jann, New York: Academic Press Inc.; 1965(5):83-91.

The dry bacterial mass was suspended in water at a ratio of 5 g bacterial mass per 100 ml water, then the suspension was stirred with an equal volume of 90% phenol heated to 65°C and stirred at this temperature in water bath for 15 mins. After cooling, the mixture was centrifuged to separate the phases (5000 x g, 20 mins), the aqueous phase was collected, the phenol phase was supplemented to the previous volume with pure water and extracted again at 65°C. The material was centrifuged as before, the aqueous phase was collected, and the extraction process was repeated with another portion of water. The combined aqueous phases were dialysed in a dialysis bag (cut-off mass of 12 kDa) until the phenol odour was lost. The dialysate was centrifuged (5000 g, 10 mins) to remove precipitated particles, then LPS was deposited by ultra centrifugation (100,000 x g, 2h). The LPS precipitate was suspended in 0.2M NaCI (25 mg LPS/ml) and a nuclease (RNase and DNase) in the amount of 50pg/ml was added, the mixture was incubated for 2h at 37°C. Subsequently, the LPS was ultracentrifuged twice in water to remove fragments of nucleic acids and nucleotides. The purity of the preparation (absence of nucleic acids) was controlled spectrophotometrically, the absence of peak at wavelengths of 260 and 280 nm indicated the absence of contamination with proteins and nucleic acids.

Example 2. Preparation of DMSO-soluble LPS

50 mg LPS was dissolved in 2 mL 2% SDS with an addition of EDTA (50 pi 0.5 M solution), centrifuged to remove insoluble impurities (16000 x g for 5 mins) and precipitated with 4 volumes of ethanol. The material was centrifuged (16000 x g for 30 mins), the supernatant was collected and discarded, the precipitate was suspended in ethanol (95%) and centrifuged (16000 x g for 30 mins), the step was repeated. The precipitate was dissolved in 2 mL water and passed through a column filled with a Dowex 50Wx8 ion-exchanger in an acidic form. The column was washed with water, fractions with acidic pH were collected, combined and lyophilized. The procedure was used for LPS from E.coli : F470, F576, F653, F2513, W3100 and E.coli K12 C600.

Example 3. Preparation of DMSO-soluble E.coli LPS

20 mg LPS from E coli K12 C600 was dissolved in 4 ml 2% SDS, then passed through a column filled with Chelex 100 medium, the column was washed with water, an LPS-containing fraction was collected (the presence of LPS was detected with a phenol-sulfuric acid colorimetric test). The LPS was precipitated with 4 vol. of ethanol and recovered by centrifugation (16000 g for 30mins), the supernatant was collected and discarded, the precipitate was suspended in ethanol (95%) and centrifuged (16000 g for 30 mins), the step was repeated. The precipitate was dissolved in 2 mL water and passed through a column filled with a Dowex 50Wx8 ion-exchanger in an acidic form. The column was washed with water, LPS fractions with acidic pH were collected, combined and lyophilized. This procedure was also used to obtain acidic LPS forms: Hafnia alvei 1216, Hafnia alvei 1286.

Example 4. Preparation of DMSO-soluble Salmonella minnesota LPS

60 mg 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 (cut-off mass of 12 kDa) and dialysed into water to remove EDTA. The post-dialysis material was collected in a Falcon tube (50 mL), 2 mL Dowex 50Wx8 medium suspension in an acidic form was added, incubated for 1 hour on a serology rotator, then centrifuged on a swing-bucket rotor (5000 x g for 5 min), the aqueous phase was collected and lyophilized.

