A method for preparing a bivalent dysentery conjugate combination vaccine

Abstract

The present invention discloses a method for preparing a bivalent dysentery conjugate combination vaccine, which belongs to the technical field of biological product preparation. The vaccine includes two serotypes, 2a flexneri and sonnei. The O-specific polysaccharides of the two bacteria are covalently bound to a carrier protein according to their unique chemical structure through a corresponding chemical method to prepare a polysaccharide-protein conjugate stock solution. The polysaccharide-protein conjugate stock solution is then adsorbed with an aluminum adjuvant in an appropriate proportion to obtain a dysentery conjugate vaccine. The 2a flexneri dysentery polysaccharide is prepared as a conjugate by a reductive amine method, and the sonnei dysentery polysaccharide is prepared as a conjugate by a direct conjugation method with a spacer. In particular, the present invention no longer uses the highly toxic reagent cyanogen bromide and a high activation pH. The method is free of toxic reagents, has a mild reaction, is easy to control, and is conducive to future large-scale production. The dysentery conjugate vaccine prepared by the present invention is used for the immune prevention of infection with related serotypes of Shigella dysenteriae.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a method for preparing a bivalent dysentery conjugate combination vaccine. Background Art

[0002] Shigella dysenteriae is a Gram-negative intracellular pathogen that clinically causes bacillary dysentery. Intestinal inflammation is a key characteristic of dysentery, manifesting primarily as edema, ulcers, and inflammation of the colon. While living and sanitary conditions have improved somewhat, dysentery remains a common and increasingly difficult disease to treat due to the overuse of antibiotics and the emergence of drug-resistant strains.

[0003] Shigella dysenteriae includes four species and 48 serotypes. In Africa and Asia, approximately 90% of bacillary dysentery cases are caused by Shigella flexneri and Shigella sonnei. Shigella flexneri 2a is the predominant serotype.

[0004] Polysaccharide-protein conjugate vaccines are designed to covalently bind polysaccharide antigens to carrier proteins through chemical methods. They can induce effective immune responses and immune memory in children ≤ 2 years old, the elderly, and those with immunodeficiency. The induced protective antibodies are primarily IgG, which is more effective than IgM induced by polysaccharide vaccines and can be sustained for a longer period of time, offering unparalleled advantages over polysaccharide vaccines. Polysaccharide-protein conjugate vaccines covalently bind polysaccharide antigens to carrier proteins through chemical methods. There are three main methods: direct conjugation, reductive amine method, and cyano group activation. With the development of conjugation technology, new conjugation methods have been developed based on these three methods. For example, the molecular arm adipic acid dihydrazide (ADH) is introduced between the polysaccharide and protein to promote binding, or new linking groups are introduced to promote the binding reaction.

[0005] At present, the commonly used carrier proteins used for conjugate vaccines in China are mainly tetanus toxoid (TT) and diphtheria toxoid (DT). The molecular weights of TT and DT are 151 kilodaltons (kD) and 58kD, respectively. Toxoids have been successfully used in a variety of polysaccharide-protein conjugate vaccines.

[0006] Shigella conjugate vaccines are a new approach to developing dysentery vaccines that emerged in the 1990s. Polysaccharide-protein conjugate vaccines, prepared by linking the Shigella dysenteriae O antigen to a protein carrier, are activated with cyanogen bromide. However, cyanogen bromide-activated and EDAC-mediated conjugates have shown poor protective efficacy. This may be because the cyanide activation process requires a pH of 10.5, an alkaline environment that affects the oxyacetyl groups on the polysaccharide, potentially impacting the vaccine's immune response. Furthermore, the conjugates exhibit a cross-linked network structure, resulting in a relatively large molecular weight, which hinders chromatographic purification and sterile filtration.