Example 5. Preparation of DMSO-soluble C. jejuni NCTC 11168 LPS

12 mg C. jejuni NCTC 11168 LPS prepared as described in Example 1 was suspended in 0.5 mL milli-Q water in an eppendorf tube. 100 pL Chelex 100 ion-exchange resin suspension in a sodium form (Sigma-Aldrich) was added and stirred on a serology rotator overnight (approx. 14 hours). After this time, the sample was centrifuged (5 mins, 14000xg), the supernatant was collected and transferred to a fresh tube, and then 100 pi Dowex 50Wx8 medium suspension in an acidic form was added and incubated for an hour on a serology rotator. The sample was centrifuged (5 mins, 14000xg), the aqueous phase was collected and lyophilized. The method for producing an acidic form of C. jejuni LPS is a modification of the methods described in Examples 3 and 4 useful when a small amount of material is being processed.

The above methods of Examples 2-5 depicting the preparation of LPS from bacterial cells are alternative and equivalent methods, the basis for their use is the removal of divalent metal ions from the LPS and then converting it into an acidic form, which is an essential step of the procedure. These methods can be used interchangeably for any lipopolysaccharides isolated from various bacteria. The choice of the technique does not depend on the origin and constitution of the lipopolysaccharide.

Example 6. Preparation of the Salmonella R60 LPS/cow's milk casein complex

20 mg DMSO-soluble LPS was dissolved in 2 mL DMSO. 10 mg cow's milk casein was dissolved in 2 mL DMSO, the suspension was mildly sonicated until a clear solution was obtained. The solutions were mixed and incubated overnight with stirring at room temperature. Then, the mixture was slowly diluted with water, while stirring continuously. The rate at which water was to be added was set in such a way as to avoid an increase in the temperature of the mixture by more than 10°C. After a 1:1 ratio had been reached, the mixture was transferred to a dialysis bag and dialysed into water until DMSO was removed. The material was transferred to a centrifuge tube and centrifuged to recover the LPS/protein complex (16000 x g, for 20 mins). The complex was collected and dissolved in PBS. Subsequently, the LPS content in both the supernatant and complex fractions was assayed using the phenol-sulfuric acid colorimetric method 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 for Salmonella R60 LPS in the range of 0-100 pg LPS was made (shown in Figure 1) and the LPS content in both fractions was determined; it was shown that the complex-containing fraction contained 66.2% LPS.

Example 7. Preparation of Salmonella R60 LPS /cow's milk casein and H. alvei 1216/cow's milk casein, H. alvei 1286/cow's milk casein complexes.

Each sample of 10 mg LPS in a DMSO-soluble form was dissolved in 1 mL DMSO and mixed with 1 mL DMSO containing dissolved cow's milk casein. The solutions were incubated overnight with stirring at room temperature. Next, the mixtures were slowly diluted with water, while constantly stirring, the rate at which water was to be added was set in such a way as to avoid an increase in the temperature of the mixture by more than 10°C. After a 1:1 ratio had been reached, the mixtures were transferred to dialysis bags and dialysed into water until DMSO was removed. The material was transferred to a centrifuge tube and centrifuged to recover the LPS/protein complex (16000 x g for 20 mins). The complexes were collected and dissolved in PBS, the presence of LPS was confirmed by colorimetric method.

Example 8. Preparation of complexes of E.coli LPS: F470 , F576, F653, F2513 and K12 with cows' milk casein.

Samples of 50 mg of E.coli: F470, F576, F653, F2513 and K12 LPS were dissolved in 5 mL DMSO and mixed with 5 mL DMSO containing 50 mg cows' milk casein. The solutions were incubated overnight with stirring at room temperature. Next, the mixtures were slowly diluted with water, while constantly stirring, the rate at which water was to be added was set in such a way as to avoid an increase in the temperature of the mixture by more than 10°C. After a 1:1 ratio had been reached, the mixtures were transferred to dialysis bags and dialysed into water until DMSO was removed. The material was transferred to a centrifuge tube and centrifuged to recover the LPS/protein complex (16000g for 20 mins). The complexes were collected and dissolved in PBS, the presence of LPS was confirmed by colorimetric method.

Example 9. Preparation of the C. jejuni NCTC 11168 LPS/cow's milk casein complex

10 mg C jejuni LPS was dissolved in 1 mL DMSO. 5 mg cow's milk casein was dissolved in 1 mL DMSO, the suspension was treated with ultrasound until a clear solution was obtained. The solutions were mixed and incubated overnight at room temperature on a serology rotator. Then, the mixture was slowly diluted with water, while stirring continuously. The rate at which water was to be added was set in such a way as to avoid an increase in the temperature of the mixture by more than 10°C. After a 1:1 ratio had been reached, the mixture was transferred to a dialysis bag and dialysed into water until DMSO was removed. The material was transferred to a centrifuge tube and centrifuged to recover the LPS/protein complex (16000 x g, for 20 mins). The complex was collected and dissolved in PBS. The presence of LPS in the resulting complex was confirmed by colorimetric method as described in Example 6.

Example 10. Preparation of bovine serum albumin (BSA) protein soluble in DMSO.

100 mg BSA was dissolved in 10 mL water, 1 mL 0.5 M EDTA pH 8.0 was added and dialysed in a dialysis bag to remove EDTA. Then the protein solution was passed through a column packed with Dowex 50Wx8 medium, the column was washed with water, protein-containing fractions were collected and lyophilized.

Example 11. Preparation of the Salmonella R60 LPS/BSA complex.

10 mg BSA obtained in Example 8 was dissolved in 2 mL DMSO, 10 mg Salmonella R60 LPS was dissolved separately in 1 mL DMSO. Both solutions were mixed and incubated overnight with stirring at room temperature. Next, the mixture was slowly diluted with water at a 1:1 ratio, while constantly stirring, the rate at which water was to be added was set in such a way 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 dialysed into water until DMSO was removed and then to PBS.

Example 12. Immunization of mice with the Hafnia alvei 1216/cow's milk casein complex.

Balb/c strain mice were immunized with the complex of Hafnia alvei 1216 and casein in PBS in the amount equivalent to 20pg LPS per dose. The complex was administered intraperitoneally without an additional adjuvant. The animals were immunized 3 times at 3-week intervals. Control groups were administered either LPS alone or casein alone (20 pg per mouse). 10 days after the last vaccination, blood samples were taken, serum was prepared and analysed by ELISA for reactivity with the antigen used for immunization. A serum titre for Hafnia alvei 1216 LPS is shown in the table below.

The titre of antibodies against Hafnia alvei LPS in mouse serum is shown in Table 1.

Table 1 Example 13. Immunization of mice with the C. jejuni NCTC 11168 LPS/cow's milk casein complex.

Balb/c strain mice were immunized with the complex of C. jejuni LPS/cow's milk casein in PBS in the amount equivalent to 20pg LPS per dose. The complex was administered intraperitoneally without an additional adjuvant. The animals were immunized 2 times at 2-week intervals. A control group was administered casein (20 pg per mouse). 10 days after the first and 10 days after the last vaccination, blood samples were taken, and serum was prepared and analysed by ELISA for reactivity with C. jejuni LPS. The titre of antibodies against Campylobacter jejuni NCTC 11168 LPS in mouse serum is shown in Table 2.

Table 2.

Example 14. Immunogenicity testing of the Salmonella R60 LPS /cow's milk casein complex in hen.

5 laying hens were immunized twice in the pectoral muscle with the Salmonella R60 LPS/casein complex in the amount of 200 pg of the complex in PBS without an additional adjuvant. Prior to immunization, blood samples were taken. The interval between vaccinations was 3 weeks. Blood samples were taken again 14 days after the second vaccination. The sera were analysed by ELISA by testing a number of serum dilutions, the titre was calculated on the basis of the curves obtained, defining it as the highest dilution at which the signal was > 0.2 unit (absorbance units) above the background level. The serum titre is shown in the table below.

The IgG antibody titre in the sera of hens in response to the administration of the Salmonella R60 LPS/casein complex is shown in Table 3.