[0007] Research and development of vaccines targeting dysentery have been ongoing for decades; unfortunately, to date, no licensed vaccine has been developed. The present invention discloses a method for preparing a bivalent dysentery conjugate combination vaccine. The vaccine comprises two serotypes, 2a flexneri and 2a sonnei. The O-specific polysaccharides of the two bacteria are covalently bound to a carrier protein based on their unique chemical structures using corresponding chemical methods to prepare a polysaccharide-protein conjugate stock solution. The 2a flexneri dysentery polysaccharide is prepared using the reductive amine method. Because the polysaccharide structure does not contain carboxyl groups and cannot directly react with ADH or the carrier protein, but contains adjacent hydroxyl groups, the polysaccharide is first oxidized with sodium periodate to form an aldehyde group, which then reacts with one amino group of ADH to form a derivative. This is then reduced with sodium borocyanide to form a polysaccharide derivative. Finally, the other end of the ADH reacts with the active amino group of the carrier protein to form a stable covalent conjugate. Sonne's dysentery polysaccharide is prepared using a direct conjugation method. Sonne's dysentery polysaccharide contains a carboxyl group, which directly reacts with one amino group in ADH to form a polysaccharide derivative. Finally, the other end of ADH reacts with the active amino group of the carrier protein to form a stable covalent conjugate. The polysaccharide-protein conjugate concentrate is then adsorbed with an aluminum adjuvant in an appropriate ratio to produce a dysentery conjugate vaccine. Summary of the Invention

[0008] The present invention relates to a method for preparing a bivalent Shigella dysenteriae conjugate combination vaccine. The combination vaccine is composed of two Shigella dysenteriae polysaccharide-protein conjugates, wherein the conjugates are polysaccharide-protein conjugates prepared by selecting different conjugation methods between Shigella dysenteriae polysaccharides of corresponding serotypes and physiologically acceptable carrier proteins. The polysaccharide-protein conjugate stock solution is then adsorbed with an aluminum adjuvant in an appropriate proportion to obtain the Shigella dysenteriae conjugate vaccine. The conjugation method adopts two or more conjugation methods, including direct conjugation method, reductive amine method, cyano activation method and other methods.

[0009] Preferably, the dysentery combination vaccine is prepared from a combination of dysentery polysaccharide conjugates selected from Shigella flexneri serotype 2a and Shigella sonnei.

[0010] The preferred combination vaccine of the present invention is prepared as follows: wherein the conjugate is prepared from the dysentery 2a polysaccharide using the reductive amine method, and the conjugate is prepared from the dysentery sonnei polysaccharide using the direct conjugation method.

[0011] The preparation method of the combination vaccine of the present invention comprises the following steps:

[0012] (1) The flexneri 2a polysaccharide protein conjugate was prepared by the reductive amine method;

[0013] (2) Sonne's dysenteriae polysaccharide protein conjugates were prepared by direct conjugation method with spacer;

[0014] (3) Mix the 2a polysaccharide-protein conjugate of Escherichia coli and the polysaccharide-protein conjugate of E. sonnei, and adsorb them with an appropriate amount of aluminum phosphate adjuvant to obtain the vaccine.

[0015] Among them, (1) the flexneri 2a polysaccharide-protein conjugate is prepared as follows:

[0016] Step 1: The dysentery 2a polysaccharide is oxidized by sodium periodate, and then ultrafiltration and dialysis are performed to obtain the oxidized polysaccharide;

[0017] Step 2: Derivatizing the oxidized polysaccharide with adipic dihydrazide (ADH), and obtaining the polysaccharide derivative after ultrafiltration and dialysis;

[0018] Step 3: preparing a conjugate by reacting the polysaccharide derivative with the carrier protein under the action of carbodiimide (EDAC) condensation;

[0019] Step 4: The conjugate is purified by chromatography and sterilized by filtration to obtain the stock solution of the flexneri 2a polysaccharide-protein conjugate.