Table 3

Example 15. Testing the effectiveness of the vaccine in hens experimentally infected with Salmonella.

In the test, 6-week-old hens were immunized with the Salmonella minnesota LPS/casein complex with the use of 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 4 weeks apart. After the second vaccination, the animals were subjected to experimental infection with a Salmonella field isolate. The infection was performed 2 times on days 28 and 62 after the second vaccination. Approximately one week after the last infection, the animals were euthanized and, for microbiological examinations, the following organs were collected: liver, bursa of Fabricius, spleen as well as blood samples and cloaca swab samples.

The results of the organ analysis are summarized in the following Table 4. The results of microbiological examination of the organs of immunized hens. (-) - absence of Salmonella bacteria in the organ, (+) - presence of Salmonella bacteria in the organ.

The swabs from the cloaca taken on days 1, 56 and 98 did not show the presence of Salmonella in this organ. The presence of Salmonella in the organs collected from the post-slaughter birds was also tested (day 98). In the case of liver of unvaccinated hens, the presence of bacteria was confirmed in 60% hens, and in the groups of vaccinated hens - 20%. Similarly, in the case of testing for the presence of Salmonella in the spleen - 30% in unvaccinated hens and 10% in vaccinated hen groups. To check the immunogenicity of the preparations made, an analysis of the level of IgG antibodies in the serum of hens following vaccination was performed with the use of ELISA (Figure 2 - LPS Salmonella and Figure 3 - carrier protein).

A significant increase in antibodies against Salmonella LPS and carrier protein was confirmed. Example 16. Testing the effectiveness of the vaccine in pigs.

The tests were aimed to test the activity of a developed vaccine against colibacteriosis and confirm its effectiveness in preventing this disease in piglets. A total of 9 sows and 27 piglets, progeny of these sows (3 piglets from a litter), were used in the test. In the experiment, 3 experimental groups of 3 sows in each group were formed.

To obtain the animal material for the tests, 26 DanBred hybrid gilts were purchased from a Danish breeding facility. DanBred Hybrid is the first cross between DanBred Landrace and DanBred Yorkshire. It is an optimal maternal breed, which ensures high production efficiency by combining the best traits.

Test groups:

Group D - sows vaccinated twice with an experimental vaccine and their piglets infected with an infectious dose of E. coli F4 (K88) strain producing the Stx2e toxin (lxlO ⁹ CFU/lml in a volume of 5 ml/piglet).

Group K1 - non-vaccinated sows, infected piglets identical to the piglets from group D Group K2 - unvaccinated sows and piglets, sows and piglets of this group were not infected intentionally, infections in this group occurred spontaneously, similarly as on farms.

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

The composition of the immunization mixture (single dose):

0.5 ml mixture of LPS/casein complexes in PBS containing a total of 150 pg LPS 0.5 mL incomplete Freund's adjuvant

Pregnant sows were vaccinated intramuscularly at 3-week intervals (week 8 and 11 of gestation). The piglets were infected at 7 days of age. Subsequently, clinical observations, temperature measurements, health checks were carried out and production results were assessed on the basis of weight gain. The piglets were euthanized at 21 days of age, and histopathological material was collected: samples of jejunum, duodenum, ileum and colon. The same samples constituted the material for microbiological tests.

The level of antibodies was analysed in the serum of vaccinated sows, piglets and colostrum. Test Results

The most frequently reported infection symptom was diarrhoea of varying severity observed in all the test groups.

Table 5 below shows clinical observations for piglets subjected to an experimental infection. The piglets were infected at 7 days of age with a suspension of bacteria at a dose of lxlO ⁶ CFU/lml 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 baseline piglets, i.e., as of the day before the recorded symptom. Abbreviations: A - stiffness and apathy, lack of appetite; B - diarrhoea; C - weight loss; K - lameness; U - the animal dies (death).