[0020] Preferably, (1) the flexneri 2a polysaccharide-protein conjugate is prepared as follows:

[0021] Step 1: After dissolving the dysentery 2a polysaccharide, sodium periodate is added to a concentration of 1-10 mmol / L, reacting for 1-24 hours, and then purified by ultrafiltration and dialysis to obtain oxidized polysaccharide;

[0022] Step 2: The oxidized polysaccharide is mixed with adipic dihydrazide (ADH), sodium borocyanide is added, and the pH is adjusted to about 4.5-6.0 to a final concentration of 10-80 mg / ml of ADH and 2-10 mg / ml of sodium borocyanide. The mixture is reacted at room temperature for 3 days, and purified by ultrafiltration and dialysis to obtain a polysaccharide derivative.

[0023] Step 3: The polysaccharide derivative is mixed with the protein, and carbodiimide (EDAC) is added to 0.02-0.05 mol / L, and the mixture is reacted for 120-240 minutes to prepare a conjugate.

[0024] Step 4: The conjugate was purified by Sepharose 4FF chromatography, and the elution peak near V0 was collected. After sterile filtration, the concentrate was obtained as the flexneri 2a dysenteriae polysaccharide-protein conjugate stock solution.

[0025] Among them, the preparation method of (2) the sonnei dysentery polysaccharide protein conjugate is as follows:

[0026] Step 1: Derivatizing the sonnei dysenteriae polysaccharide with adipic dihydrazide (ADH), and then ultrafiltration and dialysis to obtain a polysaccharide derivative;

[0027] Step 2: preparing a conjugate by reacting the polysaccharide derivative with the carrier protein under the action of carbodiimide (EDAC) condensation;

[0028] Step 3: The conjugate is purified by chromatography and sterilized by filtration to obtain the sonnei dysenteriae polysaccharide-protein conjugate stock solution.

[0029] Preferably, (2) the preparation method of the Sonne's dysentery polysaccharide-protein conjugate is as follows:

[0030] Step 1: After the sonnei dysentery polysaccharide is dissolved, 10-80 mg / ml ADH and 1-5 mg / ml EDAC are added and mixed evenly. The pH is adjusted to about 4.5-5.6. The mixture is reacted at room temperature for 120-240 minutes. The polysaccharide derivative is obtained after ultrafiltration purification.

[0031] Step 2: The polysaccharide derivative is mixed with the protein, EDAC is added to a concentration of 0.02 to 0.05 mol / L, and the mixture is reacted for 120 to 240 minutes to prepare a conjugate.

[0032] Step 3: The conjugate is purified by Sepharose 4FF chromatography, and the elution peak near V0 is collected. After sterile filtration, it is the sonnei dysenteriae polysaccharide-protein conjugate stock solution.

[0033] The polysaccharide-protein conjugate preparation method of the present invention uses the reductive amine method to prepare the conjugate of 2a dysenteriae polysaccharide. It is known that the structural composition of dysenteriae O-specific polysaccharide is composed of three Rha and one GlcNAc units in sequence. The specific chemical structure is as follows. The chemical reaction formula for the preparation of 2a dysenteriae polysaccharide conjugate is as follows:

[0034]

[0035] The method for preparing a polysaccharide-protein conjugate of the present invention, wherein the conjugate is prepared by a direct conjugation method using the sonnei dysentery polysaccharide. Among the known dysentery O-specific polysaccharide structures, the sonnei dysentery polysaccharide structural units are AltNAc and FucNAc, respectively. The specific chemical structure is as follows. The chemical reaction formula for preparing the sonnei dysentery polysaccharide conjugate is as follows:

[0036]

[0037] The polysaccharide-protein conjugate preparation methods described herein both involve adding a spacer (ADH) during the polysaccharide derivatization process to introduce amino groups. Finally, the conjugate is formed with a carrier protein under the action of EDAC condensation. The two methods differ only in the formation of the derivative. The conjugate is prepared using the reductive amination method for the 2a-Synaecium flexneri polysaccharide. The adjacent hydroxyl groups on the polysaccharide are oxidized with sodium periodate to form aldehyde groups. These groups then react with the amino group at one end of ADH to form a derivative. This is then reduced with sodium borocyanide to form a polysaccharide derivative. Finally, the other end of ADH reacts with an active amino group on the carrier protein to form a stable covalent conjugate. The conjugate is prepared using the direct conjugation method for the sonnei dysenteriae polysaccharide. Because the sonnei dysenteriae polysaccharide contains carboxyl groups, it directly reacts with the amino group at one end of ADH to form a polysaccharide derivative. Finally, the other end of ADH reacts with an active amino group on the carrier protein to form a stable covalent conjugate.

[0038] The ratio of ADH content to polysaccharide content in the 2a dysenteriae polysaccharide derivatives and the sonnei polysaccharide derivatives of the present invention is 20 to 60 μg / mg. The ADH content is controlled by adjusting the amount of ADH added in the derivatization reaction and process parameters such as the reaction pH, thereby avoiding excessive cross-linking of the polysaccharide-protein conjugate and ensuring smooth chromatographic purification and sterilization filtration.

[0039] The carrier proteins used in the present invention are physiologically acceptable carrier proteins, including diphtheria toxoid (DT) and its cross-reactive substance (CRM), tetanus toxoid (TT), pertussis toxoid (PT), pneumolysin (PLY), Haemophilus influenzae protein D, and outer membrane protein complex (OMPC). DT, TT, and CRM are preferred carrier proteins, with TT being most preferred.

[0040] In the unit dose of the combined vaccine of the present invention, the content of each type of dysentery polysaccharide is 0.1-50 μg, preferably 10 μg.

[0041] The present invention may comprise an adjuvant, wherein the adjuvant is selected from aluminum adjuvants, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc., preferably aluminum phosphate. The aluminum ion content in a unit dose of the combination vaccine is 0.1-0.3 mg.

[0042] The dysentery conjugate combination vaccine of the present invention can be prepared into various preparation forms for clinical use, wherein the preparation is selected from liquid preparation form and freeze-dried preparation form, and the liquid preparation form is preferred.

[0043] The dysentery conjugate combination vaccine of the present invention can be administered by intramuscular injection, subcutaneous injection and intradermal injection.

[0044] Most preferably, the preparation method of the present invention comprises the following steps:

[0045] (1) Preparation of conjugates of 2a dysentery polysaccharide by the reductive amine method: After dissolving 2a dysentery polysaccharide, sodium periodate was added for oxidation reaction, and the oxidized polysaccharide was obtained after ultrafiltration purification; the oxidized polysaccharide was mixed with 40 mg / ml adipic dihydrazide (ADH), 10 mg / ml sodium borocyanide was added, the pH was adjusted to around 6.0, and the reaction was carried out at room temperature for 3 days. The polysaccharide derivative was obtained after ultrafiltration purification; the polysaccharide derivative was mixed with the carrier protein TT, carbodiimide (EDAC) was added to 0.05 mol / L, and the reaction was carried out for 120 minutes to prepare the conjugate. The conjugate was purified by Sepharose 4FF chromatography, and the elution peak near V0 was collected. After sterile filtration, the 2a dysentery polysaccharide-protein conjugate stock solution was obtained.

[0046] (2) Preparation of a conjugate of Sonne's dysentery polysaccharide using a direct conjugation method with a spacer: After dissolving Sonne's dysentery polysaccharide, 40 mg / ml ADH and 0.05 mol / L EDAC were added and mixed. The pH was adjusted to around 5.6, and the mixture was reacted at room temperature for 120 minutes. The polysaccharide derivative was obtained after ultrafiltration purification. The polysaccharide derivative was mixed with the carrier protein TT, and EDAC was added to 0.05 mol / L. The mixture was reacted for 120 minutes to prepare a conjugate. The conjugate was purified by Sepharose 4FF chromatography, and the elution peak near V0 was collected. After sterile filtration, the solution of Sonne's dysentery polysaccharide-protein conjugate was obtained.