Table 5

The mortality of piglets following 7 days of age (i.e., after the experimental infection) was the lowest for the offspring of vaccinated sows (a total of 5.88% without piglets dead due to manipulation during blood collection), and the highest for the piglets infected experimentally and being the offspring of non-vaccinated sows (34.28%). The mortality of piglets being the offspring of non-vaccinated and non-infected sows during the 14-day observation period was (13.51%). This means a reduction of deaths by about 28% as compared to the piglets which have been subjected to experimental infection.

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

Table 6 presents the body weight of experimental piglets at individual stages of the study (kg). AB - a = 0.01 ab - a = 0.05; * mean values of the traits in group D were assumed to be 100%.

AB - a 0,01 ab - a 0,05; * _mean values of traits in group D were assumed to be 100%

Table 6

According to the data presented in Table 6, piglets from group D, which were the offspring of vaccinated sows, in all testing periods, were characterized by significantly (a = 0.05) and highly significantly (a = 0.01) higher body weight in comparison to the piglets from other groups.

The analysis of the rate of weight gain shows that piglets of vaccinated sows show significantly higher weight gains than the piglets from other groups.

Table 7 shows the rate of weight gain of piglets of immunized sows (D) and control sows (K1 and K2).

Table 7 The protective effect correlates with the high titre of antibodies in the serum and colostrum of the immunized sows. The piglets acquired passive immunity owing to the antibodies transmitted in the colostrum, which is reflected in the level of antibodies in the serum of the piglets being the offspring of the immunized sows.

Table 8 shows the level of IgG antibodies in the colostrum of immunized sows (D) and control sows (K1 and K2).

Table 8

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

Table 9 Table 10 shows the level of IgG antibodies in the serum of piglets of immunized sows (D) and control groups (K1 and K2) at various stages of the experiment.

Table 10

The piglets of immunized sows also showed a reduction in the population of E. coli strain used for infection primarily in the jejunum and colon.

Table 11 shows the results of microbiological examination of sections of the gastrointestinal tract of piglets being the offspring of immunized sows (D) and control sows (K1 and K2).

Table 11

Summing up the conducted experiment, it was found that:

The preparation used stimulates a specific humoral response in sows, resulting in an increase in anti-E coli LPS IgG antibodies. The piglets being the offspring of immunized sows acquire passive immunity through the transfer of antibodies via the colostrum, which results in their presence in the serum of the piglets. The piglets being the offspring of the immunized sows tolerated the experimental infection better, as reflected in faster weight gains and reduced bacteria counts in the examined sections of the gastrointestinal tract, and fewer deaths as compared to the control groups.

Example 17. ELISA procedure for the assay of antibody levels in serum and colostrum samples.

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

Coating buffer:

Proportions: 8 mL 0.2 mL Na2CC>3 + 17 mL 0.2 M NaHCC>3 - 32 mL Na2COsand 68 mL NaHCOs were mixed

2) Then the entire buffer was manually removed from the plate

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

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

5) The plate was incubated at a temperature of 37°C for 1 h

6) Then the entire serum was manually removed from the plate

7) The plate was washed three times with TBS-T solution (TBS with 0.1% TWEEN 20) with Tecan washer, using ELISA_384_TL programme

8) A conjugate of anti-lgG antibody with HRP diluted in TBS buffer with 1% BSA was applied to the plate, 35 mI per well

In the case of pigs: goat anti-pig IgG antibodies from Jackson ImmunoResearch at 5000x dilution.

9) The plate was incubated at a temperature of 37°C for 1 h

10) After an hour, the entire 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) with Tecan washer, using ELISA_384_TL programme

12) 35 mI/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 mins.

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

13) The reaction was stopped by adding 35 mI 1M H3PO4 solution per well

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

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 polysaccharide of their lipopolysaccharides. Clin. Infect. Dis.15, 346-361.

2. Svenson, S.B. and Lind berg, 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 Lind berg, 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 childrenin 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 0157 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