[0047] (3) The stock solutions of the 2a dysentery polysaccharide-protein conjugate and the sonnei dysentery polysaccharide-protein conjugate are mixed, and adsorbed with an appropriate amount of aluminum phosphate adjuvant to prepare a semi-finished product with a content of 20 μg / ml of each type of dysentery polysaccharide and an aluminum ion content not higher than 0.6 mg / ml, and packaged in 0.5 ml / vial to prepare the dysentery bivalent combination vaccine.

[0048] The beneficial effects of the present invention are further illustrated by experimental data below:

[0049] Immunogenicity of combined vaccine against dysentery of flexneri and sonnei in mice

[0050] NIH mice were divided into a normal saline negative control group, a Sonneli dysenteriae polysaccharide group (before immunization), a flexneri 2a polysaccharide group (before immunization), and a combination vaccine group. Three subcutaneous immunizations were performed, two weeks apart, with a dose of 0.1 ml. Blood was collected 14 days after each immunization, serum was separated, and specific serum titers were determined. The average A450 value of the serum from the negative control mice was calculated at a dilution of 1 / 100. The cutoff value was 2.1 times the average A value of the negative control. Samples with an A value greater than the cutoff value were considered positive, and the highest dilution was the antibody titer of the serum. The mouse immunization experiment compared the immune response to the polysaccharide and combination vaccines before and after immunization, as well as the positive conversion rate (see Table 1).

[0051] Table 1 Immune responses of mice to combined vaccines and dysentery polysaccharides of dysentery fowleri

[0052]

[0053] Mouse immunization experiments revealed that, in the combination vaccine of the present invention, the antibody levels of the dysentery polysaccharide and conjugate combination vaccines after immunization showed that the geometric mean titer (GMT) of the antibodies produced after immunization with the dysentery polysaccharide was relatively low, while the GMT in the serum of mice increased to varying degrees after the first, second, and third doses of the conjugate combination vaccine. After the first dose, the positive conversion rate was 80%, and after the second dose, the positive conversion rate was 100%.

[0054] Compared with the existing preparation methods, the present invention has the following advantages:

[0055] (1) The present invention no longer uses the highly toxic reagent cyanogen bromide and high activation pH, thus avoiding the use of highly toxic reagents and ensuring the safety of operators; it also avoids the potential impact of high pH on the immune effect of the vaccine. The method has a mild reaction and is easy to control, which is conducive to future large-scale production.

[0056] (2) The reaction route is designed based on the chemical structure of the polysaccharide, which has a clear purpose, reduces the occurrence of side reactions, and is conducive to the production of the target product.

[0057] (3) ADH was used as a spacer and its content was controlled. The ADH content directly affects the degree of cross-linking of the polysaccharide protein; the higher the ADH content, the greater the degree of cross-linking. To avoid excessive cross-linking of the polysaccharide protein conjugate, the ADH content was controlled to ensure smooth ultrafiltration purification, chromatography purification, and sterile filtration, making it easier to scale up production.

[0058] (4) After the oxidation reaction or derivatization reaction, the product is directly subjected to ultrafiltration purification and stored in liquid form at 2-8°C, eliminating the original steps of membrane dialysis, column chromatography, freeze-drying, etc., saving time and cost. DETAILED DESCRIPTION

[0059] The present invention is further described below by way of examples. This example is merely an example and one of the solutions of the present invention, and the present invention is not limited to the examples.

[0060] Example 1: Preparation of conjugates using the reductive amine method using 2a dysenteriae polysaccharide

[0061] After dissolving the 2a dysenteriae polysaccharide, sodium periodate was added for oxidation reaction, and ultrafiltration was performed to obtain the oxidized polysaccharide. The oxidized polysaccharide was mixed with 40 mg / ml adipic dihydrazide (ADH), 10 mg / ml sodium borocyanide was added, and the pH was adjusted to around 6.0. The mixture was reacted at room temperature for 3 days, and ultrafiltration was performed to obtain the polysaccharide derivative. The polysaccharide derivative was mixed with protein, and carbodiimide (EDAC) was added to 0.05 mol / L, and the reaction was carried out for 120 minutes to prepare the conjugate. The conjugate was purified by Sepharose 4FF chromatography, and the peak eluting near V0 was collected. After sterile filtration, the 2a dysenteriae polysaccharide-protein conjugate solution was obtained.

[0062] Example 2: Preparation of conjugates using a direct conjugation method with a spacer for Dysenteria sonnei polysaccharide

[0063] After dissolving the D. sonnei polysaccharide, 40 mg / ml ADH and 0.05 mol / L EDAC were added and mixed. The pH was adjusted to around 5.6, and the mixture was reacted at room temperature for 120 minutes. The polysaccharide derivative was purified by ultrafiltration. The polysaccharide derivative was mixed with the protein, and EDAC was added to 0.05 mol / L, and the mixture was reacted for 120 minutes to prepare the conjugate. The conjugate was purified by Sepharose 4FF chromatography, and the peak eluting near V0 was collected. After sterile filtration, the D. sonnei polysaccharide-protein conjugate solution was obtained.

[0064] Example 3: Detection results of ADH content in 2a dysenteriae polysaccharide derivatives and sonnei dysenteriae polysaccharide derivatives

[0065] The polysaccharide derivatives containing the spacer ADH were obtained after oxidation and derivatization of the 2a polysaccharide of Fusarium flexneri. The polysaccharide derivatives containing the spacer ADH were obtained after derivatization of the sonnei dysentery polysaccharide. The content of ADH was controlled by the process parameters in the derivatization reaction. Three batches of polysaccharide derivatives were prepared for each type. The test results of each batch of polysaccharide derivatives are shown in Table 2.

[0066] Table 2 Detection results of ADH content in 2a dysenteriae polysaccharide derivatives and sonnei dysenteriae polysaccharide derivatives

[0067]

[0068] Example 4: Test results of Shigella flexneri 2a polysaccharide-protein conjugate and Shigella sonnei polysaccharide-protein conjugate

[0069] The conjugates of the dysentery polysaccharide of flexneri 2a were prepared by the reductive amine method, and the conjugates of the dysentery polysaccharide of sonnei were prepared by the direct conjugation method. Three batches of conjugates were prepared for each type. The test results of each batch of conjugates are shown in Tables 3 and 4.

[0070] Table 3 Test results of Shigella flexneri 2a polysaccharide protein conjugate

[0071]

[0072]

[0073] Table 4 Sonnei's dysentery polysaccharide protein conjugate test results

[0074]

[0075] Example 5: Preparation of a bivalent combination vaccine for dysentery

[0076] The original solutions of dysentery polysaccharide-protein conjugate of 2a flexneri and dysentery polysaccharide-protein conjugate are mixed in a certain proportion, adsorbed with an appropriate amount of aluminum phosphate adjuvant, and prepared into a semi-finished product with each type of dysentery polysaccharide at 20ug / ml and an aluminum ion content not higher than 0.6mg / ml, and packaged in 0.5ml / vial to prepare the dysentery bivalent combination vaccine of flexneri and dysentery.

[0077] Example 6: Test results of the bivalent combination vaccine for dysentery

[0078] Three batches of finished products of the bivalent combination vaccine for dysentery with flexneri and sonnei were produced consecutively and tested, see Table 5.

[0079] Table 5 Test results of finished product of dysentery bivalent combination vaccine

[0080]

[0081] Example 7: Immunogenicity of the bivalent combination vaccine against dysentery in mice

[0082] NIH mice were divided into a saline negative control group, a Sonneli dysenteriae polysaccharide group, a flexneri 2a polysaccharide group, and three combined vaccine groups. Three subcutaneous immunizations were administered, two weeks apart, with a dose of 0.1 ml. Blood was collected 14 days after each immunization, serum was separated, and specific serum titers were determined. The average A450 value at a 1 / 100 dilution of serum from the negative control mice was used to determine the mean. The cutoff value was 2.1 times the average A value of the negative control. Samples with an A value greater than the cutoff value were considered positive, and the highest dilution was used as the antibody titer of the serum. The mouse immunization experiment compared the immune responses to the polysaccharide and combined vaccines before and after immunization, as well as the positive conversion rate.

[0083] Table 6 Immune responses of mice to three consecutive batches of dysentery bivalent combination vaccine and dysentery polysaccharide

[0084]

[0085] Mouse immunization experiments revealed that, in the combination vaccine of the present invention, the antibody levels of the dysentery polysaccharide and conjugate combination vaccines after immunization showed that the geometric mean titer (GMT) of the antibodies produced after dysentery polysaccharide immunization was relatively low, while the GMT in the serum of mice increased to varying degrees after the first, second, and third doses of the conjugate combination vaccine. After the second dose, the positive conversion rate was 90%, and after the third dose, the positive conversion rate was 100%.

[0086] Example 8, control test

[0087] The preparation method of the flexneri 2a derivative in the document "Preparation and Immunological Properties of Shigella flexneri 2a O-Specific Polysaccharide and Tetanus Toxoid Conjugate Vaccine" (Progress in Microbiology and Immunology, Vol. 29, No. 1, 2001, pp. 1-7) is as follows: the polysaccharide is dissolved, the pH is adjusted to 11.0±0.2 with alkali, an equal weight of CNBr is added, and the reaction is carried out for 6 minutes; an equal volume of 0.5 mol / L ADH is added, and the pH is maintained at 8.5±0.2. The reactants are kept at 2-8°C overnight and then dialyzed at 2-8°C for 2 days; the reactants are concentrated by ultrafiltration and passed through a chromatography column, and the elution peak within the V0 volume is collected and dialyzed in water for injection at 2-8°C for 2 days. The dialyzate is taken out and lyophilized to obtain the derivative, which is then sealed and stored at -20°C.

[0088] Domestic and foreign literature use the above method to prepare flexneri derivatives. The preparation method of the present invention is as follows: after dissolving flexneri 2a dysentery polysaccharide, sodium periodate is added for oxidation reaction, and the oxidized polysaccharide is obtained by ultrafiltration purification; the oxidized polysaccharide is mixed with 40 mg / ml of adipic dihydrazide (ADH), 10 mg / ml of sodium borocyanide is added, the pH is adjusted to approximately 6.0, and the reaction is carried out at room temperature for 3 days. The polysaccharide derivative is obtained by ultrafiltration purification.

[0089] The present invention has the following advantages: (1) the use of highly toxic reagents such as cyanogen bromide and high activation pH is no longer required, thus avoiding the use of highly toxic reagents and ensuring the safety of operators; the potential influence of high pH on the immune effect of the vaccine is avoided, the method has a mild reaction and is easy to control, and is conducive to large-scale production in the future; (2) after the oxidation reaction and the derivatization reaction, ultrafiltration technology is directly used for dialysis purification, and the liquid state is stored at 2 to 8°C, eliminating the original membrane dialysis, column chromatography, freeze-drying and other steps, saving time and cost.

[0090] Example 9: Clinical trial immunogenicity

[0091] A Phase II clinical trial was conducted on a bivalent combination vaccine for dysentery caused by dysentery. Since there is no registered and marketed dysentery vaccine, no control group was set up. The subjects of the clinical efficacy trial of the bivalent dysentery combination vaccine obtained by the present invention are infants and young children aged 3 months to 5 years. Three age groups were set up: 3 to 6 months (3 injections), 6 to 12 months (2 injections), and 1 to 5 years (1 injection), and a placebo was also provided.

[0092] No serious adverse reactions occurred in all vaccine recipients. Blood samples were collected 30 days after vaccination to test the positive conversion rate and IgG antibody concentration (GMC) of serum IgG antibodies against two dysentery polysaccharides, which produced immune responses of varying degrees, indicating that the vaccine is safe and immunogenic in infants and young children.

[0093] Table 7 IgG antibody positive conversion rate and IgG antibody concentration GMC (EU / mL) in three age groups

[0094]

[0095] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing a bivalent dysentery conjugate combination vaccine, characterized in that: The combined vaccine is composed of two polysaccharide-protein conjugates of Shigella dysenteriae. The conjugates are prepared by combining the corresponding serotypes of Shigella dysenteriae polysaccharides with carrier proteins through different binding methods. The polysaccharide-protein conjugate stock solution is then adsorbed with aluminum adjuvant in an appropriate proportion to obtain the Shigella dysenteriae combined vaccine. The carrier protein is tetanus toxoid; The bivalent Shigella conjugate combination vaccine is prepared by combining the Shigella dysenteriae polysaccharide-protein conjugates of Shigella dysenteriae serotype 2a and Shigella sonnei. The conjugates of 2a dysenteriae polysaccharide were prepared by the reductive amine method, and the conjugates of sonnei dysenteriae polysaccharide were prepared by the direct conjugation method with a spacer. The preparation method of the 2a dysenteriae polysaccharide conjugate stock solution is as follows: after dissolving the 2a dysenteriae polysaccharide, sodium periodate is added for oxidation reaction, and the oxidized polysaccharide is obtained by ultrafiltration purification; the oxidized polysaccharide is mixed with 40 mg / mL adipic dihydrazide, 10 mg / mL sodium borocyanide is added, the pH is adjusted to 6.0, and the reaction is carried out at room temperature for 3 days, and the polysaccharide derivative is obtained by ultrafiltration purification; the polysaccharide derivative is mixed with protein, carbodiimide (EDAC) is added to 0.05 mol / L, and the reaction is carried out for 120 minutes to prepare the conjugate; The preparation method of the sonnei dysenteriae polysaccharide conjugate stock solution is as follows: after dissolving the sonnei dysenteriae polysaccharide, 40 mg / mL of adipic dihydrazide and 0.05 mol / L of EDAC are added and mixed evenly, the pH is adjusted to 5.6, the mixture is reacted at room temperature for 120 minutes, and the polysaccharide derivative is obtained after ultrafiltration purification; the polysaccharide derivative is mixed with protein, EDAC is added to 0.05 mol / L, and the mixture is reacted for 120 minutes to prepare the conjugate; In the combination vaccine, the sodium chloride content is 7.5-10.0 g / L, the aluminum ion content is 0.2-0.6 mg / mL, the content of various types of dysentery polysaccharides is 20±6 μg / mL, and the pH value of the combination vaccine is 5.0-7.

0.

2. The preparation method according to claim 1, wherein the 2a flexneri polysaccharide conjugate is purified by Sepharose 4FF chromatography, and the elution peak near V0 is collected and sterilized and filtered to obtain the 2a flexneri polysaccharide-protein conjugate stock solution.

3. The preparation method according to claim 1, wherein the sonnei dysenteriae polysaccharide conjugate is purified by Sepharose 4FF chromatography, and the elution peak near V0 is collected and sterilized and filtered to obtain the sonnei dysenteriae polysaccharide conjugate stock solution.

4. The preparation method according to claim 1, characterized in that Here are the steps: (1) The flexneri 2a polysaccharide protein conjugate was prepared by the reductive amine method; (2) Sonne's dysenteriae polysaccharide protein conjugates were prepared by direct conjugation method with spacer; (3) Mix the 2a polysaccharide-protein conjugate of Escherichia coli and the polysaccharide-protein conjugate of E. sonnei, and adsorb them with an appropriate amount of aluminum phosphate adjuvant to obtain the vaccine.

5. The preparation method according to any one of claims 1 to 4, characterized in that The content of adipic dihydrazide in the flexneri 2a polysaccharide-protein conjugate and the sonnei dysenteriae polysaccharide-protein conjugate is 20-60 μg / mg.

6. The bivalent dysentery conjugate combination vaccine prepared according to the preparation method of any one of claims 1 to 5.

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